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Sample records for mitomycin c-treated cryopreserved

  1. Enhanced healing of mitomycin C-treated healing-impaired wounds in rats with PRP-containing fragmin/protamine microparticles (PRP&F/P MPs).

    Science.gov (United States)

    Takikawa, Megumi; Ishihara, Masayuki; Takabayashi, Yuki; Sumi, Yuki; Takikawa, Makoto; Yoshida, Ryuichi; Nakamura, Shingo; Hattori, Hidemi; Yanagibayashi, Satoshi; Yamamoto, Naoto; Kiyosawa, Tomoharu

    2015-04-13

    The purpose of this study was to evaluate the accelerating effects of platelet-rich plasma-containing (PRP&) fragmin/protamine microparticles (F/P MPs) for repairing mitomycin C-treated healing-impaired wounds. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL-staining) showed that apoptosis of dermal fibroblast cells (DFCs) and epidermal keratinocyte cells (EKCs) were significantly induced in the skin of the mitomycin C-treated rats. Full-thickness skin defects were made on the back of rats and mitomycin C was applied on the wounds to prepare a healing-impaired wound. After washing out the mitomycin C, saline (control), F/P MPs alone, PRP alone, and PRP&F/P MPs were injected around the wounds. The rats were later euthanised and histological sections of the wounds were then prepared at indicated time periods after the treatment. These results indicated the numbers of large, medium, and small capillary lumens 7 days after injection of PRP&F/P MPs were significantly higher than those after injection of PRP or F/P MPs alone. Furthermore, epithelium and granulation tissue formations were significantly stimulated in the healing-impaired wounds treated with PRP&F/P MPs 3, 7 and 14 days after injection of PRP&F/P MPs.

  2. Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin C-treated monkey cells

    International Nuclear Information System (INIS)

    Roilides, E.; Munson, P.J.; Levine, A.S.; Dixon, K.

    1988-01-01

    When monkey cells were treated with mitomycin C 24 h before transfection with UV-irradiated pZ189 (a simian virus 40-based shuttle vector), there was a twofold increase in the frequency of mutations in the supF gene of the vector. These results suggest the existence of an enhancible mutagenesis pathway in mammalian cells. However, DNA sequence analysis of the SupF- mutants suggested no dramatic changes in the mechanisms of mutagenesis due to mitomycin C treatment of the cells

  3. Toll-like receptor 6 and connective tissue growth factor are significantly upregulated in mitomycin-C-treated urothelial carcinoma cells under hydrostatic pressure stimulation.

    Science.gov (United States)

    Chen, Shao-Kuan; Chung, Chih-Ang; Cheng, Yu-Che; Huang, Chi-Jung; Chen, Wen-Yih; Ruaan, Ruoh-Chyu; Li, Chuan; Tsao, Chia-Wen; Hu, Wei-Wen; Chien, Chih-Cheng

    2014-06-01

    Urothelial carcinoma (UC) is the most common histologic subtype of bladder cancer. The administration of mitomycin C (MMC) into the bladder after transurethral resection of the bladder tumor (TURBT) is a common treatment strategy for preventing recurrence after surgery. We previously applied hydrostatic pressure combined with MMC in UC cells and found that hydrostatic pressure synergistically enhanced MMC-induced UC cell apoptosis through the Fas/FasL pathways. To understand the alteration of gene expressions in UC cells caused by hydrostatic pressure and MMC, oligonucleotide microarray was used to explore all the differentially expressed genes. After bioinformatics analysis and gene annotation, Toll-like receptor 6 (TLR6) and connective tissue growth factor (CTGF) showed significant upregulation among altered genes, and their gene and protein expressions with each treatment of UC cells were validated by quantitative real-time PCR and immunoblotting. Under treatment with MMC and hydrostatic pressure, UC cells showed increasing apoptosis using extrinsic pathways through upregulation of TLR6 and CTGF.

  4. Enhanced mutagenesis of UV-irradiated simian virus 40 occurs in mitomycin C-treated host cells only at a low multiplicity of infection

    International Nuclear Information System (INIS)

    Sarasin, A.; Benoit, A.

    1986-01-01

    Treatment of monkey kidney cells with mitomycin C (MMC) 24 h prior to infection with UV-irradiated simian virus 40 (SV40) enhanced both virus survival and virus mutagenesis. The use of SV40 as a biological probe has been taken as an easy method to analyse SOS response of mammalian cells to the stress caused by DNA damage or inhibition of DNA replication. The mutation assay we used was based on the reversion from a temperature-sensitive phenotype (tsA58 mutant) to a wild-type phenotype. The optimal conditions for producing enhanced survival and mutagenesis in the virus progeny were determined with regard to the multiplicity of infection (MOI). Results showed that the level of enhanced mutagenesis observed for UV-irradiated virus grown in MMC-treated cells was an inverse function of the MOI, while enhanced survival was observed at nearly the same level regardless of the MOI. For the unirradiated virus, almost no increase in the mutation of virus progeny issued from MMC-treated cells was observed, while a small amount of enhanced virus survival was obtained. These results show that enhanced virus mutagenesis and enhanced virus survival can be dissociated under some experimental conditions. Enhanced virus mutagenesis, analogous to the error-prone replication of phages in SOS-induced bacteria, was observed, at least for SV40, only when DNA of both virus and host cells was damaged and when infection occurred with a small number of viral particles. We therefore hypothesize that an error-prone replication mode of UV-damaged templates is observed in induced monkey kidney cells

  5. Transconjunctival penetration of mitomycin C

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    Velpandian T

    2008-01-01

    Full Text Available Aims: The study was performed to estimate transconjunctival penetration of mitomycin C (MMC to Tenon′s tissue following application over the intact conjunctiva before routine trabeculectomy. Settings and Design: Institution-based case series. Materials and Methods: In 41 eyes of 41 patients, MMC (0.4 mg/ml for 3 min was applied over the intact conjunctiva before beginning trabeculectomy. Tenon′s capsule directly beneath the site of application was excised during trabeculectomy and was homogenized, centrifuged and MMC concentrations were analyzed using high-performance liquid chromatography (HPLC. Statistical Analysis Used: Statistical analysis was performed using stata0 8.0 version software (STATA Corporation, Houston, TX, USA. In this study, P -values less than 0.05 were considered as statistically significant. Results: The average weight of the sample of Tenon′s tissue excised was 5.51 ± 4.42 mg (range: 0.9-17.1 and the average estimated MMC concentration found to be present in Tenon′s tissue using HPLC was 18.67 ± 32.36 x 10−6 moles/kg of the tissue (range: 0.38-197.05 x 10−6 . In 36 of the 41 patients (87.80%, the MMC concentration reached above 2 x 10−6 moles/kg of the tissue concentration required to inhibit human conjunctival fibroblasts. Conclusions: Mitomycin C does permeate into the subconjunctival tissue after supraconjunctival application for 3 min. Application of MMC over the conjunctiva may be a useful alternative to subconjunctival or subscleral application during routine trabeculectomy and as an adjunct for failing blebs.

  6. Cryopreservation of crane semen

    Science.gov (United States)

    Gee, G.F.; Harris, James

    1991-01-01

    The method for the cryopreservation of crane semen at Patuxent Wildlife Research Center is described in detail. Cryopreservation is useful for the long-term storage of crane semen and for specialized propagation needs. A 50% fertility rate from most sandhill cranes, Grus canadensis, inseminated with frozen-thawed semen can be expected. Additional research should improve the fertility rate and determine how applicable the technique is to other crane species.

  7. Trabeculectomy with mitomycin-c

    International Nuclear Information System (INIS)

    Baig, M.S.A.; Ali, M.A.

    2008-01-01

    To study the efficacy and safety of trabeculectomy augmented with intra-operative application of Mitomycin- C (MMC) in various types of refractory glaucomas. Forty patients with a number of refractory glaucomas who underwent trabeculectomies with MMC. MMC 0.02% (0.2mg/ml) was applied to 52 eyes of 40 patients being operated for trabeculectomies under the partial thickness scleral flap with an exposure time of three minutes. Visual outcome, control of intraocular pressure (IOP) and complications were evaluated. There were 25 males and 15 females in the study with an average age of 41.3 (range 1-65) years and an average follow up of 26.4 months (range 12-60 months). The average preoperative IOP was 32.5mm Hg (range 24- 52 mmHg) on an average of 1.34 medications, and the mean postoperative IOP after one year in successful cases was 14.42mmHg (range 12-18 mm Hg) without any medication (qualified success) in 45(86.5%) cases; five patients required one medication. Early postoperative complications included hypotony of more than one month duration in 11(21.2%) cases, hyphema of more than one week duration in 7(13.5%) and epithelial defect in 3(5.76%) cases. Late complication included cataract in 21(40.4%) cases, uncontrolled intraocular pressure despite medical therapy in two (3.84%) cases who further underwent Ahmed glaucoma valve implantation. One case (1.92%) of hypotonous maculopathy was noted. Trabeculectomy with MMC is a promising, safe and effective modality in the management of refractory glaucomas. As the complications related to bleb thinning increases with time, hence further study is required to determine the long term complications. (author)

  8. Cryopreservation of Human Mucosal Leukocytes.

    Directory of Open Access Journals (Sweden)

    Sean M Hughes

    Full Text Available Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible.To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension.Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

  9. [Cryopreservation of teeth].

    Science.gov (United States)

    Zimmerli, Melanie; Filippi, Andreas

    2010-01-01

    After tooth loss dental implants or fixed prosthetic restorations are not indicated in children and adolescents due to incomplete maxillary and mandibular development. Cryopreservation is a method for long-term storage of healthy teeth which were removed for orthodontic reasons or due to traumatic origin. These preserved teeth can be used as autogenous replants or transplants after tooth loss. During transport to and from the freezing facilities prior to freezing the teeth are stored in a cell culture medium. The tooth is transferred into a freezing tube containing cell culture medium and cryoprotectant DMSO. Teeth autotransplanted after cryopreservation show vitality of the PDL cells. Usually no enamel and/or dentinal cracks can be observed. After tooth loss transplantation of cryopreserved teeth could be an effective and biological therapy for tooth replacement.

  10. Cryopreservation of oocytes

    International Nuclear Information System (INIS)

    Jadoon, S.

    2015-01-01

    Various approaches have been utilized in attempting to cryopreserve oocytes, beginning with slow cooling and more recently the advent of technique of vitrification. Now it seems that oocyte cryopreservation is no longer an experimental technique and it is being increasingly utilized in clinics around the world. As successful outcome in oocyte cryopreservation can be assessed by survival through the freeze-thaw process, potential for fertilization, embryo development and dynamics of meiotic spindles. This study aimed to analyse these features in context of vitrification and slow freezing. Methods: In this laboratory based study, mature MII mouse oocytes from F1(C57BL6/J X CBA) mice (n=43) were divided randomly into two groups of equal numbers and were cryopreserved by slow freezing and by vitrification. Upon re-warming these oocytes were assessed for survival and for fertilization potential. Oocytes were fixed and stained to compare the effect of both protocols on spindle reassembly and chromosome configuration 10min, 1h and 3h after warming. Unfrozen oocytes were used as controls. Results: A greater number of vitrified oocytes survived cryopreservation than slow frozen oocytes (70.3% vs. 12.5%, p=0.024). After insemination, fertilization rates were higher for vitrified oocytes as compared to slow frozen oocytes (15.86% vs. 4.6%, p=0.046). Morphology of the meiotic spindle was found to be in a disorganized configuration in slow frozen oocytes at all-time points 10 mins, 1 h and 3h), whereas in vitrified oocytes the spindles were found to be aligned at all-time points. Chromosomes were seen to be displaced from equatorial region in both groups. Conclusion: Cryopreservation of mouse oocytes was conducted with greater success using vitrification, compared to slow freezing, with survival, fertilization, and spindle assembly more favourable to a successful outcome in this model. (author)

  11. Cryopreservation of Parathyroid Glands

    Directory of Open Access Journals (Sweden)

    Marlon A. Guerrero

    2010-01-01

    Full Text Available The risk of permanent hypoparathyroidism following thyroid and parathyroid surgery is around 1% in the hands of experienced endocrine surgeons. Although this complication is rare, rendering a patient permanently aparathyroid has significant consequences on the health and quality of life of the patient. Immediate autotransplantation of parathyroid glands that are injured or unintentionally removed offers the best possibility of graft viability and functionality. However, since the majority of cases of hypoparathyroidism are transient, immediate autotransplantation can complicate postoperative surveillance in certain patients, especially those with primary hyperparathyroidism. Cryopreservation of parathyroid tissue is an alternate technique that was developed to treat patients with permanent hypoparathyroidism. This method allows for parathyroid tissue to be stored and then autotransplanted in a delayed fashion once permanent hypoparathyroidism is confirmed. This article provides a contemporary review on cryopreservation of parathyroid tissue and its current role in thyroid and parathyroid surgery.

  12. Structural characterization of the mitomycin 7-O-methyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Shanteri; Chang, Aram; Goff, Randal D.; Bingman, Craig A.; Grüschow, Sabine; Sherman, David H.; Phillips, Jr., George N.; Thorson, Jon S. (Michigan); (UW)

    2014-10-02

    Mitomycins are quinone-containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9{beta}- and C9{alpha}-configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR-S-adenosylhomocysteine (SAH) binary complex and MmcR-SAH-mitomycin A (MMA) ternary complex at resolutions of 1.9 and 2.3 {angstrom}, respectively. The study revealed MmcR to adopt a common S-adenosyl-L-methionine-dependent O-methyltransferase fold and the presence of a structurally conserved active site general acid-base pair is consistent with a proton-assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins.

  13. Embryo cryopreservation and preeclampsia risk.

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    Sites, Cynthia K; Wilson, Donna; Barsky, Maya; Bernson, Dana; Bernstein, Ira M; Boulet, Sheree; Zhang, Yujia

    2017-11-01

    To determine whether assisted reproductive technology (ART) cycles involving cryopreserved-warmed embryos are associated with the development of preeclampsia. Retrospective cohort study. IVF clinics and hospitals. A total of 15,937 births from ART: 9,417 singleton and 6,520 twin. We used linked ART surveillance, birth certificate, and maternal hospitalization discharge data, considering resident singleton and twin births from autologous or donor eggs from 2005-2010. We compared the frequency of preeclampsia diagnosis for cryopreserved-warmed versus fresh ET and used multivariable logistic regression to adjust for confounders. Among pregnancies conceived with autologous eggs resulting in singletons, preeclampsia was greater after cryopreserved-warmed versus fresh ET (7.51% vs. 4.29%, adjusted odds ratio = 2.17 [95% CI 1.67-2.82]). Preeclampsia without and with severe features, preeclampsia with preterm delivery, and chronic hypertension with superimposed preeclampsia were more frequent after cryopreserved-warmed versus fresh ET (3.99% vs. 2.55%; 2.95% vs. 1.41%; 2.76 vs. 1.48%; and 0.95% vs. 0.43%, respectively). Among pregnancies from autologous eggs resulting in twins, the frequency of preeclampsia with severe features (9.26% vs. 5.70%) and preeclampsia with preterm delivery (14.81% vs. 11.74%) was higher after cryopreserved versus fresh transfers. Among donor egg pregnancies, rates of preeclampsia did not differ significantly between cryopreserved-warmed and fresh ET (10.78% vs. 12.13% for singletons and 28.0% vs. 25.15% for twins). Among ART pregnancies conceived using autologous eggs resulting in live births, those involving transfer of cryopreserved-warmed embryos, as compared with fresh ETs, had increased risk for preeclampsia with severe features and preeclampsia with preterm delivery. Copyright © 2017 American Society for Reproductive Medicine. All rights reserved.

  14. Cryopreservation of mammalian semen.

    Science.gov (United States)

    Curry, Mark R

    2007-01-01

    Mammalian spermatozoa were among the very first cells to be successfully cryopreserved and over the last five decades the use of frozen-thawed semen for artificial insemination has come to play an important role in domestic livestock production. More recently, semen freezing has increasingly been utilized in the establishment of genetic resource banks for endangered species. Semen is collected, most commonly either by use of an artificial vagina or by electroejaculation of an anaesthetized animal, and basic sperm parameters assessed. Semen is extended using a TEST-egg yolk-glycerol diluent, packaged in 0.25-mL plastic straws and slowly cooled to 5 degrees C over a period of 1-2 h. Cooled straws are frozen by suspending within liquid nitrogen vapor above the liquid nitrogen surface before plunging into the liquid phase. Straws are thawed briefly in air before immersing in a 35 degrees C water bath for 15 s, and often are used directly for insemination without any further processing.

  15. Teratogenic interactions between methylmercury and mitomycin-C in mice

    Energy Technology Data Exchange (ETDEWEB)

    Inouye, Minoru; Kajiwara, Yuji

    1988-01-01

    Pregnant mice were given p.o. various nonteratogenic doses (0, 2.5 and 10 mg/kg) of methylmercuric chloride on day 9 of pregnancy, and then injected i.p. with a teratogenic dose (4 mg/kg) of mitomycin-C on day 10. Major malformations produced by mitomycin-C alone were cervical rib and vertebral anomaly, polydactyly of the hindlimb and tail anomaly. Combined treatment significantly increased the incidence of these malformations, showing the dose-effect relationship of methylmercury, whereas methylmercury alone is known not to produce such malformations. When mitomycin-C treatment alone was performed on day 9.5 of pregnancy, only vertebral anomalies increased in incidence. Therefore, mitomycin-C teratogenicity in terms of the manifestation of cervical rib, polydactyly and tail anomaly, but not vertebral anomaly, was suggested to be enhanced by methylmercury. A considerable number of foetuses showed cleft palate involvement following combined treatments, but not by either chemical alone. Cleft palate is known to be a major malformation in mice that is caused by methylmercury, and mitomycin-C also induces cleft palate. Therefore, the two chemicals might have affected foetuses additively and thereby induced cleft palate. (orig.)

  16. The mitogenic response of cryopreserved human lymphocytes in a microculture system.

    Science.gov (United States)

    Steel, C M; Ennis, M; Levin, A G; Wasunna, A

    1977-01-01

    Fresh blood lymphocytes from nine health donors have been compared with samples from the same donors, recovered after period of 2 to 21 months storage in liquid nitrogen, for the capacity to respond to a range of mitogens in vitro. A microculture assay was used, requireing aliquots of only 25,000 cells. The mean levels of 14C-thymidine uptake for fresh and frozen samples were closely comparable when the cells had been stimulated by PHA, Pokeweed or mitomycin-C-treated allogeneic lymphoblastoid cells. Lymphocytes from six East African donors, frozen by a very simple technique, were recovered after 3 or more years storage in liquid nitrogen. Five of the samples were in good condition as judged by cell viability and the capacity to form spontaneous 'E' rosettes with sheep erythrocytes. These five samples also responded extremely well to PHA, PWM and mitomycin-C-treated allogeneic lymphoblastoid cells using the microculture assay. This study extends the range of applications of cell banks in which small aliquots of blood lymphocytes are stored in liquid nitrogen for periods of several years.

  17. Uses and complications of mitomycin C in ophthalmology.

    Science.gov (United States)

    Mearza, Ali A; Aslanides, Ioannis M

    2007-01-01

    Mitomycin C is a chemotherapeutic agent that acts by inhibiting DNA synthesis. Its use and application in ophthalmology has been increasing in recent years because of its modulatory effects on wound healing. Current applications include pterygium surgery, glaucoma surgery, corneal refractive surgery, cicatricial eye disease, conjunctival neoplasia and allergic eye disease. Although it has been used successfully in these conditions, it has also been associated with significant complications. This article reviews the current trends and uses of mitomycin C in the eye and its reported complications.

  18. Repair system and mitomycin mutagenesis in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Otsuji, N; Murayama, I [Kyushu Univ., Fukuoka (Japan). Faculty of Pharmaceutical Sciences

    1974-03-01

    Ultraviolet light sensitive mutants of E. coli defective at the uvrA, uvrB or uvrC locus showed increased sensitivity to the lethal effects of monofunctional mitomycin, 7-methoxymitosene (7-MMT) or decarbamoyl mitomycin C (DCMTC), as well as mitomycin C (MTC). (1) Treatment of wild type bacteria with these monofunctional mitomycins resulted in the production of single-strand breaks in DNA, which were repaired upon incubation in a growth medium. Such breaks in DNA were not produced in the UvrA and the UvrB mutants. In the UvrC mutant, single-strand breaks were produced by 7-MMT or by DCMTC, but these breaks were not repaired upon incubation. (2) Exposure of E. coli to MTC caused cross-linkage between DNA strands, which converted to a normally denaturable form during further incubation after treatment. This was not occurred in the UvrB strain. In the UvrC mutant, a portion of the cross-links was removed upon incubation. (3) 7-MMT and DCMTC induced mutation in UV sensitive UvrB and UvrC mutants with much higher frequency than in wild type bacteria, while MTC did not induce mutation in these Uvr-strains.

  19. Mitomycin C application in resistant caustic esophageal stricture

    African Journals Online (AJOL)

    procedures and in laryngotracheal stenosis [4]. Topical application of .... replacement [1], stents with its high incidence of failure and morbidity [5], and .... 8 Fröhlich T, Greess H, Köhler H. Topical mitomycin C treatment of a benign oesophageal ...

  20. Dacryocystorhinostomy without intubation with intraoperative mitomycin-c

    International Nuclear Information System (INIS)

    Rahman, A.; Channa, S.; Memon, M.S.; Niazi, J.H.

    2006-01-01

    To evaluate the success rate and complications of intraoperative Mitomycin-C in dacryocystorhinostomy surgery. This study included total 90 eyes of 90 patients fulfilling the inclusion criteria.The surgical procedure of external DCR done with intraoperative Mitomycin-C with a neurosurgical cottonoid soaked with 0.2mg/ml. Mitomycin C was applied to the anastomosed flaps and osteotomy site for 10 minutes, without Silicon tube intubation. Surgery was done under local as well as general anesthesia. Patients were followed for 6 months. Out of 90 patients included in this study, only 2 patients complained of persistent epiphora after 6 months follow-up and were labeled as failed DCR. Remaining 88 had either no tearing or significant improvement of tearing after 6 months follow up and patent lacrimal system by syringing without pressure. Success rate in this procedure was 97.77% (p-value< 0.001). This study showed very high rate of success. Only complication noted was excessive nasal bleeding which was easily controlled. Intraoperative Mitomycin-C application in external DCR is safe, effective, cheap adjunct that helps to achieve good results of DCR surgery. (author)

  1. Repair system and mitomycin mutagenesis in Escherichia coli

    International Nuclear Information System (INIS)

    Otsuji, Nozomu; Murayama, Ichiko

    1974-01-01

    Ultraviolet light sensitive mutants of E. coli defective at the uvrA, uvrB or uvrC locus showed increased sensitivity to the lethal effects of monofunctional mitomycin, 7-methoxymitosene (7-MMT) or decarbamoyl mitomycin C (DCMTC), as well as mitomycin C (MTC). (1) Treatment of wild type bacteria with these monofunctional mitomycins resulted in the production of single-strand breaks in DNA, which were repaired upon incubation in a growth medium. Such breaks in DNA were not produced in the UvrA and the UvrB mutants. In the UvrC mutant, single-strand breaks were produced by 7-MMT or by DCMTC, but these breaks were not repaired upon incubation. (2) Exposure of E. coli to MTC caused cross-linkage between DNA strands, which converted to a normally denaturable form during further incubation after treatment. This was not occurred in the UvrB strain. In the UvrC mutant, a portion of the cross-links was removed upon incubation. (3) 7-MMT and DCMTC induced mutation in UV sensitive UvrB and UvrC mutants with much higher frequency than in wild type bacteria, while MTC did not induce mutation in these Uvr-strains. (author)

  2. Coconut (Cocos nucifera l.) pollen cryopreservation.

    Science.gov (United States)

    Karun, A; Sjini, K K; Niral, V; Amarnth, C H; Remya, P; Rajesh, M K; Samsudeen, K; Jerard, B A; Engelmann, F

    2014-01-01

    Coconut genetic resources are threatened by pests and pathogens, natural hazards and human activities. Cryopreservation is the only method allowing the safe and cost-effective long-term conservation of recalcitrant seed species such as coconut. The objective of this work was to test the effect of cryopreservation and of cryostorage duration on coconut pollen germination and fertility. Pollen of two coconut varieties (West Coast Tall WWCTW and Chowghat Orange Dwarf CODC) was collected in March-May over three successive years, desiccated to 7.5 % moisture content (FW) and cryopreserved by direct immersion in liquid nitrogen. Germination and pollen tube length (PTL) of desiccated and cryopreserved pollen were not significantly different for both WCT and COD over the three harvest months of the three consecutive years of study. Pollen germination ranged from 24 to 32 % in desiccated pollen whereas it was between 26 and 29 % in cryopreserved COD pollen. In the case of WCT, germination ranged from 30 to 31 % in desiccated pollen, while it was between 28 and 32 % in cryopreserved pollen. PTL of cryopreserved pollen ranged between 224-390 nm and 226-396 mm for COD and WCT, respectively. Germination of COD pollen varied between 29.0 and 44.1 % after 4 years and 1.0/1.5 years cryostorage, respectively. Germination of WCT pollen did not change significantly between 0 and 6 years cryostorage, being comprised between 32 (24 h) and 40 % (1.5 years). Germination and vigour of cryopreserved pollen were generally higher compared to that of pollen dried in oven and non-cryopreserved. Normal seed set was observed in COD and WCT palms using pollen cryostored for 6 months and 4 years. Cryopreserved pollen of five Tall and five Dwarf accessions displayed 24-31 % and 25-49 % germination, respectively. These results show that it is now possible to establish pollen cryobanks to contribute to coconut germplasm long-term conservation.

  3. Cryopreservation of microencapsulated canine sperm.

    Science.gov (United States)

    Shah, Shambhu; Otsuki, Tsubasa; Fujimura, Chika; Yamamoto, Naoki; Yamashita, Yasuhisa; Higaki, Shogo; Hishinuma, Mitsugu

    2011-03-01

    The objective was to develop a method for cryopreserving microencapsulated canine sperm. Pooled ejaculates from three beagle dogs were extended in egg yolk tris extender and encapsulated using alginate and poly-L-lysine at room temperature. The microcapsules were cooled at 4 °C, immersed in pre-cooled extender (equivalent in volume to the microcapsules) to reach final concentration of 7% (v/v) glycerol and 0.75% (v/v) Equex STM paste, and equilibrated for 5, 30 and 60 min at 4 °C. Thereafter, microcapsules were loaded into 0.5 mL plastic straws and frozen in liquid nitrogen. In Experiment 1, characteristics of microencapsulated canine sperm were evaluated after glycerol addition at 4 °C. Glycerol exposure for 5, 30 and 60 min did not significantly affect progressive motility, viability, or acrosomal integrity of microencapsulated sperm compared with pre-cooled unencapsulated sperm (control). In Experiment 2, characteristics of frozen-thawed canine microencapsulated sperm were evaluated at 0, 3, 6, and 9 h of culture at 38.5 °C. Pre-freeze glycerol exposure for 5, 30, and 60 min at 4 °C did not influence post-thaw quality in unencapsulated sperm. Post-thaw motility and acrosomal integrity of microencapsulated sperm decreased more than those of unencapsulated sperm (P < 0.05) following glycerol exposure for 5 min. However, motility, viability and acrosomal integrity of microencapsulated sperm after 30 and 60 min glycerol exposure were higher than unencapsulated sperm cultured for 6 or 9 h (P < 0.05). In conclusion, since microencapsulated canine sperm were successfully cryopreserved, this could be a viable alternative to convention sperm cryopreservation in this species. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Advances in Stallion Semen Cryopreservation.

    Science.gov (United States)

    Alvarenga, Marco Antonio; Papa, Frederico Ozanam; Ramires Neto, Carlos

    2016-12-01

    The use of stallion frozen semen minimizes the spread of disease, eliminates geographic barriers, and preserves the genetic material of the animal for an unlimited time. Significant progress on the frozen thawed stallion semen process and consequently fertility has been achieved over the last decade. These improvements not only increased fertility rates but also allowed cryopreservation of semen from "poor freezers." This article reviews traditional steps and new strategies for stallion semen handling and processing that are performed to overcome the deleterious effects of semen preservation and consequently improve frozen semen quality and fertility. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Conjunctival dysfunction and mitomycin C-induced hypotony.

    Science.gov (United States)

    Sihota, R; Dada, T; Gupta, S D; Sharma, S; Arora, R; Agarwal, H C

    2000-10-01

    To determine the role of a physically intact conjunctiva in the development of chronic hypotony after mitomycin C-enhanced trabeculectomy. Three patients with mitomycin C-related hypotonic maculopathy, but without a leak on Siedel test, had a thorough evaluation of the bleb area and an anterior segment fluorescein angiography. The bleb was excised and a pedicle flap, rotated from the temporal conjunctiva, was sutured to cover the defect superiorly. The scleral flap and its sutures were not disturbed. The excised bleb was subjected to light and electron microscopy. The Seidel test result was negative in all patients, but late phases of the anterior segment angiography showed a generalized seepage of aqueous from the bleb. After revision of the bleb, there was a gradual increase in the intraocular pressure, a reversal of the hypotonic maculopathy, and consequent improvement in visual acuity in all three patients, stable up to a minimum follow-up of 18 months. On histopathologic examination, the basement membrane was thickest under thin areas of the epithelium and thinnest below thicker epithelial layers. A dysfunctional conjunctival barrier, as evidenced by the "sweating" of the bleb and histopathologic alterations in the epithelial barrier, could be responsible for the hypotonic maculopathy in these patients. Excision of the conjunctiva alone and replacement by a pedicle conjunctival graft offers a safe and effective method of treating chronic hypotony after mitomycin C-augmented trabeculectomy in such patients.

  6. Mature Oocyte Cryopreservation for Fertility Preservation.

    Science.gov (United States)

    Liang, Tina; Motan, Tarek

    2016-01-01

    In recent decades, advances in cancer treatment have led to a dramatic improvement in long term survival. This has led to an increasing focus on quality of life after surviving cancer treatment, with fertility being an important aspect. Given the known reproductive risks of cancer therapies, there has been a growing interest in the field of fertility preservation (also referred to as oncofertility). Mature oocyte cryopreservation is no longer considered experimental and has become a realistic option for reproductive aged women prior to undergoing cancer treatment. Additionally, as cryopreservation techniques improve, mature oocyte cryopreservation is increasing being marketed to healthy women without cancer wishing to delay child bearing, also termed "social egg freezing". This chapter provides a review of the current technology, use, and outcomes of mature oocyte cryopreservation. It also outlines the ethical debate surrounding social egg freezing and directions for future research in female fertility preservation.

  7. Protectants used in the cryopreservation of microorganisms

    Czech Academy of Sciences Publication Activity Database

    Hubálek, Zdeněk

    2003-01-01

    Roč. 46, č. 3 (2003), s. 205-229 ISSN 0011-2240 Institutional research plan: CEZ:AV0Z6093917 Keywords : cryopreservation * cryoprotectants Subject RIV: EE - Microbiology, Virology Impact factor: 1.445, year: 2003

  8. Mitomycin C dissolved in a reversible thermosetting gel: target tissue concentrations in the rabbit eye.

    Science.gov (United States)

    Ichien, K; Yamamoto, T; Kitazawa, Y; Oguri, A; Ando, H; Kondo, Y

    1997-01-01

    To determine whether a new, reversible thermosetting gel enhances mitomycin C transfer to target ocular tissues in the rabbit eye. A 0.1 ml solution of mitomycin C containing 0.22 microgram, 2.9 micrograms, or 28 micrograms of the agent dissolved in a reversible thermosetting gel consisting of methylcellulose, citric acid, and polyethylene glycol was injected subconjunctivally in 30 New Zealand albino rabbits. Scleral and conjunctival tissues were excised at 0.5, 1, 2, 4, or 24 hours after the injection and mitomycin C concentrations in these tissues were determined by high performance liquid chromatography. The concentration over time was approximated to a single exponential curve, and initial mitomycin C concentrations, time constants, and half life values were determined. Finally, the areas under the curves (AUCs) between 0.5 and 24 hours were calculated. The mitomycin C concentrations in the target tissues were dose dependent and decreased rapidly over 24 hours. Both the initial mitomycin C concentrations as well as AUCs in these eyes treated with mitomycin C, dissolved in a reversible thermosetting gel, were higher than those in eyes treated similarly in a previous study in which the gel was not used. Applied subconjunctivally in the rabbit eye, mitomycin C dissolved in the reversible thermosetting gel enhanced transfer of the agent to the sclera and the conjunctiva.

  9. Delayed healing at transurethral resection of bladder tumour sites after immediate postoperative mitomycin C instillation

    DEFF Research Database (Denmark)

    Aagaard, Mark F; Mogensen, Karin; Hermann, Gregers G

    2014-01-01

    The most common reactions to mitomycin C are dysuria and drug-related palmar and genital desquamation. This report describes two cases of delayed healing of the mucosa at resection sites after transurethral resection of bladder tumours, most likely due to immediate postoperative mitomycin C...

  10. Effect of Intraoperative Mitomycin C on the Re-currence rate of ...

    African Journals Online (AJOL)

    ... effectif dans les cas recurrents. L'utilisation intra-operative du mitomycin C est fortement recommend après excision du pterygium du type primaire chez les Nigerian pour reduire le taux de recurrence. Mot clés: Pterygium, recurrence, mitomycin. West African Jnl of Pharmacology and Drug Research Vol.18 2002: 17-20 ...

  11. [The destiny of cryopreserved embryos].

    Science.gov (United States)

    Karpel, L; Achour-Frydman, N; Frydman, R; Flis-Trèves, M

    2007-12-01

    To know the psychological motivations of couples who keep their embryos so long (five years and more) and do not make a decision about them. We studied 84 couples refrained from making a decision on their cryopreserved embryos for at least five years. They were invited to fill out a questionnaire focusing on three points: the reasons of the indecision, their own representation of the cryopreserved embryos and their choice for the future: donation to another couple, to research, pregnancy or no solution for the moment. Mean (S.D.) women's and men's age were respectively, 38.8 (2.5)- and 41.3 (2.5)-years old. On average, three (1-9) embryos are preserved since 7.5 (5-12) years. Most of couples are parents. Four major reasons explain their attitudes: feeling of being too aged (25%), fear of a multiple pregnancy (45%), disagreement between members of couple (20%) and fear of failure (42.5%). Multiple choices were given to the future of the embryos: 25% wanted a pregnancy, 8% wanted to give them to infertile couples, 20% to research and 27.5% did not find any solution. Twenty percent were hesitating. The representation of those embryos is more symbolic than material. Most of the time, they see them like a potential child, a hope for the future or a brother or sister of their alive children. Those embryos are symbolized. They are a proof of fertility, a hope for another child. So, whatever the legal statement, couples will be in a dilemma because it is never easy for an infertile person to renounce to embryos, and the hope for children.

  12. Cryopreservation and revival of mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Kastrup, Jens

    2011-01-01

    initiated. As there has been a precedent for the use of bone marrow stem cells in the treatment of hematological malignancies and ischemic heart diseases through randomized clinical safety and efficacy trials, the development of new therapies based on culture-expanded human mesenchymal stromal cells (MSCs......Over the past few years, the pace of preclinical stem cell research is astonishing and adult stem cells have become the subject of intense research. Due to the presence of promising supporting preclinical data, human clinical trials for stem cell regenerative treatment of various diseases have been......) opens up new possibilities for cell therapy. To facilitate these applications, cryopreservation and long-term storage of MSCs becomes an absolute necessity. As a result, optimization of this cryopreservation protocol is absolutely critical. The major challenge during cellular cryopreservation...

  13. Karyotype of cryopreserved bone marrow cells

    Directory of Open Access Journals (Sweden)

    M.L.L.F. Chauffaille

    2003-07-01

    Full Text Available The analysis of chromosomal abnormalities is important for the study of hematological neoplastic disorders since it facilitates classification of the disease. The ability to perform chromosome analysis of cryopreserved malignant marrow or peripheral blast cells is important for retrospective studies. In the present study, we compared the karyotype of fresh bone marrow cells (20 metaphases to that of cells stored with a simplified cryopreservation method, evaluated the effect of the use of granulocyte-macrophage colony-stimulating factor (GM-CSF as an in vitro mitotic index stimulator, and compared the cell viability and chromosome morphology of fresh and cryopreserved cells whenever possible (sufficient metaphases for analysis. Twenty-five bone marrow samples from 24 patients with hematological disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, megaloblastic anemia and lymphoma (8, 3, 3, 8, 1, and 1 patients, respectively were selected at diagnosis, at relapse or during routine follow-up and one sample was obtained from a bone marrow donor after informed consent. Average cell viability before and after freezing was 98.8 and 78.5%, respectively (P < 0.05. Cytogenetic analysis was successful in 76% of fresh cell cultures, as opposed to 52% of cryopreserved samples (P < 0.05. GM-CSF had no proliferative effect before or after freezing. The morphological aspects of the chromosomes in fresh and cryopreserved cells were subjectively the same. The present study shows that cytogenetic analysis of cryopreserved bone marrow cells can be a reliable alternative when fresh cell analysis cannot be done, notwithstanding the reduced viability and lower percent of successful analysis that are associated with freezing.

  14. Karyotype of cryopreserved bone marrow cells.

    Science.gov (United States)

    Chauffaille, M L L F; Pinheiro, R F; Stefano, J T; Kerbauy, J

    2003-07-01

    The analysis of chromosomal abnormalities is important for the study of hematological neoplastic disorders since it facilitates classification of the disease. The ability to perform chromosome analysis of cryopreserved malignant marrow or peripheral blast cells is important for retrospective studies. In the present study, we compared the karyotype of fresh bone marrow cells (20 metaphases) to that of cells stored with a simplified cryopreservation method, evaluated the effect of the use of granulocyte-macrophage colony-stimulating factor (GM-CSF) as an in vitro mitotic index stimulator, and compared the cell viability and chromosome morphology of fresh and cryopreserved cells whenever possible (sufficient metaphases for analysis). Twenty-five bone marrow samples from 24 patients with hematological disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, megaloblastic anemia and lymphoma (8, 3, 3, 8, 1, and 1 patients, respectively) were selected at diagnosis, at relapse or during routine follow-up and one sample was obtained from a bone marrow donor after informed consent. Average cell viability before and after freezing was 98.8 and 78.5%, respectively (P < 0.05). Cytogenetic analysis was successful in 76% of fresh cell cultures, as opposed to 52% of cryopreserved samples (P < 0.05). GM-CSF had no proliferative effect before or after freezing. The morphological aspects of the chromosomes in fresh and cryopreserved cells were subjectively the same. The present study shows that cytogenetic analysis of cryopreserved bone marrow cells can be a reliable alternative when fresh cell analysis cannot be done, notwithstanding the reduced viability and lower percent of successful analysis that are associated with freezing.

  15. Sperm cryopreservation in fish and shellfish.

    Science.gov (United States)

    Tiersch, Terrence R; Yang, Huiping; Jenkins, Jill A; Dong, Qiaoxiang

    2007-01-01

    Initial success in sperm cryopreservation came at about the same time for aquatic species and livestock. However, in the 50-plus years since then cryopreserved sperm of livestock has grown into a billion-dollar global industry, while despite work in some 200 species with well over 200 published reports, cryopreservation of aquatic species sperm remains essentially a research activity with little commercial application. Most research has focused on large-bodied culture and sport fishes, such as salmonids, carps, and catfishes, and mollusks such as commercially important oyster and abalone species. However, only a handful of studies have addressed sperm cryopreservation in small fishes, such as zebrafish, and in endangered species. Overall, this work has yielded techniques that are being applied with varying levels of success around the world. Barriers to expanded application include a diverse and widely distributed literature base, technical problems, small sperm volumes, variable results, a general lack of access to the technology, and most importantly, the lack of standardization in practices and reporting. The benefits of cryopreservation include at least five levels of improvements for existing industries and for creation of new industries. First, cryopreservation can be used to improve existing hatchery operations by providing sperm on demand and simplifying the timing of induced spawning. Second, frozen sperm can enhance efficient use of facilities and create new opportunities in the hatchery by eliminating the need to maintain live males, potentially freeing resources for use with females and larvae. Third, valuable genetic lineages such as endangered species, research models, or improved farmed strains can be protected by storage of frozen sperm. Fourth, cryopreservation opens the door for rapid genetic improvement. Frozen sperm can be used in breeding programs to create improved lines and shape the genetic resources available for aquaculture. Finally

  16. Strategies for commercialization of cryopreserved fish semen

    Directory of Open Access Journals (Sweden)

    Terrence R. Tiersch

    2008-07-01

    Full Text Available Initial success in sperm cryopreservation occurred at about the same time for aquatic species and livestock. However, in the 50 plus years since then cryopreserved sperm of livestock has grown into a billion-dollar global industry, while cryopreserved sperm of aquatic species remains a research activity with little commercial application despite work in more than 90 species and more than 200 published reports. Most research work has focused on large-bodied culture and sport fishes, such as salmon, trout, carp, and catfish, and mollusks such as commercially important oyster and abalone species. However, only a few studies have addressed sperm cryopreservation in small fishes such as zebrafish, or in endangered species. Overall, this work has yielded techniques that are being applied with varied levels of success around the world. Barriers to expanded application include a diverse and widely distributed literature base, technical problems, small sperm volumes, variable results, a general lack of access to the technology, and most importantly, a lack of standardization in practices and reporting. The benefits of cryopreservation include at least five levels of improvements for existing industries and for creation of new industries. First, cryopreservation can be used to improve existing hatchery operations by providing sperm on demand and simplifying the timing of induced spawning. Second, frozen sperm can enhance efficient use of facilities and create new opportunities in the hatchery by eliminating the need to maintain live males, potentially freeing resources for use with females and larvae. Third, valuable genetic lineages such as endangered species, research models or improved farmed strains can be protected by storage of frozen sperm. Fourth, cryopreservation opens the door for rapid genetic improvement. Frozen sperm can be used in breeding programs to create improved lines and shape the genetic resources available for aquaculture. Finally

  17. Effects of low dose mitomycin C on experimental tumor radiotherapy

    International Nuclear Information System (INIS)

    Yang Jianzheng; Liang Shuo; Qu Yaqin; Pu Chunji; Zhang Haiying; Wu Zhenfeng; Wang Xianli

    2001-01-01

    Objective: To evaluate the possibility of low dose mitomycin C(MMC) as an adjunct therapy for radiotherapy. Methods: Change in tumor size tumor-bearing mice was measured. Radioimmunoassay was used to determine immune function of mice. Results: Low dose Mac's pretreatment reduced tumor size more markedly than did radiotherapy only. The immune function in mice given with low dose MMC 12h before radiotherapy was obviously higher than that in mice subjected to radiotherapy only (P<0.05), and was close to that in the tumor-bearing mice before radiotherapy. Conclusion: Low dose MMC could improve the radiotherapy effect. Pretreatment with low dose MMC could obviously improve the immune suppression state in mice caused by radiotherapy. The mechanism of its improvement of radiotherapeutic effect by low dose of MMC might be due to its enhancement of immune function and induction of adaptive response in tumor-bearing mice

  18. LASIK flap buttonhole treated immediately by PRK with mitomycin C.

    Science.gov (United States)

    Kymionis, George D; Portaliou, Dimitra M; Karavitaki, Alexandra E; Krasia, Maria S; Kontadakis, Georgios A; Stratos, Aimilianos; Yoo, Sonia H

    2010-03-01

    To describe the visual outcomes of three patients who had LASIK flap buttonhole and were treated immediately with photorefractive keratectomy (PRK) and topical mitomycin C (MMC) 0.02%. Three patients underwent bilateral LASIK with the SCHWIND Carriazo-Pendula 90 microm head microkeratome. In all three cases, a buttonhole flap occurred in the left eye. The flap was repositioned and phototherapeutic keratectomy for 50 microm was used for epithelial removal while immediate PRK with MMC was performed to treat the buttonhole flap. Three months after the procedure, uncorrected distance visual acuity and corrected distance visual acuity were 20/20 with regular topographic findings. Using PRK with MCC immediately after the occurrence of the LASIK flap buttonhole may be an effective treatment.

  19. Impact of procedural steps and cryopreservation agents in the cryopreservation of chlorophyte microalgae.

    Directory of Open Access Journals (Sweden)

    Tony V L Bui

    Full Text Available The maintenance of traditional microalgae collections based on liquid and solid media is labour intensive, costly and subject to contamination and genetic drift. Cryopreservation is therefore the method of choice for the maintenance of microalgae culture collections, but success is limited for many species. Although the mechanisms underlying cryopreservation are understood in general, many technical variations are present in the literature and the impact of these are not always elaborated. This study describes two-step cryopreservation processes in which 3 microalgae strains representing different cell sizes were subjected to various experimental approaches to cryopreservation, the aim being to investigate mechanistic factors affecting cell viability. Sucrose and dimethyl sulfoxide (DMSO were used as cryoprotectants. They were found to have a synergistic effect in the recovery of cryopreserved samples of many algal strains, with 6.5% being the optimum DMSO concentration. The effect of sucrose was shown to be due to improved cell survival and recovery after thawing by comparing the effect of sucrose on cell viability before or after cryopreservation. Additional factors with a beneficial effect on recovery were the elimination of centrifugation steps (minimizing cell damage, the reduction of cell concentration (which is proposed to reduce the generation of toxic cell wall components and the use of low light levels during the recovery phase (proposed to reduce photooxidative damage. The use of the best conditions for each of these variables yielded an improved protocol which allowed the recovery and subsequent improved culture viability of a further 16 randomly chosen microalgae strains. These isolates included species from Chlorellaceae, Palmellaceae, Tetrasporaceae, Palmellopsis, Scenedesmaceae and Chlamydomonadaceae that differed greatly in cell diameter (3-50 µm, a variable that can affect cryopreservation success. The collective improvement

  20. NEEDLE REVISION WITH MITOMYCIN-C IN ENCAPSULATED BLEBS

    Directory of Open Access Journals (Sweden)

    R Zarei

    2008-08-01

    Full Text Available "nThe most common cause of failure during the first trimester after trabeculectomy is encapsulated bleb and needling bleb revision is a less invasive method in the management of refractory cases. The purpose of this study is to determine the efficacy and safety of mitomycin-C (MMC augmented bleb revision of failed filtration surgery. This study is a before-after (paired observation. 33 patients with failed trabeculectomy because of bleb encapsulation, whose intraocular pressure (IOP was not reduced under 21 mmHg despite of medications and digital massage , underwent needling bleb revision and subconjunctival injection of 0.1 ml MMC (0.4 mg/ml.The mean follow-up time was 9.24 ± 5.27 months (1-20 months. Statistical analysis of the data included the paired two-tailed Student's t test for preoperative and postoperative IOP and number of medications. 36 needling procedures (mean, 1.09 ± 0.21 revisions per eye were performed on 33 eyes. Patients were between 10-80 years old (mean, 45.67 ± 22.41 years and mean follow-up was 9.24 ± 5.27 months. IOP decreased from 29.06 ± 5.03 mmHg to 18.21 ± 6.76 mmHg at last follow-up (P= 0.000. Antiglaucoma medications decreased from 2.18 ± 0.58 to 1.36 ± 0.29 at last follow-up (P= 0.000.Overall, 6 (18.2% of 33 cases achieved a complete success and 20 (60.6% of cases achieved a qualified success. The complications of this procedure were subconjunctival hemorrhage (17 cases, hyphema (5 cases and conjunctival button hole (2 cases. Needling bleb revision with mitomycin-C appears to be an effective and relatively safe way to revive failed filtration surgery.

  1. Embryo transfer using cryopreserved Boer goat blastocysts ...

    African Journals Online (AJOL)

    The aim of this trial was to evaluate the effect of embryo cryopreservation techniques on the survivability of embryos and fertility following transfer to Boer goat does. The oestrous cycles of 27 mature recipients Boer goat does were synchronised using controlled internal drug release dispensers (CIDR's) for 16 days. At CIDR ...

  2. Changing rooster sperm membranes to facilitate cryopreservation

    Science.gov (United States)

    Cryopreservation damages rooster sperm membranes. Part of this damage is due to membrane transitioning from the fluid to the gel state as temperature is reduced. This damage may be prevented by increasing membrane fluidity at low temperatures by incorporating cholesterol or unsaturated lipids into t...

  3. Preliminary investigation of cryopreservation by encapsulation ...

    African Journals Online (AJOL)

    Protocorm-like bodies (PLBs) of Brassidium Shooting Star, a new commercial ornamental orchid hybrid, were cryopreserved by an encapsulation-dehydration technique. The effects of PLB size, various sucrose concentrations in preculture media and sodium alginate concentration for encapsulation were the main ...

  4. Cryopreservation and Cryotherapy of Citrus Cultivars

    Science.gov (United States)

    Long-term conservation of Citrus clones can be accomplished by cryopreservation. Shoot tips will survive liquid nitrogen exposure and storage when appropriately desiccated and treated with cryoprotectant solutions. In our research, vegetative Citrus budwood is shipped from Riverside to Fort Collin...

  5. Cryopreservation of medicinal plants: role of melatonin

    Science.gov (United States)

    Many useful plant species found in Canada are of conservation concern. In vitro storage and cryopreservation techniques guarantees safety of these species and have potential applications which may result in sustainable agriculture. Shoot tips of in vitro-grown plantlets of American elm, St John’s Wo...

  6. Cryopreservation of South African indigenous goat semen

    African Journals Online (AJOL)

    use

    2011-12-05

    Dec 5, 2011 ... sperm cell motility rate of 83.1%, progressive sperm cell motility of 49.3% ... cells can be stored and used after a long period of time. ... artificial insemination to enhance improvement of .... Effect of cryopreservation on semen quality (mean ± S.E) of South African .... Successful pregnancies with directional.

  7. Viability of ectomycorrhizal fungi following cryopreservation.

    Science.gov (United States)

    Crahay, Charlotte; Declerck, Stéphane; Colpaert, Jan V; Pigeon, Mathieu; Munaut, Françoise

    2013-02-01

    The use of ectomycorrhizal (ECM) fungi in biotechnological processes requires their maintenance over long periods under conditions that maintain their genetic, phenotypic, and physiological stability. Cryopreservation is considered as the most reliable method for long-term storage of most filamentous fungi. However, this technique is not widespread for ECM fungi since many do not survive or exhibit poor recovery after freezing. The aim of this study was to develop an efficient cryopreservation protocol for the long-term storage of ECM fungi. Two cryopreservation protocols were compared. The first protocol was the conventional straw protocol (SP). The mycelium of the ECM isolates was grown in Petri dishes on agar and subsequently collected by punching the mycelium into a sterile straw before cryopreservation. In the second protocol, the cryovial protocol (CP), the mycelium of the ECM isolates was grown directly in cryovials filled with agar and subsequently cryopreserved. The same cryoprotectant solution, freezing, and thawing process, and re-growth conditions were used in both protocols. The survival (positive when at least 60 % of the replicates showed re-growth) was evaluated before and immediately after freezing as well as after 1 week, 1 m, and 6 m of storage at -130 °C. Greater survival rate (80 % for the CP as compared to 25 % for the SP) and faster re-growth (within 10 d for the CP compared to the 4 weeks for the SP) were observed for most isolates with the CP suggesting that the preparation of the cultures prior to freezing had a significant impact on the isolates survival. The suitability of the CP for cryopreservation of ECM fungi was further confirmed on a set of 98 ECM isolates and displayed a survival rate of 88 % of the isolates. Only some isolates belonging to Suillus luteus, Hebeloma crustuliniforme, Paxillus involutus and Thelephora terrestris failed to survive. This suggested that the CP is an adequate method for the ultra-low cryopreservation of

  8. Cryopreservation of peach palm zygotic embryos.

    Science.gov (United States)

    Steinmacher, Douglas A; Saldanha, Cleber W; Clement, Charles R; Guerra, Miguel P

    2007-01-01

    Cryopreservation is a safe and cost-effective option for long-term germplasm conservation of non-orthodox seed species, such as peach palm (Bactris gasipaes). The objective of the present study was to establish a cryopreservation protocol for peach palm zygotic embryos based on the encapsulation-dehydration technique. After excision, zygotic embryos were encapsulated with 3 percent sodium alginate plus 2 M glycerol and 0.4 M sucrose, and pre-treated or not with 1 M sucrose during 24 h, followed by air-drying. Fresh weight water contents of beads decreased from 83 percent and 87 percent to 18 percent and 20 percent for pre-treated or non-pretreated beads, respectively, after 4 h of dehydration. Sucrose pre-treatment at 1 M caused lower zygotic embryo germination and plantlet height in contrast to non-treated beads. All the variables were statistically influenced by dehydration time. Optimal conditions for recovery of cryopreserved zygotic embryos include encapsulation and dehydration for 4 h in a forced air cabinet to 20 percent water content, followed by rapid freezing in liquid nitrogen (-196 degree C) and rapid thawing at 45 degree C. In these conditions 29 percent of the zygotic embryos germinated in vitro. However, plantlets obtained from dehydrated zygotic embryos had stunted haustoria and lower heights. Histological analysis showed that haustorium cells were large, vacuolated, with few protein bodies. In contrast, small cells with high nucleus:cytoplasm ratio formed the shoot apical meristem of the embryos, which were the cell types with favorable characteristics for survival after exposure to liquid nitrogen. Plantlets were successfully acclimatized and showed 41+/-9 percent and 88+/-4 percent survival levels after 12 weeks of acclimatization from cryopreserved and non-cryopreserved treatments, respectively.

  9. Cryopreservation of Mycobacterium bovis isolates

    Directory of Open Access Journals (Sweden)

    Cássia Yumi Ikuta

    2016-11-01

    Full Text Available Research, development of new biotechnological methods, diagnostic tests, confirmation of results, and reinvestigations are possible because of the availability of well-preserved living organisms maintained without any changes. Cryopreservation is a simpler, more reliable and long-term stable method for culture maintenance. Storage temperature and composition of the suspending vehicle are factors that affect the viability of mycobacterial strains. Three vehicles and three storage temperatures were evaluated to define a suitable cryoprotective medium for the preservation of Mycobacterium bovis strains. Colonies of sixteen M. bovis isolates were used to prepare the suspensions, which were then added to three vehicles: sterile 0.85% saline solution (SS, Middlebrook 7H9 broth (7H9, and Middlebrook 7H9 broth with sodium pyruvate (7H9p replacing glycerol. Aliquots of these suspensions were frozen by three different methods, directly in the -20°C freezer, directly in the -80°C freezer, and at -196°C by immersion in liquid nitrogen (LN. The frozen aliquots were thawed at room temperature after 45, 90 and 120 days. Mycobacterial viability was assessed by counting the living cells on plates of Stonebrink medium before and after the freezing procedure. Storage at -20°C exhibited a lower recovery of M. bovis compared to storage at -80°C (Dunn’s test, p=0.0018 and LN (Dunn’s test, p=0.0352. There was no statistically significant difference between storage at -80°C and in LN (Dunn’s test, p=0.1403, yet -80°C showed better results than LN. All three suspending vehicles showed no statistically significant difference in terms of viability (Friedman’s test, p=0.7765. Given the low loss proportion of 5% during storage at -20°C and the high cost equipment required for storage at -80°C and LN, we recommend storage at -20°C or -80°C, when this is available, for preservation of M. bovis field strains.

  10. Intra-arterial mitomycin C treatment of unresectable liver tumours

    International Nuclear Information System (INIS)

    Starkhammar, H.; Haakansson, L.; Morales, O.; Svedberg, J.; Linkoeping Univ.; Linkoeping Univ.

    1987-01-01

    Regional chemotherapy might be more efficient if the cytostatic drug is injected together with degradable starch microspheres (DSM), which induce temporary blockage of arterioles and trap the co-injected drug in tumour. Eighteen patients with non-resectable liver cancer were included. Mitomycin C (15 mg/m 2 ) was injected intra-arterially mixed with 900 mg of DSM every six weeks. For estimation of the effect of DSM in the liver a radiolabelled tracer was injected via the same route. Its passage through the liver to the systemic circulation was continuously measured by a detector situated over peripheral blood vessels. The effect of DSM on the tracer passage varied considerably between different patients. The study also indicated opening of new vascular pathways some minutes after the initial injection. The dose of DSM for total blockage of the arterial blood flow, indicated by angiography, also varied. In some patients 540 mg induced total occlusion. In others neither angiographic nor tracer passage were affected by the microspheres although 900 mg (or even more) were injected. Factors such as size of the vascular bed, portal and arterial blood flow and arterio-venous shunting seemed to be of great importance and should be controlled in order to optimize the use of DSM in conjunction with chemotherapy of liver tumours. (orig.)

  11. Cryopreserved semen in ecotoxicological bioassays: sensitivity and reliability of cryopreserved Sparus aurata spermatozoa.

    Science.gov (United States)

    Fabbrocini, Adele; D'Adamo, Raffaele; Del Prete, Francesco; Langellotti, Antonio Luca; Rinna, Francesca; Silvestri, Fausto; Sorrenti, Gerarda; Vitiello, Valentina; Sansone, Giovanni

    2012-10-01

    The aim of this study was to evaluate the feasibility of using cryopreserved S. aurata semen in spermiotoxicity tests. Cryopreservation is a biotechnology that can provide viable gametes and embryos on demand, rather than only in the spawning season, thus overcoming a limitation that has hindered the use of some species in ecotoxicological bioassays. Firstly, the sperm motility pattern of cryopreserved semen was evaluated after thawing by means of both visual and computer-assisted analyses. Motility parameters in the cryopreserved semen did not change significantly in the first hour after thawing, meaning that they were maintained for long enough to enable their use in spermiotoxicity tests. In the second phase of the research, bioassays were performed, using cadmium as the reference toxicant, in order to evaluate the sensitivity of cryopreserved S. aurata semen to ecotoxicological contamination. The sensitivity of the sperm motility parameters used as endpoints (motility percentages and velocities) proved to be comparable to what has been recorded for the fresh semen of other aquatic species (LOECs from 0.02 to 0.03 mg L(-1)). The test showed good reliability and was found to be rapid and easy to perform, requiring only a small volume of the sample. Moreover, cryopreserved semen is easy to store and transfer and makes it possible to perform bioassays in different sites or at different times with the same batch of semen. The proposed bioassay is therefore a promising starting point for the development of toxicity tests that are increasingly tailored to the needs of ecotoxicology and environmental quality evaluation strategies. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Cryopreservation of mutton snapper ( Lutjanus analis sperm

    Directory of Open Access Journals (Sweden)

    EDUARDO G. SANCHES

    2013-09-01

    Full Text Available This study aimed to develop a protocol of semen cryopreservation of the mutton snapper Lutjanus analis. The interaction between three extenders ( pH 6.1; 7.8 and 8.2 , two concentrations of dimethyl sulfoxide ( DMSO, 5 and 10% and three cooling rates ( -90; -60 and -30°C.min−1 on the sperm motility rate and motility time were analyzed by a factorial experiment. A sample of 30 fishes ( 1,261 ± 449 g collected in the nature was kept in floating net cages. The semen was frozen by using cryogenic straws, in nitrogen vapour and transferred, later, to liquid nitrogen. Fertilization test was accomplished to evaluate the viability of the cryopreserved sperm. The highest sperm motility rate and motility time ( P < 0.05 was achieved by combining extender C ( pH 8.2 with DMSO ( 10% and cooling rate of -60°C.min−1 ( P < 0.05 . The use of cryopreserved sperm presented fertilization rates higher than 59% validating the present protocol for mutton snapper.

  13. Cryopreservation of Leishmania Species in Manisa Province.

    Science.gov (United States)

    Çavuş, İbrahim; Ocak, Fulya; Kaya, Tuğba; Özbilgin, Ahmet

    2017-09-01

    It was aimed to assess the success of the cryopreservation process which is carried out in order to preserve the genetic material and the virulence of the Leishmania species that are an important health problem in our region. Leishmania tropica, L. infantum, L. major, and L. donovani strains in Novy-MacNeal-Nicolle (NNN) medium in MCBU were used. Promastigotes cultured in the NNN medium were transferred to RPMI 1640 medium; promastigotes in the logarithmic phase were washed three times with PBS, and 15% dimethylsulfoxide (DMSO) was added. Leishmania species were transferred to 12 separate tubes. The tubes were stored at -86°C for one night by placing them in Coolcell boxes. The tubes were transferred into a liquid nitrogen tank. One cryotube per Leishmania strain is thawed monthly and cultured in NNN medium. For the duration of study it was observed that each Leishmania isolate preserved 60-65% of their viability and entered the logarithmic phase on the 7th day following the inoculation in the NNN medium. Abnormalities in the structures and movements of the promastigotes were not observed in microscopic examinations. The following conclusions were made: cryopreservation is important for studies planned related to leishmaniasis and cryopreservation with DMSO is successful.

  14. Cryopreservation of Arachis pintoi (leguminosae) somatic embryos.

    Science.gov (United States)

    Rey, H Y; Faloci, M; Medina, R; Dolce, N; Engelmann, F; Mroginski, L

    2013-01-01

    In this study, we successfully cryopreserved cotyledonary somatic embryos of diploid and triploid Arachis pintoi cytotypes using the encapsulation-dehydration technique. The highest survival rates were obtained when somatic embryos were encapsulated in calcium alginate beads and precultured in agitated (80 rpm) liquid establishment medium (EM) with daily increasing sucrose concentration (0.50, 0.75, and 1.0 M). The encapsulated somatic embryos were then dehydrated with silica gel for 5 h to 20% moisture content (fresh weight basis) and cooled either rapidly (direct immersion in liquid nitrogen, LN) or slowly (1 degree C per min from 25 degree C to -30 degree C followed by immersion in LN). Beads were kept in LN for a minimum of 1 h and then were rapidly rewarmed in a 30 degree C water-bath for 2 min. Finally, encapsulated somatic embryos were post-cultured in agitated (80 rpm) liquid EM with daily decreasing sucrose concentration (0.75 and 0.5 M) and transferred to solidified EM. Using this protocol, we obtained 26% and 30% plant regeneration from cryopreserved somatic embryos of diploid and triploid cytotypes. No morphological abnormalities were observed in any of the plants regenerated from cryopreserved embryos and their genetic stability was confirmed with 10 isozyme systems and nine RAPD profiles.

  15. Efficacy of External Dacryocystorhinostomy (DCR) with and without Mitomycin-C in Chronic Dacryocystitis

    International Nuclear Information System (INIS)

    Mukhtar, S. A.; Jamil, A. Z.; Ali, Z.

    2014-01-01

    Objective: To compare the efficacy of external dacryocystorhinostomy (DCR) with and without intraoperative application of mitomycin-C in patients with chronic dacryocystitis. Study Design: An experimental study. Place and Duration of Study: Department of Ophthalmology, Bahawal Victoria Hospital (BVH), Bahawalpur, from September 2009 to December 2010. Methodology: One hundred and sixty patients with chronic dacryocystitis undergoing external DCR were divided into two groups comprising of 80 patients each. Group A included patients, who underwent external DCR with intraoperative use of mitomycin-C. Group B included those patients who were not administered intraoperative mitomycin-C. Sociodemographic information and the data regarding the patency of the lacrimal drainage system by irrigation with normal saline were collected at the end of the third month after the surgery. Chi-square test was used, at 95% confidence level, as the test of significance to compare the success of surgery between the two groups. Results: Surgical success rate (efficacy) in group 'A' was found to be 97.5% and 86.25% in group 'B'. The difference in success rate was statistically significant (p=0.017). Conclusion: External dacryocystorhinostomy with intraoperative mitomycin-C is more efficacious in achieving lacrimal system patency than external dacryocystorhinostomy without mitomycin-C. (author)

  16. Comparison of mean corneal endothelial cell loss after trabeculectomy with and without mitomycin C

    International Nuclear Information System (INIS)

    Shaheer, M.; Amjad, A.; Ahmed, N.

    2018-01-01

    Objective:To compare mean endothelial cell loss in patients of primary open angle glaucoma undergoing trabeculectomy with and without mitomycin C. Study Design:Randomised control study. Place and Duration of Study:Institute of Ophthalmology, Mayo Hospital, Lahore, from May 2016 to April 2017. Methodology:Patients with primary open angle glaucoma, not controlled with medication, were selected from the outpatient department. Patients with secondary glaucomas and concomitant ocular disease were excluded. Selected patients were divided into two groups of 30 each. Group A patients underwent trabeculectomy with adjunctive mitomycin C while group B patients underwent trabeculectomy alone. Pre- and post-trabeculectomy endothelial cell counts were recorded with the help of specular microsope and entered on proforma. Results:Median cell loss was 283 (66.50) when trabeculectomy was done with the use of adjunctive mitomycin C in group A while median endothelial cell loss was 72.50 (19.25) when the trabeculectomy was done without the use of adjunctive mitomycin C in group B. Conclusion:Use of mitomycin C causes more endothelial cell loss during trabeculectomy as compared to when done without it. (author)

  17. Effect of Intraoperative mitomycin-c application in outcome of external dacryocystorhinostomy

    International Nuclear Information System (INIS)

    Jawad, M.; Ali, Z.; Aftab, H.

    2015-01-01

    Background: Patients develop postoperative fibrosis at the site of operation after dacryocystorhinostomy (DCR) which results in impairment of the osteum patency. This quasi-experimental study was undertaken to determine the role of intraoperative Mitomycin C (MMC) application in maintaining postoperative patency of the osteum. Methodology: The present study was conducted at the Eye department of Ayub Medical College, Abbottabad on patients in whom routine DCR was indicated. Subjects were divided into mitomycin C (Test) and non mitomycin C (Control) groups. In test group, Mitomycin C was applied to the anastomosed flaps and osteotomy site for 30 minutes. Postoperative patients were followed for up to 6 months and outcome of patency was documented. Results: A.total of 73 patients were included, divided into test (30) and control (43) groups. An overall success rate of 86.3 percentage was obtained for patent ostia; this was based on 96.67 percentage success in test group compared to 79.1 percentage in the control group (p=0.031). Conclusion: Intraoperative application of Mitomycin-C significantly improves the success rate in external dacryocystorhinostomy. (author)

  18. Medium-term cryopreservation of rabies virus samples

    Directory of Open Access Journals (Sweden)

    Tereza D'avila de Freitas Aguiar

    2013-12-01

    Full Text Available Introduction The cryopreservation of rabies virus has been described in detail in the literature. To date, little information is available on the use of cryoprotective agents for cold preservation of this virus, and the available data focus only on short-term virus preservation. In this study, we investigated the medium-term cryopreservation of samples of rabies virus using different cryopreservation protocols. Methods The cryopreservation protocols for the rabies virus samples were performed at -20°C and were divided according to the variables of time and cryoprotectant type used. The laboratory tests (intracerebral inoculation of mice, viral titration and direct immunofluorescence were performed at regular intervals (360 and 720 days to assess the viability of the viral samples according to the different preservation techniques used. Results After 1 year of cryopreservation, the fluorescence intensity of intracellular corpuscles of the rabies virus and the median survival time of the mice differed between the positive controls and the treatments with the cryoprotectants. After 2 years, most of the samples subjected to the cryopreservation protocols (including the controls did not produce fluorescence. However, the virus samples exposed to the cryoprotectant sucrose (68% solution responded positively in the direct immunofluorescence assay and in the intracerebral inoculation of the mice. Conclusions Medium-term cryopreservation of the rabies virus inactivates the viral sample. However, the cryoprotectant agent sucrose (68% produces a preservative effect in cryopreserved rabies virus samples.

  19. [Effect of mitomycin C dissolved in a reversible thermosetting gel on outcome of filtering surgery in the rabbit].

    Science.gov (United States)

    Ichien, K; Sawada, A; Yamamoto, T; Kitazawa, Y; Shiraki, R; Yoh, M

    1999-04-01

    Based on our previous report that showed enhanced transfer of mitomycin C to the sclera and the conjunctiva by dissolving the antiproliferative in a reversible thermo-setting gel, we conducted a study to investigate the efficacy of the mitomycin C-gel in the rabbit. We subconjunctivally injected 0.1 ml of the mitomycin C-gel solution containing several amounts of the drug. Trephination was performed in the injected region 24 hours later. Intraocular pressure measurement, and photography and ultrasound biomicroscopic examination of the filtering bleb were done 1, 2, and 4 weeks postoperatively. The gel containing 3.0 micrograms or more mitomycin C significantly enhanced bleb formation in addition to reducing the intraocular pressure. The reversible thermo-setting gel seems to facilitate filtration following glaucoma filtering surgery in the rabbit and deserves further investigation as a new method of mitomycin C application.

  20. Variability of Streptomyces narbonesis producing ν-1,3/1,4 glucanglucan hydrolasi induced by UV and mitomycin C

    International Nuclear Information System (INIS)

    Elinov, N.P.; Pronina, M.I.; Zaikina, N.A.; Azizov, P.I.

    1982-01-01

    Effect of mitomycin c and ultraviolet rays on spores and spore sprouts of 2 hour old of Streptomyces narbonensis of 2a producer of ν-1.3/1.4 glucanglucanhydrolase (ν-gluconase) has been studied. It is shown that the lethal effect of mitomycin c on spore sprouts is considerably higher than on producer spores. Mutageneous activity of mitomycin C is closely related to the process of spore germination. Under the effect of mitomycin C on spore sprouts noted is higher mutability as compared with the effect on spores. Frequency of morphological mutations is lower under the effect of ultraviolet rays as compared with the effect of mitomycin C. Under ultraviolet rays produced were mutants synthesizing during fermentation on medium without inductor of exotype ν-gluconase

  1. Effect of Mitomycin C on Myopic versus Astigmatic Photorefractive Keratectomy

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    Ashwag A. Almosa

    2017-01-01

    Full Text Available Purpose. Long-term mitomycin C (MMC effects on photorefractive keratectomy (PRK were compared in simple myopic and astigmatic patients. Methods. In this observational cohort study, subjects were selected based on preoperative and postoperative data collected from medical records; they were divided into simple myopia with/without MMC and myopic astigmatism with/without MMC groups. Haze, uncorrected visual acuity (UCVA, best-corrected visual acuity (BCVA, subjective refraction, and K-reading were evaluated at 1-, 3-, 6-, and 12-month follow-ups. Results. One hundred fifty-nine eyes of 80 subjects (34 women and 46 men; mean age, 26.81 ± 7.74 years; range, 18–53 years; spherical powers, −0.50 to −8.00 DS; and cylindrical powers, −0.25 to −5.00 DC were enrolled. One year postoperatively, the simple myopia with/without MMC groups showed no difference in UCVA (P=0.187, BCVA (P=0.163, or spherical equivalent (P=0.163 and a significant difference (P=0.0495 in K-reading; the haze formation difference was nonsignificant (P=0.056. Astigmatic groups with/without MMC showed a significant difference in K-reading (P<0.0001. MMC groups had less haze formation (P<0.0001. Conclusion. PRK with intraoperative MMC application showed excellent visual outcomes. MMC’s effect on astigmatic patients was significantly better with acceptable safety and minimal side effects.

  2. Topical Mitomycin C and Radiation Induce Conjunctival DNA-Polyploidy

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    Olaf Cartsburg

    2001-01-01

    Full Text Available Introduction: Atypical cell changes often occur following treatment of premalignant or malignant conjunctival neoplasias with topical mitomycin C (MMC and/or radiation. These reactive, non‐neoplastic alterations of the conjunctival epithelium can be a differential diagnostic problem. Our aim was to investigate changes in the nuclear DNA‐distribution of conjunctival epithelial cells after MMC‐ and radiation therapy by DNA‐image‐cytometry. Methods: Conjunctival brush smears were obtained from 13 patients (13 eyes with squamous cell carcinomas and six patients (6 eyes with conjunctival malignant melanomas in situ before, during and after treatment. The patients were treated with MMC‐drops (0.02% or 0.04% alone (n=12, with radiation therapy (n=3 or both (n=4. At first, the obtained brush smears were evaluated by cytology. Secondly, after Feulgen restaining, the DNA content of reactively changed cells was determined using the AutoCyte‐QUIC‐DNA® workstation. Results: We observed euploid DNA‐polyploidy and cytomorphological changes in all patients (19/19. We considered these alterations as reactive to treatment. Four patients showed their greatest DNA‐stemline at 4c and 15 patients at 8c. This effect was observed during and following MMC‐drops and/or radiation and remained stable in 94% of all patients after a mean follow‐up of 22.5 months (SD 15.4. In five cases image cytometry additionally demonstrated DNA‐stemline aneuploidy as an evidence of tumor recurrence. Conclusion: Measurements of DNA‐content revealed euploid polyploidisation of morphological suspicious but benign squamous cells which is the biologic correlate of well known secondary morphologic changes following topical chemotherapy and/or radiation. DNA‐image‐cytometry is a useful tool in the differention of euploid polyploidization as a sign of reactive cell changes following treatment and tumor recurrences.

  3. The effect of mitomycin-c in keloid fibroblast cultures

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    Ishandono Dachlan

    2016-12-01

    Full Text Available ABSTRACT Keloid occurs due to hyperactivity of keloid fibroblast (KF in proliferation, migration, collagen deposition, together with low rates of collagen degradation. These are under the responsibility of TGF-b. Mitomycin C (MC is used for treating keloid by a topical application during surgery at the level of 0.02% to 0.08%. Unfortunately, the lowest effective level of MC for keloid has not been determined yet. We aimed to determine the lowest effective level of MC in the suppression of KF activities. Various levels of MC diluted in growth medium were administered on KF that were isolated from six patients. After 24 hours and 72 hours of incubation, cellular proliferation, collagen deposition, cellular migration and level of TGF-b, were analyzed. Application of 120 uM MC on KF culture for 24 hours could significantly reduce TGF-b production from 1265.74 ± 274.81 pg/mL to 265.17 ± 12.20 pg/mL; proliferation index from 100% to 84.01 ± 12.91%; inhibit cellular migration to 64.38 ± 3.66%; but reduce collagen depositions from 100% to only 91.13 ± 10.19%. The lowest MC level is on 30 uM or equal with 0.001%. In conclusion, the lowest level of MC can suppress the activities of KF is 0.001%. Moreover, due to low activity in inhibiting collagen deposition, MC would be better as an adjuvant drug for keloid surgery.

  4. Prospective randomized comparison of mitomycin C application in endoscopic and external dacryocystorhinostomy.

    Science.gov (United States)

    Mudhol, Rekha R; Zingade, N D; Mudhol, R S; Harugop, Anil S; Das, Amal T

    2013-08-01

    The aim of the study is to compare the subjective (relief of symptoms) and objective (endoscopic visualization of ostium patency at the time of syringing) outcomes at the end of two procedures-Endonasal DCR versus External DCR with Mitomycin C and to assess the role of Mitomycin C in maintaining patency of nasolacrimal drainage system. Prospective randomized comparative study was performed. Thirty-five patients were enrolled in each endoscopic and external dacryocystorhinostomy groups with Mitomycin C (MMC) application. The 37 eyes underwent endonasal DCR (28 unilateral primary eyes + 1 bilateral primary eyes + 5 unilateral revision eyes + 1 bilateral revision eye) while 35 eyes underwent external DCR (34 unilateral primary eyes + 1 unilateral revision eye). Mitomycin C 0.2 mg/ml was applied intra-operatively for 5 min to the ostium site at the end of endonasal or external DCR procedure. Objective assessment by syringing at the end of 1 year in the endonasal group showed 35 eyes (94%) were patent, 1 (3%) was partially blocked and 1(3%) was completely blocked; while in external group all 35 eyes (100%) were patent. Endoscopic visualization of the ostium at the time of syringing showed only one eye (3%) in the endonasal group was blocked while all the other eyes in both groups were patent. Both groups had a mean follow-up of 6-36 months. No complications were associated with use of Mitomycin C. In conclusion, intra-operative use of Mitomycin C in both endoscopic DCR and external DCR is safe and effective in increasing the success rate.

  5. Ovarian tissue cryopreservation and transplantation among alternatives for fertility preservation in the Nordic countries

    DEFF Research Database (Denmark)

    Rodriguez-Wallberg, Kenny A; Tanbo, Tom; Tinkanen, Helena

    2016-01-01

    cryopreservation to be experimental. In Iceland, embryo cryopreservation is the only option for fertility preservation. Most centers use slow-freezing methods for ovarian tissue cryopreservation. Most patients selected for ovarian tissue cryopreservation were newly diagnosed with cancer and the tissue...

  6. Cryopreservation of Embryos and Oocytes in Human Assisted Reproduction

    Directory of Open Access Journals (Sweden)

    János Konc

    2014-01-01

    Full Text Available Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification of human embryos and oocytes are summarized.

  7. Cryopreservation of embryos and oocytes in human assisted reproduction.

    Science.gov (United States)

    Konc, János; Kanyó, Katalin; Kriston, Rita; Somoskői, Bence; Cseh, Sándor

    2014-01-01

    Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification) of human embryos and oocytes are summarized.

  8. First attempts to cryopreserve red abalone (Haliotis rufescens oocytes

    Directory of Open Access Journals (Sweden)

    Ramírez Torrez, A.

    2015-01-01

    Full Text Available Overall, few advances in the cryopreservation of complex cells such as oocytes, embryo or tissue have been registered and in less quantity have been reported for aquatic species. Abalone has high economic interest worldwide and the conservation of abalone germplasm may help to enhance its culture and develop repopulation programs. In this work, we reported the cytotoxic effect of two concentration of trehalose (0.2 and 0.4 M on red abalone oocytes incubated for 10, 15 and 20 min. Also, we reported the cryopreservation of red abalone oocytes using a 3-steps cryopreservation protocol and 5 thawing protocols. Significant differences on cytotoxic effect were found (p<0.01. However, none of the cryoprotectant was optimum to cryopreserve red abalone oocyte. In conclusion, it is necessary to find an appropriate method to dehydrate or make the cryoprotectant penetrate on the abalone oocyte before proceeding to cryopreservation.

  9. Effect of Cryopreservation and Post-Cryopreservation Somatic Embryogenesis on the Epigenetic Fidelity of Cocoa (Theobroma cacao L.).

    Science.gov (United States)

    Adu-Gyamfi, Raphael; Wetten, Andy; Marcelino Rodríguez López, Carlos

    2016-01-01

    While cocoa plants regenerated from cryopreserved somatic embryos can demonstrate high levels of phenotypic variability, little is known about the sources of the observed variability. Previous studies have shown that the encapsulation-dehydration cryopreservation methodology imposes no significant extra mutational load since embryos carrying high levels of genetic variability are selected against during protracted culture. Also, the use of secondary rather than primary somatic embryos has been shown to further reduce the incidence of genetic somaclonal variation. Here, the effect of in vitro conservation, cryopreservation and post-cryopreservation generation of somatic embryos on the appearance of epigenetic somaclonal variation were comparatively assessed. To achieve this we compared the epigenetic profiles, generated using Methylation Sensitive Amplified Polymorphisms, of leaves collected from the ortet tree and from cocoa somatic embryos derived from three in vitro conditions: somatic embryos, somatic embryos cryopreserved in liquid nitrogen and somatic embryos generated from cryoproserved somatic embryos. Somatic embryos accumulated epigenetic changes but these were less extensive than in those regenerated after storage in LN. Furthermore, the passage of cryopreserved embryos through another embryogenic stage led to further increase in variation. Interestingly, this detected variability appears to be in some measure reversible. The outcome of this study indicates that the cryopreservation induced phenotypic variability could be, at least partially, due to DNA methylation changes. Phenotypic variability observed in cryostored cocoa somatic-embryos is epigenetic in nature. This variability is partially reversible, not stochastic in nature but a directed response to the in-vitro culture and cryopreservation.

  10. The effect of topical mitomycin C on full-thickness burns.

    Science.gov (United States)

    Tennyson, Heath; Helling, Eric R; Wiseman, Joseph; Dick, Edward; Lyons, Robert C

    2007-09-15

    Burns result in substantial morbidity because of fibroblast proliferation and contracture. Mitomycin C is a chemotherapeutic agent known to suppress fibroblast proliferation. It is used in ophthalmologic disorders and reduces scarring in upper aerodigestive surgery. No study of the effect of mitomycin C on cutaneous burns has been performed. This study examined burn healing in the presence of topical mitomycin C by evaluation of wound appearance, contraction, and histology in a pig model. Standardized full-thickness burns were produced on the flanks of three pigs. One animal received no further therapy and was an external control. Two animals underwent placement of topical mitomycin C, 0.4 mg/ml, on selected burn sites for 5 minutes. This was repeated 2 and 4 weeks after injury. Evaluation was performed at 2 and 6 months using a clinical assessment scale and a visual analogue scale. Scar length and histologic analysis were also evaluated. Clinical assessment scale and visual analogue scale scores showed improved appearance in the untreated external control wounds versus the untreated internal control and treated wounds (p Wound contraction was not significantly different between groups. Histologic characteristics between groups were similar except for epidermal hyperplasia, which was decreased in the untreated external control (p wounds at 0.4 mg/cc for three courses does not improve, and may worsen, clinical appearance and scarring during early healing. There is no difference in histology during the long-term healing process. Scar contraction was unchanged.

  11. Successful Oocyte Cryopreservation in Reproductive-Aged Cancer Survivors.

    Science.gov (United States)

    Druckenmiller, Sarah; Goldman, Kara N; Labella, Patty A; Fino, M Elizabeth; Bazzocchi, Antonia; Noyes, Nicole

    2016-03-01

    To demonstrate that oocyte cryopreservation is a feasible reproductive option for patients with cancer of childbearing age who require gonadotoxic therapies. This study is a university-based retrospective review of reproductive-aged cancer patient treatment cycles that included ovarian stimulation, transvaginal oocyte retrieval, oocyte cryopreservation, and, in some cases, subsequent oocyte thaw, in vitro fertilization, and embryo transfer. Outcome measures included ovarian stimulation response, number of oocytes retrieved, cryopreserved, and thawed, and pregnancy data. From 2005 to 2014, 176 reproductive-aged patients with cancer (median age 31 years, interquartile range 24-36) completed 182 oocyte cryopreservation cycles. Median time between consult request and oocyte retrieval was 12 days (interquartile range 10-14). Median peak stimulation estradiol was 1,446 pg/mL (interquartile range 730-2,687); 15 (interquartile range 9-23) oocytes were retrieved and 10 (interquartile range 5-18) metaphase II oocytes were cryopreserved per cycle. Ten patients (11 cycles) have returned to attempt pregnancy with their cryopreserved oocytes. Among thawed oocytes, the cryopreservation survival rate was 86% (confidence interval [CI] 78-94%). Nine of 11 thaw cycles resulted in embryos suitable for transfer. The embryo implantation rate was 27% (CI 8-46%) and the live birth rate was 44% (CI 12-77%) per embryo transfer. Chance for live birth with embryos created from cryopreserved oocytes was similar between the patients with cancer in this study and noncancer patients who underwent the same treatment at our center (44% [CI 12-77%] compared with 33% [CI 22-44%] per embryo transfer). Oocyte cryopreservation is now a feasible fertility preservation option for reproductive-aged patients with cancer who require gonadotoxic therapies.

  12. Cryopreservation of tissue engineered constructs for bone.

    Science.gov (United States)

    Kofron, Michelle D; Opsitnick, Natalie C; Attawia, Mohamed A; Laurencin, Cato T

    2003-11-01

    The large-scale clinical use of tissue engineered constructs will require provisions for its mass availability and accessibility. Therefore, it is imperative to understand the effects of low temperature (-196 degrees C) on the tissue engineered biological system. Initial studies used samples of the osteoblast-like cell line (SaOS-2) adhered to a two-dimensional poly(lactide-co-glycolide) thin film (2D-PLAGA) or a three-dimensional poly(lactide-co-glycolide) sintered microsphere matrix (3D-PLAGA) designed for bone tissue engineering. Experimental samples were tested for their ability to maintain cell viability, following low temperature banking for one week, in solutions of the penetrating cryoprotective agents, dimethylsulfoxide (DMSO), ethylene glycol, and glycerol. Results indicated the DMSO solution yielded the greatest percent cell survival for SaOS-2 cells adhered to both the 2D- and 3D-PLAGA scaffolds; therefore, DMSO was used to cryopreserve mineralizing primary rabbit osteoblasts cells adhered to 2D-PLAGA matrices for 35 days. Results indicated retention of the extracellular matrix architecture as no statistically significant difference in the pre- and post-thaw mineralized structures was measured. Percent cell viability of the mineralized constructs following low temperature storage was approximately 50%. These are the first studies to address the issue of preservation techniques for tissue engineered constructs. The ability to successfully cryopreserve mineralized tissue engineered matrices for bone may offer an unlimited and readily available source of bone-like materials for orthopaedic applications.

  13. Recent Progress in Cryopreservation of Bovine Oocytes

    Directory of Open Access Journals (Sweden)

    In-Sul Hwang

    2014-01-01

    Full Text Available Principle of oocyte cryoinjury is first overviewed and then research history of cryopreservation using bovine oocytes is summarized for the last two decades with a few special references to recent progresses. Various types of cryodevices have been developed to accelerate the cooling rate and applied to the oocytes from large domestic species enriched with cytoplasmic lipid droplets. Two recent approaches include the qualitative improvement of IVM oocytes prior to the vitrification and the short-term recovery culture of vitrified-warmed oocytes prior to the subsequent IVF. Supplementation of L-carnitine to IVM medium of bovine oocytes has been reported to reduce the amount of cytoplasmic lipid droplets and improve the cryotolerance of the oocytes, but it is still controversial whether the positive effect of L-carnitine is reproducible. Incidence of multiple aster formation, a possible cause for low developmental potential of vitrified-warmed bovine oocytes, was inhibited by a short-term culture of the postwarm oocytes in the presence of Rho-associated coiled-coil kinase (ROCK inhibitor. Use of an antioxidant α-tocopherol, instead of the ROCK inhibitor, also supported the revivability of the postwarm bovine oocytes. Further improvements of the vitrification procedure, combined with pre- and postvitrification chemical treatment, would overcome the high sensitivity of bovine oocytes to cryopreservation.

  14. Nucleons II: cryopreservation and metabolic activity.

    Science.gov (United States)

    Reyes, R; Flores-Alonso, J C; Rodríguez-Hernández, H M; Merchant-Larios, H M; Delgado, N M

    2001-01-01

    The establishment of intracytoplasmatic sperm injection (ICSI) as a routine procedure in assisted fertilization has been used in the treatment of male infertility. The major technical problem that has arisen with the use of immotile sperm for ICSI has been differentiating between live and dead cells. Nucleons from human, pig, hamster, mouse, rat, and bull have been able to induce their chromatin decondensation by the action of heparin/GSH. Cryopreservation is deleterious to sperm function, killing more than 50% of the spermatozoa during the process. Nucleon cryostorage was performed at 5 and -5 degrees C and analyzed for total area (mu2), perimeter (mu), width (mu), and length (mu), using Metamorph Imaging System software. On the other hand, fluorescein diacetate (FDA) is hydrolyzed by intracellular estereases to produce fluorescein, which exhibits green fluorescence when excited by blue light. This fact is a striking result since the presence of this metabolic activity opens the possibility to select the nucleons for ICSI. In the present study, the authors decided to search for a suitable metabolic test, which might reflect the metabolism and viability of these chromatin structures. This is a simple cryostorage technique that after months of cryopreservation, allow the use of nucleons for ICSI with suitable fertilization and pregnancies rates.

  15. Partial dehydration and cryopreservation of Citrus seeds.

    Science.gov (United States)

    Graiver, Natalia; Califano, Alicia; Zaritzky, Noemí

    2011-11-01

    Three categories of seed storage behavior are generally recognized among plant species: orthodox, intermediate and recalcitrant. Intermediate seeds cannot be stored in liquid nitrogen (LN) without a previous partial dehydration process. The water content (WC) of the seeds at the moment of immersion in LN must be regarded as the most critical factor in cryopreservation. The purpose of this study was to investigate the basis of the optimal hydration status for cryopreservation of Citrus seeds: C. sinensis (sweet orange), C. paradisi (grapefruit), C. reticulata (mandarin) in LN. To study the tolerance to dehydration and LN exposure, seeds were desiccated by equilibration at relative humidities between 11 and 95%. Sorption isotherms were determined and modeled; lipid content of the seeds was measured. Seed desiccation sensitivity was quantified by the quantal response model. Differential scanning calorimetry (DSC) thermograms were determined on cotyledon tissue at different moisture contents to measure ice melting enthalpies and unfrozen WC. Samples of total seed lipid extract were also analyzed by DSC to identify lipid transitions in the thermograms. The limit of hydration for LN Citrus seeds treatment corresponded to the unfrozen WC in the tissue, confirming that seed survival strictly depended on avoidance of intracellular ice formation. Copyright © 2011 Society of Chemical Industry.

  16. Evaluation of the damage in fish spermatozoa cryopreservation

    Science.gov (United States)

    Li, Jun; Liu, Qinghua; Zhang, Shicui

    2006-12-01

    Cryodamages occur during sperm cryopreservation. Cryopreservation of fish sperm usually results in marked decrease in sperm quality, such as swelling or disruption of the plasma membrane, mitochondrial dysfunction, diminished sperm motility, impaired velocity, shorter motility period, denaturation, and release of some enzymes from spermatozoa. In this paper, damages in morphology, physiology, biochemistry and metabolism, and genetic integrity of fish semen after cryopreservation are discussed. New approaches in assessment of fish thawed sperm quality such as computer assisted sperm analysis, flow cytometic analysis combined with fluorescent probes and single cell gel electrophoresis are also briefly reviewed.

  17. Exploration of two methods for quantitative Mitomycin C measurement in tumor tissue in vitro and in vivo

    DEFF Research Database (Denmark)

    Fischer, Lee MacKenzie; Vásquez, Juan Luis; Gehl, Julie

    2013-01-01

    Two methods of quantifying Mitomycin C in tumor tissue are explored. A method of ultraviolet-visible absorption microscopy is developed and applied to measure the concentration of Mitomycin C in preserved mouse tumor tissue, as well as in gelatin samples. Concentrations as low as 60 μM can...... be resolved using this technique in samples that do not strongly scatter light. A novel method for monitoring the Mitomycin C concentrations inside a tumor is developed, based on microdialysis and ultraviolet-visible spectroscopy. A pump is used to perfuse a microdialysis probe with Ringer’s solution, which...

  18. Microbial Safety and Shelf Life of UV-C Treated Freshly Squeezed White Grape Juice.

    Science.gov (United States)

    Unluturk, Sevcan; Atilgan, Mehmet R

    2015-08-01

    The effects of UV-C irradiation on the inactivation of Escherichia coli K-12 (ATCC 25253), a surrogate of E. coli O157:H7, and on the shelf life of freshly squeezed turbid white grape juice (FSWGJ) were investigated. FSWGJ samples were processed at 0.90 mL/s for 32 min by circulating 8 times in an annular flow UV system. The UV exposure time was 244 s per cycle. The population of E. coli K-12 was reduced by 5.34 log cycles after exposure to a total UV dosage of 9.92 J/cm(2) (1.24 J/cm(2) per cycle) at 0.90 mL/s flow rate. The microbial shelf life of UV-C treated FSWGJ was extended up to 14 d at 4 °C. UV exposure was not found to alter pH, total soluble solid, and titratable acidity of juice. There was a significant effect (P shelf life of FSWGJ was doubled after UV-C treatment, whereas the quality of juice was adversely affected similarly observed in the control samples. © 2015 Institute of Food Technologists®

  19. [Autoimmunity in children with chronic hepatitis C treated with interferon alpha and ribavirin].

    Science.gov (United States)

    Gora-Gebka, Magdalena; Liberek, Anna; Bako, Wanda; Raczkowska-Kozak, Janina; Sikorska-Wisniewska, Grazyna; Korzon, Maria

    2004-01-01

    The role of interferon alpha or the virus itself in the pathogenesis and the risk of autoimmunological disorders in patients infected with HCV, still remain unknown, especially in children. The aim of the study was to evaluate the incidence of autoantibodies and the risk of autoimmunological disorders in children with chronic hepatitis C, treated with interferon alpha and ribavirin in the Department of Paediatrics, Paediatric Gastroenterology and Oncology in Gdansk. In the studied group of 12 patients, in 4 cases autoantibodies were present in low titers prior to the treatment and they had no prognostic value for the response to the therapy or the risk of autoimmunological disorders. Positive response for the treatment was achieved in 4 cases; in 3 cases indications for discontinuation of the therapy were established. During the therapy with interferon alpha and ribavirin, in 2 children elevation of serum titers of antibodies to liver-kidney microsome type 1 (anti-LKM1) (> 1:640) with normal gammaglobulin levels was noted. In none of the children autoimmunological disorders were observed.

  20. Cryopreservation of Mammalian Oocyte for Conservation of Animal Genetics

    Directory of Open Access Journals (Sweden)

    Jennifer R. Prentice

    2011-01-01

    Full Text Available The preservation of the female portion of livestock genetics has become an international priority; however, in situ conservation strategies are extremely expensive. Therefore, efforts are increasingly focusing on the development of a reliable cryopreservation method for oocytes, in order to establish ova banks. Slow freezing, a common method for cryopreservation of oocytes, causes osmotic shock (solution effect and intracellular ice crystallization leading to cell damage. Vitrification is an alternative method for cryopreservation in which cells are exposed to a higher concentration of cryoprotectants and frozen with an ultra rapid freezing velocity, resulting in an ice crystal free, solid glass-like structure. Presently, vitrification is a popular method for cryopreservation of embryos. However, vitrification of oocytes is still challenging due to their complex structure and sensitivity to chilling.

  1. Chapter 1 Historical Background on Gamete and Embryo Cryopreservation.

    Science.gov (United States)

    Ali, Jaffar; AlHarbi, Naif H; Ali, Nafisa

    2017-01-01

    This chapter describes the development of the science of cryopreservation of gametes and embryos of various species including human. It attempts to record in brief the main contributions of workers in their attempts to cryopreserve gametes and embryos. The initial difficulties faced and subsequent developments and triumphs leading to present-day state of the art are given in a concise manner. The main players and their contributions are mentioned and the authors' aim is to do justice to them. This work also attempts to ensure that credit is correctly attributed for significant advances in gamete and embryo cryopreservation. In general this chapter has tried to describe the historical development of the science of cryopreservation of gametes and embryos as accurately as possible without bias or partiality.

  2. Cryopreservation studies on the marine diatom Navicula subinflata Grun

    Digital Repository Service at National Institute of Oceanography (India)

    Redekar, P.D.; Wagh, A.B.

    of cryoprotectant decreased the growth. These experiments after cryopreservation revealed that the maximum growth was recorded in Ethylene glycol treated cells for 0 day sample and 7 days and 30 days liquid nitrogen preserved samples. No growth was observed...

  3. Vitrification versus slow freezing for women undergoing oocyte cryopreservation

    NARCIS (Netherlands)

    Glujovsky, Demián; Riestra, Barbara; Sueldo, Carlos; Fiszbajn, Gabriel; Repping, Sjoerd; Nodar, Florencia; Papier, Sergio; Ciapponi, Agustín

    2014-01-01

    Oocyte cryopreservation is a technique with considerable potential in reproductive medicine, including fertility preservation, as a way of delaying childbearing and as part of oocyte donation programs. Although the technique was relatively ineffective at first more recently numerous modifications

  4. Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos by vitrification.

    Science.gov (United States)

    Sajini, K K; Karun, A; Amamath, C H; Engelmann, F

    2011-01-01

    The present study investigates the effect of preculture conditions, vitrification and unloading solutions on survival and regeneration of coconut zygotic embryos after cryopreservation. Among the seven plant vitrification solutions tested, PVS3 was found to be the most effective for regeneration of cryopreserved embryos. The optimal protocol involved preculture of embryos for 3 days on medium with 0.6 M sucrose, PVS3 treatment for 16 h, rapid cooling and rewarming and unloading in 1.2 M sucrose liquid medium for 1.5 h. Under these conditions, 70-80 survival (corresponding to size enlargement and weight gain) was observed with cryopreserved embryos and 20-25 percent of the plants regenerated (showing normal shoot and root growth) from cryopreserved embryos were established in pots.

  5. Early investigation on cryopreservation of Dendrobium sonia-28 ...

    African Journals Online (AJOL)

    User

    2011-05-02

    May 2, 2011 ... This study was conducted to determine the potential of cryostoring and regenerating Dendrobium ... Key words: Orchid, protocorm-like bodies, cryopreservation. ... technology is now taken as an ideal alternative propa-.

  6. Respiration shutoff in Escherichia coli K12 strains is induced by far ultraviolet radiations and by mitomycin C

    International Nuclear Information System (INIS)

    Swenson, P.A.; Norton, I.L.

    1984-01-01

    Near ultraviolet radiations (UV) cause respiration to shutoff in Escherichia coli B/r. It has been reported that E. coli K12 strains do not shut off respiration after UV. It is also reported that mitomycin C did not cause this 'SOS' response. In this paper it is reported that higher UV fluences than were previously used will cause respiration shutoff in K12 strain W3110 and that cyclic AMP increases the sensitivity of respiration shutoff of irradiated cell suspensions. Also mitomycin C shuts off respiration in this strain. Neither UV nor mitomycin C causes respiration shutoff in the recA56 derivative of W3110. Thus respiration shutoff is a recA dependent response to UV and mitomycin C in E. coli K12 strains. (Auth.)

  7. Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT

    Directory of Open Access Journals (Sweden)

    Morse Michael A

    2005-07-01

    Full Text Available Abstract Background Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC, and ELISPOT. In this study, we compared the performance of these assays on leukapheresed PBMC shipped overnight in medium versus cryopreserved PBMC from matched donors. Results Using CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p ≤ 0.001. The differences in response magnitude between cryopreserved and shipped PBMC specimens were not significant for most antigens and assays. There was significant correlation between CFC and ELISPOT assay using pp65 peptide pool stimulation, in both shipped and cryopreserved samples (p ≤ 0.001. Strong correlation was observed between CFC (using HLA-A2-restricted pp65 peptide stimulation and tetramer staining (p Conclusion We conclude that all three assays show concordant results on shipped versus cryopreserved specimens, when using a peptide-based readout. The assays are also concordant with each other in pair wise comparisons using equivalent antigen systems.

  8. Respiratory analysis of coupled mitochondria in cryopreserved liver biopsies

    Directory of Open Access Journals (Sweden)

    Mercedes García-Roche

    2018-07-01

    Full Text Available The aim of this work was to develop a cryopreservation method of small liver biopsies for in situ mitochondrial function assessment. Herein we describe a detailed protocol for tissue collection, cryopreservation, high-resolution respirometry using complex I and II substrates, calculation and interpretation of respiratory parameters. Liver biopsies from cow and rat were sequentially frozen in a medium containing dimethylsulfoxide as cryoprotectant and stored for up to 3 months at −80 °C. Oxygen consumption rate studies of fresh and cryopreserved samples revealed that most respiratory parameters remained unchanged. Additionally, outer mitochondrial membrane integrity was assessed adding cytochrome c, proving that our cryopreservation method does not harm mitochondrial structure. In sum, we present a reliable way to cryopreserve small liver biopsies without affecting mitochondrial function. Our protocol will enable the transport and storage of samples, extending and facilitating mitochondrial function analysis of liver biopsies. Keywords: Cryopreservation, Mitochondria, Biopsy, Oxygen consumption rate, High-resolution respirometry, Mitochondrial function

  9. Oocyte cryopreservation for donor egg banking.

    Science.gov (United States)

    Cobo, Ana; Remohí, José; Chang, Ching-Chien; Nagy, Zsolt Peter

    2011-09-01

    Oocyte donation is an efficient alternative to using own oocytes in IVF treatment for different indications. Unfortunately, 'traditional' (fresh) egg donations are challenged with inefficiency, difficulties of synchronization, very long waiting periods and lack of quarantine measures. Given the recent improvements in the efficiency of oocyte cryopreservation, it is reasonable to examine if egg donation through oocyte cryopreservation has merits. The objective of the current manuscript is to review existing literature on this topic and to report on the most recent outcomes from two established donor cryobank centres. Reports on egg donation using slow freezing are scarce and though results are encouraging, outcomes are not yet comparable to a fresh egg donation treatment. Vitrification on the other hand appears to provide high survival rates (90%) of donor oocytes and comparable fertilization, embryo development, implantation and pregnancy rates to traditional (fresh) egg donation. Besides the excellent outcomes, the ease of use for both donors and recipients, higher efficiency, lower cost and avoiding the problem of synchronization are all features associated with the benefit of a donor egg cryobank and makes it likely that this approach becomes the future standard of care. Oocyte donation is one of the last resorts in IVF treatment for couples challenged with infertility problems. However, traditional (fresh) egg donation, as it is performed today, is not very efficient, as typically all eggs from one donor are given to only one recipient, it is arduous as it requires an excellent synchronization between the donor and recipient and there are months or years of waiting time. Because of the development of an efficient oocyte cryopreservation technique, it is now possible to cryo-store donor (as well as non-donor) eggs, maintaining their viability and allowing their use whenever there is demand. Therefore, creating a donor oocyte cryobank would carry many advantages

  10. First production of larvae using cryopreserved sperm: Effects of preservation temperature and cryopreservation on European eel sperm fertilization capacity

    DEFF Research Database (Denmark)

    Asturiano, J.F.; Sørensen, Sune Riis; Perez, L.

    2016-01-01

    Sperm cryopreservation is a useful tool in captive fish reproduction management, that is to synchronize gamete production, especially in the case of species as the European eel, where the time of female spawning readiness is unpredictable. Several protocols to cryopreserve sperm of this species....... Fertilization of two egg batches was attempted. Diluted sperm caused a similar percentage of fertilized eggs and a similar number of embryos and larvae, independently of storage temperature (4 or 20°C). The cryopreserved sperm resulted in a lower percentage of fertilized eggs, but embryos developed and a few...... larvae ('cryolarvae') were obtained 55 h after fertilization in one of the two egg batches. This result evidences that the tested cryopreservation protocol is applicable for eel reproduction management, although improvements will be required to enhance fertilization success...

  11. Bentall Procedure Using Cryopreserved Valved Aortic Homografts

    Science.gov (United States)

    Christenson, Jan T.; Sierra, Jorge; Trindade, Pedro T.; Didier, Dominique; Kalangos, Afksendiyos

    2004-01-01

    The Bentall procedure is the standard operation for patients who have lesions of the ascending aorta associated with aortic valve disease. In many cases, however, mechanical prosthetic conduits are not suitable. There are few reports in the English-language medical literature concerning the mid- to long-term outcome of Bentall operations with cryopreserved homografts. Therefore, we reviewed our experience with this procedure and valved homografts. From January 1997 through December 2002, 21 patients underwent a Bentall operation with cryopreserved homografts at our institution. There were 14 males and 7 females; the mean age was 36 ± 21 years (range, 15–74 years). Eleven patients had undergone previous aortic valve surgery. All patients had aortic dilatation or aneurysms involving the ascending aorta. Indications for surgery included aortic valve stenosis or insufficiency, and aortic valve endocarditis (native valve or prosthetic). One patient had Takayasu's arteritis and 3 had Marfan syndrome. There was 1 hospital death (due to sepsis), but no other major postoperative complications. The mean hospital stay was 14 ± 7 days. Follow-up echocardiographic and computed tomographic scans were performed yearly. The mean follow-up was 34 months (6–72 months). Follow-up imaging revealed no calcifications or degenerative processes related to the homograft. Four patients had minimal valve regurgitation. Two patients died during follow-up. The 3-year actuarial survival rate was 85.7%. Our data suggest that the Bentall procedure with a valved homograft conduit is a safe procedure with excellent mid- to long-term results, comparable to results reported with aortic valve replacement with a homograft. PMID:15745290

  12. Validation and use of microdialysis for determination of pharmacokinetic properties of the chemotherapeutic agent mitomycin C - an experimental study

    International Nuclear Information System (INIS)

    Sørensen, Olaf; Andersen, Anders; Olsen, Harald; Alexandr, Kristian; Ekstrøm, Per Olaf; Giercksky, Karl-Erik; Flatmark, Kjersti

    2010-01-01

    Mitomycin C is a chemotherapeutic agent used in the treatment of peritoneal surface malignancies, administered as hyperthermic intraperitoneal chemotherapy after cytoreductive surgery. Pharmacokinetic studies have been based on analyses of blood, urine and abdominal perfusate, but actual tissue concentrations of the drug have never been determined. Microdialysis is an established method for continuous monitoring of low-molecular substances in tissues, and in the present study microdialysis of mitomycin C was studied in vitro and in vivo. Using in vitro microdialysis, relative recovery was determined when varying drug concentration, temperature and perfusion flow rate. In vivo microdialysis was performed in rats to verify long-term stability of relative recovery in four compartments (vein, peritoneum, extraperitoneal space and hind leg muscle). Subsequently, intravenous and intraperitoneal bolus infusion experiments were performed and pharmacokinetic parameters were calculated. In vitro, compatibility of mitomycin C and microdialysis equipment was demonstrated, and relative recovery was stable over an adequate concentration range, moderately increased by raising medium temperature and increased when flow rate was reduced, all according to theory. In vivo, stable relative recovery was observed over seven hours. Mitomycin C exhibited fast and even distribution in rat tissues, and equal bioavailability was achieved by intravenous and intraperitoneal infusion. The half-life of mitomycin C calculated after intravenous infusion was 40 minutes. Mitomycin C concentration can be reliable monitored in vivo using microdialysis, suggesting that this technique can be used in pharmacokinetic studies of this drug during hyperthermic intraperitoneal chemotherapy

  13. Induced chromosomal aberrations in somatic cells of Nigella sativa L. by mitomycin C.

    Science.gov (United States)

    Kumar, P; Nizam, J

    1978-01-01

    A cytological study was carried out on root tips of Nigella sativa L. by treatment with Mitomycin C at 0.001% for six time intervals (10, 15, 20, 30, 40, and 50 min). The chromosomal abnormalities were increasingly proportionate to the increase in time of treatment. The seedlings treated with a 0.001% concentration of Mitomycin C for 10 min. did not show any significant effect. At other time intervals, the effect was observed to be quite significant. Beyond 40 min. treatment almost all the cells would become sticky. Thirty minutes' treatment showed significant effect, inducing various types of chromosomal aberrations in the anaphase, such as bridges and fragments of 34.13% and 48.07%, respectively.

  14. Radical radiation therapy with 5-fluorouracil infusion and mitomycin C for oesophageal squamous carcinoma

    International Nuclear Information System (INIS)

    Keane, T.J.; Harwood, A.R.; Elhakim, T.; Rider, W.D.; Cummings, B.J.; Ginsberg, R.J.; Cooper, J.C.

    1985-01-01

    Thirty-five patients with clinically staged non-metastatic squamous carcinoma of the oesophagus were treated with radiation combined with mitomycin C, and 5-fluorouracil (5-FUra) infusion. Twenty patients were planned for a split course regimen 2250-2500 cGy in 10 fractions and chemotherapy. This dose of radiation to be repeated with another course of chemotherapy after 4 weeks rest. Fifteen patients were planned for a single course 4500-5000 cGy in 20 fractions and a single course of chemotherapy. Patients were matched for age, sex, TNM stage, and total radiation dose. There was a significant difference in survival p = 0.004 and local relapse-free rate p = 0.05 for patients receiving radiation and chemotherapy. We conclude that radiation combined with mitomycin C, and 5-FUra infusion appears to have a significant benefit compared to radiation as a single modality in the management of oesophageal squamous carcinoma. (orig.)

  15. Controlled release of mitomycin C from PHEMAH-Cu(II) cryogel membranes.

    Science.gov (United States)

    Bakhshpour, Monireh; Yavuz, Handan; Denizli, Adil

    2018-02-19

    Molecular imprinting technique was used for the preparation of antibiotic and anti-neoplastic chemotherapy drug (mitomycin C) imprinted cryogel membranes (MMC-ICM). The membranes were synthezied by using metal ion coordination interactions with N-methacryloyl-(l)-histidine methyl ester (MAH) functional monomer and template molecules (i.e. MMC). The 2-hydroxyethyl methacrylate (HEMA) monomer and methylene bisacrylamide (MBAAm) crosslinker were used for the preparation of mitomycin C imprinted cryogel membranes by radical suspension polymerization technique. The imprinted cryogel membranes were characterized by scanning electron microscopy (SEM), Brunauer-Emmett-Teller (BET), Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) and swelling degree measurements. Cytotoxicity of MMC-ICMs was investigated using mouse fibroblast cell line L929. Time-dependent release of MMC was demonstrated within 150 h from cryogel membranes. Cryogels demonstrated very high MMC loading efficiency (70-80%) and sustained MMC release over hours.

  16. Modification of trout sperm membranes associated with activation and cryopreservation. Implications for fertilizing potential

    Science.gov (United States)

    Abstract We investigated the effects of two trout sperm activation solutions on sperm physiology and membrane organization prior to and following cryopreservation using flow cytometry and investigated their impact on in vitro fertility. Cryopreservation caused greater phospholipid disorder (high pl...

  17. Determination of metabolic stability using cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss)

    Science.gov (United States)

    Standard protocols for isolating, cryopreserving, and thawing rainbow trout hepatocytes are described, along with procedures for using fresh or cryopreserved hepatocytes to assess chemical metabolic stability in fish by means of a substrate depletion approach. Variations on thes...

  18. Pulse radiolysis study of the reduction mechanism of an antitumor antibiotic, mitomycin C

    International Nuclear Information System (INIS)

    Machtalere, G.; Houee-Levin, C.; Gardes-Albert, M.; Ferradini, C.; Hickel, B.

    1988-01-01

    Mitomycin C is a quinonic antitumor metabolized in vivo by one-electron reduction. We have studied the mechanism of the one-electron reduction of this drug by pulse radiolysis using C00 .- free radicals as reductants. Semiquinonic and hydroquinonic intermediates are formed. The hydroquinonic form undergoes a methanol elimination leading to a transient which can disappear in one of two ways: by either internal redox reaction or hydrolysis of the aziridine. 17 refs [fr

  19. Evaluation of ebselen supplementation on cryopreservation medium in human semen.

    Science.gov (United States)

    Khodayari Naeini, Zohreh; Hassani Bafrani, Hassan; Nikzad, Hossein

    2014-04-01

    An effect of cryopreservation on human sperm is sublethal cryodamage, in which cell viability post-thaw is lost more rapidly at later times than in fresh cells. This study examined whether the addition of an antioxidant to cryopreservation medium could improve the post-thaw parameters and evaluation of sperm chromatin quality of cryopreserved human spermatozoa from men with normal semen parameters. Semen samples (n=35) were collected by masturbation and assessed following WHO standards. Individual samples were classified as two portions. One portion (n=10) was for elucidate the concentration of ebselen.Then the samples(n=25) were divided in to 5groups.The first aliquot remained fresh.The second aliquots was mixed with cryopreservation medium.The third aliquots were mixed with cryopreservation medium containing solvent of ebselen.The forth and fifth aliquots were mixed with cryopreservation medium containing 1.25 and 2.5 µm of ebselen.Samples were frozen and thawed samples were assessed for sperm parameters.Three-way ANOVA Multivariate measures were used to assess. According to this assesment the differences are observed in existent groups in post-thaw count, motility index, vitality staining, and morphology and DNA fragmentation. After freezing the media containing of ebselen, DNA fragmentation is significantly different in comparison with control group. ebselen with 1.25 µm dose was significantly associated with post-thaw DNA fragmentation (p=0.047). Similarly ebselen with 2.5 µm dose was significantly associated with post-thaw DNA fragmentation (p=0.038). But other parameters were not altered. These results suggest that the addition of ebselen to cryopreservation medium doesnot improve post-thaw parameters and DNA fragmentation of sperm.

  20. Sensitivity of pathogenic and free-living Leptospira spp. to UV radiation and mitomycin C

    International Nuclear Information System (INIS)

    Stamm, L.V.; Charon, N.W.

    1988-01-01

    The habitats for the two major Leptospira spp. differ. The main habitat of L. biflexa is soil and water, whereas L. interrogans primarily resides in the renal tubules of animals. We investigated whether these two species, along with L. illini (species incertae sedis), differ with respect to their sensitivity to UV radiation. The doses of UV resulting in 37, 10 and 1% survival were determined for representive serovars from each species. L. interrogans serovar pomona was 3.0 to 4.8 times more sensitive to UV than the other Leptospira species under the 37, 10, and 1% survival parameters. In comparison to other bacteria, L. interrogans serovar pomona is among the most sensitive to UV. In a qualitative UV sensitivity assay., L. interrogans serovars were found to be in general more sensitive than L. biflexa serovars. All three species were found to have a photoreactivation DNA repair mechanism. Since organisms that are resistant to UV are often resistant to the DNA cross-linking agent mitomycin C, we tested the relative sensitivity of several Leptospira serovars to this compound. With few exceptions, L. biflexa and L. illini serovars were considerably more resistant to mitomycin C than the L. interrogans serovars. The mitomycin C sensitivity assay could be a useful addition to current characterization tests used to differentiate the Leptospira species

  1. Sensitivity of pathogenic and free-living Leptospira spp. to UV radiation and mitomycin C

    Energy Technology Data Exchange (ETDEWEB)

    Stamm, L.V.; Charon, N.W.

    1988-03-01

    The habitats for the two major Leptospira spp. differ. The main habitat of L. biflexa is soil and water, whereas L. interrogans primarily resides in the renal tubules of animals. We investigated whether these two species, along with L. illini (species incertae sedis), differ with respect to their sensitivity to UV radiation. The doses of UV resulting in 37, 10 and 1% survival were determined for representive serovars from each species. L. interrogans serovar pomona was 3.0 to 4.8 times more sensitive to UV than the other Leptospira species under the 37, 10, and 1% survival parameters. In comparison to other bacteria, L. interrogans serovar pomona is among the most sensitive to UV. In a qualitative UV sensitivity assay., L. interrogans serovars were found to be in general more sensitive than L. biflexa serovars. All three species were found to have a photoreactivation DNA repair mechanism. Since organisms that are resistant to UV are often resistant to the DNA cross-linking agent mitomycin C, we tested the relative sensitivity of several Leptospira serovars to this compound. With few exceptions, L. biflexa and L. illini serovars were considerably more resistant to mitomycin C than the L. interrogans serovars. The mitomycin C sensitivity assay could be a useful addition to current characterization tests used to differentiate the Leptospira species.

  2. Tetrathiomolybdate sensitizes ovarian cancer cells to anticancer drugs doxorubicin, fenretinide, 5-fluorouracil and mitomycin C

    International Nuclear Information System (INIS)

    Kim, Kyu Kwang; Lange, Thilo S; Singh, Rakesh K; Brard, Laurent; Moore, Richard G

    2012-01-01

    Our recent study showed that tetrathiomolybdate (TM), a drug to treat copper overload disorders, can sensitize drug-resistant endometrial cancer cells to reactive oxygen species (ROS)-generating anticancer drug doxorubicin. To expand these findings in the present study we explore TM efficacy in combination with a spectrum of ROS-generating anticancer drugs including mitomycin C, fenretinide, 5-fluorouracil and doxorubicin in ovarian cancer cells as a model system. The effects of TM alone or in combination with doxorubicin, mitomycin C, fenretinide, or 5-fluorouracil were evaluated using a sulforhodamine B assay. Flow cytometry was used to detect the induction of apoptosis and ROS generation. Immunoblot analysis was carried out to investigate changes in signaling pathways. TM potentiated doxorubicin-induced cytotoxicity and modulated key regulators of apoptosis (PARP, caspases, JNK and p38 MAPK) in SKOV-3 and A2780 ovarian cancer cell lines. These effects were linked to the increased production of ROS, as shown in SKOV-3 cells. ROS scavenging by ascorbic acid blocked the sensitization of cells by TM. TM also sensitized SKOV-3 to mitomycin C, fenretinide, and 5-fluorouracil. The increased cytotoxicity of these drugs in combination with TM was correlated with the activity of ROS, loss of a pro-survival factor (e.g. XIAP) and the appearance of a pro-apoptotic marker (e.g. PARP cleavage). Our data show that TM increases the efficacy of various anticancer drugs in ovarian cancer cells in a ROS-dependent manner

  3. Cryopreservation of citrus seed via dehydration followed by immersion in liquid nitrogen

    Science.gov (United States)

    An important method for plant germplasm conservation is offered by a biotechnology-based approach of cryopreservation. Cryopreservation refers to the storage of plant material at ultralow temperatures in liquid nitrogen. A procedure for cryopreservation of polyembryonic seeds was improved for select...

  4. Cryopreservation of canine semen - new challenges.

    Science.gov (United States)

    Farstad, W

    2009-07-01

    Egg yolk (EY) protects cell membranes against cold shock, and it prevents or restores the loss of phospholipids from the membrane. EY has been widely used in semen extenders. It has been added to Tris-Glucose buffer and has been widely used for cooling and cryopreservation of canine semen. EY is not a defined entity, but a complex biological compound containing proteins, vitamins, phospholipids, glucose and antioxidants which are all potentially useful for cell membrane integrity. Unfortunately, it also is a biologically hazardous compound. Hence, whole EY needs to be replaced by other chemically defined components for semen processing in dogs. Freezing poor semen does not improve its quality, so attention must be focused on how to cope with dogs whose semen does not freeze well, and on designing individual freezing extenders for semen from such males. Furthermore, differences have been found among canid species in the ability of their spermatozoa to withstand freezing. There are differences in sperm membrane fatty acid composition among species, which may explain part of these differences. If the presence of long-chained polyunsaturated fatty acids contributes to increased membrane fluidity, this relationship may be biphasic, i.e. either too much membrane fluidity, or too little, could compromise successful sperm cryopreservation. An increase in fluidity of the outer leaflet of the plasma membrane has been shown in frozen thawed dog spermatozoa. The protective effect of exogenous lipids may lie in close association with the membrane rather than in modification or rearrangement of the membrane. This also points at lipids as an important, if not entirely new group of substances, which may substitute standard EY-based diluents in preserving sperm survival during freezing. EY-derived phospholipids or lecithin could be used to replace whole EY. Vegetable lecithin is currently investigated to avoid using substances of animal origin. EY also contains antioxidants which

  5. Dehydration improves cryopreservation of coconut (Cocos nucifera L.).

    Science.gov (United States)

    Sisunandar; Sopade, Peter A; Samosir, Yohannes M S; Rival, Alain; Adkins, Steve W

    2010-12-01

    Cryopreservation of coconut can be used as a strategy to back up the establishment of living collections which are expensive to maintain and are under constant threat from biotic and abiotic factors. Unfortunately, cryopreservation protocols still need to be developed that are capable of producing a sizeable number of field-grown plants. Therefore, we report on the development of an improved cryopreservation protocol which can be used on a wide range of coconut cultivars. The cryopreservation of zygotic embryos and their recovery to soil-growing plants was achieved through the application of four optimised steps viz.: (i) rapid dehydration; (ii) rapid cooling; (iii) rapid warming and recovery in vitro and (iv) acclimatization and soil-supported growth. The thermal properties of water within the embryos were monitored using differential scanning calorimetry (DSC) in order to ensure that the freezable component was kept to a minimum. The feasibility of the protocol was assessed using the Malayan Yellow Dwarf (MYD) cultivar in Australia and then tested on a range of cultivars which were freshly harvested and studied in Indonesia. The most efficient protocol was one based on an 8-h rapid dehydration step followed by rapid cooling step. Best recovery percentages were obtained when a rapid warming step and an optimised in vitro culture step were used. Following this protocol, 20% (when cryopreserved 12 days after harvesting) and 40% (when cryopreserved at the time of harvest) of all MYD embryos cryopreserved could be returned to normal seedlings growing in soil. DSC showed that this protocol induced a drop in embryo fresh weight to 19% and significantly reduced the amount of water remaining that could produce ice crystals (0.1%). Of the 20 cultivars tested, 16 were found to produce between 10% and 40% normal seedlings while four cultivars generated between 0% and 10% normal seedlings after cryopreservation. This new protocol is applicable to a wide range of coconut

  6. Oocyte cryopreservation beyond cancer: tools for ethical reflection.

    Science.gov (United States)

    Linkeviciute, Alma; Peccatori, Fedro A; Sanchini, Virginia; Boniolo, Giovanni

    2015-08-01

    This article offers physicians a tool for structured ethical reflection on challenging situations surrounding oocyte cryopreservation in young healthy women. A systematic literature review offers a comprehensive overview of the ethical debate surrounding the practice. Ethical Counseling Methodology (ECM) offers a practical approach for addressing ethical uncertainties. ECM consists of seven steps: (i) case presentation; (ii) analysis of possible implications; (iii) presentation of ethical question(s); (iv) explanation of ethical terms; (v) presentation of the ethical arguments in favor of and against the procedure; (vi) examination of the individual patient's beliefs and wishes; and (vii) conclusive summary. The most problematic aspects in the ethical debate include the distinction between medical and non-medical use of oocyte cryopreservation, safety and efficiency of the procedure, and marketing practices aimed at healthy women. Female empowerment and enhanced reproductive choices (granted oocyte cryopreservation is a safe and efficient technique) are presented as ethical arguments supporting the practice, while ethical reservations towards oocyte cryopreservation are based on concerns about maternal and fetal safety and wider societal implications. Oocyte cryopreservation is gaining popularity among healthy reproductive age women. However, despite promised benefits it also involves risks that are not always properly communicated in commercialized settings. ECM offers clinicians a tool for structured ethical analysis taking into consideration a wide range of implications, various ethical standpoints, and patients' perceptions and beliefs.

  7. Cryopreservation of human colorectal carcinomas prior to xenografting

    International Nuclear Information System (INIS)

    Linnebacher, Michael; Maletzki, Claudia; Ostwald, Christiane; Klier, Ulrike; Krohn, Mathias; Klar, Ernst; Prall, Friedrich

    2010-01-01

    Molecular heterogeneity of colorectal carcinoma (CRC) is well recognized, forming the rationale for molecular tests required before administration of some of the novel targeted therapies that now are rapidly entering the clinics. For clinical research at least, but possibly even for future individualized tumor treatment on a routine basis, propagation of patients' CRC tissue may be highly desirable for detailed molecular, biochemical or functional analyses. However, complex logistics requiring close liaison between surgery, pathology, laboratory researchers and animal care facilities are a major drawback in this. We here describe and evaluate a very simple cryopreservation procedure for colorectal carcinoma tissue prior to xenografting that will considerably reduce this logistic complexity. Fourty-eight CRC collected ad hoc were xenografted subcutaneously into immunodeficient mice either fresh from surgery (N = 23) or after cryopreservation (N = 31; up to 643 days). Take rates after cryopreservation were satisfactory (71%) though somewhat lower than with tumor tissues fresh from surgery (74%), but this difference was not statistically significant. Re-transplantation of cryopreserved established xenografts (N = 11) was always successful. Of note, in this series, all of the major molecular types of CRC were xenografted successfully, even after cryopreservation. Our procedure facilitates collection, long-time storage and propagation of clinical CRC specimens (even from different centres) for (pre)clinical studies of novel therapies or for basic research

  8. Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy.

    Directory of Open Access Journals (Sweden)

    Daniel Rodríguez-Martínez

    Full Text Available Cryopreservation protocols are essential for stem cells storage in order to apply them in the clinic. Here we describe a new standardized cryopreservation protocol for GABAergic neural precursors derived from the medial glanglionic eminence (MGE, a promising source of GABAergic neuronal progenitors for cell therapy against interneuron-related pathologies. We used 10% Me2SO as cryoprotectant and assessed the effects of cell culture amplification and cellular organization, as in toto explants, neurospheres, or individualized cells, on post-thaw cell viability and retrieval. We confirmed that in toto cryopreservation of MGE explants is an optimal preservation system to keep intact the interneuron precursor properties for cell transplantation, together with a high cell viability (>80% and yield (>70%. Post-thaw proliferation and self-renewal of the cryopreserved precursors were tested in vitro. In addition, their migration capacity, acquisition of mature neuronal morphology, and potency to differentiate into multiple interneuron subtypes were also confirmed in vivo after transplantation. The results show that the cryopreserved precursor features remained intact and were similar to those immediately transplanted after their dissection from the MGE. We hope this protocol will facilitate the generation of biobanks to obtain a permanent and reliable source of GABAergic precursors for clinical application in cell-based therapies against interneuronopathies.

  9. Social oocyte cryopreservation: a portrayal of Brazilian women.

    Science.gov (United States)

    Santo, Elisangela V Espirito; Dieamant, Felipe; Petersen, Claudia G; Mauri, Ana L; Vagnini, Laura D; Renzi, Adriana; Zamara, Camila; Oliveira, João Batista A; Baruffi, Ricardo L R; Franco, José G

    2017-06-01

    This study aimed to determine what Brazilian childless women of reproductive age think about oocyte cryopreservation to postpone pregnancy and their reasons for performing or not performing this procedure. Women of reproductive age were randomly selected from the general population using different e-mail lists and were invited to participate in the study by completing an online web survey regarding social oocyte cryopreservation. The survey was also distributed through social media to women of reproductive age. Although most of the responders had a partner (86.9%) and had already planned the pregnancy of their first child (69.6%), 85.4% (379) considered the potential of social oocyte freezing to improve their chances of giving birth later in life. Those that had already planned pregnancy were two times more likely to intend to freeze their oocytes (p=0.03). The most important barrier for not undergoing oocyte cryopreservation was cost. The women who indicated that they could not currently undergo the procedure now because of cost were two times (p=0.03) more likely to intend to cryopreserve their oocytes than women who thought that they would not need to delay pregnancy. Brazilian women who think that they are not ready to have a family are discovering the option of oocyte cryopreservation. Most participants considered safeguarding their reproductive potential. Making the procedure more accessible could give women the opportunity to make proactive decisions about the future of their fertility.

  10. The effect of the melatonin on cryopreserved mouse testicular cells

    Directory of Open Access Journals (Sweden)

    Ghasem Saki

    2016-01-01

    Full Text Available Background: After improvements in various cancer treatments, life expectancy has been raised, but success in treatment causes loss of fertility in many of the survived young men. Cryopreservation of immature testicular tissues or cells introduced as the only way to preserve fertility. However, freezing has some harmful effects. Melatonin, a pineal gland hormone, has receptors in reproductive systems of different species. It is assumed that melatonin has free radical scavenger properties. Objective: The aim of this study was to evaluate the effects of melatonin on the cryopreserved testicular cells in mouse. Materials and Methods: Cells from 7- 10 days old NMRI mice testes were isolated using two step enzymatic digestion. The testicular cells were divided into two groups randomly and cryopreserved in two different freezing media with and without the addition of 100 μm melatonin. Finally, apoptosis of the cells was assayed by flow cytometry. Also, lactate dehydrogenase activity test was performed to assess the cytotoxicity. Results: The results of lactate dehydrogenase showed the nearly cytotoxic effect of melatonin. The results of flow cytometry showed increase in apoptosis in the cryopreserved cells in the media containing melatonin compared to the control group. Conclusion: The present study shows that melatonin has an apoptotic effect on cryopreserved mouse testicular cells.

  11. Cryopreservation of Tetraclinis articulata (vahl.) Masters.

    Science.gov (United States)

    Serrano-Martinez, Francisco; Casas, José Luis

    2011-01-01

    Tetraclinis articulata shoot tips excised from in vitro grown shoots were cryopreserved using a modified PVS2-based vitrification protocol. Preliminary experiments with non-cryostored shoot tips showed that the high concentrations of sucrose in loading (LS), vitrification (PVS2) and unloading (US) solutions employed in the protocol were very toxic for the explants. Replacement of sucrose by sorbitol in equal molar concentration in all these solutions enhanced survival of shoot tips after all treatments. However, cold-hardening of donor shoots before shoot tip excision was strictly required to obtain post-rewarming survival. Therefore, the protocol was outlined as follows: pre-conditioning of explants at 4 degree C for 3 weeks in the dark; excision of 1 mm long shoot tips; loading for 20 min in modified LS at room temperature; dehydration in modified PVS2 at 0 degree C for 60 min; immersion in liquid nitrogen (LN); rewarming at 40 degree C for 2 min and subsequent transfer of shoot tips in modified US for 20 min.

  12. Cryopreservation of canine ovarian and testicular fibroblasts.

    Science.gov (United States)

    Yu, Il-Jeoung; Leibo, S P; Songsasen, Nucharin; Dresser, Betsy L; Kim, In-Shik

    2009-01-01

    To derive a practical procedure to store canine somatic cells, fibroblasts isolated from testicular or ovarian tissues were cryopreserved in 1.2 M ethylene glycol or in 1.2 M dimethylsulfoxide prepared in Dulbecco's Modified Eagle Medium as cryoprotectants, and were frozen either in plastic straws or vials. Thawed cells were cultured for 24 hr at 38.5 degree C in a humidified atmosphere of 5 percent CO2 95 percent air, and then their membrane integrity was assayed with a double fluorescent stain, Fertilight. In addition, frozen-thawed fibroblasts were cultured for 4 days, and then their functional survival was measured after staining small colonies with trypan blue. After freezing and thawing, membrane integrity of testicular fibroblasts was 55-70 percent and functional survival ranged from 20-40 percent. With frozen-thawed ovarian cells, the average membrane integrity was 55-75 percent and the average functional survival was 35-40 percent. When frozen in ethylene glycol, functional survival of ovarian fibroblasts was significantly higher than that of testicular cells (P less than 0.05). These methods should prove useful to preserve cells collected from canids in the wild.

  13. Eggs on Ice. Imaginaries on Eggs and Cryopreservation in Denmark

    DEFF Research Database (Denmark)

    Herrmann, Janne Rothmar; Kroløkke, Charlotte

    2018-01-01

    While Denmark is widely known as a global exporter of cryopreserved sperm, Danish women’s eggs follow very different trajectories. This paper combines legal and rhetorical analyses with the concept of sociotechnical imaginaries (Jasanoff, 2015). In establishing the genealogy of the sociotechnical...... imaginaries that shaped the Danish regulation on the cryopreservation of eggs, we analyze the relevant Acts, Bills, preparatory work and readings in Parliament along with the concurrent public and ethical debates that in time relaxed the legal limit for the cryopreservation of eggs to the current 5 years...... and today continue to ignite discussions on elective egg freezing. We rely on welfare state perspectives to discuss why reproduction, in the Danish context, is seen as a legitimate and appropriate sphere to regulate and we turn to feminist theorizing to discuss their gendered implications captured...

  14. Proximate composition analysis posterior to the cryopreservation of Chaetoceros calcitrans

    Directory of Open Access Journals (Sweden)

    Joan Salas-Leiva

    2015-12-01

    Full Text Available Objective. The effect of cryopreservation on the proximate composition of microalgae Chaetoceros calcitrans was evaluated. Materials and methods. C. calcitrans was cultured and cryopreserved using 5% (v/v dimethylsulfoxide as cryoprotectant. The freezing was controlled at a rate of 3°C/min, up to -60°C and then the microalgae were immersed in liquid nitrogen (-196°C. After storage in liquid nitrogen, microalgae were rapidly thawed out and subcultured. The percentage of proteins, lipids and carbohydrates was analyzed using absorption spectrophotometry and the organic matter was studied by gravimetric analysis. Results. There was no significant variation between the proximate composition of C. calcitrans cryopreserved and the controls (p>0.05. Conclusion. Our results show that, despite low cell recovery after the preservation of this organism at low temperatures, there is no apparent loss of nutritional characteristics caused by the storing process at low temperatures.

  15. Cryopreservation of roe deer abomasal nematodes for morphological identification.

    Science.gov (United States)

    Beraldo, Paola; Pascotto, Ernesto

    2014-02-01

    Conventional methods to preserve adult nematodes for taxonomic purposes involve the use of fixative or clearing solutions (alcohol, formaldehyde, AFA and lactophenol), which cause morphological alterations and are toxic. The aim of this study is to propose an alternative method based on glycerol-cryopreservation of nematodes for their subsequent identification. Adults of trichostrongylid nematodes from the abomasum of roe deer (Capreolus capreolus Linnaeus) were glycerol-cryopreserved and compared with those fixed in formaldehyde, fresh and frozen without cryoprotectans. Morphology, transparency and elasticity of the anterior and posterior portion of male nematodes were compared, especially the caudal cuticular bursa and genital accessories. The method presented is quick and easy to use, and the quality of nematode specimens is better than that of nematodes fixed by previously used fixatives. Moreover, glycerol cryopreserved nematodes can be stored for a long time at -20 degrees C in perfect condition and they could be suitable for further analyses, such as histological or ultrastructural examinations.

  16. Fertility in cancer patients after cryopreservation of one ovary

    DEFF Research Database (Denmark)

    Schmidt, K T; Andersen, Anders Nyboe; Greve, T

    2013-01-01

    This questionnaire study describes the fertility and ovarian function in 143 adult female cancer survivors with only one ovary due to cryopreservation of the other. The women were asked about their ovarian function (as defined by the presence of a spontaneous menstrual cycle), pregnancies...... and their outcome. The mean follow-up time was 58months after cryopreservation (range 24-129months). The risk of premature ovarian failure was high in the group of patients with leukaemia (13/15; 87%) but low in the breast cancer group (5/54; 9%). Fifty-seven women had actively tried to become pregnant after end...

  17. Fixation effects on the release of copper, chromium and arsenic from CCA-C treated marine piles

    Science.gov (United States)

    Stan Lebow

    1999-01-01

    This study sought to determine the effect of fixation time and temperature on the release of copper, chromium and arsenic from treated marine piles immersed in seawater under "worst case" conditions. Sections of piles were CCA-C treated to a target retention of 2.5 lbs/ft3) (40 kg/m3) and then allowed to Condition at 36°F (2°C) for either 3, 7 or 20 days. As...

  18. One-electron reduction of mitomycin c by rat liver : role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

    NARCIS (Netherlands)

    Vromans, R M; Van de Straat, R; Groeneveld, M.; Vermeulen, N P

    1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of

  19. Detection of tumor-associated cells in cryopreserved peripheral blood mononuclear cell samples for retrospective analysis.

    Science.gov (United States)

    Zhu, Peixuan; Stanton, Melissa L; Castle, Erik P; Joseph, Richard W; Adams, Daniel L; Li, Shuhong; Amstutz, Platte; Tang, Cha-Mei; Ho, Thai H

    2016-07-02

    Cryopreserved peripheral blood mononuclear cells (PBMCs) are commonly collected in biobanks. However, little data exist regarding the preservation of tumor-associated cells in cryopreserved collections. The objective of this study was to determine the feasibility of using the CellSieve™ microfiltration assay for the isolation of circulating tumor cells (CTCs) and circulating cancer-associated macrophage-like cells (CAMLs) from cryopreserved PBMC samples. Blood samples spiked with breast (MCF-7), prostate (PC-3), and renal (786-O) cancer cell lines were used to establish analytical accuracy, efficiency, and reproducibility after cryopreservation. The spiked samples were processed through Ficoll separation, and cryopreservation was followed by thawing and microfiltration. MCF-7 cells were successfully retrieved with recovery efficiencies of 90.5 % without cryopreservation and 87.8 and 89.0 %, respectively, on day 7 and day 66 following cryopreservation. The corresponding recovery efficiencies of PC-3 cells were 83.3 % without cryopreservation and 85.3 and 84.7 %, respectively, after cryopreservation. Recovery efficiencies of 786-O cells were 92.7 % without cryopreservation, and 82.7 and 81.3 %, respectively, after cryopreservation. The recovered cells retained the morphologic characteristics and immunohistochemical markers that had been observed before freezing. The protocols were further validated by quantitation of CAMLs in blood samples from two patients with renal cell carcinoma (RCC). The recovery rates of CTCs and CAMLs from cryopreserved samples were not statistically significant different (P > 0.05) from matched fresh samples. To our knowledge, this is the first report that CAMLs could be cryopreserved and analyzed after thawing with microfiltration technology. The application of microfiltration technology to cryopreserved samples will enable much greater retrospective study of cancer patients in relation to long-term outcomes.

  20. Cryopreservation of Sambar deer semen in Thailand.

    Science.gov (United States)

    Vongpralub, Thevin; Chinchiyanond, Wittaya; Hongkuntod, Pornchai; Sanchaisuriya, Pitcharat; Liangpaiboon, Sanan; Thongprayoon, Areeya; Somphol, Noppadon

    2015-01-01

    Little is known of the different freezing and thawing techniques for post-thaw survival of spermatozoa in Sambar deer. So, this study determined the effect of seminal plasma, egg yolk and glycerol extenders and their concentrations, plus cooling, freezing, and thawing protocols on the post-thaw quality of their semen. Semen samples were collected by electro-ejaculation from four Thai Sambar deer stags (Cervus unicolor equinus). As evaluated by post-thaw progressive motility and acrosome integrity removal of seminal plasma was beneficial; Tris-egg yolk was the most efficient extender; a 20% egg yolk concentration was better than the 0%, 10%, or 30%; and a 3% glycerol concentration was better than 5%, 7%, or 9%. Using the optimum dilution techniques, semen was loaded in 0.5 ml plastic straws. Cooling times from ambient temperature to 5°C in 3 hr resulted in higher post-thaw progressive motility and acrosome integrity than 1, 2, or 4 hr. Suspending the straws 4 cm above the surface for 15 min before plunging into liquid nitrogen was better than suspending at 2 or 6 cm. For thawing frozen semen, an intermediate thawing (50°C, 8 sec) protocol was more effective than the slower (37°C, 10 sec) or faster (70°C, 5 sec) thawing rates. Timed insemination following estrus synchronization of 10 hinds resulted in six confirmed pregnancies at 60 days. Five hinds delivered live fawns. This study provides an effective approach for semen cryopreservation and artificial insemination (AI), which should be valuable to scientists for genetics and reproductive management of Sambar deer in developing countries. © 2015 Wiley Periodicals, Inc.

  1. VEGF111b, a new member of VEGFxxxb isoforms and induced by mitomycin C, inhibits angiogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Gu, Fang; Li, Xiuli [Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing (China); Kong, Jian [Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing (China); Pan, Bing [The Institute of Cardiovascular Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Sun, Min [Department of Obstetrics and Gynecology, Tangdu Hospital, Fourth Military Medical University, Xian (China); Zheng, Lemin, E-mail: zhengl@bjmu.edu.cn [The Institute of Cardiovascular Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing (China); Yao, Yuanqing, E-mail: yqyao@126.com [Department of Obstetrics and Gynecology, Chinese PLA General Hospital, Beijing (China)

    2013-11-08

    Highlights: •We discovered a new member of VEGFxxxb family-VEGF111b. •We found VEGF111b mRNA and protein can be induced by mitomycin C. •We confirmed VEGF111b over-expression inhibits angiogenesis. •VEGF111b inhibits angiogenesis through inhibiting VEGF-R2/PI3K/Akt and VEGF-R2/ERK1/2 phosphorylation. -- Abstract: Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA{sub 3.1} empty vector, pcDNA{sub 3.1}-VEGF111b or pcDNA{sub 3.1}-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.

  2. VEGF111b, a new member of VEGFxxxb isoforms and induced by mitomycin C, inhibits angiogenesis

    International Nuclear Information System (INIS)

    Gu, Fang; Li, Xiuli; Kong, Jian; Pan, Bing; Sun, Min; Zheng, Lemin; Yao, Yuanqing

    2013-01-01

    Highlights: •We discovered a new member of VEGFxxxb family-VEGF111b. •We found VEGF111b mRNA and protein can be induced by mitomycin C. •We confirmed VEGF111b over-expression inhibits angiogenesis. •VEGF111b inhibits angiogenesis through inhibiting VEGF-R2/PI3K/Akt and VEGF-R2/ERK1/2 phosphorylation. -- Abstract: Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA 3.1 empty vector, pcDNA 3.1 -VEGF111b or pcDNA 3.1 -VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth

  3. Detecting Lactococcus lactis Prophages by Mitomycin C-Mediated Induction Coupled to Flow Cytometry Analysis

    Directory of Open Access Journals (Sweden)

    Joana Oliveira

    2017-07-01

    Full Text Available Most analyzed Lactococcus lactis strains are predicted to harbor one or more prophage genomes within their chromosome; however, the true extent of the inducibility and functionality of such prophages cannot easily be deduced from sequence analysis alone. Chemical treatment of lysogenic strains with Mitomycin C is known to cause induction of temperate phages, though it is not always easy to clearly identify a lysogenic strain or to measure the number of released phage particles. Here, we report the application of flow cytometry as a reliable tool for the detection and enumeration of released lactococcal prophages using the green dye SYTO-9.

  4. Application of Mitomycin C after dilation of an anastomotic stricture in a newborn with necrotizing enterocolitis

    Directory of Open Access Journals (Sweden)

    Jonathan Green

    2016-01-01

    Full Text Available Necrotizing enterocolitis (NEC is a common life-threatening condition in premature infants. Bacterial translocation, localized inflammation and subsequent perforation often require surgery for source control and definitive treatment. Small and large intestinal strictures may result from either creation of a surgical anastomosis or the disease process itself. Current methods to treat strictures include, balloon dilation and surgical resection with or without anastomosis. We report the diagnosis and surgical management of a premature infant treated for NEC, who developed an anastomotic stricture and was successfully treated with topical Mitomycin C after balloon stricturoplasty.

  5. Seasonal and cryopreservation impacts on semen quality in boars

    Science.gov (United States)

    Seasonal boar infertility occurs worldwide and contributes to economic loss to the pork industry. The current study evaluated cooled vs cryopreserved semen quality of 11 Duroc boars collected in June (cool season) and August 2014 (warm season). Semen was cooled to 16°C (cooled) or frozen over liquid...

  6. Effect of season on fresh and cryopreserved stallion semen

    Science.gov (United States)

    The objective of this study was to determine the effect of season on sperm quality parameters, expression of the fertility-related protein SP22 and selected mRNA transcripts infresh and cryopreserved stallion sperm. Four stallions were collected in each of the four seasons: summ...

  7. Sperm cryopreservation before cancer treatment: a 15-year monocentric experience.

    Science.gov (United States)

    Bizet, P; Saias-Magnan, J; Jouve, E; Grillo, J M; Karsenty, G; Metzler-Guillemain, C; Perrin, J

    2012-03-01

    Sperm banking is an important procedure to preserve fertility before cancer therapy. The aim of this study was to comprehensively analyse cryopreservation activity retrospectively for 1080 patients referred to the sperm bank for sperm cryopreservation before cancer treatment. This study included 1007 patients diagnosed with testicular cancer (TC) (41.7%), lymphoma (26%), other haematological cancers (9.4%) or other types of cancer (22.8%); of these, 29 patients did not produce any semen sample and cryopreservation was impossible for 67 patients. Semen characteristics before treatment were within normal ranges, except moderate asthenospermia. Sperm concentration was significantly lower in TC than in non-TC. Straws from 57 patients (6.3%) were used in assisted reproductive technologies, which led to a 46.8% cumulative birth rate. Straws were destroyed for 170 patients (18.7%) and 140 patients performed semen analyses after cancer therapy. After an average delay of 22.5 months after the end of therapy, 43 patients (30.7%) exhibited azoospermia. This study of a large population of cancer patients revealed a high level of successful sperm storage. Utilization of cryopreserved spermatozoa led to good chances of fatherhood. Nevertheless, sperm banks should be aware of the low rates of straw use and straw destruction by cancer patients. Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  8. Cryopreservation of human skeletal muscle impairs mitochondrial function

    DEFF Research Database (Denmark)

    Larsen, Steen; Wright-Paradis, C; Gnaiger, E

    2012-01-01

    functionality after long term cryopreservation (1 year). Skeletal muscle samples were preserved in dimethyl sulfoxide (DMSO) for later analysis. Human skeletal muscle fibres were thawed and permeabilised with saponin, and mitochondrial respiration was measured by high-resolution respirometry. The capacity...

  9. In vitro propagation and cryopreservation of Aerides odorata Lour. (Orchidaceae).

    Science.gov (United States)

    Hongthongkham, J; Bunnag, S

    2014-05-01

    An efficient method for in vitro propagation and cryopreservation of Aerides odorata was established. Leaf segments were cultured on New Dogashima (ND) mediums supplemented with various concentrations of Benzyladenine (BA) (0-5 mg L(-1)) combined with Naphthaleneacetic Acid (NAA) (0-2 mg L(-1)). The optimal treatment for inducing Protocorm-like Bodies (PLBs) from leaf segments was obtained from the combination of 1 or 3 mg L(-1) BA and 0.5 or 1 mg L(-1) NAA; whereas, the addition of BA or NAA alone induced shoot and/or root initiation rather than PLB or callus formation. Shoots rapidly developed on ND mediums containing 5 mg L(-1) BA. Cryopreservation of leaf segment-derived PLBs was successful using the encapsulation-dehydration method. The maximum survival percentage of Cryopreserved (Cryp) PLBs was achieved by encapsulating PLBs with 2% Na-alginate combined with 2 M glycerol and 0.4 M sucrose. The encapsulated PLBs were then precultured in 0.75 M sucrose for 24 h and dehydrated for 6 h before plunging into liquid nitrogen. Genetic stability of Cryp PLBs after regrowth was assessed by flow cytometry. The findings showed no different patterns of ploidy levels and morphology between Cryp and non-cryopreserved (Ncryp) control plantlets.

  10. Semen cryopreservation in fish: effects on sperm motility and fertility

    International Nuclear Information System (INIS)

    Martinez, Jose Gregorio; Pardo Carrasco, Sandra

    2010-01-01

    The cryopreservation of semen in fish, as in many species even shows effects that decrease sperm quality and directly engage cell ability to successfully participate in the processes of fertilization and embryonic development. the characteristics such as mobility and fertilizing capacity of fertilization of sperm are considered to be quality criteria that allow to measure the success or failure of the process, since they are considered integrative variables, being indicators that depend not on a single factor, but on the stability and welfare of all structures, enzymes and subcellular functional compounds that give place to these spermatic characteristics. membrane damage (Adenylate cyclase, ion channels, grouping of other proteins, among others) and their implication in the route of signaling pathway leading to spermatic activation, ATP degradation and fragmentation of nuclear and mitochondrial DNA (genome), degradation of kinase enzymes and other cytosolic proteins (proteome) are considered today, as some of the molecular factors that most affect during cryopreservation and markedly decreasing the fertilizing capacity and mobility of sperm in fish. Proposals on the molecular mechanisms, by which these subcellular factors interact and act as consequence of cryopreservation, are some of the topics covered in this review. Understanding the principles and factors that are involved in the origin of such damages, will allow to improved cryopreservation processes, making them less harmful and more efficient.

  11. Development of a cryopreservation protocol for type A spermatogonia

    NARCIS (Netherlands)

    Izadyar, Fariborz; Matthijs-Rijsenbilt, J. J.; den Ouden, Krista; Creemers, Laura B.; Woelders, Henri; de Rooij, Dirk G.

    2002-01-01

    The aim of this study was to develop a cryopreservation protocol for type A spermatogonia. Testes from 5- to 7-month-old calves were collected, and type A spermatogonia were isolated using two-step enzymatic digestion and Percoll separation. Cells were resuspended in minimum essential medium (MEM)

  12. Fertility rate of daily collected and cryopreserved fowl semen

    NARCIS (Netherlands)

    Voorst, van A.; Leenstra, F.R.

    1995-01-01

    Semen was collected for 4 consecutive d individually from experimental broiler breeder males that had not been massaged for 7 d. The semen was mixed and diluted with the Beltsville Poultry Semen Extender with dimethylsulfoxide (DMSO) as cryoprotectant and cryopreserved. After thawing of the semen,

  13. Cryopreservation of in vitro -grown shoot tips of apricot ( Prunus ...

    African Journals Online (AJOL)

    In vitro grown apricot (Prunus armeniaca L.) cv. El-Hamawey shoot tips were successfully cryopreserved using an encapsulation-dehydration procedure. Shoot tips were encapsulated in calcium-alginate beads before preculture on woody plant (WP) medium supplemented with different sucrose concentrations; 0.1, 0.3, 0.5, ...

  14. Midterm Results of Aortic Valve Replacement with Cryopreserved Homografts

    Directory of Open Access Journals (Sweden)

    Emre Özker

    2012-06-01

    Full Text Available Objective: The aim of this study was to analyze the midterm clinical results of aortic valve replacement with cryopreserved homografts.Materials and Methods: Aortic valve replacement was performed in 40 patients with cryopreserved homograft. The indications were aortic valve endocarditis in 20 patients (50%, truncus arteriosus in 6 patients (15%, and re-stenosis or regurtitation after aortic valve reconstruction in 14 (35% patients. The valve sizes ranged from 10 to 27mm. A full root replacement technique was used for homograft replacement in all patients.Results: The 30-day postoperative mortality rate was 12.5% (5 patients. There were four late deaths. Only one of them was related to cardiac events. Overall mortality was 22.5%. Thirty-three patients were followed up for 67±26 months. Two patients needed reoperation due to aortic aneurysm caused by endocarditis. The mean transvalvular gradient significantly decreased after valve replacement (p<0.003. The last follow up showed that the 27 (82% patients had a normal left ventricular function.Conclusion: Cryopreserved homografts are safe alternatives to mechanical valves that can be used when there are proper indications. Although it has a high perioperative mortality rate, cryopreserved homograft implantation is an alternative for valve replacement, particularly in younger patients and for complex surgical problems such as endocarditis that must be minimalized.

  15. Comparison of glycerolisation with cryopreservation methods on HIV-1 inactivation

    International Nuclear Information System (INIS)

    Van Baare, J.; Pagnon, J.; Cameron, P.; Vardaxis, N.; Middlekoop, E.; Crowe, S.

    1999-01-01

    Cryopreservation and glycerolisation are two successful long-term preservation methods for human cadaveric donor skin, which is used in the treatment of bum patients. High concentrations of glycerol has been shown to be antibacterial and virucidal. Because fear of possible transmission of HIV-1 following allograft transplantation, this study was undertaken to investigate whether HIV can be effectively eliminated from skin explants. HIV-1 Ba-L, which has been shown to infect monocytes in skin explants and also dendritic cells, was. For the experiments we used cell-free virus, exogenously HIV infected peripheral blood mononuclear cells (PBMCs) and exogenously HIV infected cadaver split skin. Different concentrations of glycerol at various temperatures and the glycerolisation procedure as used by the Euro Skin Bank were used to determine the effects on HIV-1 Ba-L infectivity. For the cryopreservation technique we used 10% DMSO and a controlled rate freezer. HIV-1 Ba-L transfer was determined by adding uninfected PBMCs to the infected material and reverse transcriptase was measured. Cell-free HIV-1 Ba-L was not inactivated by 50% glycerol but was effectively inactivated within 30 minutes by 70% and 85% glycerol at 4 degree C, room temperature and 37 degree C. In contrast, cell-free HIV-1 Ba-L was not inactivated by cryopreservation. Most importantly, we have shown that HIV-1 Ba-L present in split skin is inactivated by incubating skin in 70% glycerol for three hours at 37-C. HIV in exogenously infected skin was not inactivated by cryopreservation. High concentrations of glycerol effectively inactivates free HIV-1 Ba-L and intracellular HIV-1 Ba-L. Also the current glycerolisation procedure carried out by the Euro Skin Bank effectively inactivates infectious virus. However, the cryopreservation technique did not show any reduction in HIV-1 Ba-L infectivity

  16. Cryopreservation of preimplantation embryos of cattle, sheep, and goats.

    Science.gov (United States)

    Youngs, Curtis R

    2011-08-05

    Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 - 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6°C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5°C/min, enabling further dehydration, to a temperature of -34°C before being plunged into liquid nitrogen (-196°C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37°C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

  17. Successful endoscopic management with Mitomycin C application for sinusitis with orbital cellulitis

    Directory of Open Access Journals (Sweden)

    Anil S Harugop

    2013-01-01

    Full Text Available Background: Sinusitis with orbital complication is a potentially fatal disease that has been known since the days of Hippocrates. Primary sinus infection is the most common cause of orbital cellulitis. It is an emergency that threatens not only vision but also life from complications such as meningitis, cavernous sinus thrombosis, and brain abscess. Surgical intervention is mandatory whenever antibiotic treatment fails. There are two surgical options for the drainage, an external approach via a Lynch incision and an intranasal endoscopic procedure. Materials and Methods: Five patients with orbital cellulitis secondary to acute on chronic rhinosinusitis were included in the study from the period of 2010 - 2011. All five patients did not respond to medical management and hence underwent endoscopic sinus surgery with treatment of orbital pathology. At the end of the surgical procedure Mitomycin C in a concentration of 0.4mg/ml was applied with a cottonoid for a period of 4 minutes to prevent chance of adhesion formation. Results: In this series 3 females and 2 male patient with orbital cellulitis secondary to acute on chronic rhinosinusitis underwent endoscopic sinus surgery with treatment of orbital pathology. All 5 patients showed subjective and objective improvement within one week of endoscopic management. Conclusion: Though antibiotics have altered the course of sinusitis, its grave complications still persist in our environment. The excellent results and the absence of any major complications of endoscopic sinus surgery and drainage of abscess with application of Mitomycin C can be recommended as the preferred surgical technique.

  18. A review of the efficacy of mitomycin C in glaucoma filtration surgery

    Directory of Open Access Journals (Sweden)

    Al Habash A

    2015-10-01

    Full Text Available Ahmed Al Habash,1,2 Leyla Ali Aljasim,1 Ohoud Owaidhah,1 Deepak P Edward1,3 1King Khaled Eye Specialist Hospital, Riyadh, Kingdom of Saudi Arabia; 2Department of Ophthalmology, University of Dammam, Dammam, Saudi Arabia; 3Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA Abstract: The success of trabeculectomy, which is considered the gold standard in the surgical treatment of glaucoma, depends on the wound healing response. The introduction of antiproliferative agents such as mitomycin C (MMC has increased the success rates of trabeculectomy. However, complications due to these agents can be challenging to manage. Hence, it is important to determine the most efficacious dose and duration of exposure. Multiple studies suggest that many factors, including but not limited to MMC preparation, different concentrations, different exposure times, and method of application may affect success rate, and these factors were reviewed in this article. We concluded that lower concentrations of MMC that are prepared and applied in a standardized fashion, such as that using the Mitosol® kit (for 2–3 minutes during trabeculectomy, could potentially provide trabeculectomy success rates similar to that reported with off-label preparations, and that such a treatment regime could result in in lower complication rates than higher doses of MMC. Keywords: mitomycin C, Mitosol, filtering surgery, trabeculectomy, outcomes

  19. Comparative study of encapsulated blebs following Ahmed glaucoma valve implantation and trabeculectomy with mitomycin-C.

    Science.gov (United States)

    Bae, Kunho; Suh, Wool; Kee, Changwon

    2012-08-01

    To compare the histopathologic and morphologic findings of encapsulated blebs following Ahmed glaucoma valve implantation and primary standard trabeculectomy with mitomycin-C. We reviewed the records of patients with otherwise uncontrollable glaucoma who had undergone Ahmed glaucoma valve implantation or trabeculectomy with mitomycin-C. Five eyes that underwent Ahmed valve implantation and three eyes that underwent trabeculectomy needed surgical revision of the initial surgery due to encapsulated bleb development with total loss of function. The surgically removed encapsulated blebs were analyzed macroscopically and microscopically. Removal of the encapsulated bleb was performed at a mean follow-up time of 26.6 ± 19.4 weeks in the Ahmed valve implantation group and 12.0 ± 11.4 weeks in the trabeculectomy group. The fibrotic wall of the encapsulated blebs had an overall thickness of 2.48 ± 0.42 mm in the Ahmed valve implantation group and 1.62 ± 0.37 mm in the trabeculectomy group. Macroscopically, the coconut flesh-like smooth surface was split into two layers, and the wall of the capsule was thicker in the Ahmed valve implantation group than in the trabeculectomy group. Histopathologically, the fibrotic capsule was composed of an inner fibrodegenerative layer and an outer fibrovascular layer, and there were no histopathological differences between the two groups. The fibrotic capsule wall was thicker in the Ahmed valve group, but there were no differences in histological findings between the two groups.

  20. Rescue of Mitomycin C- or Psoralen-Inactivated Micrococcus Radiodurans by Additional Exposure to Radiation or Alkylating Agents

    DEFF Research Database (Denmark)

    Hansen, M. Trier

    1982-01-01

    The processing of damaged DNA was altered in a mitomycin C-sensitive mutant (mtcA) of Micrococcus radiodurans. Even though the mutant retained resistance to 254-nm UV radiation, it did not, in contrast to the wild-type strain, show any excessive DNA degradation or cell death when incubated...

  1. Treatment of multiple-level tracheobronchial stenosis secondary to endobronchial tuberculosis using bronchoscopic balloon dilatation with topical mitomycin-C.

    Science.gov (United States)

    Faisal, Mohamed; Harun, Hafaruzi; Hassan, Tidi M; Ban, Andrea Y L; Chotirmall, Sanjay H; Abdul Rahaman, Jamalul Azizi

    2016-04-14

    Tracheobronchial stenosis is a known complication of endobronchial tuberculosis. Despite antituberculous and steroid therapy, the development of bronchial stenosis is usually irreversible and requires airway patency to be restored by either bronchoscopic or surgical interventions. We report the use of balloon dilatation and topical mitomycin-C to successful restore airway patency. We present a 24-year old lady with previous pulmonary tuberculosis and laryngeal tuberculosis in 2007 and 2013 respectively who presented with worsening dyspnoea and stridor. She had total left lung collapse with stenosis of both the upper trachea and left main bronchus. She underwent successful bronchoscopic balloon and manual rigid tube dilatation with topical mitomycin-C application over the stenotic tracheal segment. A second bronchoscopic intervention was performed after 20 weeks for the left main bronchus stenosis with serial balloon dilatation and topical mitomycin-C application. These interventions led to significant clinical and radiographic improvements. This case highlights that balloon dilatation and topical mitomycin-C application should be considered in selected patients with tracheobronchial stenosis following endobronchial tuberculosis, avoiding airway stenting and invasive surgical intervention.

  2. Influence of intraperitoneal therapy with mitomycin C adsorbed on activated carbon on anastomotic and wound healing in rats

    NARCIS (Netherlands)

    Jansen, M; Jansen, PL; Fass, J; Langejurgen, E; Forsch, S; Tietze, L; Schumpelick, [No Value

    In an effort to prevent intraperitoneal dissemination of gastric carcinoma, local chemotherapy with mitomycin C adsorbed to activated carbon (MMC-CH) has been implemented. Results of clinical studies showed improved survival and a reduced systemic toxicity after the use of prophylactic treatment

  3. Lithium chloride attenuates mitomycin C induced necrotic cell death in MDA-MB-231 breast cancer cells via HMGB1 and Bax signaling.

    Science.gov (United States)

    Razmi, Mahdieh; Rabbani-Chadegani, Azra; Hashemi-Niasari, Fatemeh; Ghadam, Parinaz

    2018-07-01

    The clinical use of potent anticancer drug mitomycin C (MMC) has limited due to side effects and resistance of cancer cells. The aim of this study was to investigate whether lithium chloride (LiCl), as a mood stabilizer, can affect the sensitivity of MDA-MB-231 breast cancer cells to mitomycin C. The cells were exposed to various concentrations of mitomycin C alone and combined with LiCl and the viability determined by trypan blue and MTT assays. Proteins were analyzed by western blot and mRNA expression of HMGB1 MMP9 and Bcl-2 were analyzed by RT-PCR. Flow cytometry was used to determine the cell cycle arrest and percent of apoptotic and necrotic cells. Concentration of Bax assessed by ELISA. Exposure of the cells to mitomycin C revealed IC 50 value of 20 μM, whereas pretreatment of the cells with LiCl induced synergistic cytotoxicity and IC 50 value declined to 5 μM. LiCl combined with mitomycin C significantly down-regulated HMGB1, MMP9 and Bcl-2 gene expression but significantly increased the level of Bax protein. In addition, the content of HMGB1 in the nuclei decreased and pretreatment with LiCl reduced the content of HMGB1 release induced by MMC. LiCl increased mitomycin C-induced cell shrinkage and PARP fragmentation suggesting induction of apoptosis in these cells. LiCl prevented mitomycin C-induced necrosis and changed the cell death arrest at G2/M-phase. Taking all together, it is suggested that LiCl efficiently enhances mitomycin C-induced apoptosis and HMGB1, Bax and Bcl-2 expression may play a major role in this process, the findings that provide a new therapeutic strategy for LiCl in combination with mitomycin C. Copyright © 2018 Elsevier GmbH. All rights reserved.

  4. Hatchery-scale trials using cryopreserved spermatozoa of black-lip pearl oyster, Pinctada margaritifera

    OpenAIRE

    Hui, Belinda; Vonau, Vincent; Moriceau, Jacques; Tetumu, Roger; Vanaa, Vincent; Demoy-schneider, Marina; Suquet, Marc; Le Moullac, Gilles

    2011-01-01

    Cryopreservation is a valuable tool for genetic improvement programs. Several bivalve mollusc species have already been the subject of such programs and the Tahitian black pearl oyster industry is now planning the development of selective breeding for desirable traits in Pinctada margaritifera. The ability to cryopreserve spermatozoa would, therefore, offer significant benefits to the cultured black pearl industry. Spermatozoa were cryopreserved with CPA 0.7 M trehalose in 0.8 M Me2SO and...

  5. Data on antioxidant activity in grapevine (Vitis vinifera L.) following cryopreservation by vitrification

    OpenAIRE

    María Fernanda Lazo-Javalera; Martín Ernesto Tiznado-Hernández; Irasema Vargas-Arispuro; Elisa Valenzuela-Soto; María del Carmen Rocha-Granados; Marcos Edel Martínez-Montero; Marisela Rivera-Domínguez

    2015-01-01

    Cryopreservation is used for the long-term conservation of plant genetic resources. This technique very often induces lethal injury or tissue damage. In this study, we measured indicators of viability and cell damage following cryopreservation and vitrification-cryopreservation in Vitis vinifera L. axillary buds cv. ?Flame seedless? stored in liquid nitrogen (LN) for: three seconds, one hour, one day, one week and one month; after LN thawed at 38??C for three minutes. The enzymatic activity o...

  6. A randomized study of accelerated fractionation radiotherapy with and without mitomycin C in the treatment of locally advanced head and neck cancer

    DEFF Research Database (Denmark)

    Ezzat, M.; Shouman, T.; Zaza, K.

    2005-01-01

    Objectives: This single-institution study evaluates the feasibility of accelerated fractionation radiotherapy (AF) with and without mitomycin C (MMC) in the treatment of locally advanced head and neck cancer. Patients and Methods: Between May 1998 and October 2001, sixty patients with locally...... advanced stage III and IV of head and neck cancer were randomized into three treatment arms: (1) conventional fractionation radiotherapy (CF) (5 fractions per week); (2) accelerated fractionation radiotherapy (AF) (6 fractions per week); and (3) AF plus Mitomycin C (MMC). Results: The 2-year overall....... Key Words: Head and Neck cancer , Radiotherapy , Altered fractionation , Mitomycin C....

  7. A new strategy and system for the ex vivo ovary perfusion and cryopreservation: An innovation.

    Science.gov (United States)

    Ali Mohamed, Mohamed Shehata

    2017-06-01

    Children and young adults, who suffer from cancer, receive gonadotoxic therapy, which destroys their fertile abilities after survival. Ovarian cryopreservation and transplantation provide the promising solution to this problem, where the ovary can be removed before the gonadotoxic therapy and reimplanted after patient's survival, where the ovary is to be cryopreserved during the period of the therapy. However, cryopreservation of the whole ovary is still facing great obstacles, namely the ischemic reperfusion injury and the defective cryopreservation related to the defective ability to universally deliver the cryopreservation/warming solutions through the ovarian vascular bed. Meanwhile, the currently applied technique of ovarian tissue cryopreservation provides limited follicular recovery because many follicles are lost until the development of revascularization post-transplantation. To solve the problems, an innovative system has been developed to insure immediate and universal delivery of the cryopreservation/warming solutions to the graft, in addition to keeping the graft under continuous perfusion before and after cryopreservation, minimizing any chance for microthrombi formation or ischemia-reperfusion. This innovative system can be applied in the following surgical and clinical interventions: 1) Allogeneic ovarian transplantation; 2) Preservation of fertility after systemic chemotherapy or bone marrow transplantation in young females, where the ovaries could be removed before the therapy and exposed to the adequate cryopreservation provided by the system till re-implantation after the patient's survival; 3) The system is also suitable for the corresponding applications on the testicles.

  8. Optimization of Artificial Propagation in Piracanjuba Fish Brycon orbignyanus Using Cryopreserved Semen.

    Science.gov (United States)

    Felizardo, V O; Melo, C C V; Murgas, L D S; Andrade, E S; Navarro, R D; Ftreitas, T F

    BACKGROUND: Cryopreserved semen could facilitate procedures during the artificial reproduction in fish. Factors affecting cryopreservation efficiency are important to define efficient protocols. This study investigated the application of cryoprotectants on the quality of piracanjuba fish semen, the sperm concentration required for oocyte fertilization and spermatic activation. We evaluated two intracellular cryoprotectant solutions (DMSO and methanol) and two extracellular cryoprotectant solutions (egg yolk and lactose) to cryopreserved piracanjuba semen. Sperm motility rate, motility duration and spermatic alterations were assessed. The protocol for piracanjuba semen cryopreservation can use solutions including either DMSO or methanol as intracellular cryoprotectant and egg yolk or lactose as extracellular cryoprotectants.

  9. Efficacy of postal communication with patients who have cryopreserved pre-embryos.

    Science.gov (United States)

    Brzyski, R G

    1998-11-01

    To compare the characteristics of patients who did and did not respond to a request for information regarding their cryopreserved pre-embryos. Mail survey. Academic-assisted reproductive technology program. One hundred thirty-six patients with cryopreserved pre-embryos. Patients were surveyed by first-class mail regarding their plans for their cryopreserved pre-embryos and their interest in embryo donation. Age, number of stored pre-embryos, and duration of storage of responders and nonresponders at 6 weeks after mailing. Eighty-three patients (62%) did not respond to the survey. Compared with responders, nonresponders were significantly older at the time of embryo cryopreservation, had fewer pre-embryos cryopreserved, and had the pre-embryos cryopreserved for a longer duration. Five responders (9%) expressed an interest in embryo donation. Three patients requested disposal of pre-embryos. Sixteen surveys (12%) were returned as undeliverable. As a group, these patients had the fewest pre-embryos cryopreserved and had the longest duration of storage. A disturbing number of patients with cryopreserved pre-embryos ignored efforts by our program to maintain contact. Older patients with few cryopreserved pre-embryos may require special attention to avoid abandonment.

  10. A questionnaire survey on attitude toward sperm cryopreservation among hematologists in Japan.

    Science.gov (United States)

    Kobayashi, Tomohiro; Shin, Takeshi; Nishio, Kojiro; Shimomura, Yukihito; Iwahata, Toshiyuki; Suzuki, Keisuke; Miyata, Akane; Kobori, Yoshitomo; Arai, Gaku; Okada, Hiroshi

    2017-03-01

    Advances in multimodal treatment have led to dramatic improvement in cancer treatment outcomes. It is now necessary to consider cancer patients' holistic quality of life. Fertility preservation is the top concern for cancer survivors of reproductive age. Sperm cryopreservation before treatment is recommended for postpubescent men, but many patients lose fertility without having been informed about options for fertility preservation. To determine how sperm cryopreservation is perceived and practiced in Japan, we surveyed hematologists who often treat young males. A questionnaire about sperm cryopreservation was sent to 45 major hematology institutions. A total of 22 institutions responded before the deadline. All institutions but one responded that they felt sperm cryopreservation is necessary. Only 15 institutions responded that they inform patients about sperm cryopreservation, and 12 institutions responded that they perform sperm cryopreservation before chemotherapy. A total of 213 young males started their first course of chemotherapy during the survey period, of whom 61 (28.6%) had their sperm cryopreserved. Although almost all hematologists stated that sperm cryopreservation is necessary for fertility preservation, not all institutions informed patients about it. Our findings indicate that, to promote fertility preservation in Japan, it will be necessary to systematize sperm cryopreservation and build inter-hospital networks.

  11. Determination of in vivo behavior of mitomycin C-loaded o/w soybean oil microemulsion and mitomycin C solution via gamma camera imaging.

    Science.gov (United States)

    Kotmakçı, Mustafa; Kantarcı, Gülten; Aşıkoğlu, Makbule; Ozkılıç, Hayal; Ertan, Gökhan

    2013-09-01

    In this study, a microemulsion system was evaluated for delivery of mitomycin C (MMC). To track the distribution of the formulated drug after intravenous administration, radiochemical labeling and gamma scintigraphy imaging were used. The aim was to evaluate a microemulsion system for intravenous delivery of MMC and to compare its in vivo behavior with that of the MMC solution. For microemulsion formulation, soybean oil was used as the oil phase. Lecithin and Tween 80 were surfactants and ethanol was the cosurfactant. To understand the whole body localization of MMC-loaded microemulsion, MMC was labeled with radioactive technetium and gamma scintigraphy was applied for visualization of drug distribution. Radioactivity in the bladder 30 minutes after injection of the MMC solution was observed, according to static gamma camera images. This shows that urinary excretion of the latter starts very soon. On the other hand, no radioactivity appeared in the urinary bladder during the 90 minutes following the administration of MMC-loaded microemulsion. The unabated radioactivity in the liver during the experiment shows that the localization of microemulsion formulation in the liver is stable. In the light of the foregoing, it is suggested that this microemulsion formulation may be an appropriate carrier system for anticancer agents by intravenous delivery in hepatic cancer chemotherapy.

  12. Germination, carbohydrate composition and vigor of cryopreserved Caesalpinia echinata seeds

    Directory of Open Access Journals (Sweden)

    Rafael Fonsêca Zanotti

    2012-10-01

    Full Text Available The present study investigated the germination and vigor of Caesalpinia echinata (Brazilwood seeds stored at negative temperatures. Recently harvested seeds were cryopreserved at -18º or -196ºC and periodically evaluated for germination, seed vigor and carbohydrate composition. The temperatures did not influence the germination percentages or vigor. The germination percentage decreased from 88% in recently harvested seeds to 60% after 730 days of storage. The different temperature and storage times tested did not affect the vigor seed germination as indicated by the measures of plant growth and survival. The different temperatures used did not cause changes in the carbohydrate composition. The tegument cell walls were rich in lignin, arabinose and xylose. The cytoplasm of the cotyledons and embryos had high levels of glucose, fructose, and sucrose. The cryopreservation technique here presented was effective in the conservation of Brazilwood seeds for the medium term.

  13. Fertilization and Embryo Development of Fresh and Cryopreserved Sibling Oocytes

    Directory of Open Access Journals (Sweden)

    Robert F. Casper

    2010-01-01

    Full Text Available Background: Oocyte cryopreservation is potentially the best way to preserve female fertility forunmarried women or young girls at risk of losing ovarian function. The aim of this study was tocompare fertilization and embryo development in frozen-thawed oocytes to their fresh siblings inwomen undergoing in vitro fertilization (IVF and embryo transfer (ET.Materials and Methods: Eleven infertile women undergoing infertility treatment, between theages of 24 to 37 years (mean ± SD = 31.6 ± 3.5, were included in this study. Mature oocytesfrom each patient were randomized into cryopreserved and fresh groups prior to intracytoplasmicsperm injection (ICSI. One hundred and thirty nine oocytes were retrieved, of which 105 were atmetaphase II (MII. Forty- five fresh MII oocytes were kept in culture whereas their sibling 60 MIIoocytes were cryopreserved using a slow cooling protocol. The frozen oocytes remained in LN2for 2 hours before thawing. ICSI was performed 1-2 hours after thawing for frozen oocytes and 4-5hours after retrieval for fresh oocytes. Fertilization and embryo development were compared.Results: Following thawing, 31 oocytes (51.6 % survived and 22 fertilized (79% while 32 freshoocytes fertilized upon ICSI (71%. The mean ± SE scores for embryos developing from frozenthawedoocytes were significantly lower at 48 and 72 hours post-ICSI than for embryos resultingfrom fresh oocytes (p<0.05.Conclusion: Our data demonstrated that oocyte freezing resulted in acceptable survival ratesfollowing cryopreservation, and similar fertilization rates following ICSI as compared to the freshsibling oocytes. However the number of blastomeres and the embryo quality on day three wassuperior in embryos from fresh oocytes when compared to the frozen oocytes.

  14. Cryopreservation of zebrafish (Danio rerio) oocytes by vitrification.

    Science.gov (United States)

    Guan, M; Rawson, D M; Zhang, T

    2010-01-01

    Cryopreservation of fish oocytes is challenging because these oocytes have low membrane permeability to water and cryoprotectant and are highly chilling sensitive. Vitrification is considered to be a promising approach for their cryopreservation as it involves rapid freezing and thawing of the oocytes and therefore minimising the chilling injury. In the present study, vitrification properties and the toxicity of a range of vitrification solutions containing different concentrations of Me2SO, methanol, propylene glycol and ethylene glycol were investigated. Two different base media and vitrification methods were compared. The effect of different post-thaw dilution solutions together with incubation periods on oocyte viability were also investigated. Stage III zebrafish oocytes were equilibrated in increasing concentrations of cryoprotectants for 30 min in 3 steps. Oocytes were thawed rapidly in a water bath and cryoprotectants were removed in 4 steps. Oocyte viability was assessed using trypan blue staining. The results showed that vitrification solutions V3 and V4 in KCl buffer had low toxicity and vitrified well. The survivals of oocytes after stepwise dilution using solutions containing permeable cryoprotectants were significant higher than those diluted in 0.5M glucose, and the use of CVA65 vitrification system improved oocyte survival when compared with plastic straws after 30 min at 22 degrees C post-thawing. Cryopreservation of zebrafish oocytes by vitrification is reported here for the first time, although oocyte survivals after cryopreservation assessed by trypan blue staining were relatively high shortly after thawing, they became swollen and translucent after incubation in KCl buffer. Further studies are needed to optimise the post-thaw culturing conditions.

  15. Apoptosis in fresh and cryopreserved cardiac valves of pig samples.

    Science.gov (United States)

    Rendal Vázquez, M Esther; Díaz Román, T M; Rodríguez Cabarcos, M; Zavanella Botta, C; Domenech García, N; González Cuesta, M; Sánchez Dopico, M J; Pértega Díaz, S; Andión Núñez, C

    2008-06-01

    To analyse the influence of cold ischemic time (CIT) (2-24 h) and of cryopreservation (liquid phase) on the viability of the valvular fibroblasts and in the presence of apoptosis. Cardiac valves from 10 pigs were evaluated by anatomo-pathological study of the wall, muscle and leaflet. At the same time, the presence of cellular death due to apoptosis was investigated in two ways; directly on tissue by Apodetec system and by two-colour flow cytometry assay analyzing a suspension of fibroblast from valve leaflets using Anexina V and propidium iodure (PI). We established three groups of samples to compare different experimental conditions: 2 h of ischemia (group 1), 24 h of ischemia (group 2), and a programme of cryopreservation (-1 degrees C/min) after 2 h of ischemia, followed by storage in liquid nitrogen during a week and thawing was performed (group 3). The analysis of viabilities showed slight differences between all three groups. The results indicated CIT of 24 h undergoing more structural affectation than CIT of 2 h. Flow cytometry analysis did not show important differences between groups; however cryopreserved samples (group 3) slightly less viability and a higher percentage of death by apoptosis than group 1 and 2 using flow cytometry. Apoptosis was confirmed on tissue from all valves but mainly in samples of group 2 and group 3. In summary, the viability of the valves in the case of ischemic times of 2 h, 24 h or after cryopreservation/thawing differs slightly. The death of the cells is mainly mediated by necrosis and not by apoptosis.

  16. Effective Condition for Whole Testis Cryopreservation of Endangered Miho Spine Loach (Cobitis choii) Through the Optimization of Mud Loach (Misgurnus mizolepis) Whole Testis Cryopreservation Condition.

    Science.gov (United States)

    Kim, J J; Nam, Y K; Bang, I C; Gong, S P

      BACKGROUND: Miho spine loach (Cobitis choii) is an endangered Korean endemic fish. Whole testis cryopreservation is a good way for species preservation, but needs to the sacrifice of a large number of fish to optimize the freezing condition. Considering this limitation, a surrogate fish species was used for the protocol development. This study was to establish the effective condition for Miho spine loach whole testis cryopreservation by optimizing the conditions for whole testis cryopreservation in an allied species, mud loach (Misgurnus mizolepis). The condition for whole testis cryopreservation was optimized in mud loach first, and then the optimal condition was applied to Miho spine loach testes. The optimal condition for mud loach testis cryopreservation consists of the freezing medium containing 1.3 M dimethyl sulfoxide, 6% fetal bovine serum and 0.3 M trehalose, -1 C/min cooling rate and 26 degree C thawing temperature, which also permits effective cryopreservation of Miho spine loach testes. An effective cryopreservation condition for whole testis of the endangered Miho spine loach has been established by using mud loach as a surrogate fish.

  17. Human oocyte cryopreservation and the fate of cortical granules.

    Science.gov (United States)

    Ghetler, Yehudith; Skutelsky, Ehud; Ben Nun, Isaac; Ben Dor, Liah; Amihai, Dina; Shalgi, Ruth

    2006-07-01

    To examine the effect of the commonly used oocyte cryopreservation protocol on the cortical granules (CGs) of human immature germinal vesicle (GV) and mature metaphase II (MII) oocytes. Laboratory study. IVF unit. Unfertilized, intracytoplasmic sperm injected (ICSI) oocytes, and immature oocytes were cryopreserved using a slow freezing-rapid thawing program with 1,2-propanediol (PROH) as a cryoprotectant. Cortical granule exocytosis (CGE) was assessed by either confocal microscopy or transmission electron microscopy (TEM). The survival rates of frozen-thawed oocytes (mature and immature) were significantly lower compared with zygotes. Both mature and immature oocytes exhibited increased fluorescence after cryopreservation, indicating the occurrence of CGE. Mere exposure of oocytes to cryoprotectants induced CGE of 70% the value of control zygotes. The TEM revealed a drastic reduction in the amount of CGs at the cortex of frozen-thawed GV and MII oocytes, as well as appearance of vesicles in the ooplasm. The commonly used PROH freezing protocol for human oocytes resulted in extensive CGE. This finding explains why ICSI is needed to achieve fertilization of frozen-thawed human oocytes.

  18. Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos by vitrification.

    Science.gov (United States)

    Adu-Gyamfi, Raphael; Wetten, Andy

    2012-01-01

    Losses of cultivated cocoa (Theobroma cacao L.) due to diseases and continued depletion of forests that harbour the wild progenitors of the crop make ex situ conservation of cocoa germplasm of paramount importance. In order to enhance security of in situ germplasm collections, 2-3 mm floral-derived secondary somatic embryos were cryopreserved by vitrification. This work demonstrates the most uncomplicated clonal cocoa cryopreservation. Optimal post-cryostorage survival (74.5 percent) was achieved by 5 d preculture of SSEs on 0.5 M sucrose medium followed by 60 min dehydration in cold PVS2. To minimise free radical related cryo-injury, cation sources were removed from the embryo development solution and/or the recovery medium, the former treatment resulting in a significant benefit. After optimisation with cocoa genotype AMAZ 15, the same protocol was effective across all five additional cocoa genotypes tested. For the multiplication of clones, embryos regenerated following cryopreservation were used as explant sources, and vitrification was found to maintain their embryogenic potential.

  19. Highly efficient vitrification method for cryopreservation of human oocytes.

    Science.gov (United States)

    Kuwayama, Masashige; Vajta, Gábor; Kato, Osamu; Leibo, Stanley P

    2005-09-01

    Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in plastic handle (Cryotop method). In the second experiment, the Cryotop method, which had yielded the best results, was used to vitrify human oocytes. Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection, 52 became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescence in-situ hybridization of five blastocysts showed that all were normal diploid embryos. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies, seven healthy babies and three ongoing pregnancies. The results suggest that vitrification using the Cryotop is the most efficient method for human oocyte cryopreservation.

  20. Cryopreservation of in vitro shoot apices of Oxalis tuberosa Mol.

    Science.gov (United States)

    Gonzalez-Benito, M E; Mendoza-Condori, V H; Molina-Garcia, A D

    2007-01-01

    Oca (Oxalis tuberosa Mol.) is an under-utilized tuber crop from the Andean region. Cryopreservation would allow the safe and long-term preservation of the genetic resources of this crop. A protocol for the cryopreservation of in vitro grown shoots has been developed using the vitrification solution PVS2. Two genotypes were studied (G1 and G27). Nodal segments were cultured on MS medium and incubated at 10 degree C with 16 h photoperiod and 10 mol per square meter per second irradiance, for two weeks. Apices were then excised and cultured on MS+0.15 M sucrose for 3 days at 5 degree C in darkness. Subsequently, apices were immersed in a loading solution (liquid MS medium+2 M glycerol+0.4 M sucrose), and then treated with the vitrification solution PVS2 for 0 to 40 minutes. Cryovials were then immersed in liquid nitrogen. Four weeks after rewarming and culture on recovery medium, genotype G1 showed approximately 60 percent recovery (normal growth) with 20 min PVS2 treatment. Genotype G27 showed lower recovery (30 percent). Differential scanning calorimetry yielded a Tg midpoint for PSV2 solution of ca. -120 degree C. Calorimetric studies on apices at different stages of the cryopreservation protocol showed a change in calorimetric parameters consistent with a decrease in the amount of frozen water as the protocol advanced.

  1. [Prospects for Application of Gases and Gas Hydrates to Cryopreservation].

    Science.gov (United States)

    Shishova, N V; Fesenko, E E

    2015-01-01

    In the present review, we tried to evaluate the known properties of gas hydrates and gases participating in the formation of gas hydrates from the point of view of the mechanisms of cryoinjury and cryoprotection, to consider the papers on freezing biological materials in the presence of inert gases, and to analyze the perspectives for the development of this direction. For the purpose, we searched for the information on the physical properties of gases and gas hydrates, compared processes occured during the formation of gas hydrates and water ice, analyzed the influence of the formation and growth of gas hydrates on the structure of biological objects. We prepared a short review on the biological effects of xenon, krypton, argon, carbon dioxide, hydrogen sulfide, and carbon monoxide especially on hypothermal conditions and probable application of these properties in cryopreservation technologies. The description of the existing experiments on cryopreservation of biological objects with the use of gases was analyzed. On the basis of the information we found, the most perspective directions of work in the field of cryopreservation of biological objects with the use of gases were outlined. An attempt was made to forecast the potential problems in this field.

  2. Cryopreservation: a cold look at technology for fertility preservation.

    Science.gov (United States)

    Gosden, Roger

    2011-08-01

    To outline the history of cryopreservation technology and its contributions to reproductive medicine, including fertility preservation. A search of the relevant literature using Medline and other online tools. Research and laboratory protocol development. The biology of preserving cells at low temperatures is complex and still being unraveled. Principles were first established more than half a century ago, with progress being driven empirically and often by trial and error. The protocols vary widely, and practice is still heavily dependent on operator skill, accounting for wide differences in the success rates between centers. No single protocol fits all specimen types, and differential vulnerability to cryoinjury remains a major obstacle. Nevertheless, semen cryopreservation has long been established, embryo banking is now highly effective, and vitrification appears to overcome problems with oocytes. Protocols in the future, although specific to the cell type and tissue, are likely to evolve toward generally acknowledged standards. But heterogeneity between patients and even within samples implies that each cell may have its own peculiar optimum for minimizing cryoinjury; because protocols are therefore compromises, "perfect" preservation may be unattainable. Cryopreservation has become a mainstay in the assisted reproduction laboratory and underpins fertility preservation for patients with cancer and other conditions. The practice is currently evolving from slow freezing methods toward more vitrification, and future technology is likely to reduce dependence on operator skill, which should raise success rates to higher, more uniform levels. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  3. Newly diagnosed hepatocellular carcinoma in patients with advanced hepatitis C treated with DAAs: A prospective population study.

    Science.gov (United States)

    Romano, Antonietta; Angeli, Paolo; Piovesan, Sara; Noventa, Franco; Anastassopoulos, Georgios; Chemello, Liliana; Cavalletto, Luisa; Gambato, Martina; Russo, Francesco Paolo; Burra, Patrizia; Vincenzi, Valter; Scotton, Pier Giorgio; Panese, Sandro; Tempesta, Diego; Bertin, Tosca; Carrara, Maurizio; Carlotto, Antonio; Capra, Franco; Carolo, Giada; Scroccaro, Giovanna; Alberti, Alfredo

    2018-03-16

    Direct-acting antiviral agents (DAAs) are safe and effective in patients with hepatitis C. Conflicting data were reported on the risk of hepatocellular carcinoma (HCC) during/after therapy with DAAs. The aim of this study was to evaluate the incidence of newly diagnosed HCC and associated risk factors in patients with advanced hepatitis C treated with DAAs. The study is based on the NAVIGATORE platform, a prospectively recording database of all patients with hepatitis C receiving DAAs in the Veneto region of Italy. The inclusion criteria were: fibrosis stage ≥F3. The exclusion criteria were: Child-Turcotte-Pugh (CTP)-C, liver transplantation before DAAs, history or presence of HCC, follow-up hepatocarcinoma during the first year is not higher, and might be lower, than that of untreated patients. The risk further declines thereafter. Early hepatocarcinoma appearance may reflect pre-existing, microscopic, undetectable tumors. Hepatocellular carcinoma is one of the complications of hepatitis C related cirrhosis. Treating patients with advanced hepatitis C with the new interferon-free direct-acting antiviral agents has been associated with improvement in liver function and survival, while more conflicting data have been reported regarding the risk of hepatocellular carcinoma. We report the results of a prospective population study on the incidence of newly diagnosed hepatocellular carcinoma in patients with advanced hepatitis C treated with direct-acting antiviral agents, clearly indicating that the residual hepatocellular carcinoma risk is reduced and declines progressively with time after a sustained virological response. Development of a liver tumor during/after therapy was associated with known risk factors and with virological failure. Copyright © 2018. Published by Elsevier B.V.

  4. Mitomycin C induced alterations in antioxidant enzyme levels in a model insect species, Spodoptera eridania.

    Science.gov (United States)

    Batcabe, J P; MacGill, R S; Zaman, K; Ahmad, S; Pardini, R S

    1994-05-01

    1. An insect species, the southern armyworm Spodoptera eridania, was used as an in vivo model to examine mitomycin C's (MMC) pro-oxidant effect reflected in alterations of antioxidant enzymes. 2. Following a 2-day exposure to 0.01 and 0.05% w/w dietary concentrations, MMC only induced superoxide dismutase activity. All other enzyme activities were not affected, indicating oxidative stress was mild. 3. Following a 5-day exposure to 0.05% w/w dietary MMC, the activities of superoxide dismutase, glutathione-S-transferase and its peroxidase activity and DT-diaphorase were induced. GR activity was not altered. The high constitutive catalase activity was also not affected. These responses of S. eridania's antioxidant enzymes are analogous to those of mammalian systems in alleviating MMC-induced oxidative stress. 4. S. eridania emerges as an appropriate non-mammalian model for initial and cost-effective screening of drug-induced oxidative stress.

  5. Micronucleus formation in cultured human keratinocytes following exposure to mitomycin C and cyclophosphamide.

    Science.gov (United States)

    van Pelt, F N; Haring, R M; Overkamp, M J; Weterings, P J

    1991-02-01

    A method is described to investigate the induction of micronuclei in cultured human keratinocytes after short-term exposure to known clastogenic agents. The cytokinesis-block method was applied to facilitate the scoring of micronucleated cells. Mitomycin C, a direct-acting compound, caused a 5-20-fold increase in micronuclei over the controls at the highest concentration tested (1 microgram/ml). Cyclophosphamide, an agent requiring metabolic activation, did not induce the formation of micronuclei in cultured keratinocytes. However, after pretreatment of the keratinocyte cultures with Aroclor 1254 for 72 h, exposure to cyclophosphamide resulted in a 3-fold increase in micronucleus frequency over the controls. No cytogenetic effect of Aroclor 1254 was observed in control experiments.

  6. Preclinical Evaluation of Promitil, a Radiation-Responsive Liposomal Formulation of Mitomycin C Prodrug, in Chemoradiotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Xi; Warner, Samuel B.; Wagner, Kyle T.; Caster, Joseph M. [Laboratory of Nano- and Translational Medicine, Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, Carolina Center for Cancer Nanotechnology Excellence, Carolina Institute of Nanomedicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (United States); Zhang, Tian [Division of Medical Oncology, Department of Medicine, Duke Cancer Institute, Duke University Medical Center, Durham, North Carolina (United States); Ohana, Patricia [Lipomedix Pharmaceuticals, Jerusalem (Israel); Gabizon, Alberto A. [Lipomedix Pharmaceuticals, Jerusalem (Israel); Shaare Zedek Medical Center, Jerusalem (Israel); Wang, Andrew Z., E-mail: zawang@med.unc.edu [Laboratory of Nano- and Translational Medicine, Department of Radiation Oncology, Lineberger Comprehensive Cancer Center, Carolina Center for Cancer Nanotechnology Excellence, Carolina Institute of Nanomedicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (United States)

    2016-11-01

    Purpose: To examine the effect of radiation on in vitro drug activation and release of Promitil, a pegylated liposomal formulation of a mitomycin C (MMC) lipid-based prodrug; and examine the efficacy and toxicity of Promitil with concurrent radiation in colorectal cancer models. Methods and Materials: Promitil was obtained from Lipomedix Pharmaceuticals (Jerusalem, Israel). We tested the effects of radiation on release of active MMC from Promitil in vitro. We next examined the radiosensitization effect of Promitil in vitro. We further evaluated the toxicity of a single injection of free MMC or Promitil when combined with radiation by assessing the effects on blood counts and in-field skin and hair toxicity. Finally, we compared the efficacy of MMC and Promitil in chemoradiotherapy using mouse xenograft models. Results: Mitomycin C was activated and released from Promitil in a controlled-release profile, and the rate of release was significantly increased in medium from previously irradiated cells. Both Promitil and MMC potently radiosensitized HT-29 cells in vitro. Toxicity of MMC (8.4 mg/kg) was substantially greater than with equivalent doses of Promitil (30 mg/kg). Mice treated with human-equivalent doses of MMC (3.3 mg/kg) experienced comparable levels of toxicity as Promitil-treated mice at 30 mg/kg. Promitil improved the antitumor efficacy of 5-fluorouracil–based chemoradiotherapy in mouse xenograft models of colorectal cancer, while equitoxic doses of MMC did not. Conclusions: We demonstrated that Promitil is an attractive agent for chemoradiotherapy because it demonstrates a radiation-triggered release of active drug. We further demonstrated that Promitil is a well-tolerated and potent radiosensitizer at doses not achievable with free MMC. These results support clinical investigations using Promitil in chemoradiotherapy.

  7. [Intraoperative chemotherapy against peritoneal dissemination of gastric cancer with intraperitoneal activated carbon particles adsorbing mitomycin C].

    Science.gov (United States)

    Hagiwara, A; Takahashi, T; Sawai, K; Yamaguchi, T; Iwamoto, A; Yoneyama, C

    1989-02-01

    For prevention and therapy of peritoneal dissemination, a new dosage from (MMC-CH) comprising carbon particles adsorbing mitomycin C was given to 44 patients (the MMC-CH group) undergoing gastrectomy for gastric cancer, of which advancing stage was classified into the category of H0, and S2 or S3, and P0, P1, P2 or P3 according to the General Rules for the Gastric Cancer Study. MMC-CH, principally at 50 mg person in terms of mitomycin C was administered intraperitoneally before the surgical wound was closed. Historical control group was composed of 53 patients not given MMC-CH, who underwent gastrectomy for gastric cancer in the same advancing stage as those of the 44 patients. There was statistically no significant difference of age, sex, depth of infiltration, macroscopically and microscopically defined progression of lymph-nodal metastases, between the MMC-CH group and the historical control group. The survival rate of the overall patients, and each group of the patients with the lesion defined as P0, P1, P2, or P3 was compared with Kaplan-Meier's method between the MMC-CH group and the historical control group. In the MMC-CH group, the survival rates of the overall patients and the patients with P0, P1, or P2 lesion were statistically significantly higher than those in the historical control group. However, the rate of the P3 patients in the MMC-CH group was statistically significantly lower than in the historical control group.

  8. Subconjunctival bevacizumab to augment trabeculectomy with mitomycin C in the management of failed glaucoma surgery

    Directory of Open Access Journals (Sweden)

    Saeed AM

    2014-09-01

    Full Text Available Ahmed M Saeed, Tarek Tawfeek AboulNasrOphthalmology Department, Benha University, Egypt Purpose: To provide a feasible solution to the problem of failed glaucoma surgery. The aim was to evaluate the efficacy and safety of the additional effects of a combined surgical approach. This approach augments the application of trabeculectomy with mitomycin C (MMC by adding subconjunctival bevacizumab injection. The results were compared with those of trabeculectomy with only adjunctive MMC. Methods: A randomized controlled prospective clinical trial included 28 eyes diagnosed with failed scarred bleb of a previous trabeculectomy. The eyes were divided into two equal groups: combined group A, “trabeculectomy with adjunctive MMC and subconjunctival bevacizumab,” and control group B, “trabeculectomy with adjunctive MMC only.” The main outcome results included the cumulative probability of surgical success, intraocular pressure (IOP values, and number of IOP-lowering medications needed to achieve the target IOP.Results: Group A achieved a cumulative probability of complete success of 0.769 and of qualified success of 0.231 at the end of the 24 month study period; group B achieved cumulative probabilities of 0.538 and 0.308, respectively. Group A achieved a lower mean IOP value than group B, with fewer antiglaucoma drugs at all postoperative visits, but this lower value did not reach a statistically significant level (P>0.05. There was no statistically significant difference between both groups regarding best corrected visual acuity, visual field parameters, operative and/or postoperative complications, and additional interventions. No significant adverse effects were caused by this combined approach.Conclusion: Bevacizumab was not found to add much to the favorable long-term outcome of conventional trabeculectomy with MMC as a solution to the problem of scarred failed bleb. Keywords: glaucoma, bevacizumab, mitomycin C, failed trabeculectomy

  9. Reconstruction of delayed scleral flap melting with bovine pericardium after trabeculectomy with mitomycin C

    Directory of Open Access Journals (Sweden)

    Coutinho, Inês

    2017-06-01

    Full Text Available Aim: To present a challenging case of hypotony after trabeculectomy and its treatment. Case description: A 22-year-old woman with juvenile glaucoma underwent a conventional trabeculectomy with mitomycin C on the right eye (OD. In the immediate postoperative period, we observed a hyperfiltration bleb with hypotony refractory to conservative measures leading to hypotony maculopathy.A surgical revision with scleral flap resuture and conjunctival graft was performed with a satisfactory result and resolution of hypotony maculopathy. After two years, the patient complained of low visual acuity (VA of the OD. During examination, we observed a fine and avascular bleb with Seidel and visualization of the underlying uveal tissue, an intraocular pressure (IOP of 5 mmHg, and chorioretinal folds. A new revision of the trabeculectomy was performed. During the procedure, it was not possible to identify the scleral flap, so the fistula was closed with a patch of collagenous membrane derived from bovine pericardium (Tutopatch graft. A good clinical evolution occurred. After 2 months, IOP was 15 mmHg without Seidel or changes in the fundus and VA was 20/20. After of follow-up, the IOP remains stable without further complaints. Conclusion: This case illustrates the difficulties faced in the management of a common complication of trabeculectomy and highlights some of the options available for its treatment. There are few reports of scleral melting after trabeculectomy. However, trauma and scleral necrosis associated with mitomycin are listed as the main causes.The use of a scleral patch derived from bovine pericardium allows effective suturing and closure of the aqueous leak.

  10. Locoregional mitomycin C injection for esophageal stricture after endoscopic submucosal dissection.

    Science.gov (United States)

    Machida, H; Tominaga, K; Minamino, H; Sugimori, S; Okazaki, H; Yamagami, H; Tanigawa, T; Watanabe, K; Watanabe, T; Fujiwara, Y; Arakawa, T

    2012-06-01

    This prospective study aimed to evaluate the feasibility and safety of locoregional mitomycin C (MMC) injection to treat refractory esophageal strictures after endoscopic submucosal dissection (ESD) for superficial esophageal carcinoma. Patients with dysphagia and strictures that were refractory to repeated endoscopic balloon dilation (EBD) were eligible. After EBD, MMC was injected into the dilated site. Between June 2009 and August 2010, five patients were recruited. The treatment was performed once in two patients and twice in three patients with recurrent dysphagia or restenosis. In all patients, passing a standard endoscope through the site was easy and the dysphagia grade improved (grade 3→1 in 3 patients, grade 4→2 in 2 patients). No serious complications were noted. During the observation period of 4.8 months, neither recurrent dysphagia nor re-stricture appeared in any of the patients. The combination of locoregional MMC injections and EBD is feasible and safe for the treatment of esophageal strictures after ESD.Recently, endoscopic submucosal dissection (ESD) has been developed and accepted as a new endoscopic treatment for gastrointestinal tumors. ESD is a promising treatment for superficial esophageal carcinoma (SEC), and it has a reliable en bloc resection rate. However, the application of ESD for widespread lesions is challenging because of the high risk of the development of severe strictures, which lead to a low quality of life after ESD. Although endoscopic balloon dilation (EBD) is effective for benign strictures, it needs to be performed frequently until the dysphagia disappears 1. Mitomycin C (MMC), which is a chemotherapeutic agent derived from some Streptomyces species 2, reduces scar formation when topically applied to a surgical lesion. MMC has been applied to treat strictures in a variety of anatomical locations, including a variety of organs 3. The aim of this study was to prospectively evaluate both the feasibility and the safety of

  11. Intraoperative Injection vs Sponge-applied Mitomycin C during Trabeculectomy: One-year Study.

    Science.gov (United States)

    S Khouri, Albert; Huang, Grace; Y Huang, Linda

    2017-01-01

    To determine the safety and efficacy of intraoperative injection of mitomycin C (MMC) against conventional sponge-applied MMC during trabeculectomy. This study was a retrospective, comparative case series. Thirty eyes with primary open-angle glaucoma underwent consecutive trabeculectomies with MMC injection (injection group), and thirty eyes with sponge-applied MMC were as controls (sponge group). Data were collected preoperatively and postoperatively at 1 day, 1 week, 1 month, 3 months, 6 months, and 1 year after surgery. Demographic data, applanation intraocular pressure (IOP), best-corrected visual acuity (VA), number of glaucoma medications, postoperative interventions, postoperative complications, and number of visits within 3 months were recorded. In order to stratify data, proportion of eyes achieving >30% IOP reduction from baseline with or without glaucoma medications was calculated and defined as surgical success. Mean IOP reduction at 1 year was significant in both the injection and sponge groups from baseline (46.8 and 37.8% respectively). The injection group had overall lower postoperative IOP and comparable complete treatment success, defined as achieving >30% IOP reduction without glaucoma medications (p = 0.941). The number of postoperative visits within 3 months and the proportion of eyes needing 5-fluorouracil (5-FU) intervention were significantly lower in the injection group (p = 0.03, p = 0.04 respectively). Injection of MMC was as safe and effective as sponge application with comparable estimated complete treatment success, less need for visits within 3 months, and 5-FU intervention. Surgeons may consider intraopera-tive injection of MMC in appropriate patient cohorts given comparable safety and efficacy and several advantages over traditional sponge application. Further study in a prospective, larger, long-term manner is necessary to assess this modality. How to cite this article: Khouri AS, Huang G, Huang LY. Intraoperative Injection vs

  12. Survival, growth and reproduction of cryopreserved larvae from a marine invertebrate, the Pacific oyster (Crassostrea gigas.

    Directory of Open Access Journals (Sweden)

    Marc Suquet

    Full Text Available This study is the first demonstration of successful post-thawing development to reproduction stage of diploid cryopreserved larvae in an aquatic invertebrate. Survival, growth and reproductive performances were studied in juvenile and adult Pacific oysters grown from cryopreserved embryos. Cryopreservation was performed at three early stages: trochophore (13±2 hours post fertilization: hpf, early D-larvae (24±2 hpf and late D-larvae (43±2 hpf. From the beginning (88 days at the end of the ongrowing phase (195 days, no mortality was recorded and mean body weights did not differ between the thawed oysters and the control. At the end of the growing-out phase (982 days, survival of the oysters cryopreserved at 13±2 hpf and at 43±2 hpf was significantly higher (P<0.001 than those of the control (non cryopreserved larvae. Only the batches cryopreserved at 24±2 hpf showed lower survival than the control. Reproductive integrity of the mature oysters, formely cryopreserved at 13±2 hpf and 24±2 hpf, was estimated by the sperm movement and the larval development of their offspring in 13 crosses gamete pools (five males and five females in each pool. In all but two crosses out of 13 tested (P<0.001, development rates of the offspring were not significantly different between frozen and unfrozen parents. In all, the growth and reproductive performances of oysters formerly cryopreserved at larval stages are close to those of controls. Furthermore, these performances did not differ between the three initial larval stages of cryopreservation. The utility of larvae cryopreservation is discussed and compared with the cryopreservation of gametes as a technique for selection programs and shellfish cryobanking.

  13. Utilization and quality of cryopreserved red blood cells in transfusion medicine

    NARCIS (Netherlands)

    Henkelman, S.; Noorman, F.; Badloe, J. F.; Lagerberg, J. W. M.

    Cryopreserved (frozen) red blood cells have been used in transfusion medicine since the Vietnam war. The main method to freeze the red blood cells is by usage of glycerol. Although the usage of cryopreserved red blood cells was promising due to the prolonged storage time and the limited cellular

  14. Cryopreservation of human embryos and its contribution to in vitro fertilization success rates

    NARCIS (Netherlands)

    Wong, Kai Mee; Mastenbroek, Sebastiaan; Repping, Sjoerd

    2014-01-01

    Cryopreservation of human embryos is now a routine procedure in assisted reproductive technologies laboratories. There is no consensus on the superiority of any protocol, and substantial differences exist among centers in day of embryo cryopreservation, freezing method, selection criteria for which

  15. Cryopreservation of in vitro-grown shoot tips of apricot (Prunus ...

    African Journals Online (AJOL)

    akpobome

    2013-03-20

    Mar 20, 2013 ... Thus, they can be preserved in such a state for a long period (Sakai, 1995). Many new cryopreservation ..... formation of intracellular ice crystals during rapid cooling in LN (Wilkinson et al., 1998). Effect preculture and ..... Cryopreservation of protocorm-like bodies. (PLBs) of Phalaenopsis bellina. (Rchb.f.) ...

  16. Cryopreservation techniques and their application in vegetatively propagated crop plants in Finland

    Directory of Open Access Journals (Sweden)

    A. NUKARI

    2008-12-01

    Full Text Available Cryopreservation protocols have been introduced as techniques for germplasm preservation of vegetatively propagated horticultural and staple food crops. In Finland, cryopreservation has been studied since 1990’s, beginning with cryopreservation of forest tree breeding material and since 2004 on cryopreservation of genetic resources of horticultural plants and potato. Priority was given to cryopreservation of raspberry (Rubus ideaus L., strawberry (Fragaria x ananassa Duch. and potato (Solanum tuberosum L. and the possibility to use cryotherapy in eradication of raspberry bushy dwarf virus (RBDV from in vitro cultures were studied on raspberry. Modified droplet vitrification cryopreservation protocols were designed for raspberry and strawberry and cryotherapy combined with thermotherapy was proven to be a successful application to eliminate RBDV from infected raspberries. Cryotherapy method can be applied for a large scale elimination of viruses from plant germplasm and from candidate nuclear stock in a certified plant production scheme. Routine use of cryotechniques in germplasm preservation of vegetatively propagated horticultural plants was started. Besides for long term germplasm preservation, cryopreservation techniques can be applied also for maintenance of mother stocks in certified plant production schemes and in commercial plant production. Cryopreservation of potato shoot tips needs additional detailed research to obtain sufficient recovery and regrowth rates.;

  17. Parental desire and acceptability of spermatogonial stem cell cryopreservation in boys with cancer

    NARCIS (Netherlands)

    van den Berg, H.; Repping, S.; van der Veen, F.

    2007-01-01

    BACKGROUND: In the near future, a substantial proportion of adults will be childhood cancer survivors. The cryopreservation and transplantation of spermatogonial stem cells (SSCs) is currently successful in animals; application in humans seems likely in the near future. Cryopreserving SSCs might

  18. CHARACTERISTICS AND FERTILITY OF SUMATRAN TIGER SPERMATOZOA CRYOPRESERVED WITH DIFFERENT SUGARS.

    Science.gov (United States)

    Wayan Kurniani Karja, Ni; Fahrudin, Mokhamad; Setiadi, Mohamad Agus; Tumbelaka, Ligaya Ita; Sudarwati, Retno; Hastuti, Yohana Tri; Mulia, Bongot Huas; Widianti, Ardyta; Sultan, Keni; Terazono, Tsukasa; Namula, Zhao; Taniguchi, Masayasu; Tanihara, Fuminori; Takemoto, Tatsuya; Kikuchi, Kazuhiro; Sato, Yoko; Otoi, Takeshige

    Cryopreservation of semen is one of the most important methods for the preservation of endangered tigers. This study evaluated the effects of sugar supplementation on the cryosurvival of spermatozoa from Sumatran tigers (Panthera tigris sumatrae). The post-thaw characteristics and fertility of spermatozoa cryopreserved with different sugars (glucose, lactose, and trehalose) were evaluated using heterologous in-vitro fertilisation with cat oocytes. All parameters of post-thaw spermatozoa significantly decreased as compared with those of fresh spermatozoa. The index of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose. The percentage of total fertilisation for tiger spermatozoa cryopreserved with trehalose was significantly lower than that for control cat spermatozoa. Our findings indicated that supplementation with lactose or glycerol as the main sugar in the egg yolk extender resulted in a better motility and fertility potential for post-thawed spermatozoa.

  19. Impact of starting material (fresh versus cryopreserved marrow) on mesenchymal stem cell culture.

    Science.gov (United States)

    Kaplan, Alesia; Sackett, Katie; Sumstad, Darin; Kadidlo, Dianne; McKenna, David H

    2017-09-01

    Mesenchymal stem cells (MSCs) continue to be investigated in multiple clinical trials as potential therapy for different disorders. There is ongoing controversy surrounding the clinical use of cryopreserved versus fresh MSCs. However, little is known about how cryopreservation affects marrow as starting material. The growth kinetics of MSC cultures derived from fresh versus cryopreserved marrow were compared. Data were reviewed on the growth kinetics of MSCs derived from fresh versus cryopreserved marrow of nine donors. Marrow harvested from each donor was separated into four aliquots (one fresh and three cryopreserved for culture). Data on the date of mononuclear cell cryopreservation/thaw, MSC counts at Passages 1 and 2, MSC doubling, MSC fold expansion, viability (of mononuclear cells and final MSCs), and on flow cytometry markers of mononuclear cells and final MSCs were analyzed for the fresh and cryopreserved marrow groups. In total, 21 MSC lots (seven fresh and 14 cryopreserved) were obtained. The average age of cryopreserved mononuclear cell product was 295 days (range, 18-1241 days). There were no significant differences between MSC numbers at Passage 1 (p = 0.1), final MSC numbers (p = 0.5), MSC doubling (p = 0.7), or MSC fold expansion (p = 0.7). A significant difference was observed in viability by flow cytometry for both mononuclear cells (p = 0.002) and final MSCs (p = 0.009), with higher viability in the fresh marrow group. This study demonstrates that MSCs derived from cryopreserved marrow have the same growth characteristics as fresh marrow-derived MSCs. Further studies are needed to explore potential differences in clinical efficacy. © 2017 AABB.

  20. Electrochemical monitoring of the interaction between mitomycin C and DNA at chitosan--carbon nanotube composite modified electrodes

    OpenAIRE

    CANAVAR, Pembe Ece; EKŞİN, Ece; ERDEM, Arzum

    2015-01-01

    Single-walled carbon nanotube (CNT) and chitosan composite (chitosan*CNT) based sensors were developed as DNA biosensors, and then they were applied for electrochemical investigation of the interaction between the anticancer drug mitomycin C (MC) and DNA. The oxidation signals of MC and guanine were monitored before and after the interaction process by differential pulse voltammetry (DPV). The DPV results were in good agreement with those of electrochemical impedance spectroscopy (EIS)....

  1. Mitomycin-C-Induced TTP/HUS Treated Successfully with Rituximab: Case Report and Review of the Literature

    Directory of Open Access Journals (Sweden)

    Gunjan Shah

    2013-01-01

    Full Text Available Microangiopathic hemolytic anemia (MAHA, thrombocytopenia, fever, renal failure, and neurologic symptoms comprise the cardinal features of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Etiologies can include medications, infections, cancers, or transplantation. We present a patient with a history of rectal cancer treated with mitomycin-C who developed MAHA, acute kidney injury, and thrombocytopenia 6 months after completing therapy and to did not respond the plasmapheresis or steroids. She was treated with four weekly doses of rituximab with full recovery.

  2. Preoperative infusion of mitomycin-C in the bronchial artery in squamous cell carcinoma of the lung

    Energy Technology Data Exchange (ETDEWEB)

    Hellekant, C; Boijsen, E; Svanberg, L [Departments of Diagnostic Radiology and Thoracic Sugery, Malmoe Allmaenna Sjukhus, Malmoe, Sweden

    1978-01-01

    Bronchial angiography was performed in 9 patients with squamous cell carcinoma of the lung. The tumour-feeding vessel was identified and infused with 10 mg of mitomycin-C (MMC) diluted in saline. At operation after 28 to 48 days complete remission of the tumour had occurred in 2 patients, almost complete in one, partial remission in 2 and a marked regression in 2. In 2 patients no change was noted. No side effects except moderate malaise and slight fever occurred.

  3. [Response of HeLa cells to mitomycine C. III. The analysis of nucleoli of mother and daughter cells].

    Science.gov (United States)

    Petrov, Iu P; Neguliaev, Iu A; Tsupkina, N V

    2014-01-01

    The comparative analysis of the number of nucleoli in cells of the established HeLa-M line was carried out before and after exposure to mitomycin C in a concentration of 10 μg/ml for 2 h. Using time-lapse microscopy, nucleoli in mother and their respective daughter cells were computed. It has been shown that the average number of nucleoli per cell is generally higher in daughter cells than in mother cells, and a standard deviation, on the contrary, decreases. An average number of nucleoli in daughter cells, whose mother cells had been treated with mitomycin C, was higher than in corresponding cells of control group. The separate analysis has been performed for the cells having from 1 to 4 nucleoli. Nonrandom complete coincidence of the number of nucleoli in mather and daughter cells has been typicaly shown for about 1/7 of the total cell population. Mitomycin C reduces this value of about 1.5 times.

  4. USE OF DIFFERENT EXTENDERS TO CRYOPRESERVATION OF MANGALARGA MARCHADOR SPERM

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    Jessica Neri Nascimento

    2015-07-01

    Full Text Available The techniques applied to animal reproduction such as artificial insemination, transfer and in vitro production of embryos, heat synchronization and induction, and gametes cryopreservation have been more utilized in veterinary practice each day. Nevertheless, some techniques have not achieved their full technical capacity within the equine reproduction field, such as semen cryopreservation. The aim of this study was to evaluate the characteristics of post-thawing semen (total motility, strength, plasmatic and acrosomal membrane integrity of Mangalarga Marchador breed stallions, using three different extenders (Botucrio, FR5 and FR6. Ejaculates from five stallions were collected and the gel-free semen was diluted in a 1:1 dilution in skim milk extender, and centrifuged at 600 g for 10 minutes. After the centrifugation the supernatant was removed and sperm pellet was divided and re-suspended using three different extenders to a concentration of 200 x 106 cells/mL. The samples were packed into 0.5 mL straws, placed in a stainless steel support and kept inside the refrigerator (5 oC for one hour. Subsequently, these straws were kept at a height of 6 cm from liquid nitrogen for 15 minutes in an isotherm box and, after that, plunged into liquid nitrogen (-196 oC and stored in a liquid nitrogen holding tank. There were no differences in the parameters evaluated when extenders using mixed amides and glycerol (Botucrio and FR6 were used (P > 0.05. All parameters evaluated were lower for the extender containing only glycerol (P<0.05. The use of cryoprotectants (methylformamide and dimethylformamide in association with glycerol concentrations around 1 to 2% is an alternative for semen cryopreservation of Mangalarga Marchador breed stallions.

  5. Two-piece cryopreserved tracheal allotransplantation: an experimental study.

    Science.gov (United States)

    Iyikesici, Tuncel; Tuncozgur, Bulent; Sanli, Maruf; Isik, Ahmet Feridun; Meteroglu, Fatih; Elbeyli, Levent

    2009-10-01

    For successful reconstruction with tracheal allotransplants following long tracheal resections, problems related to the preservation and vascularisation of the tracheal graft have to be solved. In this study, instead of using a long-segment single-piece graft, we used a graft that has been split into two. The aim was to use this graft after cryopreservation in order to ease neo-vascularisation and to maintain tracheal integrity by transplanting it to two separate regions of the dog cervical trachea. This experimental study was conducted in animal laboratories of the medical school on 11 half-blood dogs. The trachea obtained from the first dog was 8 cm in length; it was split into two pieces of 4 cm each and stored in the preservation solution at -80 degrees C for 4 weeks. Following this, the dog was sacrificed. Two 2 cm portions of cervical trachea were excised from the second dog. These parts were then reconstructed with two tracheal grafts of the same length as the cryopreserved ones. Ten dogs that were grouped into five groups of two dogs each underwent the same procedure. The subjects had a bronchoscopic evaluation on the third postoperative week. Anastomosis regions of the test tracheas were resected to be examined histopathologically. Seven subjects were found to have third-degree obstructions during bronchoscopy; two had close to fourth-degree obstructions. In the histopathological examination, contrary to the findings of the bronchoscopies, 75% of the anastomoses had intact epithelium. The cartilage was seen to have well-preserved structural characteristics in all the anastomoses. Twelve anastomoses had moderate, seven mild and one had severe inflammation. All anastomoses had either good or very good level of vascularisation. The integrity of the tracheal epithelium can be maintained with cryopreservation and split anastomosis technique. The cartilage preserves its structural characteristics despite losing its viability, thereby offering an advantage to

  6. Cryopreservation of mouse embryos by ethylene glycol-based vitrification.

    Science.gov (United States)

    Mochida, Keiji; Hasegawa, Ayumi; Taguma, Kyuichi; Yoshiki, Atsushi; Ogura, Atsuo

    2011-11-18

    Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s(1), then followed by vitrification methods developed in the late 1980s(2). Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4°C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained(3), and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature(4). Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos(5). It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and

  7. Cryopreservation of spermatozoa of black marlin, Makaira indica (Teleostei: Istiophoridae).

    Science.gov (United States)

    van der Straten, K M; Leung, L K-P; Rossini, R; Johnston, S D

    2006-01-01

    As a first step towards the development of a method for the cryopreservation of black marlin spermatozoa, this study investigated the effect of dimethylsulfoxide (DMSO) concentration and pellet size on post-thaw spermatozoal motility. Spermatozoa were recovered from the spermatic duct of testes retrieved post-mortem from four adult black marlin caught in the Coral Sea spawning grounds of Australia. Undiluted spermatozoa were stored on ice for 4 to 10 hours during transport to shore, then evaluated for motility after activation in seawater (1:10 v:v). Spermatozoa were prepared for cryopreservation in pellets by extension (1:3 v:v) in a defined fish Ringer's solution to give two final DMSO concentrations of 2.5% or 5.0%. Diluted spermatozoa were frozen directly on a dry ice block in pellet sizes of either 0.25 ml or 0.50 ml. Frozen pellets were thawed in a water bath at 40 degrees C for 60 seconds and assessed for post-thaw motility following activation in seawater. Spermatozoa recovered within 50 minutes of death and chilled on ice for 4 to 10 hours showed a mean (+/- SEM) motility immediately following activation of 91.6 +/- 7.9%. 50% of the spermatozoa remained motile for approximately 4 to 5 minutes. Following cryopreservation, mean motility declined significantly across all cryoprotectant and pellet size combinations (P < 0.001) but spermatozoa frozen in 2.5% DMSO showed higher motility than those frozen in 5.0% DMSO (P = 0.014). Pellet size had no effect on post-thaw motility (P = 0.179).

  8. [Intraoperative chemotherapy with intraperitoneal activated carbon particles adsorbing mitomycin C against peritoneal dissemination of gastric cancer].

    Science.gov (United States)

    Iwamoto, A; Takahashi, T; Sasabe, T; Itoh, M; Kondoh, S; Seiki, K; Yoneyama, C; Shimotsuma, M; Hagiwara, A; Yamaguchi, T

    1989-08-01

    A new form of dosage (MMC-CH) was composed of activated carbon particles adsorbing mitomycin C. Intraperitoneal administration of MMC-CH was tested clinically for prophylactic and therapeutic effects on peritoneal carcinomatosis of gastric cancer. The criteria of MMC-CH's administration were equal or less than 70 years old, more than 40 kg in body weight, no disfunction of liver and kidney, no particular findings in electrocardiography, S2 or S3 in the grade of serosal invasion, P0, P1, P2 or P3 in the grade of peritoneal dissemination, according to the General Rules for the Gastric Cancer Study in Surgery and Pathology by the Japanese Research Society for Gastric Cancer. MMC-CH was given to 44 patients undergoing gastrectomy for gastric cancer in our department from 1985 to 1988. The 44 patients were composed of 12 patients with P0 findings (P0 patients), 8 patients with P1 findings (P1 patients), 12 patients with P2 findings (P2 patients), and 12 patients with P3 findings (P3 patients). MMC-CH at 50 mg/person in terms of mitomycin C was administered intraperitoneally before the operation wound was closed. Fifty-seven patients in our department from 1983 to 1987 for whom the same criteria were applicable and did not receive MMC-CH therapy, served as the control group. The 57 patients were composed of 23 P0 patients, 21 P1 patients, 10 P2 patients, and 3 P3 patients. There was statistically with chi 2 test no significant difference of age, sex, depth of infiltration macroscopically and microscopically defined progression of lymph-nodal metastases between the MMC-CH group and the control group. Survival rate was calculated with Kaplan-Meier's method in the overall patients in each of the MMC-CH group or the control group. The overall survival rate in the MMC-CH group was statistically significantly (p less than 0.01-0.05) higher from day 460 to day 552 and from day 736 to day 800 than that in the control group. Next, the patients were classified into two subgroups

  9. Partial Tenon’s capsule resection with adjunctive mitomycin C in Ahmed glaucoma valve implant surgery

    Science.gov (United States)

    Susanna, R

    2003-01-01

    Aim: To verify if partial intraoperative Tenon’s capsule resection (PTCR) with adjunctive mitomycin C is effective in developing thin, avascular blebs in eyes undergoing Ahmed glaucoma valve insertion, and to assess the efficacy and safety of this procedure. Methods: A multicentre, prospective, alternating case assignment, investigator unmasked, parallel group, comparative interventional study was conducted in four Latin American countries (Argentina, Brazil, Colombia, and Peru). Ahmed glaucoma valve implant insertion with PTCR (group A) and without PCTR (group B) was performed in neovascular glaucomatous eyes without previous surgery. Adjunctive mitomycin C (MMC) was used in both groups. Patients were examined 1 day, 10 days, 1 month, 2 months, 3 months, 6 months, and 1 year following the surgery. Intraocular pressure (IOP) and the appearance of the bleb were evaluated at each examination. Appearance of the bleb was classified at both the 1 month mark and last examinations into one of three groups: flat and vascularised; elevated avascular; or elevated and not avascular. Results: 92 eyes from 92 patients were included in the study. The preoperative mean IOP was 50.0 (SD 10.5) mm Hg in group A and 48.4 (11.7) in group B (p>0.05). Statistically significant IOP reductions were observed at all periods of follow up. 12 months after surgery, the mean IOP was 17.2 (5.0) mm Hg in group A and 18.3 (8.7) mm Hg in group B (p>0.05). A hypertensive phase occurred in 40.0% in group A and in 46.8% in group B (p>0.05). At the 1 month and the final follow up, the blebs in all eyes were considered elevated and not avascular. The success rate (IOP⩽21 mm Hg) at 1 year after surgery was 70.4% in group A and 77.7% in group B (p>0.05). Overall, 74.2% of the patients achieved an IOP ⩽21 mm Hg and 55.2% an IOP⩽17 mm Hg, with or without additional medication administered to lower IOP. The incidence of complications was similar in both groups. Conclusions: In eyes undergoing Ahmed

  10. Partial Tenon's capsule resection with adjunctive mitomycin C in Ahmed glaucoma valve implant surgery.

    Science.gov (United States)

    Susanna, R

    2003-08-01

    To verify if partial intraoperative Tenon's capsule resection (PTCR) with adjunctive mitomycin C is effective in developing thin, avascular blebs in eyes undergoing Ahmed glaucoma valve insertion, and to assess the efficacy and safety of this procedure. A multicentre, prospective, alternating case assignment, investigator unmasked, parallel group, comparative interventional study was conducted in four Latin American countries (Argentina, Brazil, Colombia, and Peru). Ahmed glaucoma valve implant insertion with PTCR (group A) and without PCTR (group B) was performed in neovascular glaucomatous eyes without previous surgery. Adjunctive mitomycin C (MMC) was used in both groups. Patients were examined 1 day, 10 days, 1 month, 2 months, 3 months, 6 months, and 1 year following the surgery. Intraocular pressure (IOP) and the appearance of the bleb were evaluated at each examination. Appearance of the bleb was classified at both the 1 month mark and last examinations into one of three groups: flat and vascularised; elevated avascular; or elevated and not avascular. 92 eyes from 92 patients were included in the study. The preoperative mean IOP was 50.0 (SD 10.5) mm Hg in group A and 48.4 (11.7) in group B (p>0.05). Statistically significant IOP reductions were observed at all periods of follow up. 12 months after surgery, the mean IOP was 17.2 (5.0) mm Hg in group A and 18.3 (8.7) mm Hg in group B (p>0.05). A hypertensive phase occurred in 40.0% in group A and in 46.8% in group B (p>0.05). At the 1 month and the final follow up, the blebs in all eyes were considered elevated and not avascular. The success rate (IOP0.05). Overall, 74.2% of the patients achieved an IOP glaucoma, PCTR with MMC augmentation showed no additional benefits or complications over MMC augmentation alone; no avascular bleb was obtained with this technique. The incidence of a hypertensive phase was lower than reported in previous studies.

  11. Trabeculectomy with double low dose of mitomycin C – two years of follow-up

    Directory of Open Access Journals (Sweden)

    Errico D

    2011-12-01

    Full Text Available Donato Errico1, Francesca Scrimieri1, Roberta Riccardi1, Romolo Fedeli1, Giancarlo Iarossi21Opthalmology Unit, Glaucoma Service, Azienda Ospedaliera, Cardinale G Panìco, Tricase (Le, Italy; 2Department of Opthalmology, Ospedale Pediatrico Bambino Gesù, Rome, ItalyPurpose: To evaluate the efficacy and the safety of a surgical technique in which classic trabeculectomy ab externo is performed with a double application of low-dose mitomycin C (MMC in uncontrolled open-angle glaucoma (OAG patients.Method: A consecutive series of 43 white patients (43 eyes with uncontrolled primary OAG underwent trabeculectomy surgery. A double application of MMC (0.1% was performed: the first under the Tenon's capsule for 3 minutes, and the second below the scleral flap for 1 or 2 minutes, according to the risk factors. Complete success was defined as intraocular pressure (IOP <14 mmHg without any additional glaucoma surgery or medication. Qualified success was defined as IOP <14 mmHg with additional needling revision.Results: Mean preoperative IOP was 29.9 mmHg (SD 3.8 for all eyes evaluated. At 1 day postoperative, mean IOP was 6.7 mmHg (SD 1.26. At the end of the first 2 weeks postoperative, mean IOP was 8.6 mmHg (SD 1.7, at 12 months mean IOP was 11.3 mmHg (SD 1.4; P < 0.0001 and at 24 months mean IOP was 11.4 mmHg (SD 1.5; P < 0.0001. At 3 months, two eyes (5.4% underwent needling of the bleb for cystic blebs formation.Conclusion: In this study we presented the results after 2 years of follow-up of OAG undergoing trabeculectomy with dual administration of MMC (0.1 mg/mL. After 24 months, complete success was achieved in 93% of patients and a qualified success in 100% of patients.Keywords: glaucoma, trabeculectomy, mitomycin C

  12. Pre- and intraoperative mitomycin C for recurrent pterygium associated with symblepharon

    Directory of Open Access Journals (Sweden)

    Mohammed I

    2013-01-01

    Full Text Available Isyaku MohammedDepartment of Ophthalmology, Aminu Kano Teaching Hospital, Kano, NigeriaBackground: Treatment of recurrent pterygium associated with symblepharon usually involves the use of tissue grafting and/or the intraoperative application of mitomycin C (MMC. For the graft, a conjunctival/limbal autograft and/or amniotic membrane may be used. This generally requires extra technical skills and assistance, an increase in the cost and duration of surgery, and a more extensive anesthesia (a complete eye block or general anesthesia. Although widely used, safety concerns have been raised over MMC in the treatment of pterygia.Objective: The objective of this case report is to report the successful use of preoperative subconjunctival injection of low-dose (0.02% MMC one month before bare sclera excision of a multirecurrent pterygium, as well as the concomitant intraoperative application of MMC to the conjunctival fornix of the same eye after the excision of an associated symblepharon.Case report: A 31-year-old man from Kano, Northern Nigeria, presented to the eye clinic with a recurrent pterygium associated with an upper lid symblepharon in his right eye. He has had five previous pterygium excisions, with the last surgery involving conjunctival autografting and subconjunctival steroid injection. He was subsequently given 0.1 mL of 0.02% MMC as a subpterygial injection; one month later he had an alcohol-assisted bare sclera pterygium excision and a symblepharolysis with the intraoperative application of 0.02% MMC for 1 minute to the upper conjunctival fornix. Except for a Tenon granuloma that was simply excised, there has been no recurrence or other complications up to a year after surgery.Conclusion: As a cheaper and technically easier treatment option, a preoperative subconjunctival MMC injection followed by bare sclera pterygium excision was found to be effective in this patient with a recurrent pterygium. As at one-year follow-up, low

  13. Potential link between fruit yield, quality parameters and phytohormonal changes in preharvest UV-C treated strawberry.

    Science.gov (United States)

    Xu, Yanqun; Charles, Marie Thérèse; Luo, Zisheng; Roussel, Dominique; Rolland, Daniel

    2017-07-01

    Preharvest ultraviolet-C (UV-C) treatment of strawberry is a very new approach, and little information is available on the effect of this treatment on plant growth regulators. In this study, the effect of preharvest UV-C irradiations at three different doses on strawberry yield, fruit quality parameters and endogenous phytohormones was investigated simultaneously. The overall marketable yield of strawberry was not affected by the preharvest UV-C treatments, although more aborted and misshapen fruits were found in UV-C treated groups than in the untreated control. The fruits in the high dose group were firmer and had approximately 20% higher sucrose content and 15% higher ascorbic acid content than the control, while fruits from the middle and low dose groups showed no significant changes in these parameters. The lower abscisic acid (ABA) content found in the fruits in the high UV-C group may be associated with those quality changes. The citric acid content decreased only in the low dose group (reduction of 5.8%), with a concomitant 37% reduction in jasmonic acid (JA) content, compared to the control. The antioxidant status of fruits that received preharvest UV-C treatment was considered enhanced based on their oxygen radical absorbance capacity (ORAC) and malondialdehyde (MDA) content. In terms of aroma, three volatile alcohols differed significantly among the various treatments with obvious activation of alcohol acyltransferase (AAT) activity. The observed synchronous influence on physiological indexes and related phytohormones suggests that preharvest UV-C might affect fruit quality via the action of plant hormones. Crown Copyright © 2017. Published by Elsevier Masson SAS. All rights reserved.

  14. Effectiveness of human spermatozoa biomarkers as indicators of structural damage during cryopreservation.

    Science.gov (United States)

    Gómez-Torres, María José; Medrano, Llanos; Romero, Alejandro; Fernández-Colom, Pedro José; Aizpurúa, Jon

    2017-10-01

    Human spermatozoa cryopreservation techniques are used to maintain and protect male fertility in cases such as infertility and malignancy treatments. However, during cryopreservation, the spermatozoa's metabolic rate is reduced and they undergo dramatic functional and structural changes owing to exposure to cryoprotectants and freezing-thawing procedures. While the effects of cryopreservation on cells are documented, to date the induced cryodamage on structural and/or functional sperm biomarkers is not well established at multivariate scale. To address this question, we performed basic sperm analysis, sperm DNA fragmentation assessment, spontaneous acrosome reaction measurement, and cytoskeleton evaluation after thawing samples from subjects with normal and low-quality semen. A cryodamage rate was used to determine the effects of the freeze-thaw process on spermatozoa. In addition, a Principal Component Analysis (PCA) was used for data reduction and to evaluate sperm-specific patterns during the cryopreservation process. We found that the vitality, progressive motility and sperm count from low-quality samples after cryopreservation show higher damage rates (≥40%) than in normal sperm samples. However, cytoskeleton, DNA, tail and mid-piece and acrosome display the highest cryodamage rates (∼50-99%) and are equally susceptible to cryopreservation-induced damage in both low- and normal-quality semen samples. Overall, the evaluation of these parameters provides meaningful information about different aspects of sperm functionality after cryopreservation. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Cryopreservation at -75 °C of Agaricus subrufescens on wheat grains with sucrose

    Directory of Open Access Journals (Sweden)

    Lienine Luiz Zaghi Júnior

    Full Text Available Abstract Agaricus subrufescens is a basidiomycete which is studied because of its medicinal and gastronomic importance; however, less attention has been paid to its preservation. This study aimed to evaluate the effect of sucrose addition to substrate and cryotube on the viability of Agaricus subrufescens cryopreserved at -20 °C and at -75 °C for one and two years. Zero, 10% or 20% sucrose was added to potato dextrose agar or wheat grain. The mycelia were cryopreserved in the absence of cryoprotectant or with sucrose solutions at 15%, 30% or 45%. After one or two years at -75 °C or at -20 °C, mycelia were thawed and evaluated about viability, initial time of growth, colony diameter and genomic stability. Cryopreservation at -20 °C is not effective to keep mycelial viability of this fungus. Cryopreservation at -75 °C is effective when sucrose is used in substrates and/or cryotubes. Without sucrose, cryopreservation at -75 °C is effective only when wheat grains are used. Physiological characteristic as mycelial colony diameter is negatively affected when potato dextrose agar is used and unaffected when wheat grain is used after two-year cryopreservation at -75 °C. The fungus genome does not show alteration after two-year cryopreservation at -75 °C.

  16. Cryopreservation of somatic embryogenic cultures of Pinus pinaster: effects on regrowth and embryo maturation.

    Science.gov (United States)

    Álvarez, José M; Cortizo, Millán; Ordás, Ricardo J

    2012-01-01

    Pinus pinaster is one of the most economically important conifers in the world. Somatic embryogenesis is a powerful tool in breeding programmes because it allows the generation of a great number of different clonal lines from seeds of superior genotypes. Unfortunately, embryogenic competence decreases with the age of cultures. Therefore, it is necessary to have a cryopreservation protocol that ensures a continuous supply of juvenile mass while allowing good maturation and conversion rates into vigorously growing plants. In this work we studied the influence of several cryopreservation parameters, such as cryoprotectant solution and pre-cooling temperature, on embryogenic culture regrowth and embryo maturation. Recovery of rewarmed samples after cryopreservation in a -150 degree C freezer depended on the cooling temperature reached prior to plunging the tubes into liquid nitrogen. As a result, we present an optimised cryopreservation protocol that ensures high recovery and embryo maturation rates. The protocol presented is a simple and fast alternative and enabled successful cryopreservation and recovery of 100 percent of the lines tested. Cryopreserved lines presented the same maturation rates as non-cryopreserved controls.

  17. Protective effects of steroidal alkaloids isolated from Solanum paniculatum L. against mitomycin cytotoxic and genotoxic actions

    Directory of Open Access Journals (Sweden)

    PABLINE M. VIEIRA

    2013-06-01

    Full Text Available Solanum paniculatum L. is a plant species widespread throughout tropical America, especially in the Brazilian Cerrado region. It is used in Brazil for culinary purposes and in folk medicine to treat liver and gastric dysfunctions, as well as hangovers. Previous studies with S. paniculatum ethanolic leaf extract or ethanolic fruit extract demonstrated that they have no genotoxic activity neither in mice nor in bacterial strains, although their cytotoxicity and antigenotoxicity were demonstrated in higher doses. In order to assess the possible compounds responsible for the activities observed, we fractionated the ethanolic fruit extract of S. paniculatum, characterized by 1H and 13C NMR spectra, and evaluated two fractions containing steroidal alkaloids against mitomycin C (MMC using the mouse bone marrow micronucleus test. Swiss mice were orally treated with different concentrations (25, 50, or 100 mg.kg−1 of each fraction simultaneously with a single intraperitonial dose of MMC (4 mg.kg−1. Antigenotoxicity was evaluated by using the frequency of micronucleated polychromatic erythrocytes (MNPCE, whereas anticytotoxicity was assessed by the polychromatic and normochromatic erythrocytes ratio (PCE/NCE. Our results demonstrated that steroidal alkaloids isolated from S. paniculatum strongly protected cells against MMC aneugenic and/or clastogenic activities as well as modulated MMC cytotoxic action.

  18. Modulation of mitomycin C-induced genotoxicity by acetyl- and thio- analogues of salicylic acid.

    Science.gov (United States)

    Pawar, Amol Ashok; Vikram, Ajit; Tripathi, Durga Nand; Padmanabhan, Shweta; Ramarao, Poduri; Jena, Gopabandhu

    2009-01-01

    Recent reports regarding acetylsalicylic acid (ASA) and its metabolites suggest suppressive effects against mitomycin C (MMC)-induced genotoxicity in a mice chromosomal aberration assay. Keeping this in mind, the potential anti-genotoxic effect of the thio-analogue of salicylic acid namely thio-salicylic acid (TSA) was speculated upon. The present study investigated and compared the anti-genotoxic potential of ASA and TSA. The study was performed in male swiss mice (20+/-2 g) using single-cell gel electrophoresis and a peripheral blood micronucleus assay. ASA and TSA (5, 10 or 20 mg/kg) were administered 15 minutes after MMC (1 mg/kg) once daily for 3 or 7 days. Both ASA and TSA significantly decreased the DNA damage induced by MMC as indicated by a decrease in the comet parameters in bone marrow cells and decreased frequencies of micronucleated reticulocytes in peripheral blood. The results clearly demonstrate the anti-genotoxic potential of ASA and TSA.

  19. p53-Independent thermosensitization by mitomycin C in human non-small cell lung carcinoma cells

    International Nuclear Information System (INIS)

    Jin, Z.-H.; Matsumoto, H.; Hayashi, S.; Shioura, H.; Kitai, R.; Kano, E.; Hatashita, M.

    2003-01-01

    The combined treatment with hyperthermia and chemotherapeutic drugs such as cisplatin (CDDP), doxorubicin (DOX) and mitomycin C (MMC) has been widely adopted as a strategy of interdisciplinary cancer therapy to obtain greater therapeutic benefits. However, the involved mechanisms of the interactive cytotoxic effects of hyperthermia and MMC remain unclear. To elucidate the relationship between p53 functions and the interactive effects of the combined treatment with mild-hyperthermia and MMC, we examined the potentiation of cytotoxic effects, the induction of apoptosis, the changes in cell cycles and the accumulation of Hsp72 after the combined treatment with hyperthermia at 42 degree C and MMC using human non-small cell lung carcinoma H1299 transfectants with either null, wild-type (wt) or mutant (m) p53 gene. H1299/null, H1299/wtp53 and H1299/mp53 cells showed similar sensitivities to either hyperthermia at 42 degree C alone or MMC alone. The combined treatment resulted in a synergistically enhanced cytotoxicity in H1299 transfectants in a p53-independent manner. The mechanisms involved an enhancement of heat-induced apoptosis and a modulation of the cell cycle distribution by the combined treatment. The accumulation of Hsp72 was not suppressed by the combined treatment, as is not the case of the combined treatment with hyperthermia and either CDDP (1) or bleomycin (2). Our findings demonstrate a p53-independent mechanism for a synergistically cytotoxic enhancement by the combined treatment with mild-hyperthermia and MMC

  20. Prophylactic Effects of Mitomycin-C on Regression and Haze Formation in Photorefractive Keratectomy

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    Hassan Hashemi

    2008-12-01

    Full Text Available

    PURPOSE: To study the effect of prophylactic application of mitomycin-C on regression and corneal haze formation after photorefractive keratectomy (PRK for high myopia. METHODS: Fifty-four eyes of 28 high myopic patients were enrolled in this prospective study. All eyes underwent PRK with application of 0.02% mitomycin-C for two minutes and irrigation with 15-20 ml of normal saline. Follow-up visits were scheduled for the first 7 days and 1, 3 and 6 months after surgery. Hanna grading (in the scale of 0 to 4+ was used to assess corneal haze. RESULTS: Mean spherical equivalent refraction (SE was -7.08 ± 1.11 diopters (D, preoperatively. All eyes were examined on the first 7 days and one month after surgery; 48 eyes (88.9% were evaluated 3 and 6 months post-surgery. Six months after surgery, all eyes had uncorrected visual acuity (UCVA of 20/40 or better and 37 eyes (77.1 % achieved UCVA of 20/20 or better, 45 eyes (93.7% had SE within ±1.00D of emmetropia. One month postoperatively, 2 eyes (3.7% had grade 0.5 haze, while at 3 and 6 months after surgery no visited eye had haze at all. There was no decrease in best corrected visual acuity after 6 months. In spatial frequencies of 6 and 12 cycle/degree, contrast sensitivity decreased immediately after PRK but increased to the preoperative values by the 6th postoperative month

  1. Excimer laser phototherapeutic keratectomy in conjunction with mitomycin C in corneal macular and granular dystrophies

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    Erdem Yuksel

    2016-04-01

    Full Text Available ABSTRACT Purpose: To evaluate the visual outcomes, recurrence patterns, safety, and efficacy of excimer laser phototherapeutic keratectomy (PTK in conjunction with mitomycin C (MMC for corneal macular and granular diystrophies. Methods: The patients were divided into two groups. Group 1 included patients with macular corneal dystrophy (MCD that caused superficial corneal plaque opacities, and Group 2 included patients with granular corneal dystrophy (GCD. Patients in both groups were pre-, peri-, and postoperatively evaluated. The groups were compared in terms of uncorrected visual acuity (VA, best spectacle-corrected VA, presence of mild or significant recurrence, and time of recurrence. Results: Eighteen eyes (nine with MCD and nine with GCD of 18 patients (10 men and eight women were included. PTK was performed for each eye that was included in this study. The mean ablation amount was 117.8 ± 24.4 µm and 83.5 ± 45.7 µm in MCD and GCD, respectively, (p=0.18. The postoperative improvement of the mean VA was similar between the two groups before recurrences (p>0.43 and after recurrences (p>0.71. There were no statistically significant differences in the recurrence rate and the recurrence-free period for any recurrence type. Conclusion: PTK was an effective, safe, and minimally invasive procedure for patients with MCD and GCD. PTK in conjunction with MMC was similarly effective for both groups in terms of recurrence and visual outcomes.

  2. Excimer laser phototherapeutic keratectomy in conjunction with mitomycin C in corneal macular and granular dystrophies.

    Science.gov (United States)

    Yuksel, Erdem; Cubuk, Mehmet Ozgur; Eroglu, Hulya Yazıcı; Bilgihan, Kamil

    2016-04-01

    To evaluate the visual outcomes, recurrence patterns, safety, and efficacy of excimer laser phototherapeutic keratectomy (PTK) in conjunction with mitomycin C (MMC) for corneal macular and granular diystrophies. The patients were divided into two groups. Group 1 included patients with macular corneal dystrophy (MCD) that caused superficial corneal plaque opacities, and Group 2 included patients with granular corneal dystrophy (GCD). Patients in both groups were pre-, peri-, and postoperatively evaluated. The groups were compared in terms of uncorrected visual acuity (VA), best spectacle-corrected VA, presence of mild or significant recurrence, and time of recurrence. Eighteen eyes (nine with MCD and nine with GCD) of 18 patients (10 men and eight women) were included. PTK was performed for each eye that was included in this study. The mean ablation amount was 117.8 ± 24.4 µm and 83.5 ± 45.7 µm in MCD and GCD, respectively, (p=0.18). The postoperative improvement of the mean VA was similar between the two groups before recurrences (p>0.43) and after recurrences (p>0.71). There were no statistically significant differences in the recurrence rate and the recurrence-free period for any recurrence type. PTK was an effective, safe, and minimally invasive procedure for patients with MCD and GCD. PTK in conjunction with MMC was similarly effective for both groups in terms of recurrence and visual outcomes.

  3. Studies on the anticlastogenic effect of turmeric and curcumin on cyclophosphamide and mitomycin C in vivo.

    Science.gov (United States)

    Mukhopadhyay, M J; Saha, A; Mukherjee, A

    1998-01-01

    Turmeric and its main constituent curcumin were assessed in vivo for their anticlastogenic potential. In one experimental set, Swiss albino male mice were given turmeric (8, 12 and 16 mg/kg body weight) or curcumin (2, 4 and 8 mg/kg body weight) as a single intraperitoneal injection. In another set, the mice were given 8 mg/kg body weight of turmeric or one of three concentrations of curcumin (2, 4 and 8 mg/kg body weight) as a dietary supplement by gavage for 7 consecutive days. 30 min after the last dose the mice were administered a single acute dose of two known clastogens, cyclophosphamide (CP) (20 mg/kg body weight) or mitomycin C (MMC) (1.5 mg/kg body weight). After 18 hr, chromosome preparations were made from bone marrow cells. The endpoints studied were chromosome aberrations and damaged cells. Clastogenicity of the chemicals was compared using turmeric- or curcumin-primed and non-primed animals. As single agents turmeric and curcumin were not clastogenic even after 7 days of priming. Turmeric/curcumin could not inhibit CP- or MMC-induced clastogenicity. Although curcumin is reported to be the active chemopreventive principle in turmeric effective against a number of potential carcinogens in several experimental systems, it was virtually ineffective against the clastogenicity of CP or MMC at the doses tested.

  4. Genome-Wide Mutational Signature of the Chemotherapeutic Agent Mitomycin C in Caenorhabditis elegans.

    Science.gov (United States)

    Tam, Annie S; Chu, Jeffrey S C; Rose, Ann M

    2015-11-12

    Cancer therapy largely depends on chemotherapeutic agents that generate DNA lesions. However, our understanding of the nature of the resulting lesions as well as the mutational profiles of these chemotherapeutic agents is limited. Among these lesions, DNA interstrand crosslinks are among the more toxic types of DNA damage. Here, we have characterized the mutational spectrum of the commonly used DNA interstrand crosslinking agent mitomycin C (MMC). Using a combination of genetic mapping, whole genome sequencing, and genomic analysis, we have identified and confirmed several genomic lesions linked to MMC-induced DNA damage in Caenorhabditis elegans. Our data indicate that MMC predominantly causes deletions, with a 5'-CpG-3' sequence context prevalent in the deleted regions of DNA. Furthermore, we identified microhomology flanking the deletion junctions, indicative of DNA repair via nonhomologous end joining. Based on these results, we propose a general repair mechanism that is likely to be involved in the biological response to this highly toxic agent. In conclusion, the systematic study we have described provides insight into potential sequence specificity of MMC with DNA. Copyright © 2016 Tam et al.

  5. Genome-Wide Mutational Signature of the Chemotherapeutic Agent Mitomycin C in Caenorhabditis elegans

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    Annie S. Tam

    2016-01-01

    Full Text Available Cancer therapy largely depends on chemotherapeutic agents that generate DNA lesions. However, our understanding of the nature of the resulting lesions as well as the mutational profiles of these chemotherapeutic agents is limited. Among these lesions, DNA interstrand crosslinks are among the more toxic types of DNA damage. Here, we have characterized the mutational spectrum of the commonly used DNA interstrand crosslinking agent mitomycin C (MMC. Using a combination of genetic mapping, whole genome sequencing, and genomic analysis, we have identified and confirmed several genomic lesions linked to MMC-induced DNA damage in Caenorhabditis elegans. Our data indicate that MMC predominantly causes deletions, with a 5′-CpG-3′ sequence context prevalent in the deleted regions of DNA. Furthermore, we identified microhomology flanking the deletion junctions, indicative of DNA repair via nonhomologous end joining. Based on these results, we propose a general repair mechanism that is likely to be involved in the biological response to this highly toxic agent. In conclusion, the systematic study we have described provides insight into potential sequence specificity of MMC with DNA.

  6. Protective Effect of Melatonin Against Mitomycin C-Induced Genotoxic Damage in Peripheral Blood of Rats

    Directory of Open Access Journals (Sweden)

    S. Ortega-Gutiérrez

    2009-01-01

    Full Text Available Mitomycin C (MMC generates free radicals when metabolized. We investigated the effect of melatonin against MMC-induced genotoxicity in polychromatic erythrocytes and MMC-induced lipid peroxidation in brain and liver homogenates. Rats (N = 36 were classified into 4 groups: control, melatonin, MMC, and MMC + melatonin. Melatonin and MMC doses of 10 mg/kg and 2 mg/kg, respectively, were injected intraperitoneally. Peripheral blood samples were collected at 0, 24, 48, 72, and 96 hours posttreatment and homogenates were obtained at 96 hours posttreatment. The number of micronucleated polychromatic erythrocytes (MN-PCE per 1000 PCE was used as a genotoxic marker. Malondialdehyde (MDA plus 4-hydroxyalkenal (4-HDA levels were used as an index of lipid peroxidation. The MMC group showed a significant increase in MN-PCE at 24, 48, 72, and 96 hours that was significantly reduced with melatonin begin coadministrated. No significant differences were found in lipid peroxidation. Our results indicate that MMC-induced genotoxicity can be reduced by melatonin.

  7. Mitomycin-C in dacryocystorhinostomy: From experimentation to implementation and the road ahead: A review

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    Akshay Gopinathan Nair

    2015-01-01

    Full Text Available Dacryocystorhinostomy (DCR is the procedure of choice in patients with epiphora due to primary acquired nasolacrimal duct obstruction. The evolution of surgical tools, fiber-optic endoscopes, effective anesthesia techniques, and the adjunct use of antimetabolites intraoperatively; namely mitomycin-C (MMC have significantly contributed to the advancement of DCR surgery. MMC is a systemic chemotherapeutic agent derived from Streptomyces caespitosus that inhibits the synthesis of DNA, cellular RNA, and protein by inhibiting the synthesis of collagen by fibroblasts. Even the cellular changes in the human nasal mucosal fibroblasts induced by MMC at an ultrastructural level have been documented. There, however, seems to be a lack of consensus regarding MMC: The dosage, the route of delivery/application, the time of exposure and subsequently what role each of these variables plays in the final outcome of the surgery. In this review, an attempt is made to objectively examine all the evidence regarding the role of MMC in DCR. MMC appears to improve the success rate of DCR.

  8. Mitomycin C-augmented trabeculectomy: subtenon injection versus soaked sponges: a randomised clinical trial.

    Science.gov (United States)

    Pakravan, Mohammad; Esfandiari, Hamed; Yazdani, Shahin; Douzandeh, Azadeh; Amouhashemi, Nassim; Yaseri, Mehdi; Pakravan, Parto

    2017-09-01

    To compare the efficacy and safety of subtenon injection of mitomycin C (MMC) with that of conventional application of MMC-soaked sponges in trabeculectomy. In this multicentre randomised clinical trial, 80 consecutive open-angle glaucoma cases were randomised into two groups; group 1 received a subtenon injection of 0.1 mL of 0.01% MMC, while group 2 received 0.02% MMC-soaked sponges. Primary outcome measure was intraocular pressure (IOP), and secondary outcome measures were endothelial cell count (ECC) changes and bleb morphology according to the Indiana Bleb Appearance Grading Scale. Outcome measures were compared at 1, 3 and 6 months postoperatively. Complete and qualified success was defined as IOP within 6-15 mm Hg without and with medications at month 6, respectively. Mean preoperative IOP was 21.8±5.1 in group 1, which reduced to 10.3±3.7 mm Hg at final visit (pinjection of MMC is a safe and effective alternative to the conventional soaked sponge method. This method produces more favourable bleb morphology after trabeculectomy. NCT02385370, Post-results. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  9. Hydrostatic pressure enhances mitomycin C induced apoptosis in urothelial carcinoma cells.

    Science.gov (United States)

    Chen, Shao-Kuan; Chung, Chih-Ang; Cheng, Yu-Che; Huang, Chi-Jung; Ruaan, Ruoh-Chyu; Chen, Wen-Yih; Li, Chuan; Tsao, Chia-Wen; Hu, Wei-Wen; Chien, Chih-Cheng

    2014-01-01

    Urothelial carcinoma (UC) of the bladder is the second most common cancer of the genitourinary system. Clinical UC treatment usually involves transurethral resection of the bladder tumor followed by adjuvant intravesical immunotherapy or chemotherapy to prevent recurrence. Intravesical chemotherapy induces fewer side effects than immunotherapy but is less effective at preventing tumor recurrence. Improvement to intravesical chemotherapy is, therefore, needed. Cellular effects of mitomycin C (MMC) and hydrostatic pressure on UC BFTC905 cells were assessed. The viability of the UC cells was determined using cellular proliferation assay. Changes in apoptotic function were evaluated by caspase 3/7 activities, expression of FasL, and loss of mitochondrial membrane potential. Reduced cell viability was associated with increasing hydrostatic pressure. Caspase 3/7 activities were increased following treatment of the UC cells with MMC or hydrostatic pressure. In combination with 10 kPa hydrostatic pressure, MMC treatment induced increasing FasL expression. The mitochondria of UC cells displayed increasingly impaired membrane potentials following a combined treatment with 10 μg/ml MMC and 10 kPa hydrostatic pressure. Both MMC and hydrostatic pressure can induce apoptosis in UC cells through an extrinsic pathway. Hydrostatic pressure specifically increases MMC-induced apoptosis and might minimize the side effects of the chemotherapy by reducing the concentration of the chemical agent. This study provides a new and alternative approach for treatment of patients with UC following transurethral resection of the bladder tumor. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Cryopreserved irradiated tracheal homograft reconstruction for subglottic-tracheal stenosis

    International Nuclear Information System (INIS)

    Somyos Kunachak; Yongyudh Vajaradul; Boonchu Kulapaditharom

    1999-01-01

    Subglottic-tracheal stenosis is a common clinical entity. Handling on severe case is often problematic. Various tracheal replacement techniques have been used with varying degree of success and dispute. In this study we worked on cryopreserved irradiated tracheal homograft, of which its use in human has not been reported. The tracheas were harvested from donor cadavers within 24 hours of death in a sterile condition. After 1-2 weeks of preservation at -70 degree C, the grafts were irradiated at 25 kGy, then stored at -70 degree C until used. Four patients, 2 males and 2 females (aged 2-40 years, mean 16 years) with severe subglottic-tracheal stenosis underwent segmental tracheal graft reconstruction using this graft. Immunosuppressant was not given in any patient. The follow up period ranged from 11-1 5 months. Three patients were successfully decapulated, 1 patient developed local infection and dislodgement of intraluminal stent with subsequent restenosis. Postoperative tracheal lumen appeared near normal with histologic evidence of normal respiratory epithelium at the grafted site. In conclusion, cryopreserved irradiated tracheal homograft is a valuable alternative for tracheal transplant or reconstruction, without the need of immunosuppression

  11. Fertility in cancer patients after cryopreservation of one ovary.

    Science.gov (United States)

    Schmidt, K T; Nyboe Andersen, A; Greve, T; Ernst, E; Loft, A; Yding Andersen, C

    2013-03-01

    This questionnaire study describes the fertility and ovarian function in 143 adult female cancer survivors with only one ovary due to cryopreservation of the other. The women were asked about their ovarian function (as defined by the presence of a spontaneous menstrual cycle), pregnancies and their outcome. The mean follow-up time was 58months after cryopreservation (range 24-129months). The risk of premature ovarian failure was high in the group of patients with leukaemia (13/15; 87%) but low in the breast cancer group (5/54; 9%). Fifty-seven women had actively tried to become pregnant after end of treatment; of these, 41 women obtained a total of 68 pregnancies resulting in 45 live births and five ongoing pregnancies, 15 spontaneous abortions, one ectopic pregnancy and two elective abortions. In the remaining 86 women without a pregnancy wish, there had been five elective abortions. Ninety-three per cent of the pregnancies were after natural conception and only four cases were a result of fertility treatment. The overall risk of premature ovarian failure was low (22%). Patients who retain their ovarian function after treatment of a malignant disease have a good chance of becoming pregnant. Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  12. NEW BIOTECHNOLOGICAL METHODS FOR CRYOPRESERVATION OF REPRODUCTIVE CELLS OF STURGEON

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    E. N. Ponomareva

    2016-01-01

    Full Text Available The aim of the research is to increase the survivability of reproductive cells of sturgeon at cryopreservation and developing reliable technology suitable for use on an industrial scale.Methods. We have used standard methods of freezing, thawing reproductive cells, fertilization and incubation of eggs and larval rearing of sturgeon. Fundamentally new is cryoprotective composition: for sperm we have adjusted the composition of cryoprotective medium (for beluga 3% of dimethyl sulfoxide, for Russian sturgeon 4% of dimethyl sulfoxide; for freezing the eggs we have used cryoprotective mixture of unrefined vegetable and animal oils.Results. Survivability of defrosted sperm sturgeon has been increased: for Beluga it is up to 20%, for Russian sturgeon - 47%. At insemination of cryopreserved eggs of Russian sturgeon with native sperm the fertilization rate has made 41%.Main conclusions. The research proves the effectiveness of reducing the toxic effect of cryoprotective substances, thus leading to increased survivability of reproductive cells of sturgeon. During the insemination of eggs, stored in liquid nitrogen, the resulting offspring were viable and by the reactivity of the central nervous system and receptor complex it does not differ from the young obtained by conventional technology.

  13. Predictors of sperm recovery after cryopreservation in testicular cancer

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    James M Hotaling

    2016-01-01

    Full Text Available Our objective was to identify predictors of improved postthaw semen quality in men with testicular cancer banking sperm for fertility preservation. We reviewed 173 individual semen samples provided by 67 men with testicular germ cell tumor (TGCT who cryopreserved sperm before gonadotoxic treatment between 1994 and 2010 at our tertiary university medical center. Our main outcomes measures were independent predictors for the greater postthaw total motile count (TMC in men with TGCT. Men with NSGCT were more likely to be younger (P median fresh TMC each had increased odds of a postthaw TMC greater than median postthaw TMC. Interestingly, age, advanced cancer stage (II or III, rapid freezing protocol, and motility enhancer did not show increased odds of improved postthaw TMC in our models. In conclusion, men with TGCT or poor fresh TMC should consider preserving additional vials (at least 15 vials before oncologic treatment. Density gradient purification should be routinely used to optimize postthaw TMC in men with TGCT. Larger, randomized studies evaluating cancer stage and various cryopreservation techniques are needed to assist in counseling men with TGCT regarding fertility preservation and optimizing cryosurvival.

  14. Evaluation of amides and centrifugation temperature in boar semen cryopreservation.

    Science.gov (United States)

    Bianchi, I; Calderam, K; Maschio, E F; Madeira, E M; da Rosa Ulguim, R; Corcini, C D; Bongalhardo, D C; Corrêa, E K; Lucia, T; Deschamps, J C; Corrêa, M N

    2008-03-15

    Two experiments were conducted to evaluate the use of amides as cryoprotectants and two centrifugation temperatures (15 or 24 degrees C) in boar semen cryopreservation protocols. Semen was diluted in BTS, cooled centrifuged, added to cooling extenders, followed by the addition of various cryoprotectants. In experiment 1, mean (+/-S.E.M.) sperm motility for 5% dimethylformamide (DMF; 50.6+/-1.9%) and 5% dimethylacetamide (DMA; 53.8+/-1.7%) were superior (P0.05). In experiment 2, we tested MF, DMF, and DMA at 3, 5, and 7%. Sperm motility and membrane integrity were higher for 5% DMA (53.8+/-1.7 and 50.9+/-1.9%) and 5% DMF (50.6+/-1.9 and 47.9+/-2.1%), in comparison with 7% DMF and all MF concentrations (P0.05). In conclusion, boar semen was successfully cryopreserved by replacement of glycerol with amides (especially 5% DMA) and centrifugation at 15 degrees C, with benefits for post-thaw sperm motility and membrane integrity.

  15. Data on antioxidant activity in grapevine (Vitis vinifera L.) following cryopreservation by vitrification.

    Science.gov (United States)

    Lazo-Javalera, María Fernanda; Tiznado-Hernández, Martín Ernesto; Vargas-Arispuro, Irasema; Valenzuela-Soto, Elisa; Rocha-Granados, María Del Carmen; Martínez-Montero, Marcos Edel; Rivera-Domínguez, Marisela

    2015-12-01

    Cryopreservation is used for the long-term conservation of plant genetic resources. This technique very often induces lethal injury or tissue damage. In this study, we measured indicators of viability and cell damage following cryopreservation and vitrification-cryopreservation in Vitis vinifera L. axillary buds cv. "Flame seedless" stored in liquid nitrogen (LN) for: three seconds, one hour, one day, one week and one month; after LN thawed at 38 °C for three minutes. The enzymatic activity of catalase (CAT) and superoxide dismutase (SOD), as well as the amount of malondialdehyde (MDA), total protein and viability were assayed.

  16. Serum-Free Cryopreservation of Five Mammalian Cell Lines in Either a Pelleted or Suspended State

    Directory of Open Access Journals (Sweden)

    Corsini Joe

    2004-01-01

    Full Text Available Herein we have explored two practical aspects of cryopreserving cultured mammalian cells during routine laboratory maintenance. First, we have examined the possibility of using a serum-free, hence more affordable, cryopreservative. Using five mammalian lines (Crandell Feline Kidney, MCF7, A72, WI 38 and NB324K, we found that the serum-free alternative preserves nearly as efficiently as the serum-containing preservatives. Second, we compared cryostorage of those cells in suspended versus a pellet form using both aforementioned cryopreservatives. Under our conditions, cells were in general recovered equally well in a suspended versus a pellet form.

  17. Data on antioxidant activity in grapevine (Vitis vinifera L. following cryopreservation by vitrification

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    María Fernanda Lazo-Javalera

    2015-12-01

    Full Text Available Cryopreservation is used for the long-term conservation of plant genetic resources. This technique very often induces lethal injury or tissue damage. In this study, we measured indicators of viability and cell damage following cryopreservation and vitrification-cryopreservation in Vitis vinifera L. axillary buds cv. “Flame seedless” stored in liquid nitrogen (LN for: three seconds, one hour, one day, one week and one month; after LN thawed at 38 °C for three minutes. The enzymatic activity of catalase (CAT and superoxide dismutase (SOD, as well as the amount of malondialdehyde (MDA, total protein and viability were assayed.

  18. Expanded cryopreserved mesenchymal stromal cells as an optimal source for graft-versus-host disease treatment

    Czech Academy of Sciences Publication Activity Database

    Holubová, M.; Lysák, D.; Vlas, T.; Vannucci, Luca; Jindra, P.

    2014-01-01

    Roč. 42, č. 3 (2014), s. 139-144 ISSN 1045-1056 Institutional support: RVO:61388971 Keywords : Mesenchymal stromal cells * Cryopreservation * Immunomodulation Subject RIV: EC - Immunology Impact factor: 1.209, year: 2014

  19. Improved droplet-vitrification and histological studies of cryopreserved shoot tips of cultivated Jerusalem artichoke genotypes

    Science.gov (United States)

    Germplasm conservation of Jerusalem artichoke (Helianthus tuberosus L.) is crucial to preserve genetic diversity and to secure materials for genetic improvement. Long-term conservation is accomplished through cryopreservation, storing cells or tissues at an ultralow temperature in liquid nitrogen (-...

  20. Cryopreservation of common carp (Cyprinus carpio L.) sperm induces protein phosphorylation in tyrosine and threonine residues

    Czech Academy of Sciences Publication Activity Database

    Li, P.; Hulák, M.; Li, Z.; H.; Šulc, Miroslav; Pšenička, M.; Rodina, M.; Gela, D.; Linhart, O.

    2013-01-01

    Roč. 80, č. 2 (2013), s. 84-89 ISSN 0093-691X Institutional support: RVO:61388971 Keywords : Cryopreservation * Sperm * Phosphorylation Subject RIV: CE - Biochemistry Impact factor: 1.845, year: 2013

  1. Cryopreservation of sperm bundles (spermatozeugmata) from endangered livebearing goodeids.

    Science.gov (United States)

    Liu, Yue; Torres, Leticia; Tiersch, Terrence R

    2018-04-14

    More than half of fishes in the family Goodeidae are considered to be endangered, threatened, or vulnerable. Sperm cryopreservation is an effective tool for conserving genetic resources of imperiled populations, but development of protocols with livebearing fishes faces numerous challenges including the natural packaging of sperm into bundles. In this study the cryopreservation of sperm bundles (spermatozeugmata) of three goodeids species was evaluated. Sperm quality was evaluated by activation with NaCl-NaOH solution (at 300 mOsmol/kg and pH 11.8), and analysis of dissociable bundles and dissociation duration. Using Redtail Splitfin (Xenotoca eiseni) as a model, the effects of cryoprotectants (dimethyl sulfoxide, methanol, and glycerol) with different concentrations (5-15% v/v %), equilibration exposure times (1-60 min), cooling rates (5-40 °C/min), concentrations (4 × 10 4 -4 × 10 6 bundles/ml), buffers (HBSS, PBS and NaCl), and buffer osmolalities (200-400 mOsmol/kg) were investigated. After cooling and thawing, sperm bundles maintained their packed form. A specific protocol was developed (10% dimethyl sulfoxide, 20-min equilibration, 10 °C/min cooling rate, 4 × 10 6 bundles/ml, and 300 mOsmol/kg HBSS). This protocol yielded 89 ± 5% of post-thaw dissociable bundles with 209 ± 10 s of dissociation duration for X. eiseni, 96 ± 9% with 814 ± 14 s for Blackfin Goodea (Goodea atripinni), and 66 ± 2% with 726 ± 25 s for Striped Goodeid (Ataeniobius toweri). This is the first study of cryopreservation of sperm within bundles for livebearing fishes and provides a basis for establishment of germplasm repositories for goodeids and other livebearers. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Corneal melanosis successfully treated using topical mitomycin-C and alcohol corneal epitheliectomy: a 3-year follow-up case report

    Directory of Open Access Journals (Sweden)

    Mehmet Balcı

    2015-08-01

    Full Text Available ABSTRACTWe report a case of primary acquired corneal melanosis without atypia associated with corneal haze in a patient with a history of limbal malignant melanoma and the effect of mitomycin-C. A 75-year-old woman with a history of limbal malignant melanoma presented with loss of vision in right eye. Corneal examination showed a patchy melanotic pigmentation with a central haze. Topical mitomycin-C improved visual acuity and corneal haze. However, the pigmented lesions persisted, and they were removed with alcohol corneal epitheliectomy. Histopathological examination demonstrated primary acquired melanosis without atypia. The lesions were successfully removed, and there were no recurrences during the follow-up period of 36 months. The association of conjunctival and corneal melanosis without atypia is a rare condition. In addition, co-existence of central corneal haze and melanosis may decrease visual acuity. Topical mitomycin-C and alcohol corneal epitheliectomy can be useful treatments in this condition.

  3. Trehalose preincubation increases mesenchymal (CD271+ stem cells post-cryopreservation viability

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    Indra Kusuma

    2016-10-01

    Full Text Available Background: Dimethyl sulfoxide (Me2SO is a common cryoprotective agent widely used in cell preservation system. Me2SO is currently known to cause epigenetic changes which are  critical in stem cells development and cellular differentiation. Therefore, it is imperative to develop cryopreservation techniques that protect cellular functions and avert Me2SO adverse effect. Trehalose was able to protect organism in extreme condition such as dehydration and cold. This study aimed to verify the protective effect of trehalose preincubation procedure in cryopreservation.Methods: The study was conducted using experimental design. Thawed mesenchymal (CD271+ stem cells from YARSI biorepository were used for the experiment. Trehalose preincubation was performed for 1 hour, internalized trehalose was confirmed by FTIR-ATR measurement. Three groups consisted of (1 cryopreserved without trehalose preincubation, (2 cryopreserved with trehalose preincubation, and (3 did not undergo cryopreservation were evaluated after 24 hours in LN2 for viability in culture. The absorbance from each group was measured at 450 nm. The analysis performed using paired student t test.Results: Viability of thawed mesenchymal (CD271+ stem cells that undergo trehalose preincubation prior cryopreservation was significantly higher (p<0.05 compared to group without trehalose preincubation. Higher viability observed between group with trehalose preincubation compared with controlled group suggests protection to trypsinization. Mesenchymal (CD271+ stem cells incubated for 1 hour in 100 mM trehalose supplemented medium  results in 15%  trehalose loading efficiency.Conclusion: These findings confirm the protective effect of trehalose preincubation in cryopreservation. Future research should be directed to elucidate the trehalose internalization mechanism and eventually the protective mechanism of trehalose in mammalian cell cryopreservation.

  4. Augmented dried versus cryopreserved amniotic membrane as an ocular surface dressing.

    Directory of Open Access Journals (Sweden)

    Claire L Allen

    Full Text Available Dried amniotic membrane (AM can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material.AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG. Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays.Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM.Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability

  5. Cryopreservation of Embryos of the Mediterranean Fruit Fly Ceratitis capitata Vienna 8 Genetic Sexing Strain.

    Directory of Open Access Journals (Sweden)

    Antonios A Augustinos

    Full Text Available The Mediterranean fruit fly, Ceratitis capitata, is one of the most serious pests of fruit crops world-wide. During the last decades, area-wide pest management (AW-IPM approaches with a sterile insect technique (SIT component have been used to control populations of this pest in an effective and environment-friendly manner. The development of genetic sexing strains (GSS, such as the Vienna 8 strain, has been played a major role in increasing the efficacy and reducing the cost of SIT programs. However, mass rearing, extensive inbreeding, possible bottleneck phenomena and hitch-hiking effects might pose major risks for deterioration and loss of important genetic characteristics of domesticated insect. In the present study, we present a modified procedure to cryopreserve the embryos of the medfly Vienna 8 GSS based on vitrification and used this strain as insect model to assess the impact of the cryopreservation process on the genetic structure of the cryopreserved insects. Forty-eight hours old embryos, incubated at 24°C, were found to be the most suitable developmental stage for cryopreservation treatment for high production of acceptable hatch rate (38%. Our data suggest the absence of any negative impact of the cryopreservation process on egg hatch rate, pupation rates, adult emergence rates and stability of the temperature sensitive lethal (tsl character on two established cryopreserved lines (flies emerged from cryopreserved embryos, named V8-118 and V8-228. Taken together, our study provides an optimized procedure to cryopreserve the medfly Vienna 8 GSS and documents the absence of any negative impact on the genetic structure and quality of the strain. Benefits and sceneries for utilization of this technology to support operational SIT projects are discussed in this paper.

  6. Cryopreservation of Embryos of the Mediterranean Fruit Fly Ceratitis capitata Vienna 8 Genetic Sexing Strain.

    Science.gov (United States)

    Augustinos, Antonios A; Rajamohan, Arun; Kyritsis, Georgios A; Zacharopoulou, Antigone; Haq, Ihsan Ul; Targovska, Asya; Caceres, Carlos; Bourtzis, Kostas; Abd-Alla, Adly M M

    2016-01-01

    The Mediterranean fruit fly, Ceratitis capitata, is one of the most serious pests of fruit crops world-wide. During the last decades, area-wide pest management (AW-IPM) approaches with a sterile insect technique (SIT) component have been used to control populations of this pest in an effective and environment-friendly manner. The development of genetic sexing strains (GSS), such as the Vienna 8 strain, has been played a major role in increasing the efficacy and reducing the cost of SIT programs. However, mass rearing, extensive inbreeding, possible bottleneck phenomena and hitch-hiking effects might pose major risks for deterioration and loss of important genetic characteristics of domesticated insect. In the present study, we present a modified procedure to cryopreserve the embryos of the medfly Vienna 8 GSS based on vitrification and used this strain as insect model to assess the impact of the cryopreservation process on the genetic structure of the cryopreserved insects. Forty-eight hours old embryos, incubated at 24°C, were found to be the most suitable developmental stage for cryopreservation treatment for high production of acceptable hatch rate (38%). Our data suggest the absence of any negative impact of the cryopreservation process on egg hatch rate, pupation rates, adult emergence rates and stability of the temperature sensitive lethal (tsl) character on two established cryopreserved lines (flies emerged from cryopreserved embryos), named V8-118 and V8-228. Taken together, our study provides an optimized procedure to cryopreserve the medfly Vienna 8 GSS and documents the absence of any negative impact on the genetic structure and quality of the strain. Benefits and sceneries for utilization of this technology to support operational SIT projects are discussed in this paper.

  7. Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa

    OpenAIRE

    N. Prapaiwan; T. Tharasanit; S. Punjachaipornpol; D. Yamtang; A. Roongsitthichai; W. Moonarmart; K. Kaeoket; S. Manee-in

    2016-01-01

    Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. Howeve...

  8. A simple improvement of the conventional cryopreservation for human ES and iPS cells

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Midori Ozawa, Yutaka Ozawa, Masashi Iemura, Arihiro Kohara, Kana Yanagihara & Miho K Furue ### Abstract In this study, a simple method for the cryopreservation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is proposed. It is based on the conventional slow-freezing method with 10% DMSO and modified mainly in a thawing protocol without specific equipment or reagents. Recovery rate of the cells cryopreserved by this method was equally high, which is compa...

  9. Effect of mitomycin c and 5-flurouracil adjuvant therapy on the outcomes of Ahmed glaucoma valve implantation.

    Science.gov (United States)

    Cui, Qi N; Hsia, Yen C; Lin, Shan C; Stamper, Robert L; Rose-Nussbaumer, Jennifer; Mehta, Nitisha; Porco, Travis C; Naseri, Ayman; Han, Ying

    2017-03-01

    To examine the effect of mitomycin c and 5-flurouracil on treatment outcomes following Ahmed glaucoma valve implantation. Retrospective consecutive case series. Fifty patients who received Ahmed glaucoma valve implantation from 1999 to 2013 in the San Francisco Veterans Administration Hospital. The +INJECTION group received intraoperative mitomycin c followed by postoperative mitomycin c and/or 5-flurouracil, whereas the -INJECTION group did not. Primary outcome was treatment success at 1 year post-implantation. Intraocular pressure, hypertensive phase, and the number of glaucoma medications were also examined. Twenty-six patients/eyes in the +INJECTION group and 24 patients/eyes in the -INJECTION group were included. Treatment success was higher in the +INJECTION compared with the -INJECTION group (86 vs. 58%; P = 0.04). Intraocular pressure was lower in the +INJECTION compared with the -INJECTION group at 1, 3, 6 and 12 months (P ≪ 0.00001, P = 0.00003, 0.0008 and 0.024). Hypertensive phase occurred less often in the +INJECTION compared with the -INJECTION group (3.8 vs. 54%; P = 0.021). The +INJECTION group required fewer medications compared with the -INJECTION group (P = 0.02, 0.002, 0.003 and 0.008 at 1, 3, 6 and 12 months). Complication rates were comparable between groups (46.2 and 54.2%; P = 0.63). Adjuvant treatment with antifibrotics following Ahmed glaucoma valve implantation decreased the hypertensive phase and improved surgical outcomes without impacting complication rates at 1 year. This study postulates a role for antifibrotics in the postoperative management of Ahmed glaucoma valves. © 2016 Royal Australian and New Zealand College of Ophthalmologists.

  10. Efficacy of adjunctive mitomycin C in transcanalicular diode laser dacryocystorhinostomy in different age groups.

    Science.gov (United States)

    Kar, Taner; Yildirim, Yildiray; Topal, Tuncay; Çolakoğlu, Kadir; Ünal, Melih Hamdi

    2016-01-01

    To evaluate the efficacy of adjunctive mitomycin C (MMC) in transcanalicular multidiode laser dacryocystorhinostomy (TCL-DCR) in different age groups. Ninety-six eyes of 96 patients who underwent TCL-DCR for the treatment of nasolacrimal duct obstruction were included in this retrospective, comparative study. Patients were divided into 4 groups based on age and intraoperative use of MMC: group 1, TCL-DCR without MMC in the 20- to 44-year age group; group 2, TCL-DCR with MMC in the 20- to 44-year age group; group 3, TCL-DCR without MMC in the 45- to 76-year age group; group 4, TCL-DCR with MMC in the 45- to 76-year age group. The postoperative evaluation consisted of calculating and comparing the success rates between groups. Success rates at the final visit were 50% for group 1, 66.66% for group 2, 79.16% for group 3, and 84.61% for group 4. The differences between group 1 and group 4, and group 1 and group 3, were significant (p = 0.01 and p = 0.038, respectively). Logistic regression showed that age group had significant effect on success rate (p = 0.013). However, use of MMC had no significant effect on success rate (p = 0.23). The success rates of the TCL-DCR with MMC application were found to be higher than those of TCL-DCR without MMC in different age groups. However, the differences did not reach statistical significance. In addition, our study demonstrated that age may be a significant factor influencing the surgical outcome of TCL-DCR.

  11. Increasing dwell time of mitomycin C in the upper tract with a reverse thermosensitive polymer.

    Science.gov (United States)

    Wang, Agnes J; Goldsmith, Zachariah G; Neisius, Andreas; Astroza, Gaston M; Oredein-McCoy, Olugbemisola; Iqbal, Muhammad W; Simmons, W Neal; Madden, John F; Preminger, Glenn M; Inman, Brant A; Lipkin, Michael E; Ferrandino, Michael N

    2013-03-01

    Abstract Background and Purpose: Topical chemotherapy for urothelial cancer is dependent on adequate contact time of the chemotherapeutic agent with the urothelium. To date, there has not been a reliable method of maintaining this contact for renal or ureteral urothelial carcinoma. We evaluated the safety and feasibility of using a reverse thermosensitive polymer to improve dwell times of mitomycin C (MMC) in the upper tract. Using a porcine model, four animals were treated ureteroscopically with both upper urinary tracts receiving MMC mixed with iodinated contrast. One additional animal received MMC percutaneously. The treatment side had ureteral outflow blocked with a reverse thermosensitive polymer plug. MMC dwell time was monitored fluoroscopically and intrarenal pressures measured. Two animals were euthanized immediately, and three animals were euthanized 5 days afterward. In control kidneys, drainage occurred at a mean of 5.3±0.58 minutes. Intrarenal pressures stayed fairly stable: 9.7±14.0 cm H20. In treatment kidneys, dwell time was extended to 60 minutes, when the polymer was washed out. Intrarenal pressures in the treatment kidneys peaked at 75.0±14.7 cm H20 and reached steady state at 60 cm H20. Pressures normalized after washout of the polymer with cool saline. Average washout time was 11.8±9.6 minutes. No histopathologic differences were seen between the control and treatment kidneys, or with immediate compared with delayed euthanasia. A reverse thermosensitive polymer can retain MMC in the upper urinary tract and appears to be safe from our examination of intrarenal pressures and histopathology. This technique may improve the efficacy of topical chemotherapy in the management of upper tract urothelial carcinoma.

  12. Functional characterization of two SOS-regulated genes involved in mitomycin C resistance in Caulobacter crescentus.

    Science.gov (United States)

    Lopes-Kulishev, Carina O; Alves, Ingrid R; Valencia, Estela Y; Pidhirnyj, María I; Fernández-Silva, Frank S; Rodrigues, Ticiane R; Guzzo, Cristiane R; Galhardo, Rodrigo S

    2015-09-01

    The SOS response is a universal bacterial regulon involved in the cellular response to DNA damage and other forms of stress. In Caulobacter crescentus, previous work has identified a plethora of genes that are part of the SOS regulon, but the biological roles of several of them remain to be determined. In this study, we report that two genes, hereafter named mmcA and mmcB, are involved in the defense against DNA damage caused by mitomycin C (MMC), but not against lesions induced by other common DNA damaging agents, such as UVC light, methyl methanesulfonate (MMS) and hydrogen peroxide. mmcA is a conserved gene that encodes a member of the glyoxalases/dioxygenases protein family, and acts independently of known DNA repair pathways. On the other hand, epistasis analysis showed that mmcB acts in the same pathway as imuC (dnaE2), and is required specifically for MMC-induced mutagenesis, but not for that induced by UV light, suggesting a role for MmcB in translesion synthesis-dependent repair of MMC damage. We show that the lack of MMC-induced mutability in the mmcB strain is not caused by lack of proper SOS induction of the imuABC operon, involved in translesion synthesis (TLS) in C. crescentus. Based on this data and on structural analysis of a close homolog, we propose that MmcB is an endonuclease which creates substrates for ImuABC-mediated TLS patches. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Amifostine Protection Against Mitomycin-induced Chromosomal Breakage in Fanconi Anaemia Lymphocytes

    Directory of Open Access Journals (Sweden)

    Miriam T. P. Lopes

    2008-08-01

    Full Text Available Fanconi anaemia (FA is a rare genetic chromosomal instability syndrome caused by impairment of DNA repair and reactive oxygen species (ROS imbalance. This disease is also related to bone marrow failure and cancer. Treatment of these complications with radiation and alkylating agents may enhance chromosomal breakage. We have evaluated the effect of amifostine (AMF on basal and mitomycin C (MMC-induced chromosomal breakage in FA blood cells using the micronucleus assay. The basal micronuclei count was higher among FA patients than healthy subjects. Pre-treatment with AMF significantly inhibited micronucleation induced by MMC in healthy subjects (23.4 ± 4.0 – MMC vs 12.3 ± 2.9 – AMF →MMC MN/1000CB, p < 0.01, one way ANOVA as well as in FA patients (80.0 ± 5.8 – MMC vs 40.1 ± 5.8 – AMF →MMC MN/1000CB, p < 0.01, ANOVA. Release of ROS by peripheral blood mononuclear cells treated with AMF →MMC and measured by chemoluminometry showed that AMF-protection was statistically higher among FA patients than in healthy individuals. Based on these results we suggest that AMF prevents chromosomal breakage induced by MMC, probably by its antioxidant effect.

  14. The Influence of Phacoemulsification on Surgical Outcomes of Trabeculectomy with Mitomycin-C for Uveitic Glaucoma.

    Directory of Open Access Journals (Sweden)

    Asaho Nishizawa

    Full Text Available To evaluate the influence of phacoemulsification after trabeculectomy on the postoperative intraocular pressure (IOP in eyes with uveitic glaucoma (UG.Kumamoto University Hospital, Kumamoto, Japan.A retrospective cohort study.The medical records of patients with UG who had trabeculectomy with mitomycin-C (MMC were reviewed. Complete and qualified surgical failures were defined by an IOP of ≥21 mmHg (condition A, ≥18 mmHg (condition B, or ≥15 mmHg (condition C without and with glaucoma eye drops, respectively. Kaplan-Meier survival analysis, generalized by the Wilcoxon test, and the Cox proportional hazards model analysis were conducted. Post-trabeculectomy phacoemulsification was treated as a time-dependent variable. In 24 (30% of the included 80 eyes, phacoemulsification was included, and they were divided into two groups: groups I (8 eyes with phacoemulsification within 1 year after trabeculectomy and group II (16 eyes after 1 year following trabeculectomy.Multivariable Cox proportional hazards model analysis showed post-trabeculectomy phacoemulsification was a significant factor in both complete success and qualified success based upon condition C (P = 0.0432 and P = 0.0488, respectively, but not for the other conditions. Kaplan-Meier survival analyses indicated significant differences in success probabilities between groups I and group II for complete success and qualified success based upon condition C (P = 0.020 and P = 0.013, respectively. There was also a significant difference for qualified success based upon condition B (P = 0.034, while there was no significant difference for the other conditions.Post-trabeculectomy phacoemulsification, especially within 1 year, can cause poor prognosis of IOP control of UG eyes after trabeculectomy with MMC.

  15. Clinical study on photorefractive keratectomy for high myopia with mitomycin C

    Directory of Open Access Journals (Sweden)

    Hao-Jiang Yang

    2013-06-01

    Full Text Available AIM: To evaluate the efficacy, safety and stability of photorefractive keratectomy(PRKfor high myopia with 0.2g/L mitomycin C(MMC. METHODS: Totally 109 patients(201 eyesafter PRK were treated with intraoperative application of 0.2g/L MMC for 20 seconds. The recovery of cornea epithelium after surgery was regularly observed. The uncorrected visual acuity(UCVA, corrected distance visual acuity(CDVA, refraction, haze, complications and endothelial cell counts 1 month, 3, 6, 12 months after PRK were compared. RESULTS:The time of corneal epithelium recovery was 3.68±0.35 days. All eyes had a significant increase in UCVA. 12 months after surgery, 189 eyes(94%achieved UCVA better than 1.0 and 153 eyes(76%had a spherical equivalent(SEwithin±0.5D. 7 eyes(3%lost one line of CDVA. No one lost 2 or more lines of CDVA. Ninety-six percent eyes changed within±0.5D when comparing 3 month and 12 month. Postoperative endothelial cell density and coefficient of variability(CVdid not show a significant difference from preoperative measurements(P1=0.71; P2=0.83. Haze of grade 1 occurred in 12 eyes(6%and haze between grade 0.5 and 1 existed in 189 eyes(94%at 12 months. No eye developed haze over grade 2. No toxic effect and complications of MMC were found after surgery. CONCLUSION: PRK with intraoperative application of MMC for 20 seconds appears to be a safe and effective method for correction of high myopia.

  16. Photorefractive Keratectomy With Mitomycin-C for High Myopia: Three Year Follow-Up Results

    Directory of Open Access Journals (Sweden)

    Hassan Hashemi

    2017-02-01

    Full Text Available Photorefractive keratectomy (PRK is a safe and effective surgical keratorefractive technique which is done with the application of mitomycin-C (MMC in cases of high myopia to prevent the formation of corneal haze This study was conducted to evaluate 3-year visual acuity and quality outcomes of PRK-MMC in high myopia. This before-after study was conducted on 20 individuals (40 eyes with myopia more than 6.0 diopter (D. Visual acuity and quality indices were evaluated before and three years after the procedure and their stability was examined between the 1st and 3rd years. At 3 years after surgery, mean uncorrected visual acuity was 0.03±0.06 in the logarithm of minimum angle of resolution (logMAR unit which showed a significant improvement when compared to baseline (P<0.001 and means best corrected visual acuity was 0.03±0.06 logMAR, which showed no significant difference (P=0.730. Manifest refraction spherical equivalent (MRSE at 3 years (-0.12±0.2D was significantly decreased when compared to baseline (P<0.001, but it did not change significantly after the 1st year and was stable (P=0.368. Mean coma and spherical aberration 3 years postoperatively were -0.54±0.26 µm and 0.46±0.19 µm, respectively, and neither parameter showed significant differences when compared to baseline (P<0.001. No significant change was found in mesopic contrast sensitivity. The long-term results of this study showed that PRK-MMC could be regarded an effective, safe, and stable procedure in patients with myopia more than 6.0 D.

  17. Mitomycin C, ceramide, and 5-fluorouracil inhibit corneal haze and apoptosis after PRK.

    Science.gov (United States)

    Kim, Tae-im; Lee, Sun Young; Pak, Jhang Ho; Tchah, Hungwon; Kook, Michael S

    2006-01-01

    To investigate the effects of mitomycin C (MMC), ceramide, and 5-fluororacil (5-FU) on haze after photorefractive keratectomy (PRK) and exposure to ultraviolet B (UVB) radiation. The right eyes of 42 New Zealand white rabbits were treated with PRK to correct -10 diopter with a 5-mm optical zone. Sponges soaked in 0.02% MMC, 10 or 40 micromol/L ceramide, or 0.5% 5-FU were applied to the right eyes of 6 rabbits each, and a tarsorrhaphy was performed. Eight weeks after complete healing, topical 0.02% MMC or 0.5% 5-FU was applied twice daily to the right eyes of 6 rabbits that had previously received PRK but no topical medication. The control group of 6 rabbits was treated only with PRK. Three weeks after PRK, all the laser-treated eyes were exposed to 100 mJ/cm UVB radiation. Corneal haze was assessed biomicroscopically every 2 weeks using the Fantes scale. Eyes were enucleated 2, 7, and 13 weeks after PRK, and tissue specimens were stained with hematoxylin and eosin and with Apostain. Corneal haze was observed in all rabbits after PRK and was aggravated by UVB irradiation. When applied immediately after PRK, MMC induced corneal opacity and apoptosis of keratocytes, but, at later times, this reagent significantly suppressed opacity, Apostain-positive keratocytes and reactivation of keratocytes, even after UVB irradiation. In contrast, ceramide and 5-FU suppressed corneal opacity after PRK, but this effect was not sustained after UVB irradiation. MMC is a potent inhibitor of haze induced by PRK and UVB irradiation. Throughout the process of corneal wound healing, the severity of apoptosis and reactivation of keratocytes was closely correlated with haze formation.

  18. The use of mitomycin-C for respiratory papillomas: Clinical, histologic and biochemical correlation

    International Nuclear Information System (INIS)

    Hamza, Ashraf H.; Nasr, Manal M.; Deghady, Akram A.

    2005-01-01

    To assess the clinical effects of mitomycin-C (MMC) on human papilloma virus (HPV)-infected tissues of the airway. We included 10 patients with previous histologic diagnosis of recurrent respiratory papillomas (RRP) in this prospective study, conducted at the Department of Otolaryngology-Head and Neck Surgery, University of Alexandria, Egypt, between January 2000 and December 2002. Under general anesthesia, each patient underwent laser excision of all visible papillomas, followed by topical application of 1 cc of 0.5 mg/ml MMC. The procedure was repeated weekly until no visible papillomas could be microscopically detected. We histopathologically studied the obtained specimens and tested for the presence of HPV DNA using polymerase chain reaction (PCR) technique. We collected blood samples from all patients and from another 10 healthy volunteers for determination of serum interleukin-2 (IL-2) level using enzyme-linked immunosorbent assay technique. We achieved clinical remission in 8 of the patients (80%), a fact that was confirmed histopathologically and by PCR data. The mean serum IL-2 levels +/- SD was significantly lower in papilloma patients (83.6 +/- 28.83 pg/ml) than in control subjects (196.3 +/- 42.46 pg/ml) (p<0.01). Among patients with RRP, serum IL-2 levels +/-SD was lower in initial samples (83.6 +/- 28.83) than follow-up (95.7 +/- 27.98 and 112.3 +/- 33.8 and 129.4 +/- 34.04) and remission samples (154 +/- 37.84 pg/ml). Our result suggests that topical application of MMC may act adjunctively to laser surgery for RRP. (author)

  19. Identification of uvrA gene mutation sites in two mitomycin-sensitive deinococcus radiodurans strains

    International Nuclear Information System (INIS)

    Du Zeji; Kong Xianrong

    1999-01-01

    Deinococcus radiodurans (Dr) possesses a prominent ability to repair the DNA injury induced by various DNA- damaging agents including mitomycin C(MC), ultraviolet light (UV) and ionizing radiation. DNA damage resistance was restored in MC sensitive (MC s ) mutants 2621 and 3021 by transforming with DNAs of four cosmids clones derived from the gene library of strain KD8301 which showed the property of wild type phenotype to DNA-damaging agents. Gene affected by mutation (mtcA or mtcB) in both mutants was cloned and its nucleotide sequence was determined. The deduced amino acid (aa) sequence of Dr uvrA gene product consists of 1016 aa and shares homology with many bacterial UvrA proteins. The mutation sites in both mutants were identified by analyzing the polymerase chain reaction (PCR) fragments derived from the genomic DNA of the mutants. A 144-base pairs (bp) deletion including the start codon for the uvr A gene was observed in DNA of the mutant 3021, causing a defect in the gene. On the other hand, an insertion sequence (IS) element intervened in the uvrA gene of the mutant 2621, suggesting the insertional inactivation of the gene. The IS element comprise 1322-bp long, flanked by 19-bp inverted terminal repeats (ITR), and generated a 6-bp target duplication (TD). Two open reading frames (ORF) were found in the IS element. The deduced aa sequences of large and small ORF show homology to a putative transposes found in IS4 of Escherichia coli (E. coli) and to a resolvent found in IS Xc5 of Xanthomonas campestris (Xc), respectively. This is the first discovery of IS element in deino-bacteria, and the IS element was designated IS2621

  20. Adjunctive Mitomycin C or Amniotic Membrane Transplantation for Ahmed Glaucoma Valve Implantation: A Randomized Clinical Trial.

    Science.gov (United States)

    Yazdani, Shahin; Mahboobipour, Hassan; Pakravan, Mohammad; Doozandeh, Azadeh; Ghahari, Elham

    2016-05-01

    To determine whether adjunctive mitomycin C (MMC) or amniotic membrane transplantation (AMT) improve the outcomes of Ahmed glaucoma valve (AGV) implantation. This double-blind, stratified, 3-armed randomized clinical trial includes 75 eyes of 75 patients aged 7 to 75 years with refractory glaucoma. Eligible subjects underwent stratified block randomization; eyes were first stratified to surgery in the superior or inferior quadrants based on feasibility; in each subgroup, eyes were randomly assigned to the study arms using random blocks: conventional AGV implantation (group A, 25 eyes), AGV with MMC (group B, 25 eyes), and AGV with AMT (group C, 25 eyes). The 3 study groups were comparable regarding baseline characteristics and mean follow-up (P=0.288). A total of 68 patients including 23 eyes in group A, 25 eyes in group B, and 20 eyes group C completed the follow-up period and were analyzed. Intraocular pressure was lower in the MMC group only 3 weeks postoperatively (P=0.04) but comparable at other time intervals. Overall success rate was comparable in the 3 groups at 12 months (P=0.217). The number of eyes requiring medications (P=0.30), time to initiation of medications (P=0.13), and number of medications (P=0.22) were comparable. Hypertensive phase was slightly but insignificantly more common with standard surgery (82%) as compared with MMC-augmented (60%) and AMT-augmented (70%) procedures (P=0.23). Complications were comparable over 1 year (P=0.28). Although adjunctive MMC and AMT were safe during AGV implantation, they did not influence success rates or intraocular pressure outcomes. Complications, including hypertensive phase, were also comparable.

  1. Trabeculectomy With Mitomycin C or Ahmed Valve Implantation in Eyes With Uveitic Glaucoma.

    Science.gov (United States)

    Bettis, Daniel I; Morshedi, Richard G; Chaya, Craig; Goldsmith, Jason; Crandall, Alan; Zabriskie, Norm

    2015-01-01

    To report and compare the results of trabeculectomy with mitomycin C (MMC) and Ahmed valve implantation in the management of uveitic glaucoma. The records of 41 eyes of 29 patients who underwent trabeculectomy with MMC or Ahmed valve implantation for uveitic glaucoma were retrospectively reviewed. Seventeen eyes underwent trabeculectomy with MMC, and 24 eyes underwent Ahmed valve implantation. Outcomes included postoperative intraocular pressure (IOP), percent reduction from preoperative IOP, postoperative number of medications, time to failure, and complications. Mean follow-up was 21.2 months in the trabeculectomy group and 23.8 months in the valve group (P=0.06). Mean IOP was reduced from 29.2 to 18.4 mm Hg in the trabeculectomy group (31.3%), compared with a reduction from 33.4 to 15.5 mm Hg in the Ahmed valve group (42.7%, P=0.53). Postoperatively, 1.76 medications were used in the trabeculectomy group, compared with 1.83 medications in the Ahmed valve group (P=0.89). Cumulative success at 1 year was 66.7% in the trabeculectomy group, compared with 100% in the Ahmed valve group (P=0.02). Mean time to failure was 8.36 months with trabeculectomy, and 21.8 months with Ahmed valve (P=0.02). Complications in both groups were typically rare and self-limited, with recurrent inflammation being most common. Although both trabeculectomy with MMC and Ahmed valve implantation are reasonable surgical options in the management of uncontrolled uveitic glaucoma, Ahmed valve implantation was associated with higher cumulative success rate at 1 year and a longer mean time to failure.

  2. Preoperative subpterygeal injection vs intraoperative mitomycin C for pterygium removal: comparison of results and complications.

    Science.gov (United States)

    Khakshoor, Hamid; Razavi, Mohammad Etezad; Daneshvar, Ramin; Shakeri, Mohammad Taghi; Ghate, Majid Farrokh; Ghooshkhanehi, Haleh

    2010-08-01

    To evaluate and compare the recurrence rates and complications between 2 therapeutic methods for primary pterygium: subconjunctival injection of mitomycin C (MMC) 1 month before bare scleral excision and conjunctival rotational flap with intraoperative MMC use. Prospective, interventional, randomized clinical trial. setting: Institutional clinical trial in a tertiary, specialty eye hospital. study population and intervention: We included 82 eyes diagnosed with primary pterygium and randomly allocated them into 2 groups. Group A consisted of 36 eyes treated with subconjunctival injection of 0.02% MMC 1 month before bare scleral excision, and group B comprised 46 eyes that underwent conjunctival rotational flap with intraoperative 0.02% MMC for 2 minutes. Follow-up periods were at least 12 months (range, 12 to 18 months). main outcome measure: Recurrence and complication rate in each arm of study. During the 1-year follow-up, 2 cases of clinical recurrence in third and sixth month of follow-up occurred in group B (recurrence rate, 4.3%). In group A, there was no clinically significant recurrence, but 2 cases of hypovascularity and whitening of sclera at the site of pterygium excision was observed. There was no other serious complication. There was no statistically significant difference between groups for recurrence rate, mean age, sex, or pterygium area. Subconjunctival injection of MMC 0.02% (0.1 ml of 0.02% solution) 1 month before bare scleral excision is a quick, easy, and safe surgical procedure and is at least as effective as conjunctival rotational flap with intraoperative MMC for 2 minutes. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  3. Effect of mitomycin-c-assisted sutureless trabeculectomy on keratometry and axial length

    Directory of Open Access Journals (Sweden)

    Anant Sharma

    2017-01-01

    Full Text Available Aim: Glaucoma is defined as chronic, progressive optic neuropathy caused by a group of ocular conditions, which leads to damage of the optic nerve with loss of visual function. This study was conducted to find out the changes in corneal curvature, axial length and intraocular pressure. Materials and Methods: The present study was conducted in the Department of Ophthalmology, S P Medical College and Associated Group of Hospitals, Bikaner. A total of fifty cases of either age- and sex-matched controls were selected for this study. These cases were divided into two groups: Group A (sutureless trabeculectomy with mitomycin-C [MMC] and Group B (sutureless trabeculectomy without MMC. Results: The mean post-operative intraocular pressure (IOP was lowered in MMC-treated eyes when compared to control at 1½ months. The IOP reduction was greater in the MMC-treated eyes (13.25 mmHg at 1½ months as compared to control Group B (16.9 mmHg at 1½ months. Comparing results with the present study, it was found that success rate in MMC-treated eyes was noted in 23 (92% individuals, in which complete success was in 22 (88% while qualified success in 1 (4% and qualified failure in 2 (8% with mean IOP of 13.2 mmHg at 1½ months, whereas in control group, success rate was noted in 19 (76% individuals, in which complete success was in 18 (72% and qualified success in 1 (4%, while qualified failure in 6 (24% with mean IOP of 16.9 mmHg at 1½ months after operation. Conclusion: Results of this study are in favour of intraoperative application of MMC during filtration surgery, especially in cases with high-risk failure.

  4. Effects of Droplet-Vitrification Cryopreservation Based on Physiological and Antioxidant Enzyme Activities of Brassidium Shooting Star Orchid

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    Safrina Rahmah

    2015-01-01

    Full Text Available Protocorm-like bodies (PLBs of Brassidium Shooting Star orchid were successfully cryopreserved using droplet-vitrification method. Vitrification based cryopreservation protocol is comprised of preculture, osmoprotection, cryoprotection, cooling, rewarming, and growth recovery and each and every step contributes to the achievement of successful cryopreservation. In order to reveal the lethal and nonlethal damage produced by cryopreservation, histological observation, scanning electron microscopy (SEM, and biochemical analysis were carried out in both cryopreserved and noncryopreserved PLBs of Brassidium Shooting Star orchid comparing with the control PLBs stock culture. Histological and scanning electron microscopy analyses displayed structural changes in cryopreserved PLBs due to the impact of cryoinjury during exposure to liquid nitrogen. Total soluble protein significantly increased throughout the dehydration process and the highest value was achieved when PLBs were stored in liquid nitrogen. Ascorbate peroxidase (APX and catalase (CAT showed the highest enzyme activities in both dehydration and cryostorage treatments indicating that stress level of PLBs was high during these stages.

  5. Radiotherapy, combined with simultaneous chemotherapy with mitomycin C and bleomycin for inoperable head and neck cancer--preliminary report

    International Nuclear Information System (INIS)

    Smid, Lojze; Lesnicar, Hotimir; Zakotnik, Brane; Soba, Erika; Budihna, Marjan; Furlan, Ladica; Zargi, Miha; Rudolf, Zvone

    1995-01-01

    Purpose: Prospectively designed randomized clinical study was undertaken to assess the efficacy of simultaneous application of irradiation, Mitomycin C, and Bleomycin in treatment of patients with inoperable head and neck carcinoma. Methods and Materials: Between March 1991 and October 1993, 49 patients with inoperable head and neck carcinoma were randomly assigned to receive either radiation therapy alone (group A) or radiotherapy combined with simultaneous application of Mitomycin C and Bleomycin (group B). Patients in both groups were irradiated five times weekly with 2 Gy to the total dose of 66-70 Gy. Chemotherapy regimen included intramuscular application of Bleomycin 5 units twice a week, with the planned dose being 70 units and Mitomycin C 15 mg/m 2 applied intravenously after delivery of 9-10 Gy of irradiation. The application of Mitomycin C was planned to be repeated on last day of radiotherapy in the dose of 10 mg/m 2 . In attempt to enhance the effect of chemotherapeutic drugs, patients in group B received also Nicotinamide, Chlorpromazine, and Dicoumarol. Results: The difference in complete response rate between both treatment groups (24% in group A and 63% in group B) was statistically significant (p = 0.015). The difference in response rate was much more pronounced in patients with oropharyngeal carcinoma only (18% in group A compared to 81% in group B; p = 0.0003), while for all other subgroups added together, there was observed no benefit of multidrug therapy. Median follow-up was 18 months. Disease-free survival of patients in group A (9%) was significantly lower then in group B (48%) (p 0.001). The difference between both treatment groups was even greater in patients with oropharyngeal carcinoma only: disease-free survival of these patients in group B was 66%, while in group A, all recurred (p = 0.00001). Conclusion: From results of our prospective randomized study it seems that the group of patients that received multidrug treatment with

  6. Conservative treatment for late-onset bleb leaks after trabeculectomy with mitomycin C in patients with ocular surface disease

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    Sagara H

    2012-08-01

    Full Text Available Hideto Sagara,1,2 Tomohiro Iida,2,3 Kimimori Saito,4 Hiroki Noji,2 Masashi Ogasawara,2 Hiroshi Oyamada21The Marui Eye Clinic, Fukushima, 2Department of Ophthalmology, Fukushima Medical University School of Medicine, Fukushima, 3Tokyo Women's Medical University, Tokyo, 4Matuki Eye Clinic, Fukushima, JapanBackground: Sodium hyaluronate and autologous serum eye drops are used to treat ocular surface disease (OSD and are reported to prevent and treat late-onset bleb leaks following trabeculectomy with mitomycin C. In this study, we evaluated the efficacy of a combination of sodium hyaluronate and autologous serum eye drops and treatment for obstructive meibomian gland dysfunction as a therapy for late-onset bleb leaks after trabeculectomy with mitomycin C.Methods: This was a retrospective, interventional, nonsimultaneous study of 12 subjects (12 eyes of mean age of 64.3 ± 18.3 years with OSD and apparent late-onset bleb leaks following trabeculectomy with mitomycin C between 1998 and 2008. We compared patients diagnosed with leakages before July 2005, who had been treated with separate eye drop solutions containing 0.1% sodium hyaluronate, 50% autologous serum, and 0.3% ofloxacin (sodium hyaluronate and autologous serum group, n = 7, with patients diagnosed from August 2005 to December 2008, who were treated with a combination of eye drops (0.1% sodium hyaluronate, 50% autologous serum, and 0.08% levofloxacin hydrate and eyelid massage and warm compresses for obstructive meibomian gland dysfunction (combination eye drop group, n = 5.Results: Leakage was resolved in one patient (14.3% in the separately treated sodium hyaluronate and autologous serum eye drop group and in five patients (100% in the combination eye drop group (P = 0.015. The period after resolution of leakage with conservative treatment was 23 months in the one eye in the sodium hyaluronate and autologous serum group and 36–61 (mean 52.4 ± 10.1 months in the five eyes in the

  7. The use of cryopreserved sea urchin embryos (Paracentrotus lividus) in marine quality assessment.

    Science.gov (United States)

    Paredes, E; Bellas, J

    2015-06-01

    We have established for first time an ecotoxicological bioassay using cryopreserved sea urchin embryos (Paracentotus lividus) and provided a comparison to the already standardized sea urchin embryo-larval bioassay, using selected (organic and inorganic) pollutants and sediment elutriates from 4 different locations from Ria de Vigo harbour (Galicia, NW Iberian Peninsula). A cryopreservation protocol was designed in order to enable the successful cryopreservation and cryobanking of gametes and embryos to be used for marine quality assessment and ensure the accessibility to high quality reproductive material all year round, as an option to conditioning adults for out of season reproduction. The calculated EC50 using the cryopreserved blastula was 53.7 μg L(-1) for copper, 81.0 μg L(-1) for lead, 300.6 μg L(-1) for BP-3 and 300.6 μg L(-1) for 4-MBC. The sensitivity of the classic sea urchin embryo-larval bioassay was compared with the bioassay conducted with cryopreserved blastula. The results showed that the use of cryopreserved blastula bioassay allows detecting lower concentrations of pollutants in comparison with the classic bioassay. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa.

    Science.gov (United States)

    Prapaiwan, N; Tharasanit, T; Punjachaipornpol, S; Yamtang, D; Roongsitthichai, A; Moonarmart, W; Kaeoket, K; Manee-In, S

    2016-05-01

    Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05) than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05). In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa.

  9. Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa

    Directory of Open Access Journals (Sweden)

    N. Prapaiwan

    2016-05-01

    Full Text Available Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05 than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05. In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa.

  10. Sperm Cryopreservation before Testicular Cancer Treatment and Its Subsequent Utilization for the Treatment of Infertility

    Directory of Open Access Journals (Sweden)

    Jana Žáková

    2014-01-01

    Full Text Available Aims. In this study we report our results with storage of cryopreserved semen intended for preservation and subsequent infertility treatment in men with testicular cancer during the last 18 years. Methods. Cryopreserved semen of 523 men with testicular cancer was collected between October 1995 and the end of December 2012. Semen of 34 men (6.5% was used for fertilization of their partners. They underwent 57 treatment cycles with cryopreserved, fresh, and/or donor sperm. Results. A total of 557 men have decided to freeze their semen before cancer treatment. Azoospermia was diagnosed in 34 men (6.1%, and semen was cryopreserved in 532 patients. Seminoma was diagnosed in 283 men (54.1% and nonseminomatous germ cell tumors in 240 men (45.9%. 34 patients who returned for infertility treatment underwent 46 treatment cycles with cryopreserved sperm. Totally 16 pregnancies were achieved, that is, 34.8% pregnancy rate. Conclusion. The testicular cancer survivors have a good chance of fathering a child by using sperm cryopreserved prior to the oncology treatment, even when it contains only limited number of spermatozoa.

  11. Recent Advances in Boar Sperm Cryopreservation: State of the Art and Current Perspectives.

    Science.gov (United States)

    Yeste, M

    2015-07-01

    While sperm cryopreservation is the best technology to store boar semen for long-term periods, only 1% of all artificial inseminations (AI) conducted worldwide are made using frozen-thawed boar sperm. With the emergence of long-term extenders for liquid storage, the use of cryopreserved sperm in routine AI is less required. However, banks of boar semen contain cryopreserved sperm and planning inseminations in AI centres may benefit from the use of frozen-thawed semen. Therefore, there is an interest in the use of this technology to preserve boar sperm. In this regard, although the first attempts to cryopreserve boar semen date back to the seventies and this technology is still considered as optimal, some relevant improvements have been made in the last decade. After giving a general picture about boar sperm cryodamage, the present review seeks to shed light on these recent cryopreservation advances. These contributions regard to protein markers for predicting ejaculate freezability, sperm selection prior to start cryopreservation procedures, additives to freezing and thawing extenders, relevance of the AI-technique and insemination-to-ovulation interval. In conclusion, most of these progresses have allowed counteracting better boar sperm cryodamage and are thus considered as forward steps for this storage method. It is also worth noting that, despite being lower than fresh/extended semen, reproductive performance outcomes following AI with frozen-thawed boar sperm are currently acceptable. © 2015 Blackwell Verlag GmbH.

  12. Micropropagation and cryopreservation of garlic (Allium sativum L.).

    Science.gov (United States)

    Keller, E R Joachim; Senula, Angelika

    2013-01-01

    Garlic (Allium sativum L.) is a very important medicinal and spice plant. It is conventionally propagated by daughter bulbs ("cloves") and bulbils from the flower head. Micropropagation is used for speeding up the vegetative propagation mainly using the advantage to produce higher numbers of healthy plants free of viruses, which have higher yield than infected material. Using primary explants from bulbs and/or bulbils (shoot tips) or unripe inflorescence bases, in vitro cultures are initiated on MS-based media containing auxins, e.g., naphthalene acetic acid, and cytokinins, e.g., 6-γ-γ-(dimethylallylaminopurine) (2iP). Rooting is accompanying leaf formation. It does not need special culture phases. The main micropropagation methods rely on growth of already formed meristems. Long-term storage of micropropagated material, cryopreservation, is well-developed to maintain germplasm. The main method is vitrification using the cryoprotectant mixture PVS3.

  13. Cryopreservation of donkey sperm using non-permeable cryoprotectants.

    Science.gov (United States)

    Diaz-Jimenez, M; Dorado, J; Ortiz, I; Consuegra, C; Pereira, B; Gonzalez-De Cara, C A; Aguilera, R; Mari, G; Mislei, B; Love, C C; Hidalgo, M

    2018-02-01

    The aim of this study was to evaluate the effect of different concentrations of sucrose combined with bovine serum albumin (BSA), as non-permeable cryoprotectants, on donkey sperm parameters after cryopreservation, in comparison to a control extender containing glycerol. Semen from five Andalusian donkeys (n = 12) were centrifuged and resuspended with a commercial extender for equine sperm (Gent A, Minitube) adding 1% BSA and different concentrations (M, mol/l) of water-diluted sucrose: 0.05, 0.1, 0.25, 0.35 and 0.45. Thereafter, semen (n = 24) were diluted in the same base extender containing 0.25 M sucrose (S25) or glycerol (GLY, Gent B). Sperm were slowly cooled, filled in 0.5 ml straws and frozen in nitrogen vapours. Post-thaw samples were assessed for sperm motility, plasma membrane and DNA integrity and results were compared by ANOVA. In Experiment 1, sperm motility was significantly higher (P < 0.001) for S25 than the remaining treatments, and no differences were found for plasma membrane or DNA integrity. In Experiment 2, no differences were found between S25 or GLY for sperm motility and DNA integrity but plasma membrane integrity was significantly higher (P < 0.05) for S25. In conclusion, the extender with sucrose 0.25 M combined with BSA can be considered as an alternative to conventional extenders with glycerol for donkey sperm cryopreservation. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Effect of N-hydroxyurea, mitomycin C and actinomycin D on tumour formation on the leaves of Kalanchoe daigremontiana

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    Józef Koawalczyk

    2014-01-01

    Full Text Available The leaves of Kalanchoe daigremontiana wounded and infected with Agrobacterium tumefaciens were treated with single doses of inhibitors (hydroxyurea - 190, mitomycin - 0.5, actinomycin - 2 µ,g per leaf. After delaying the time' of dosage of inhibitors during five days after inoculation, changes in susceptibility of the system to antitumorous activity of analysed compounds were observed. In several hours after inoculation (period of the bacteria metabolic activity in wounds all the inhibitors prevent strongly the tumour formation. At the time between 14 and 72 hours after inoculation, including the phase of tumour induction, the system becomes sensitive to the DNA synthesis inhibitors, particularly hydroxyurea. The intensified action of actinomycin appears again only about 60 hours after inoculation and lasts till the end of experiment (the initiation of the transformed plant cell proliferation. According to the literature the antitumorous effect of inhibitors could be connected with their action on the bacteria metabolism inside the host tissue. The activities of hydroxyurea and mitomycin in the second period correspond with the intensive DNA synthesis in plant cells, which is induced by wounding. The effect of actinomycin D in 60 hours after inoculation could depend upon the inhibition of the proliferation of the transformed host cells.

  15. Predictive value of early postoperative IOP and bleb morphology in Mitomycin-C augmented trabeculectomy.

    Science.gov (United States)

    Esfandiari, Hamed; Pakravan, Mohammad; Loewen, Nils A; Yaseri, Mehdi

    2017-01-01

    Background : To determine the predictive value of postoperative bleb morphological features and intraocular pressure (IOP) on the success rate of trabeculectomy. Methods : In this prospective interventional case series, we analyzed for one year 80 consecutive primary open angle glaucoma patients who underwent mitomycin-augmented trabeculectomy. Bleb morphology was scored using the Indiana bleb appearance grading scale (IBAGS). Success was defined as IOP ≤15 mmHg at 12 months. We applied a multivariable regression analysis and determined the area under the receiver operating characteristic curve (AUC). Results : The mean age of participants was 62±12.3 years in the success and 63.2±16.3 years in the failure group (P= 0.430) with equal gender distribution (P=0.911). IOPs on day 1, 7 and 30 were similar in both (P= 0.193, 0.639, and 0.238, respectively.) The AUC of IOP at day 1, day 7 and 30 for predicting a successful outcome was 0.355, 0.452, and 0.80, respectively. The AUC for bleb morphology parameters of bleb height, extension, and vascularization, on day 14 were 0.368, 0.408, and 0.549, respectively. Values for day 30 were 0.428, 0.563, and 0.654. IOP change from day 1 to day 30 was a good predictor of failure (AUC=0.838, 95% CI: 0.704 to 0.971) with a change of more than 3 mmHg predicting failure with a sensitivity of 82.5% (95% CI: 68 to 91%) and a specificity of 87.5% (95% CI: 53 to 98%). Conclusions : IOP on day 30 had a fair to good accuracy while bleb features failed to predict success except bleb vascularity that had a poor to fair accuracy.  An IOP increase more than 3 mmHg during the first 30 days was a good predictor of failure.

  16. Mitomycin-C Trabeculectomy versus Ahmed Glaucoma Implant in Pediatric Aphakic Glaucoma

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    Mohammad Pakravan

    2008-12-01

    Full Text Available

    PURPOSE: To compare the outcomes and complications of mitomycin-C trabeculectomy (MMC-T versus the Ahmed glaucoma implant (AGI for treatment of pediatric aphakic glaucoma. METHODS: In a randomized clinical trial, 30 eyes of 28 children < 16 years of age who had undergone anterior lensectomy-vitrectomy for congenital cataract were assigned to MMC-T (15 eyes of 13 children or AGI (15 eyes of 15 children. Surgical success was classified as complete (IOP 6-21 mmHg without any antiglaucoma medication and partial (IOP 6-21 mmHg with < 2 topical antiglaucoma agents in the absence of any sight-threatening complication or need for further glaucoma surgery, stable cup/disc ratios and visual loss < 2 Snellen lines. Overall success was defined as the sum of complete and partial success. RESULTS: Mean patient age was 9.1±4.1 and 10.9±5.1 years in the MMC-T and AGI groups, respectively (P=0.29. After a mean follow up of 14.8±11 and 13.1±9.7 months; complete, partial and overall success rates were 33.3%, 40% and 73.3% in the MMC-T vs 20%, 66.7% and 86.7% in the AGI groups, respectively (P= 0.361. Complication and failure rates were 40% and 26.7% in the MMC-T group vs 26.7% and 13.3% in the AGI group, respectively (P= 0.439. CONCLUSION: MMC-T and AGI seem to be comparable in terms of success and complications as the initial surgical

  17. Cryopreservation of canine sperm using egg yolk and soy bean based extenders.

    Science.gov (United States)

    Sánchez-Calabuig, María Jesús; Maillo, Verónica; Beltrán-Breña, Paula; de la Fuente Martínez, Julio; Galera-Carrillo, Silvestre; Pérez-Gutiérrez, José Félix; Pérez-Cerezales, Serafín

    2017-09-01

    Animal protein-based extenders are widely used despite being a potential source of bacterial or mycoplasma contamination. Its replacement with vegetal protein-based extenders could represent an interesting alternative for dog sperm cryopreservation. This technique could be further improved by the addition of Tris-Glucose-Citric acid (TGC) that could physically protect the spermatozoa and improve its homeostasis. The aim of this study was to evaluate a cryopreservation protocol for dog spermatozoa using a soybean-based extender (LP1 ℗ ) as well as the effects of the addition of (TGC) immediately after the semen collection. Eleven ejaculates from purebred adult dogs were collected, centrifuged in the absence or presence of TGC and processed as fresh or cryopreserved spermatozoa with: egg yolk-based extender (CaniPRO) or LP1 ℗ . Freezing the spermatozoa in LP1 ℗ reduced the amplitude of the lateral head displacement, the percentage of spermatozoa that showed the intact acrosome and the mitochondrial function (P<0.05). These samples also showed a trend towards increased percentage of apoptotic spermatozoa (P<0.05). The addition of TGC before centrifugation did not improve the seminal parameters and adversely affected motility (P<0.05) in the spermatozoa cryopreserved in CaniPRO. However, TGC did not affect motility and increased (P<0.05) the percentage of intact acrosomes in the spermatozoa cryopreserved in LP1 ℗ , reaching similar values than those cryopreserved in CaniPRO. In conclusion, LP1 ® plus TGC provide the same level of protection to dog spermatozoa cryopreservation than the egg yolk based extender CaniPRO when comparing standard post-thaw sperm quality parameters. Copyright © 2017. Published by Elsevier Urban & Partner Sp. z o.o.

  18. Safety and efficacy of cryopreserved autologous platelet concentrates in HLA-alloimmunized patients with hematologic malignancies.

    Science.gov (United States)

    Gerber, Bernhard; Alberio, Lorenzo; Rochat, Sophie; Stenner, Frank; Manz, Markus G; Buser, Andy; Schanz, Urs; Stussi, Georg

    2016-10-01

    Curative chemotherapy approaches in patients with malignancies and platelet (PLT) transfusion refractoriness due to alloimmunization may be hampered by the lack of suitable PLT donors. For these patients, transfusion of cryopreserved autologous PLTs is an option, but is time- and resource-consuming. We aimed at further simplifying this process. A retrospective single-center analysis was conducted on the transfusion of cryopreserved autologous PLTs in nine female alloimmunized, PLT transfusion-refractory patients treated for acute leukemia (n = 8) and non-Hodgkin's lymphoma (n = 1). No additional processing was used before transfusion, and most notably, washing and centrifugation steps were omitted. Clinical efficacy and safety, as well as a flow cytometric assessment of structural and functional PLT changes, were analyzed. A total of 40 autologous PLT concentrates were thawed at bedside and transfused a median of 32 (range, 9 to 994) days after cryopreservation. No major bleeds and no severe dimethyl sulfoxide toxicity were observed. The median PLT count increments did not differ 1 and 18 to 24 hours after transfusion and reached 6 × 10 9 /L (interquartile range [IQR], 3 × 10 9 -7.5 × 10 9 /L) and 6 × 10 9 /L (IQR, 2.5 × 10 9 -9.5 × 10 9 /L), respectively. Cryopreservation resulted in partial activation of one-third of the PLTs. In vitro stimulation with strong agonists induced additional full activation of cryopreserved PLTs: median, 55% (IQR, 42%-60%) after thrombin and 39% (IQR, 36%-39%) after convulxin. The transfusion of cryopreserved autologous PLTs is feasible and safe. Despite the cryopreservation process, PLT functionality is partially maintained. © 2016 AABB.

  19. Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch-based cryoprotectants.

    Science.gov (United States)

    Naaldijk, Yahaira; Johnson, Adiv A; Friedrich-Stöckigt, Annett; Stolzing, Alexandra

    2016-12-01

    Preservation of human skin fibroblasts and keratinocytes is essential for the creation of skin tissue banks. For successful cryopreservation of cells, selection of an appropriate cryoprotectant agent (CPA) is imperative. The aim of this study was to identify CPAs that minimize toxic effects and allow for the preservation of human fibroblasts and keratinocytes in suspension and in monolayers. We cryopreserved human fibroblasts and keratinocytes with different CPAs and compared them to fresh, unfrozen cells. Cells were frozen in the presence and absence of hydroxyethyl starch (HES) or dimethyl sulfoxide (DMSO), the latter of which is a commonly used CPA known to exert toxic effects on cells. Cell numbers were counted immediately post-thaw as well as three days after thawing. Cellular structures were analyzed and counted by labeling nuclei, mitochondria, and actin filaments. We found that successful cryopreservation of suspended or adherent keratinocytes can be accomplished with a 10% HES or a 5% HES, 5% DMSO solution. Cell viability of fibroblasts cryopreserved in suspension was maintained with 10% HES or 5% HES, 5% DMSO solutions. Adherent, cryopreserved fibroblasts were successfully maintained with a 5% HES, 5% DMSO solution. We conclude that skin tissue cells can be effectively cryopreserved by substituting all or a portion of DMSO with HES. Given that DMSO is the most commonly used CPA and is believed to be more toxic than HES, these findings are of clinical significance for tissue-based replacement therapies. Therapies that require the use of keratinocyte and fibroblast cells, such as those aimed at treating skin wounds or skin burns, may be optimized by substituting a portion or all of DMSO with HES during cryopreservation protocols.

  20. The effects of X-irradiation on ex vivo expansion of cryopreserved human hematopoietic stem/progenitor cells

    International Nuclear Information System (INIS)

    Hayashi, Naoki; Takahashi, Kenji; Kashiwakura, Ikuo

    2010-01-01

    In our previous study (Life Sciences 84: 598, 2009), we demonstrated that placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells have the effect to support the regeneration of freshly prepared X-irradiated hematopoietic stem/progenitor cells (HSPCs). Generally, HSPCs are supplied from companies, institutions, and cell banks that cryopreserve them for clinical and experimental use. In this study, the influence of cryopreservation on the responses of HSPCs to irradiation and co-culture with stromal cells is assessed. After cryopreservation with the optimal procedure, 2 Gy-irradiated HSPCs were cultured with or without stromal cells supplemented with combination of interleukin-3, stem cell factor, and thrombopoietin. The population of relatively immature CD34 + /CD38 - cells in cryopreserved cells was significantly higher than in fresh cells prior to cryopreservation; furthermore, the hematopoietic progenitor populations of CD34 + /CD45RA + cells and CD34 + /CD117 + cells in cryopreserved cells were significantly lower than that in fresh cells. However, the rate of expansion in the cryopreserved HSPCs was lower than in the fresh HSPCs. In the culture of cryopreserved cells irradiated with 2 Gy, the growth rates of CD34 + cells, CD34 + /CD38 - cells, and hematopoietic progenitors were greater than growth rates of their counter parts in the culture of fresh cells. Surprisingly, the effect to support the hematopoiesis in co-culture with stromal cells was never observed in the X-irradiated HSPCs after cryopreservation. The present results demonstrated that cryopreserving process increased the rate of immature and radio-resistant HSPCs but decreased the effects to support the hematopoiesis by stromal cells, thus suggesting that cryopreservation changes the character of HSPCs. (author)

  1. Resistance to mitomycin C requires direct interaction between the fanconi anemia proteins FANCA and FANCG in the nucleus through an arginine-rich domain

    NARCIS (Netherlands)

    Kruyt, FAE; Abou-Zahr, F; Mok, H; Youssoufian, H

    1999-01-01

    Fanconi anemia (FA) is a genetically heterogeneous disorder characterized by bone marrow failure, birth defects, and chromosomal instability. Because FA cells are sensitive to mitomycin C (MMC), FA gene products could be involved in cellular defense mechanisms. The FANCA and FANCG proteins deficient

  2. Milt cryopreservation for rheophilic fish threatened by extinction in the Rio Grande, Brazil.

    Science.gov (United States)

    de Andrade, Estefânia Souza; Paula, Daniella Aparecida de Jesus; Felizardo, Viviane de Oliveira; Murgas, Luis David Solis; Veras, Galileu Crovatto; Vieira e Rosa, Priscila

    2014-01-01

    Specific protocols for milt cryopreservation have been established for some freshwater fish species. However, cryopreservation reduces sperm quality, giving unsatisfactory results in reproduction. The objective of this work was to evaluate the effect of different cryoprotectants on the quality of Prochilodus lineatus, Brycon orbignyanus and Piaractus mesopotamicus milt after cryopreservation. The milt was diluted in different cryoprotectant solutions containing 10% methanol, dimethyl sulfoxide, glycerol, propylene glycol or ethylene glycol combined with the Beltsville Thawing Solution extender (5%), then placed in the vapour of a liquid nitrogen (LN) storage tank for 24 h, after which they were immersed in LN. After rewarming, the rate (%) and duration (s) of milt motility and abnormal morphology were evaluated. All of cryoprotectant solutions tested used maintained the viability of P. lineatus and P. mesopotamicus milt. However, in P. lineatus, glycerol ensured a lower percentage of abnormal morphology. In case of P. mesopotamicus, all of the cryoprotectant solutions tested may be used in the cryopreservation process, with the exception of those containing glycerol. For B. orbignyanus, cryoprotectant solutions containing methanol and ethylene glycol are recommended for use in the cryopreservation process, although they reduced the quality of sperm post-rewarming.

  3. Cryopreservation of PLBs of Brassidium Fly Away Using Encapsulation-Dehydration Technique

    Directory of Open Access Journals (Sweden)

    Arulvilee Rajasegar

    2015-12-01

    Full Text Available In vitro grown protocorm-like bodies (PLBs of Brassidium Fly Away orchid hybrid were cryopreserved using encapsulation- dehydration technique. The viability of the cryopreserved cells was determined by 2,3,5-triphenyltetrazolium chloride (TTC assay. For the preculture treatment, the PLBs were excised into two standard sizes of 1-2 and 4-5 mm and were precultured on half-strength Murashige and Skoog (MS semi solid medium supplemented with diff erent concentrations of sucrose (0, 0.2, 0.4, 0.6, 0.8 and 1.0M. The PLBs size 4-5 mm and 0.6 M sucrose concentration was selected based on highest viability obtained in TTC assay. The PLBs were encapsulated for 30 minutes using 3% (w/v liquid s odium alginate medium supplemented with 0.4M sucrose and 0.1M calcium chl oride and osmoprotected in 0.75M sucrose solution for 24 hours at 25°C. Th e beads were then dehydrated using 50g heat-sterilised silica gel for four hours , cryopreserved for 24 hours, thawed in a 40±2°C water bath for 90 seconds, and r egenerated in semi-solid half-strength. Biochemical analyses were conducted and th e cryopreserved PLBs had produced lower content of chlorophyll while the highest specifi c peroxidase activity was observed in cryopreserved PLBs

  4. Sperm harvesting and cryopreservation during vasectomy reversal is not cost effective.

    Science.gov (United States)

    Boyle, Karen E; Thomas, Anthony J; Marmar, Joel L; Hirshberg, Steven; Belker, Arnold M; Jarow, Jonathan P

    2006-04-01

    To determine whether sperm harvesting and cryopreservation at the time of vasectomy reversal is cost-effective. Model of actual costs and results at five institutions. Multicenter study comprising five centers, including university hospitals and private practices. Men undergoing vasectomy reversal. We established two models for vasectomy reversal. The first model was sperm harvesting and cryopreservation at the time of vasectomy reversal. The second model was sperm harvesting at the time of IVF only if the patient remained azoospermic after vasectomy reversal. Vasectomy reversal procedures modeled included bilateral vasovasostomy and bilateral epididymovasostomy. The costs for each procedure at the five institutions were collated and median costs determined. Median cost of procedure and calculated financial comparisons. The median cost of testicular sperm extraction/cryopreservation performed at the time of bilateral vasovasostomy was $1,765 (range, $1,025-$2,800). The median cost of microsurgical epididymal sperm aspiration or testicular sperm extraction with cryopreservation performed at the time of epididymovasostomy was $1,209 (range, $905-$2,488). The average of the median costs for percutaneous sperm aspiration or testicular sperm aspiration for those patients with a failed vasectomy reversal was $725 (range, $400-$1,455). Sperm retrieval with cryopreservation at the time of vasectomy reversal is not a cost-effective management strategy.

  5. A validation study of new cryopreservation bags for implementation in a blood and marrow transplant laboratory.

    Science.gov (United States)

    Pomper, Gregory J; Wilson, Emily; Isom, Scott; Hurd, David D

    2011-06-01

    A new cryopreservation bag for hematopoietic cell transplantation requires validation as a safe alternative to the bag currently being used in the laboratory. The new bag was validated using both laboratory and clinical criteria. Laboratory validation proceeded using paired samples of mononuclear cells processed using standard procedures. Cells cryopreserved in the new and old bags were compared for viability, cell counts, CD34 enumeration, colony-forming unit assays, and bag integrity. After completion of laboratory investigations, engraftment with the new bags was followed and compared to historical engraftment using the old bags. There were no significant differences between the old and new bags detected using laboratory studies. Bag integrity was equivalent. The validation data suggested impaired cell function after cryopreservation in the new bags, but there were no significant differences in engraftment potential using either material. Days to engraftment was longer using the new bags, but statistical analysis revealed an association with CD34 dose and not with cryopreservation bag type. The new bags were noninferior to the old bags. A change in cryopreservation bag type may appear to affect cell function and potentially affect engraftment. Multiple analyses may be needed to understand the effect of cell processing changes. © 2010 American Association of Blood Banks.

  6. Replacement of serum with ocular fluid for cryopreservation of immature testes.

    Science.gov (United States)

    Pothana, Lavanya; Devi, Lalitha; Venna, Naresh Kumar; Pentakota, Niharika; Varma, Vivek Phani; Jose, Jedy; Goel, Sandeep

    2016-12-01

    Cryopreservation of immature testis is a feasible approach for germplasm preservation of male animals. Combinations of dimethyl sulfoxide (DMSO) and foetal bovine serum (FBS) are used for testis cryopreservation. However, an alternative to FBS is needed, because FBS is expensive. Buffalo ocular fluid (BuOF), a slaughter house by-product, could be an economical option. The objective of the present study was to assess whether BuOF can replace FBS for cryopreservation of immature mouse (Mus musculus), rat (Rattus norvegicus), and buffalo (Bubalus bubalis) testes. Results showed that rodent and buffalo testes frozen in DMSO (10% for rodents and 20% for buffalo) with 20% FBS or BuOF had similar numbers of viable and DNA-damaged cells (P > 0.05). The expression of cell proliferation- (PCNA) and apoptosis-specific proteins (Annexin V and BAX/BCL2 ratio) were also comparable in mouse and buffalo testes frozen in DMSO with FBS or BuOF (P > 0.05). Interestingly, rat testis frozen in DMSO with BuOF had lower expression of Annexin V protein than testis frozen in DMSO with FBS (P  0.05). These findings provide evidence that BuOF has potential to replace FBS for cryopreservation of immature rodent and buffalo testis. Further investigation is needed to explore whether BuOF can replace FBS for testis cryopreservation of other species. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Cryopreservation of Populus trichocarpa and Salix dormant buds with recovery by grafting or direct rooting.

    Science.gov (United States)

    Bonnart, Remi; Waddell, John; Haiby, Kathy; Widrlenchner, Mark P; Volk, Gayle M

    2014-01-01

    Methods are needed for the conservation of clonally maintained trees of Populus and Salix. In this work, Populus trichocarpa and Salix genetic resources were cryopreserved using dormant scions as the source explant. We quantified the recovery of cryopreserved materials that originated from diverse field environments by using either direct sprouting or grafting. Scions (either at their original moisture content of 48 to 60% or dried to 30%) were slowly cooled to -35 degree C, transferred to the vapor phase of liquid nitrogen (LNV, -160 degree C), and warmed before determining survival. Dormant buds from P. trichocarpa clones from Westport and Boardman, OR had regrowth levels between 42 and 100%. Direct rooting of cryopreserved P. trichocarpa was also possible. Ten of 11 cryopreserved Salix accessions, representing 10 different species, exhibited at least 40% bud growth and rooting after 6 weeks when a bottom-heated rooting system was implemented. We demonstrate that dormant buds of P. trichocarpa and Salix accessions can be cryopreserved and successfully regenerated without the use of tissue culture.

  8. Challenges in cryopreservation of regulatory T cells (Tregs) for clinical therapeutic applications.

    Science.gov (United States)

    Golab, Karolina; Leveson-Gower, Dennis; Wang, Xiao-Jun; Grzanka, Jakub; Marek-Trzonkowska, Natalia; Krzystyniak, Adam; Millis, J Michael; Trzonkowski, Piotr; Witkowski, Piotr

    2013-07-01

    Promising results of initial studies applying ex-vivo expanded regulatory T cell (Treg) as a clinical intervention have increased interest in this type of the cellular therapy and several new clinical trials involving Tregs are currently on the way. Methods of isolation and expansion of Tregs have been studied and optimized to the extent that such therapy is feasible, and allows obtaining sufficient numbers of Tregs in the laboratory following Good Manufacturing Practice (GMP) guidelines. Nevertheless, Treg therapy could even more rapidly evolve if Tregs could be efficiently cryopreserved and stored for future infusion or expansions rather than utilization of only freshly isolated and expanded cells as it is preferred now. Currently, our knowledge regarding the impact of cryopreservation on Treg recovery, viability, and functionality is still limited. Based on experience with cryopreserved peripheral blood mononuclear cells (PBMCs), cryopreservation may have a detrimental effect on Tregs, can decrease Treg viability, cause abnormal cytokine secretion, and compromise expression of surface markers essential for proper Treg function and processing. Therefore, optimal strategies and conditions for Treg cryopreservation in conjunction with cell culture, expansion, and processing for clinical application still need to be investigated and defined. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Cryopreservation of in vitro grown shoot tips and apical meristems of the forage legume Arachis pintoi.

    Science.gov (United States)

    Rey, Hebe Y; Faloci, Mirta; Medina, Ricardo; Dolce, Natalia; Mroginski, Luis; Engelmann, Florent

    2009-01-01

    A cryopreservation protocol using the encapsulation-dehydration procedure was established for shoot tips (2-3 mm in length) and meristems (0.3-0.5 mm) sampled from in vitro plantlets of diploid and triploid cytotypes of Arachis pintoi. The optimal protocol was the following: after dissection, explants were precultured for 24 h on establishment medium (EM), encapsulated in calcium alginate beads and pretreated in liquid EM medium with daily increasing sucrose concentration (0.5, 0.75, 1.0 M) and desiccated to 22-23 percent moisture content (fresh weight basis). Explants were frozen using slow cooling (1 C per min from 25C to -30C followed by direct immersion in liquid nitrogen), thawed rapidly and post-cultured in liquid EM medium enriched with daily decreasing sucrose concentrations (0.75, 0.50, 0.1 M). Explants were then transferred to solid EM medium in order to achieve shoot regeneration, then on Murashige and Skoog medium supplemented with 0.05 microM naphthalene acetic acid to induce rooting of shoots. With this procedure, 53 percent and 56 percent of cryopreserved shoot tips of the diploid and triploid cytotypes, respectively, survived and formed plants. However, only 16 percent of cryopreserved meristems of both cytotypes regenerated plants. Using ten isozyme systems and seven RAPD profiles, no modification induced by cryopreservation could be detected in plantlets regenerated from cryopreserved material.

  10. Cryopreservation of turkey semen: effect of breeding line and freezing method on post-thaw sperm quality, fertilization, and hatching

    Science.gov (United States)

    Cryopreservation methods for poultry semen are not reliable for germplasm preservation, especially for turkeys, where fertility rates from frozen/thawed semen are particularly low. The objective was to evaluate cryopreservation methods for effectiveness in promoting cryosurvival and post-thaw funct...

  11. Assessment of some critical factors in the freezing technique for the cryopreservation of precision-cut rat liver slices

    NARCIS (Netherlands)

    Maas, W.J.M.; Graaf, I.A.M. de; Schoen, E.D.; Koster, H.J.; Sandt, J.J.M. van de; Groten, J.P.

    2000-01-01

    A number of studies on the cryopreservation of precision-cut liver slices using various techniques have been reported. However, the identification of important factors that determine cell viability following cryopreservation is difficult because of large differences between the various methods

  12. Effect of Antioxidants and Apoptosis Inhibitors on Cryopreservation of Murine Germ Cells Enriched for Spermatogonial Stem Cells.

    Directory of Open Access Journals (Sweden)

    Seung-Jung Ha

    Full Text Available Spermatogonial stem cells (SSCs are germline stem cells that serve as the foundation of spermatogenesis to maintain fertility throughout a male's lifetime. To treat male infertility using stem cell banking systems and transplantation, it is important to be able to preserve SSCs for long periods of time. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using antioxidants and apoptosis inhibitors in freezing medium. No differences were observed compared to controls when SSCs were cryopreserved in the presence of apoptosis inhibitors by themselves. However, mouse germ cells cryopreserved in basal medium containing the antioxidant hypotaurine (14 mM resulted in significantly greater proliferation potential and mitochondrial activity. Furthermore, treatment groups with combinations containing 200 mM trehalose and 14 mM hypotaurine showed higher proliferation rates compared to controls. In addition, several serum free conditions were evaluated for SSC cryopreservation. Treatment media containing 10% or 20% knockout serum replacement resulted in similar cryopreservation results compared to media containing FBS. SSC transplantation was also performed to confirm the functionality of SSCs frozen in 14 mM hypotaurine. Donor SSCs formed normal spermatogenic colonies and sperm in the recipient testis. These data indicate that inclusion of 14 mM hypotaurine in cryopreservation media is an effective way to efficiently cryopreserve germ cells enriched for SSCs and that knockout serum replacement can replace FBS in germ cell cryopreservation media.

  13. Recovery patterns, histological observations and genetic integrity in Malus shoot tips cryopreserved using droplet vitrification and encapsulation-dehydration procedures

    Science.gov (United States)

    A droplet-vitrification procedure is described for cryopreservation of Malus shoot tips. Survival patterns, recovery types, histological observations, and genetic integrity were compared for Malus shoot tips cryopreserved using this droplet-vitrification procedure and an encapsulation-dehydration pr...

  14. Isolation and Characterization of Human Dental Pulp Stem Cells from Cryopreserved Pulp Tissues Obtained from Teeth with Irreversible Pulpitis.

    Science.gov (United States)

    Malekfar, Azin; Valli, Kusum S; Kanafi, Mohammad Mahboob; Bhonde, Ramesh R

    2016-01-01

    Human dental pulp stem cells (DPSCs) are becoming an attractive target for therapeutic purposes because of their neural crest origin and propensity. Although DPSCs can be successfully cryopreserved, there are hardly any reports on cryopreservation of dental pulp tissues obtained from teeth diagnosed with symptomatic irreversible pulpitis during endodontic treatment and isolation and characterization of DPSCs from such cryopreserved pulp. The aim of this study was to cryopreserve the said pulp tissues to propagate and characterize isolated DPSCs. A medium consisting of 90% fetal bovine serum and 10% dimethyl sulfoxide was used for cryopreservation of pulp tissues. DPSCs were isolated from fresh and cryopreserved pulp tissues using an enzymatic method. Cell viability and proliferation were determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. DPSC migration and interaction were analyzed with the wound healing assay. Mesenchymal characteristics of DPSCs were verified by flow cytometric analysis of cell surface CD markers. The osteogenic and adipogenic potential of DPSCs was shown by von Kossa and oil red O staining methods, respectively, and the polymerase chain reaction method. We found no significant difference in CD marker expression and osteogenic and adipogenic differentiation potential of DPSCs obtained from fresh and cryopreserved dental pulp tissue. Our study shows that dental pulp can be successfully cryopreserved without losing normal characteristics and differentiation potential of their DPSCs, thus making them suitable for dental banking and future therapeutic purposes. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  15. Effects of chilled storage and cryopreservation on sperm characteristics, antioxidant enzyme activities, and lipid peroxidation in Pacific cod Gadus microcephalus

    Science.gov (United States)

    Wang, Xueying; Shi, Xuehui; Liu, Yifan; Yu, Daode; Guan, Shuguang; Liu, Qinghua; Li, Jun

    2016-07-01

    The present study evaluated the effects of chilled storage and cryopreservation on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod Gadus macrocephalus. Sperm motility and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (Gr), and lipid peroxidation (measured via malondialdehyde (MDA) content) were determined after the milt was stored at 4°C for 12 h, cryopreserved without cryoprotectant in 12% propylene glycol (PG), cryopreserved in 12% PG+0.1 mol/L trehalose, or cryopreserved in 12% PG spermatozoa but centrifuged to decant the supernatant prior to cryopreservation (only sperm cells were cryopreserved). After chilled storage or cryopreservation, the SOD, CAT and GPx activities were reduced in sperm cells and increased in seminal plasma in almost all treatments; sperm motility parameters were also decreased. However, the addition of trehalose into the cryoprotectant could significantly improve the postthaw sperm quality as revealed by the sperm average path velocity. This improvement might be attributed to the function of trehalose in scavenging reactive oxygen species. Chilled storage and cryopreservation had significant effects on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod.

  16. Effects of cryopreservation on excretory function, cellular adhesion molecules and vessel lumen formation in human umbilical vein endothelial cells.

    Science.gov (United States)

    Cai, Guoping; Lai, Binbin; Hong, Huaxing; Lin, Peng; Chen, Weifu; Zhu, Zhong; Chen, Haixiao

    2017-07-01

    Cryopreservation is widely used in regenerative medicine for tissue preservation. In the present study, the effects of cryopreservation on excretory function, cellular adhesion molecules and vessel lumen formation in human umbilical vein endothelial cells (HUVECs) were investigated. After 0, 4, 8, 12 or 24 weeks of cryopreservation in liquid nitrogen, the HUVECs were thawed. The excretory functions markers (endothelin‑1, prostaglandin E1, von Willebrand factor and nitric oxide) of HUVECs were measured by ELISA assay. The expression of intercellular adhesion molecule‑1 (ICAM‑1) in HUVECs was analyzed using flow cytometry. An angiogenesis assay was used to determine the angiogeneic capabilities of the thawed HUVECs. The results demonstrated that cryopreserved/thawed and recultivated HUVECs were unsuitable for tissue‑engineered microvascular construction. Specifically, the excretory function of the cells was significantly decreased in the post‑cryopreserved HUVECs at 24 weeks. In addition, the level of ICAM‑1 in HUVECs was significantly upregulated from the fourth week of cryopreservation. Furthermore, the tube‑like structure‑forming potential was weakened with increasing cryopreservation duration, and the numbers of lumen and the length of the pipeline were decreased in the thawed HUVECs, in a time‑dependent manner. In conclusion, the results of the present study revealed that prolonged cryopreservation may lead to HUVEC dysfunction and did not create stable cell lines for tissue‑engineered microvascular construction.

  17. Autotransplantation of cryopreserved ovarian tissue in 12 women with chemotherapy-induced premature ovarian failure: the Danish experience

    DEFF Research Database (Denmark)

    Schmidt, Kirsten Tryde; Rosendahl, Mikkel; Ernst, Erik

    2011-01-01

    To describe a cohort of 12 Danish women who received autotransplantation of frozen-thawed cryopreserved ovarian tissue because of premature ovarian failure after cancer treatment.......To describe a cohort of 12 Danish women who received autotransplantation of frozen-thawed cryopreserved ovarian tissue because of premature ovarian failure after cancer treatment....

  18. Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos does not induce morphological, cytological or molecular changes in recovered seedlings.

    Science.gov (United States)

    Sisunandar; Rival, Alain; Turquay, Patricia; Samosir, Yohannes; Adkins, Steve W

    2010-07-01

    The present study aimed at exploring the fidelity of coconut (Cocos nucifera L.) plants recovered from cryopreservation. Zygotic embryos from various different cultivars were cryopreserved following four successive steps, namely: rapid dehydration, rapid freezing, rapid thawing and in vitro recovery followed by acclimatization. At the end of the acclimatization period, the seedlings were compared to counterparts of the same age, which were produced from non-cryopreserved embryos. Both series were submitted to morphological, cytological and molecular comparisons. No significant differences in terms of growth rates could be measured. In addition, no morphological variation could be detected through the measurement of shoot elongation rates, production of opened leaves, and the number and total length of primary roots. Karyotype analysis revealed the same chromosome number (2n = 32) in all studied cultivars independently of cryopreservation. No significant differences could be observed between control and cryopreserved material concerning the type of chromosomes, the length of the long and short arms, the arm length ratio and the centromeric index. However, idiogram analysis did show a greater number of black banding on chromosomes isolated from cryopreserved material. Genetic and epigenetic fidelity was assessed through microsatellite (SSR) analysis and global DNA methylation rates; no significant differences would be observed between genomic DNAs isolated from seedlings originating from cryopreserved embryos and respective controls. In conclusion, our results suggest that the method of cryopreservation under study did not induce gross morphological, genetic or epigenetic changes, thus suggesting that it is an appropriate method to efficiently preserve coconut germplasm.

  19. The effect of mitomycin C trabeculectomy on the progression of visual field defect in normal-tension glaucoma.

    Science.gov (United States)

    Hagiwara, Y; Yamamoto, T; Kitazawa, Y

    2000-03-01

    We investigated in a prospective fashion the visual prognosis and complications in normal-tension glaucoma following unilateral trabeculectomy with adjunctive mitomycin C. Trabeculectomy with adjunctive mitomycin C was carried out unilaterally in 21 cases of normal-tension glaucoma. Intraocular pressure (IOP), visual prognosis, and complications were compared between the operated eyes and the non-operated fellow eyes. The follow-up period ranged from 2 to 7 years. The IOP dropped significantly from 14.8+/-1.8 mmHg (mean +/- SD) to 9.6+/-3.9 mmHg in the operated eyes (P=0.0002, Wilcoxon signed-rank test), but did not drop in the non-operated eyes. The mean deviation (MD) was -12.69+/-6.41 dB preoperatively and -14.70+/-5.49 dB at the last clinic visit in the operated eyes, whereas in non-operated eyes it was -7.85+/-5.65 dB and -11.15+/-5.62 dB, respectively. The MD deteriorated significantly in both operated and non-operated eyes (operated eyes P=0.0239, non-operated eyes: P=0.0002; Wilcoxon signed-rank test). The MD slope was -0.37+/-0.60 dB/year and -0.71+/-0.89 dB/year for the operated and non-operated eyes, respectively (P=0.5243, Mann-Whitney U-test). Visual field deterioration was more frequently observed in the non-operated eyes by a pointwise definition of the progression (Ptest). Visual acuity deteriorated in 6 of the operated eyes and in 5 of the non-operated eyes. Cataract developed in 6 (29%) of the 21 operated eyes, while among the non-operated eyes 4 (19%) developed cataract. Mitomycin C trabeculectomy is effective in delaying progression of visual field defect in normal-tension glaucoma, but complications may arise and cause some visual disturbance.

  20. Cryopreservation of Indian red jungle fowl (Gallus gallus murghi) semen.

    Science.gov (United States)

    Rakha, B A; Ansari, M S; Akhter, S; Hussain, I; Blesbois, E

    2016-11-01

    The population of red jungle fowl is declining and needs special attention for its conservation with suitable approaches. For ex situ in vitro conservation of Indian red jungle fowl, establishment of semen cryobank is an appropriate option, for which an extender with adequate retrieval capacity for functional spermatozoa is required. Therefore, studies were designed to evaluate a wide range of extenders for cryopreservation of Indian red jungle fowl (Gallus gallus murghi) sperm to achieve maximal post-thawed semen quality and fertility. For this purpose, semen from eight mature cocks were collected, initially evaluated (percent sperm motility, volume and concentration), pooled, assessed for motility, plasma membrane integrity, viability and acrosome integrity, and divided into six aliquots for dilution (1:5; 37°C) in Beltsville poultry, red fowl extender, Lake, EK, Tselutin poultry and chicken semen extenders. Diluted semen was cooled from 37°C to 4°C @ -0.275°C/min. Glycerol (20%) was added to chilled semen, equilibrated for 10min, filled in 0.5mL French straws, kept over LN 2 vapours for 10min and plunged into LN 2 and stored at -196°C. Percentages of motility, plasma membrane integrity, viability and acrosome integrity were higher (Psemen extender. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Migration of fresh and cryopreserved human spermatozoa in polyacrylamide gel.

    Science.gov (United States)

    Goldstein, M C; Wix, L S; Foote, R H; Feldschuh, R; Feldschuh, J

    1982-05-01

    The ability of freshly collected and frozen human spermatozoa to migrate in round capillary tubes containing specially formulated polyacrylamide gel was investigated, using 33 ejaculates from 27 donors. Each semen sample was divided; one portion was left undiluted, and the other portion was diluted to 50 x 10(6) sperm/ml. Glycerol was used as the cryoprotectant. The percentage of motile sperm cells was determined before and after freezing. Fresh semen contained a higher percentage of motile cells, which migrated farther than those of cryopreserved-thawed semen. Various correlations between the percentage of motile sperm and migration distance ranged from 0.57 to 0.62. There was a low positive correlation of migration distance with sperm cell concentration per milliliter, r = 0.25 to 0.34; and thus adjusting semen samples to a standard sperm concentration improved the accuracy of the test only slightly. The regression coefficient of migration distance on the percentage of motile sperm in fresh semen was 0.65, indicating that for each 10% increase in sperm motility, migration distance is predicted to increase 6.5 mm. Five batches of polyacrylamide gel gave uniform results, and the application of this stable gel to fertility investigations is discussed.

  2. Intracytoplasmic sperm injection outcomes with cryopreserved testicular sperm aspiration samples.

    Science.gov (United States)

    Roque, M; Valle, M; Marques, F; Sampaio, M; Geber, S

    2016-04-01

    Intracytoplasmic sperm injection (ICSI) may be performed with testicular frozen-thawed spermatozoa in patients with nonobstructive azoospermia (NOA). Sperm retrieval can be performed in advance of oocyte aspiration, as it may avoid the possibility of no recovery of spermatozoa on the day of oocyte pickup. There are few studies available in the literature concerning the use of frozen-thawed spermatozoa obtained from testicular sperm aspiration (TESA). To evaluate the effects and the outcomes of ICSI with frozen-thawed spermatozoa obtained by TESA, we performed a retrospective analysis of 43 ICSI cycles using frozen-thawed TESA. We obtained acceptable results with a fertilisation rate of 67.9%, an implantation rate (IR) of 17.1%, and clinical and ongoing pregnancy rates of 41.9% and 37.2% respectively. The results of this study suggest that performing ICSI using cryopreserved frozen-thawed testicular spermatozoa with TESA as a first option is a viable, safe, economic and effective method for patients with NOA. © 2015 Blackwell Verlag GmbH.

  3. Cryopreservation of in vitro grown nodal segments of Rauvolfia serpentina by PVS2 vitrification.

    Science.gov (United States)

    Ray, Avik; Bhattacharya, Sabita

    2008-01-01

    This paper describes the cryopreservation by PVS2 vitrification of Rauvolfia serpentina (L.) Benth ex kurz, an important tropical medicinal plant. The effects of type and size of explants, sucrose preculture (duration and concentration) and vitrification treatment were tested. Preliminary experiments with PVS1, 2 and 3 produced shoot growth only for PVS2. When optimizing the PVS2 vitrification of nodal segments, those of 0.31 - 0.39 cm in size were better than other nodal sizes and or apices. Sucrose preculture had a positive role in survival and subsequent regrowth of the cryopreserved explants. Seven days on 0.5 M sucrose solution significantly improved the viability of nodal segments. PVS2 incubation for 45 minutes combined with a 7-day preculture gave the optimum result of 66 percent. Plantlets derived after cryopreservation resumed growth and regenerated normally.

  4. Triplet pregnancy after intracytoplasmic sperm injection of cryopreserved oocytes: case report.

    Science.gov (United States)

    Young, E; Kenny, A; Puigdomenech, E; Van Thillo, G; Tiverón, M; Piazza, A

    1998-08-01

    To report a triplet pregnancy that occurred after intracytoplasmic injection of sperm into cryopreserved oocytes. Case report. Instituto de Ginecología y Fertilidad (IFER), Buenos Aires, Argentina. A 36-year-old infertile patient with premature ovarian failure and a previous term pregnancy with fresh donated oocytes. We administered leuprolide acetate for pituitary down-regulation followed by E2 valerianate in incremental doses until an endometrial lining of >8 mm was observed by ultrasound. Thawing of frozen donated oocytes, intracytoplasmic sperm injection (ICSI), and translaparoscopic fallopian tube ET also were performed. Natural micronized progesterone was administered intravaginally (600 mg/d) before ET. Ultrasound at the 8th week of gestation revealed a triplet pregnancy with active fetal heartbeats. A triple intrauterine gestation was achieved with the use of microinjection into cryopreserved oocytes. This case illustrates the feasibility of oocyte cryopreservation for clinical use in the era of ICSI.

  5. Optimizing human semen cryopreservation by reducing test vial volume and repetitive test vial sampling

    DEFF Research Database (Denmark)

    Jensen, Christian F S; Ohl, Dana A; Parker, Walter R

    2015-01-01

    OBJECTIVE: To investigate optimal test vial (TV) volume, utility and reliability of TVs, intermediate temperature exposure (-88°C to -93°C) before cryostorage, cryostorage in nitrogen vapor (VN2) and liquid nitrogen (LN2), and long-term stability of VN2 cryostorage of human semen. DESIGN......: Prospective clinical laboratory study. SETTING: University assisted reproductive technology (ART) laboratory. PATIENT(S): A total of 594 patients undergoing semen analysis and cryopreservation. INTERVENTION(S): Semen analysis, cryopreservation with different intermediate steps and in different volumes (50......-1,000 μL), and long-term storage in LN2 or VN2. MAIN OUTCOME MEASURE(S): Optimal TV volume, prediction of cryosurvival (CS) in ART procedure vials (ARTVs) with pre-freeze semen parameters and TV CS, post-thaw motility after two- or three-step semen cryopreservation and cryostorage in VN2 and LN2. RESULT...

  6. Single versus Two-Site Phacoemulsification and Mitomycin-C Trabeculectomy

    Directory of Open Access Journals (Sweden)

    Alireza Baradaran-Rafiee

    2008-11-01

    Full Text Available

    PURPOSE: To compare the outcomes of single-site versus two-site mitomycin-C (MMC augmented phacotrabeculectomy. METHODS: This matched randomized clinical trial included 34 eyes of 30 patients with visually significant cataracts and poorly controlled glaucoma. Equal numbers of eyes were randomly assigned to the single-site and two-site groups. In the single-site approach, phacoemulsification was performed under a superior scleral tunnel followed by trabeculectomy. The two-site approach included a temporal clear corneal phaco-emulsification combined with a separate superior trabeculectomy. MMC 0.2 mg/ml was similarly applied for one minute in both groups. RESULTS: Patients were followed for a mean period of 13±1.4 (range, 12 to 15 months. Mean best corrected visual acuity one year after surgery was 0.6±0.4 LogMAR in the single-site group and 0.4±0.28 LogMAR in the two-site group (P=0.12. In the single-site group, mean preoperative intraocular pressure (IOP was 26.4±6.6 mmHg which was decreased to 14.8±2.5 mmHg, one year after the operation (P < 0.001. Corresponding figures for the two-site group were 22.9±3.3 and 13.6±1.7 mmHg respectively (P < 0.001. At final follow up no significant difference in IOP existed between the study groups. Mean number of anti-glaucoma medications was 0.06±0.24 in the two-site group vs 0

  7. Corneal haze following PRK with mitomycin C as a retreatment versus prophylactic use in the contralateral eye.

    Science.gov (United States)

    Netto, Marcelo V; Chalita, Maria Regina; Krueger, Ronald R

    2007-01-01

    To report photorefractive keratectomy (PRK) treated with mitomycin C (MMC) for previous corneal haze in one eye and PRK with MMC to prevent corneal haze formation in the fellow eye. A 40-year-old woman underwent PRK with MMC to treat previous corneal haze (secondary to previous PRK without MMC) for residual refractive error of +0.50 +0.25 x 165 in the left eye and PRK with MMC to prevent corneal haze in the right eye. Postoperative slit-lamp examination revealed no haze in the right eye, but continued mild haze in the left eye. Treatment with PRK and MMC for previous corneal haze is not as effective as primary PRK with MMC in preventing postoperative corneal haze formation.

  8. Cytostatic action of aspirin and its effect on mitomycin C activity. A study in vitro under irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kammerer, Cornelia; Getoff, Nikola E-mail: nikola.getoff@univie.ac.at

    2001-04-01

    Experiments in vitro using E. coli bacteria (AB 1157) proved that aspirin possesses a cytostatic ability under various experimental condition (pH=7.4) in airfree, aerated as well as in media containing N{sub 2}O (converting e{sub aq}{sup -} into OH- radicals). In the last case the highest effect of aspirin was observed. The combination of aspirin with the well-known cytostaticum, mitomycin C (MMC) leads in airfree as well as in aerated media to a significant decrease of the MMC activity. However, the mixture of aspirin and MMC in the presence of N{sub 2}O causes a synergistic effect, resulting in an enhancement of the MMC activity by a factor of 1.5. Probable reaction steps are presented and discussed. Using the pulse radiolysis method the rate constants for the reactions of e{sub aq}{sup -}, H and OH- species with aspirin were also determined.

  9. Cytostatic action of aspirin and its effect on mitomycin C activity. A study in vitro under irradiation

    International Nuclear Information System (INIS)

    Kammerer, Cornelia; Getoff, Nikola

    2001-01-01

    Experiments in vitro using E. coli bacteria (AB 1157) proved that aspirin possesses a cytostatic ability under various experimental condition (pH=7.4) in airfree, aerated as well as in media containing N 2 O (converting e aq - into OH- radicals). In the last case the highest effect of aspirin was observed. The combination of aspirin with the well-known cytostaticum, mitomycin C (MMC) leads in airfree as well as in aerated media to a significant decrease of the MMC activity. However, the mixture of aspirin and MMC in the presence of N 2 O causes a synergistic effect, resulting in an enhancement of the MMC activity by a factor of 1.5. Probable reaction steps are presented and discussed. Using the pulse radiolysis method the rate constants for the reactions of e aq - , H and OH- species with aspirin were also determined

  10. LC-ESI-MSD fast determination of residual mitomycin C in hen aqueous humour after corneal refractive surgery.

    Science.gov (United States)

    Nozal, M J; Bernal, J L; Martín, M T; Bernal, J; Torres, R M; Merayo, J

    2006-01-23

    A simple, fast and reliable method has been developed for the assay of traces of mitomycin C (MMC) in hen aqueous humour samples. The determination was carried out by high-performance liquid chromatography with electrospray ionization mass spectrometric detection. In isocratic elution analysis, the mobile phase was a mixture of water-acetonitrile (78:22, v/v) and the chromatographic column was C(18) at 35 degrees C. The method has been validated over a range from 0.1 to 250 microg L(-1) in hen aqueous humour with correlation coefficients higher than 0.999. Limit of detection and limit of quantification for MMC based in signal to noise ratio of 3 and 10, respectively, were 20 and 71 ng L(-1). The developed method allows the analysis of MMC in hen aqueous humour samples obtained at different times and conditions in order to evaluate and compare the efficacy of the drug administration.

  11. Inference of some pharmacokinetic parameters of the C mitomycin, through the analysis of its micro nucleate polychromatic erythrocytes induction kinetics

    International Nuclear Information System (INIS)

    Morales R, P.; Vallarino K, T.; Cruz V, V.; Delgadillo H, A.

    2003-01-01

    The objective of the present work was to establish pharmacokinetic parameters of the C Mitomycin (MMC) in vivo, comparing its kinetics of induction of polychromatic micro nucleate erythrocytes (EPGMN) with that of the gamma radiation. The used doses were of 0.75; 1.5 and 3. 0 μmoles/kg of MMC. It was observed that the MMC produces MN in the first cycle of cellular division and it is independent of the cytotoxic effect. This agent requires of a relatively long period of latency that is not compatible with her great reactivity, for what the pharmacokinetic values obtained in fact reflect the time that takes the processing of leisure in the DNA and the subsequent induction of ruptures that produce MN. (Author)

  12. Testicular tissue cryopreservation in prepubertal male children: an analysis of parental decision-making.

    Science.gov (United States)

    Ginsberg, Jill P; Li, Yimei; Carlson, Claire A; Gracia, Clarisa R; Hobbie, Wendy L; Miller, Victoria A; Mulhall, John; Shnorhavorian, Margarett; Brinster, Ralph L; Kolon, Thomas F

    2014-09-01

    Infertility is an unfortunate treatment-related consequence for some pediatric malignancies as well as some non-malignant conditions treated with stem cell transplant. Unlike pubertal males, prepubertal males cannot produce semen for cryopreservation. This manuscript reports on the acceptability and safety of a multi-institutional protocol for offering testicular tissue cryopreservation to families of prepubertal male children at highest risk for infertility. Data on decision influences, decision-making control, and emotional state when considering this option are described. Prepubertal males facing gonadotoxic therapy were offered testicular cryopreservation. Post-biopsy, patients were followed for acute side effects. In addition, parents and patients were asked to complete questionnaires, whether or not they chose to cryopreserve tissue. Seventy-four prepubertal male children were approached. Fifty-seven families (77%) consented to the testicular biopsy; 48 of 57 underwent the procedure. There was one post-operative side effect. Parents who agreed to testicular cryopreservation and those that did not felt in control of this decision. Parents who consented to the biopsy and refusers were not deterred by the experimental nature of the protocol. An important decision-making influence was the risk of the biopsy. Biopsy and cryopreservation of testicular tissue from prepubertal male children was performed successfully and safely at three institutions. Parents faced with this option at diagnosis can make an informed decision and weigh carefully the risks and benefits. Although asked to make a decision soon after they were given a difficult diagnosis, parents uniformly felt in control of this decision. © 2014 Wiley Periodicals, Inc.

  13. Circulating angiogenic cells can be derived from cryopreserved peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Tanja Sofrenovic

    Full Text Available Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs.PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw or 28 (late thaw days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential.The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34(+VEGFR2(+CD133(+ population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle.Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34(+VEGFR2(+CD133(+ progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation.

  14. Semen quality before cryopreservation and after thawing in 543 patients with testicular cancer

    Science.gov (United States)

    MacKenna, Antonio; Crosby, Javier; Huidobro, Cristián; Correa, Eduardo; Duque, Gonzalo

    2017-01-01

    Objective The main objective of this study was to assess semen characteristics of patients with testicular cancer before cryopreservation and after thawing, to evaluate the consequences of this technique on sperm quality in patients with testicular cancer. Methods Five hundred eighty-nine samples from 543 patients with testicular cancer were cryopreserved between 1995 and 2015, one aliquot per patient was used for a thawing test to assess the impact of cryopreservation on sperm motility; semen analysis was performed before cryo preservation and after thawing, the result interpretation was carried out using the 2010 World Health Organization (WHO) Laboratory Manual, and consent forms were signed by the patients for freezing and when sperm was used for reproductive purposes. Results Hypospermia was observed in 28.7% of samples, the median sperm concentration was 18 million/mL with 35% oligozoospermia; twenty-two patients (4.1%) had azoospermia and 12.7% had severe oligozoospermia, the median sperm count was 31.3 million and 261 semen samples (44.3%) were normal in all parameters according to the WHO; total motile sperm count before cryopreservation and after thawing was 12 (0-412.2) and 7 (0-303.9) million sperm, respectively (p < 0.00001, 95% CI 5.48-14.91), which represents a 32% reduction; concerning the utilization of cryopreserved semen samples, only twelve patients (2.2%) used their frozen sperm for reproductive purposes. Conclusions An impairment in semen quality was found in almost half of the samples from patients with testicular cancer, only few patients had azoospermia or severe oligozoospermia; sperm cryopreservation significantly reduces sperm motility and total motile sperm count and very few patients use their frozen sperm for reproductive purposes. PMID:28333030

  15. Oxygenated thawing and rewarming alleviate rewarming injury of cryopreserved pancreatic islets.

    Science.gov (United States)

    Komatsu, Hirotake; Barriga, Alyssa; Medrano, Leonard; Omori, Keiko; Kandeel, Fouad; Mullen, Yoko

    2017-05-06

    Pancreatic islet transplantation is an effective treatment for Type 1 diabetic patients to eliminate insulin injections; however, a shortage of donor organs hinders the widespread use. Although long-term islet storage, such as cryopreservation, is considered one of the key solutions, transplantation of cryopreserved islets is still not practical due to the extensive loss during the cryopreservation-rewarming process. We have previously reported that culturing islets in a hyperoxic environment is an effective treatment to prevent islet death from the hypoxic injury during culture. In this study, we explored the effectiveness of thawing and rewarming cryopreserved islets in a hyperoxic environment. Following cryopreservation of isolated human islets, the thawing solution and culture media were prepared with or without pre-equilibration to 50% oxygen. Thawing/rewarming and the pursuant two-day culture were performed with or without oxygenation. Short-term recovery rate, defined as the volume change during cryopreservation and thawing/rewarming, was assessed. Ischemia-associated and inflammation-associated gene expressions were examined using qPCR after the initial rewarming period. Long-term recovery rate, defined as the volume change during the two-day culture after the thawing/rewarming, was also examined. Islet metabolism and function were assessed by basal oxygen consumption rate and glucose stimulated insulin secretion after long-term recovery. Oxygenated thawing/rewarming did not alter the short-term recovery rate. Inflammation-associated gene expressions were elevated by the conventional thawing/rewarming method and suppressed by the oxygenated thawing/rewarming, whereas ischemia-associated gene expressions did not change between the thawing/rewarming methods. Long-term recovery rate experiments revealed that only the combination therapy of oxygenated thawing/rewarming and oxygenated culture alleviated islet volume loss. These islets showed higher metabolism

  16. Slow cryopreservation is not superior to vitrification in human spermatozoa; an experimental controlled study

    Directory of Open Access Journals (Sweden)

    Mohamed Shehata Ali Mohamed

    2015-10-01

    Full Text Available Background: Spermatozoa cryopreservation is used for the management of infertility and some other medical conditions. The routinely applied cryopreservation technique depends on permeating cryoprotectants, whose toxic effects have raised the attention towards permeating cryoprotectants-free vitrification technique. Objective: To compare between the application of slow cryopreservation and vitrification on human spermatozoa. Materials and Methods: This was an experimental controlled study involving 33 human semen samples, where each sample was divided into three equal parts; fresh control, conventional slow freezing, and permeating cryoprotectants-free vitrification. Viability and mitochondrial membrane potential (MMP of control and post-thawing spermatozoa were assessed with the sperm viability kit and the JC-1 kit, respectively, using fluorescence-activated cell sorting analysis. Results: Significant reduction of the progressive motility, viability and MMP was observed by the procedure of freezing and thawing, while there was not any significant difference between both cryopreservation techniques. Cryopreservation resulted in 48% reduction of the percentage of viable spermatozoa and 54.5% rise in the percentage of dead spermatozoa. In addition, high MMP was reduced by 24% and low MMP was increased by 34.75% in response to freezing and thawing. Progressive motility of spermatozoa was correlated significantly positive with high MMP and significantly negative with low MMP in control as well as post-thawing specimens (r=0.8881/ -0.8412, 0.7461/ -0.7510 and 0.7603/ -0.7839 for control, slow and vitrification respectively, p=0.0001. Conclusion: Although both cryopreservation techniques have similar results, vitrification is faster, easier and associated with less toxicity and costs. Thus, vitrification is recommended for the clinical application.

  17. Collagen matrix compared with mitomycin C for treatment of primary open-angle glaucoma with trabeculectomy performed

    Directory of Open Access Journals (Sweden)

    Lei Yu

    2017-09-01

    Full Text Available AIM:To evaluate the effectiveness and safety between trabeculectomy with collagen matrix versus trabeculectomy with mitomycin C(MMCfor patients with primary open-angle glaucoma(POAG.METHODS: In this prospective randomized comparative study from January 2015 to December 2016. Thirty-two eyes presented with POAG were included in this study, 14 eyes treated by trabeculectomy with subconjunctival implant of collagen matrix(study groupand the other 18 eyes treated by trabeculectomy with mitomycin C. Postoperative IOP, the success rate of operation, number of postoperative glaucoma medications and postoperative complications were recorded. Each patient was followed up at least 6mo. RESULTS: The mean postoperative IOP was statistically different between the study group and the control group after 1d(PP>0.05, and the mean postoperative IOP was statistically different between the two groups(PP>0.05. The IOP decreased at 1d after openations compared with before, kept stable at 1wk to 6mo. IOP of study group was lowen than control. IOP was controlled by glaucoma medications in the study group by 28% compared to control group by 33% at 6mo after operation, but there was no significant difference. There was no significant difference between the study group and the control group in complications(PCONCLUSION: Trabeculectomy with collagen matrix implant is comparable to the use of MMC with a similar success rate in open-angle glaucoma and the range in reducing intraocular pressure was significantly higher than that of MMC and it can significantly avoid the occurrence of low IOP postoperatively, transient anterior chamber, conjunctival wound leakage complications has no advantages compared with the use of MMC.

  18. Cryopreservation of ovarian tissue for a decade in Denmark: a view of the technique

    DEFF Research Database (Denmark)

    Rosendahl, Mikkel; Schmidt, Kirsten Louise Tryde; Ernst, Erik

    2011-01-01

    evaluation of mouse and human ovarian tissue after freezing with four different combinations of cryoprotectants. Viability was confirmed by transplantation of frozen-thawed human ovarian tissue (n = 49) to oophorectomized Nude mice. Viability after transport of fresh tissue 4-5 h prior to freezing had...... previously been validated. Overnight transport of fresh ovarian tissue prior to cryopreservation was evaluated when human ovarian tissue was kept on ice for 20 h and then cryopreserved. The thawed ovarian tissue was transplanted to an oophorectomized Nude mouse and histology confirmed viability. In Denmark...

  19. Transplantation of cryopreserved allogeneic bone marrow after its long-term storage to lethally irradiated dogs

    International Nuclear Information System (INIS)

    Novikova, N.N.; Fedotenkov, A.G.; Sukyasyan, G.V.; Timakova, L.A.

    1982-01-01

    The study of the dog bone marrow preserved at -196 deg C during 6-12 years has shown that in the body of lethally irradiated animals (8Gy), due to the antigenic difference in the tissues of the donor and the irradiated recipients, the cells of cryopreserved allogeneic bone marrow were differentiated by the lymphoid type similar to that observed in transplantation of freshly prepared myelocaryocytes. However, their proliferative activity in the period of active lymphocyte transformation was quantitatively less manifest than in freshly transplanted cells. The results of the study evidence that the bone marrow cells cryopreserved during 6-12 years retain their functional activity

  20. Integrating Micro- or Nanoscale Encapsulation Technology with Vitreous Cryopreservation: A New Strategy to Improve Biopreservation.

    Science.gov (United States)

    Zheng, Y Y; Zhao, G

      BACKGROUND: None-uniform distributions of temperatures, limited freezing and thawing rates, and thermal stresses are three main hindering factors for successful vitreous cryopreservation of mass volume of biosamples. Micro- and nanoscale encapsulation, owning the intrinsic features to avoid those limitations introduced by the traditional approaches, has been opened up a new way for effective and high-efficiency biopreservation. This short review article summarizes recent advances in cell encapsulation technology for biopreservation by manipulating cells and biological agents in micro- or nanoscale volume droplets and microgels, and discusses its promising applications for future vitreous cryopreservation.

  1. Novel and potential application of cryopreservation to plant genetic transformation.

    Science.gov (United States)

    Wang, Biao; Zhang, Zhibo; Yin, Zhenfang; Feng, Chaohong; Wang, Qiaochun

    2012-01-01

    The world population now is 6.7 billion and is predicted to reach 9 billion by 2050. Such a rapid growing population has tremendously increased the challenge for food security. Obviously, it is impossible for traditional agriculture to ensure the food security, while plant biotechnology offers considerable potential to realize this goal. Over the last 15 years, great benefits have been brought to sustainable agriculture by commercial cultivation of genetically modified (GM) crops. Further development of new GM crops will with no doubt contribute to meeting the requirements for food by the increasing population. The present article provides updated comprehensive information on novel and potential application of cryopreservation to genetic transformation. The major progresses that have been achieved in this subject include (1), long-term storage of a large number of valuable plant genes, which offers a good potential for further development of novel cultivars by genetic transformation; (2), retention of regenerative capacity of embryogenic tissues and protoplasts, which ensures efficient plant regeneration system for genetic transformation; (3), improvement of transformation efficiency and plant regeneration of transformed cells; (4), long-term preservation of transgenic materials with stable expression of transgenes and productive ability of recombinant proteins, which allows transgenic materials to be stored in a safe manner before being analyzed and evaluated, and allows establishment of stable seed stocks for commercial production of homologous proteins. Data provided in this article clearly demonstrate that cryo-technique has an important role to play in the whole chain of genetic transformation. Further studies coupling cryotechnique and genetic transformation are expected to significantly improve development of new GM crops. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Polymyxin B effects on motility parameters of cryopreserved bull semen

    Directory of Open Access Journals (Sweden)

    Mojtaba Rashedi

    2017-01-01

    Full Text Available Objective: To evaluate the effect of adding different values of polymyxin B (PMB to bull semen on various motility parameters of post-thawed semen such as total motility, progressive motility and velocity parameters using kinetic parameters of sperm by Computer Assisted Sperm Analysis.Methods: Gram negative bacteria release lipopolysaccharide, which induces the apoptotic pathway. Antibiotics are added to semen in order to prevent bacterial contaminations in bovine semen. These antibiotics kill the bacteria especially gram negative bacteria. Therefore, their endotoxins are released during bacteriolysis and bind to the head region and midpiece of sperm. PMB is a bactericidal antibiotic against multidrug resistant gram-negative bacteria and is able to neutralize the toxic effects of the released endotoxin. This study was performed on 3-year old Taleshi bulls.Results: The results showed both positive and negative significant effects of PMB on semen quality. Total motility and progressive motility were significantly increased (P<0.000 1 by 100 μg per mL of PMB (55.2% and 48.8% respectively against the control groups (43.5% and 37.7%, respectively. Moreover, they were significantly decreased (P<0.000 1 by 1 000 μg per mL of PMB (35.2% and 28.8% respectively against the control groups (43.5% and 37.7% respectively in above-mentioned parameters. In Computer Assisted Semen Analyzer, parameter VAP was significantly decreased (P<0.04 in 1 000 μg (69.6 μm/s against the control group (78.7 μm/s. Finally, using PMB in processing cryopreserved bull semen is advised, but before using it, the rate of endotoxins must be measured.Conclusions: We advise using PMB after measuring endotoxin concentration; In vitro, in vivo and in field fertilization, adding other sperm evaluation factors such as acrosomal integrity, DNA integrity, mitochondrial function to PMB treated semen.

  3. Liposome encapsulated soy lecithin and cholesterol can efficiently replace chicken egg yolk in human semen cryopreservation medium.

    Science.gov (United States)

    Mutalik, Srinivas; Salian, Sujith Raj; Avadhani, Kiran; Menon, Jyothsna; Joshi, Haritima; Hegde, Aswathi Raju; Kumar, Pratap; Kalthur, Guruprasad; Adiga, Satish Kumar

    2014-06-01

    Cryopreservation of spermatozoa plays a significant role in reproductive medicine and fertility preservation. Chicken egg yolk is used as an extender in cryopreservation of human spermatozoa using glycerol egg yolk citrate (GEYC) buffered medium. Even though 50% survival of spermatozoa is generally achieved with this method, the risk of high levels of endotoxins and transmission pathogens from chicken egg yolk is a matter of concern. In the present study we attempted to establish a chemically defined cryopreservation medium which can replace the chicken egg yolk without affecting sperm survival. Ejaculates from 28 men were cryopreserved with GEYC based freezing medium or liposome encapsulated soy lecithin-cholesterol based freezing medium (LFM). The semen samples were subjected to rapid thawing after 14 days of storage in liquid nitrogen. Post-thaw analysis indicated significantly higher post-thaw motility and sperm survival in spermatozoa cryopreserved with LFM compared to conventional GEYC freezing medium. The soy lecithin and cholesterol at the ratio of 80:20 with sucrose showed the highest percentage of post-thaw motility and survival compared to the other compositions. In conclusion, chemically defined cryopreservation medium with liposome encapsulated soy lecithin and cholesterol can effectively replace the chicken egg yolk from human semen cryopreservation medium without compromising post-thaw outcome.

  4. Development of methods for cryopreservation of rooster sperm from the endangered breed "Gallina Valenciana de Chulilla" using low glycerol concentrations.

    Science.gov (United States)

    Blanch, E; Tomás, C; Casares, L; Gómez, E A; Sansano, S; Giménez, I; Mocé, E

    2014-06-01

    Glycerol (11%; v:v) is the cryoprotectant most often used for the cryopreservation of rooster sperm. However, chicken breeds differ in the resistance of their sperm to the cryopreservation process and endangered or local breeds usually present low fertilizing ability when conventional sperm cryopreservation protocols are used. The objective of this study was to optimize the protocol for the cryopreservation of the sperm from the endangered breed "Gallina Valenciana de Chulilla". For this purpose, 10 pools of semen from 43 roosters of this breed were cryopreserved using 8%, 7%, 6%, or 4% glycerol, and the sperm quality was determined immediately after thawing and in the insemination doses. Lohmann Brown Classic laying hens (n = 40) were used for the insemination trials. The sperm quality after cryopreservation progressively decreased as the glycerol concentration was reduced (P roosters frozen using 4% glycerol exhibited lower sperm quality but similar fertilizing ability compared with samples processed using higher glycerol concentrations. These results may provide useful information for developing cryopreservation protocols for other breeds. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Cryopreservation and other assisted reproductive technologies for the conservation of threatened amphibians and reptiles: bringing the ARTs up to speed.

    Science.gov (United States)

    Clulow, John; Clulow, Simon

    2016-06-01

    Amphibians and reptiles are experiencing serious declines, with the number of threatened species and extinctions growing rapidly as the modern biodiversity crisis unfolds. For amphibians, the panzootic of chytridiomycosis is a major driver. For reptiles, habitat loss and harvesting from the wild are key threats. Cryopreservation and other assisted reproductive technologies (ARTs) could play a role in slowing the loss of amphibian and reptile biodiversity and managing threatened populations through genome storage and the production of live animals from stored material. These vertebrate classes are at different stages of development in cryopreservation and other ARTs, and each class faces different technical challenges arising from the separate evolutionary end-points of their reproductive biology. For amphibians, the generation of live offspring from cryopreserved spermatozoa has been achieved, but the cryopreservation of oocytes and embryos remains elusive. With reptiles, spermatozoa have been cryopreserved in a few species, but no offspring from cryopreserved spermatozoa have been reported, and the generation of live young from AI has only occurred in a small number of species. Cryopreservation and ARTs are more developed and advanced for amphibians than reptiles. Future work on both groups needs to concentrate on achieving proof of concept examples that demonstrate the use of genome storage and ARTs in successfully recovering threatened species to increase awareness and support for this approach to conservation.

  6. CRYOPRESERVATION OF REPRODUCTIVE PRODUCTS AS AN EFFECTIVE METHOD FOR PRESERVING THE BIODIVERSITY OF STURGEON FISH SPECIES (REVIEW

    Directory of Open Access Journals (Sweden)

    I. Kononenko

    2016-06-01

    Full Text Available Purpose. In recent years, cryopreservation of reproductive products has widely been used as one of the accessible and in some cases the only ways of preserving and supporting the number of endangered fish species including sturgeon fish species. Currently, the method of cryopreservation is represented by various techniques and ways using individual and species approaches. Numerous publications on the issue of fish sperm cryopreservation mostly contain inconsistent results and ambiguous data. Thus, the analysis of the existing information about principles and methods of fish sperm cryopreservation is an important issue for further studies. Moreover, summarizing the existing information will enable us to plan the experiment more efficiently and reasonably and to get the desired outcomes with higher reliability. Findings. The study presents main principles of widely used methods of fish sperm cryopreservation, the analysis of main factors of influence on outcomes of freezing or unfreezing as well as the analysis of results received when using various ways and methods of cryopreservation. Besides, the paper shows the importance of forming and full functioning of fish sperm cryobanks. Originality. The paper summarizes the existing information on the issue of fish sperm low temperature cryopreservation. The information is given in the form of successive presentation of the research outcomes received at each point of freezing or unfreezing when using different techniques as well as results of different factors influence on it. Moreover, a review of achievements in the field of cryopreservation and main principles of forming fish sperm cryobanks are given. Practical value. The presented review of traditional and modern literature data in the issue of cryopreservation can be used when planning, redesigning and experimenting fish sperm freezing or unfreezing.

  7. Mitomycin C versus 5-Fluorouracil for wound healing in glaucoma surgery.

    Science.gov (United States)

    Cabourne, Emily; Clarke, Jonathan C K; Schlottmann, Patricio G; Evans, Jennifer R

    2015-11-06

    Raised intraocular pressure is a risk factor for glaucoma. One treatment option is glaucoma drainage surgery (trabeculectomy). Antimetabolites are used during surgery to reduce postoperative scarring during wound healing. Two agents in common use are mitomycin C (MMC) and 5-Fluorouracil (5-FU). To assess the effects of MMC compared to 5-FU as an antimetabolite adjunct in trabeculectomy surgery. We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (2015 Issue 9), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to October 2015), EMBASE (January 1980 to October 2015), Latin American and Caribbean Health Sciences Literature Database (LILACS) (January 1982 to October 2015), the ISRCTN registry (www.isrctn.com/editAdvancedSearch), ClinicalTrials.gov (www.clinicaltrials.gov) and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 2 October 2015. We included randomised controlled trials where wound healing had been modified with MMC compared to 5-FU. Two review authors independently selected trials and collected data. The primary outcome was failure of a functioning trabeculectomy one year after surgery. Secondary outcomes included mean intraocular pressure at one year. We considered three subgroups: high risk of trabeculectomy failure (people with previous glaucoma surgery, extracapsular cataract surgery, African origin and people with secondary glaucoma or congenital glaucoma); medium risk of trabeculectomy failure (people undergoing trabeculectomy with extracapsular cataract surgery) and low risk of trabeculectomy failure (people who have received no previous surgical eye intervention). We identified 11 trials that enrolled 687 eyes of 679 participants. The

  8. Influence of family history, irradiation and anti-cancer drug (mitomycin C) on the occurrence of multiple primary neoplasms in breast carcinoma patients

    International Nuclear Information System (INIS)

    Yoshimoto, Masataka; Sakamoto, Goi; Sugano, Haruo; Kasumi, Fujio; Fukami, Atsuo; Kuno, Keijiro.

    1984-01-01

    The influence of family history, irradiation and anti-cancer drug (Mitomycin C) on the occurrence of multiple primary neoplasms was analysed using the person-year method in 1359 Japanese breast carcinoma patients. There were 111 multiple primary neoplasms, including bilaterl breast cancer, in 109 patients; the incidence rate was 0.0072 per person-year. The incidence rate in patients with a family history of cancer was 1.29 times higher than in those without. In the bilateral breast cancer group there was about a 3 times higher frequency of family history of breast cancer. Irradiation therapy raised the occurrence of multiple primary neoplasms 1.28 fold, and Mitomycin C (40 mg) had no effect on the occurrence of neoplasms during a 10-year observation period. (author)

  9. A rapid and simple method for cryopreservation of human liver slices

    NARCIS (Netherlands)

    de Kanter, R; Olinga, Peter; Hof, I.H; de Jager, M.H; Verwillegen, W.A; Slooff, M.JH; Meijer, D.K F; Groothuis, Geny; Koster, H

    1. Precision-cut liver slices represent a suitable and convenient in vitro preparation for studying metabolism and toxicity mechanisms of drugs and toxic chemicals. Particularly in the case of human liver slices, cryopreservation would enable more efficient utilization of this scarce and irregularly

  10. Cryopreservation of third-stage larvae of Strongylus vulgaris (large strongyle of horses).

    Science.gov (United States)

    Titoy, G A; Van Rensburg, L J

    1997-06-01

    A technique for the cryopreservation of third-stage larvae of Strongylus vulgaris is described. Infective larvae of S. vulgaris were exsheathed in a 0.16% sodium hypochlorite solution and then transferred into cryotubes containing 0.09% saline. The samples were stored in the gas phase of liquid nitrogen.

  11. Directional freezing for the cryopreservation of adherent mammalian cells on a substrate

    Science.gov (United States)

    Braslavsky, Ido

    2018-01-01

    Successfully cryopreserving cells adhered to a substrate would facilitate the growth of a vital confluent cell culture after thawing while dramatically shortening the post-thaw culturing time. Herein we propose a controlled slow cooling method combining initial directional freezing followed by gradual cooling down to -80°C for robust preservation of cell monolayers adherent to a substrate. Using computer controlled cryostages we examined the effect of cooling rates and dimethylsulfoxide (DMSO) concentration on cell survival and established an optimal cryopreservation protocol. Experimental results show the highest post-thawing viability for directional ice growth at a speed of 30 μm/sec (equivalent to freezing rate of 3.8°C/min), followed by gradual cooling of the sample with decreasing rate of 0.5°C/min. Efficient cryopreservation of three widely used epithelial cell lines: IEC-18, HeLa, and Caco-2, provides proof-of-concept support for this new freezing protocol applied to adherent cells. This method is highly reproducible, significantly increases the post-thaw cell viability and can be readily applied for cryopreservation of cellular cultures in microfluidic devices. PMID:29447224

  12. Cryopreservation of porcine fetal ventral mesencephalic tissue for intrastriatal transplantation in Parkinson's disease

    NARCIS (Netherlands)

    Koopmans, J.; Hogenesch, I.; Copray, S.; Middel, B.; van Dijk, H.; Go, K-G.; Staal, M.

    2001-01-01

    In this study we examined the efficacy of cryopreserving porcine fetal mesencephalic tissue. After microscopical dissection of the ventral mesencephalon (VM) from E28 pig fetuses, the collection of explants was randomly divided into two equal parts. One part was directly prepared as cell suspension.

  13. Efficient cryopreservation of human pluripotent stem cells by surface-based vitrification

    NARCIS (Netherlands)

    Neubauer, Julia C; Beier, Axel F; Geijsen, Niels; Zimmermann, Heiko

    2015-01-01

    Efficient cryopreservation of human stem cells is crucial for guaranteeing a permanent supply of high-quality cell material for drug discovery or regenerative medicine. Conventionally used protocols usually employing slow freezing rates, however, result in low recovery rates for human pluripotent

  14. Perfusion bioreactor-based cryopreservation of 3D human mesenchymal stromal cell tissue grafts

    Czech Academy of Sciences Publication Activity Database

    Petrenko, Yuriy; Petrenko, A.; Martin, I.; Wendt, D.

    2017-01-01

    Roč. 76, jun. (2017), s. 150-153 ISSN 0011-2240 Institutional support: RVO:68378041 Keywords : cryopreservation * tissue engineering * mesenchymal stromal cells Subject RIV: FP - Other Medical Disciplines OBOR OECD: Cell biology Impact factor: 1.996, year: 2016

  15. CRYOPRESERVATION OF RAM SPERM FROM AUTOCHTHONOUS BREEDS DURING A NON-MATING SEASON

    Directory of Open Access Journals (Sweden)

    Milko SABEV

    2007-07-01

    Full Text Available It is possible to collect and successfully cryopreserve ejaculates in a non-mating season from rams of the autochthonous breeds Karakachan, Cooper-red Shumen and Karnobat-local, raised in Bulgaria. Studies are in progress aiming the elaboration of optimal cryoprotective extenders and freezing technology.

  16. Heat and mass transfer during the cryopreservation of a bioartificial liver device: a computational model.

    Science.gov (United States)

    Balasubramanian, Saravana K; Coger, Robin N

    2005-01-01

    Bioartificial liver devices (BALs) have proven to be an effective bridge to transplantation for cases of acute liver failure. Enabling the long-term storage of these devices using a method such as cryopreservation will ensure their easy off the shelf availability. To date, cryopreservation of liver cells has been attempted for both single cells and sandwich cultures. This study presents the potential of using computational modeling to help develop a cryopreservation protocol for storing the three dimensional BAL: Hepatassist. The focus is upon determining the thermal and concentration profiles as the BAL is cooled from 37 degrees C-100 degrees C, and is completed in two steps: a cryoprotectant loading step and a phase change step. The results indicate that, for the loading step, mass transfer controls the duration of the protocol, whereas for the phase change step, when mass transfer is assumed negligible, the latent heat released during freezing is the control factor. The cryoprotocol that is ultimately proposed considers time, cooling rate, and the temperature gradients that the cellular space is exposed to during cooling. To our knowledge, this study is the first reported effort toward designing an effective protocol for the cryopreservation of a three-dimensional BAL device.

  17. Stress preconditioning of rooster semen before cryopreservation improves fertility potential of thawed sperm.

    Science.gov (United States)

    Feyzi, S; Sharafi, M; Rahimi, S

    2018-03-22

    Avian semen cryopreservation is not as successful as that seen in mammals. This failure is mostly attributed to unique physiological characteristics of poultry semen that make it susceptible to cryo-damages. Utilization of sublethal oxidative stress for preconditioning of sperm, as an innovative approach, improves the cryo-survival of sperm in certain mammalian species. The purpose of this study was to investigate the effects of preconditioning of rooster semen with sublethal oxidative stress [very low concentrations of nitric oxide (NO)] before cryopreservation on the quality and fertility potential of thawed sperm. Semen samples were collected from 20 roosters, twice a wk, and different concentrations of NO [0 (NO-0), 0.01 (NO-0.01), 0.1 (NO-0.1), 1 (NO-1), 10 (NO-10), and 100 μM (NO-100)] were used to investigate the effects of controlled induction of sublethal stress before semen cryopreservation on the thawed sperm performance. A significantly higher (P 0.05) affected by different concentrations of NO. Sperm exposed to NO-1 produced the highest percentage of viable spermatozoa (Annexin-/PI-), which was significantly different from the other samples. Finally, rate of fertility after artificial insemination was significantly higher (P < 0.05) following treatment with NO-1 compared to NO-0 and NO-0.1. Application of 1 μM NO as a sublethal oxidative stress before cryopreservation of sperm efficiently increased numerous quality indices of thawed sperm as well as its fertility potential.

  18. Rooster Semen Cryopreservation: Effect of Pedigree Line and Male Age on Post-Thaw Sperm Function

    Science.gov (United States)

    The fertility rates of cryopreserved poultry semen are highly variable and not reliable for use in preservation of commercial genetic stocks. Our objective was to evaluate the cryosurvival of semen from 8 pedigreed layer lines at the onset and end of production. Semen from 160 roosters (20/line) was...

  19. Effects of alternative cryoprotectants, diluents, straw size and cholesterol addition on cryopreserved rooster sperm

    Science.gov (United States)

    Rooster sperm do not cryopreserve well. This is due in part to osmotic changes that sperm undergo during addition and removal of cryoprotectant (CPA), as well as membrane damage during the membrane phase transition from fluid at 37 degrees C to gel at low temperatures. When a CPA is removed from spe...

  20. Schistosoma mansoni: interactive effects of irradiation and cryopreservation on parasite maturation and immunization of mice

    International Nuclear Information System (INIS)

    James, E.R.; Dobinson, A.R.

    1984-01-01

    Mechanically transformed schistosomula of Schistosoma mansoni were irradiated with levels of 60Co irradiation between 2.5 and 54 krad, cryopreserved by the two-step addition of ethanediol and rapid cooling technique, and were injected intramuscularly into groups of mice which were perfused 40 days later. The schistosomula were either irradiated and then cryopreserved (IC) or cryopreserved and then irradiated in the frozen state (CI). Development into adult worms was prevented with 4 krad for IC schistosomula, but for CI schistosomula a small number of worms (1.6%) was recovered using 8.8 krad. A dose of 4 krad was sufficient to prevent development of unfrozen controls (I), but for schistosomula irradiated while exposed to ethanediol (EI), a dose of 7 krad was required. Using the different protocols, the peak levels of protection against a challenge infection were achieved with 9 (IC) and 16 krad (CI), compared to 20 krad for unfrozen schistosomula (I) reported previously. The highest level of protection (65%) was achieved with CI schistosomula. Possible interactions between the radioprotective and damaging effects of cryopreservation are discussed

  1. Cryopreservation of Precision-cut Tissue Slices for Application in Drug Metabolism Research

    NARCIS (Netherlands)

    Graaf, Inge Anne Maria de

    2002-01-01

    The research described in this thesis had two important aims. The first was to determine whether tissue slices could be used as an in vitro tool to predict the in vivo metabolism of new drugs. The second aim was to find a manner to store tissue slices for longer time periods by cryopreservation.

  2. Comparison of the efficacy of conventional slow freezing and rapid cryopreservation methods for bovine embryos

    NARCIS (Netherlands)

    Wagtendonk-de Leeuw, van A.M.; Daas, den J.H.; Kruip, T.A.; Rail, W.F.

    1995-01-01

    Day 7 bovine morulae and early blastocysts were randomly assigned to one of four cryopreservation methods: (i) a modified conventional controlled slow freezing and stepwise dilution after thawing; and three methods which enable direct transfer of the embryo into the recipient upon thawing: (ii)

  3. The potential of cryopreservation and reproductive technologies for animal genetic resources conservation strategies

    NARCIS (Netherlands)

    Hiemstra, S.J.; Lende, van der T.; Woelders, H.

    2006-01-01

    This chapter focuses on ex situ conservation. An overview of the state of the art cryopreservation and reproductive technology for farm animals and fish is followed by a discussion on the implications of ex situ conservation strategies. Ex situ conservation of genetic material from livestock and

  4. Melatonin enhances the recovery of cryopreserved shoot tips of American elm (Ulmus Americana L.)

    Science.gov (United States)

    Climate change and the global migrations of people and goods have exposed trees to new diseases and abiotic challenges that threaten the survival of species. In vitro germplasm storage via cryopreservation is an effective tool to ensure conservation of tree species, but plant cells and tissues are e...

  5. Cryopreservation of Populus trichocarpa and Salix using dormant buds with recovery by grafting or direct rooting

    Science.gov (United States)

    Populus trichocarpa and Salix can be successfully cryopreserved by using dormant scions as the source explants. These scions (either at their original moisture content of 48 to 60% or dried to 30%) were slowly cooled to –35 degree Celsius, transferred to the vapor phase of liquid nitrogen (LNV,-160...

  6. Crosstalk between Substrates and Rho-Associated Kinase Inhibitors in Cryopreservation of Tissue-Engineered Constructs

    Directory of Open Access Journals (Sweden)

    Arindam Bit

    2017-01-01

    Full Text Available It is documented that human mesenchymal stem cells (hMSCs can be differentiated into various types of cells to present a tool for tissue engineering and regenerative medicine. Thus, the preservation of stem cells is a crucial factor for their effective long-term storage that further facilitates their continuous supply and transportation for application in regenerative medicine. Cryopreservation is the most important, practicable, and the only established mechanism for long-term preservation of cells, tissues, and organs, and engineered tissues; thus, it is the key step for the improvement of tissue engineering. A significant portion of MSCs loses cellular viability while freeze-thawing, which represents an important technical limitation to achieving sufficient viable cell numbers for maximum efficacy. Several natural and synthetic materials are extensively used as substrates for tissue engineering constructs and cryopreservation because they promote cell attachment and proliferation. Rho-associated kinase (ROCK inhibitors can improve the physiological function and postthaw viability of cryopreserved MSCs. This review proposes a crosstalk between substrate topology and interaction of cells with ROCK inhibitors. It is shown that incorporation of ionic nanoparticles in the presence of an external electrical field improves the generation of ROCK inhibitors to safeguard cellular viability for the enhanced cryopreservation of engineered tissues.

  7. STABILITY IN REAL TIME OF SOME CRYOPRESERVED MICROBIAL STRAINS WITH REFERENCE TO GENETICALLY MODIFIED MICROORGANISMS

    Directory of Open Access Journals (Sweden)

    DANIELA VINTILĂ

    2007-05-01

    Full Text Available The aim of this work is to analyze the viability of microorganisms from Collection of Industrial Microorganisms from Faculty of Animal Science and Biotechnology – Timisoara, during freezing and thawing as part of cryopreservation technique. The stability in real time of 19 strains cryopreserved in 16% glycerol was evaluated during a 6-months period. The strains studied were: Escherichia coli, Lactobacillus acidophilus, Rhizobium meliloti, Saccharomyces cerevisiae, Aspergillus oryzae, Aspergillus niger, Trichoderma viride, Bacillus globigii, Bacillus licheniformis, and 9 strains of Bacillus subtilis. The strains cryopreserved at -20oC and -70oC were activated using the fast thawing protocol. A better cell recovery was achieved with the -70oC protocol reaching an average viability for E. coli of 86,3%, comparing with 78,6% in -20oC protocol. The cell recovery percentages for the other strains were: 92,4% for L. acidophilus, 93,9% for A.niger, 89% for A. oryzae, 86,7% for T. viride, 94,2% for R. meliloti, 82,1% for S. cerevisiae, 89,9% for B. licheniformis. Regarding the viability of genetically modified microorganisms, the values shows a good recovering after freezing and thawing, even after 180 days of cryopreservation. With the -20oC protocol lower viability was observed due probably to the formation of eutectic mixtures and recrystalization processes.

  8. Droplet vitrification technique for cryopreservation of different pineapple (Ananas comosus L. Merrill) accessions

    Science.gov (United States)

    Germplasm conservation of pineapple is crucial to secure the genetic variability of the genus for breeding programs and supporting new research. Long-term conservation is done through cryopreservation, by storing cells or tissues at ultra-low temperature in liquid nitrogen (LN; -196°C) or in the LN ...

  9. Droplet-vitrification and morphohistological studies of cryopreserved shoot tips of cultivated and wild pineapple genotypes

    Science.gov (United States)

    Germplasm conservation of pineapple [Ananas comosus (L.) Mer.] is crucial to preserve the genus’ genetic diversity to secure material for genetic improvement and to support innovative and new research. Long-term conservation is accomplished through cryopreservation that is done by storing cells or t...

  10. Collection, analysis and cryopreservation of semen from Malayan gaur (Bos gaurus hubbacki: A preliminary study

    Directory of Open Access Journals (Sweden)

    M.S. Khairiah

    2012-10-01

    Full Text Available The Malayan gaur (Bos gaurus hubbacki or Seladang is classified as vulnerable by the International Union for Conservation of Nature and Natural Resources (IUCN. The Malayan gaur is mainly distributed in the tropical woodlands of Peninsular Malaysia and Southern Thailand. The aim of this study was to collect, analyze and cryopreserve the semen of wild Malayan gaur. Transrectal massage (TM and electroejaculation (EEJ technique was applied in semen collection of the Malayan gaur. The semen was then cryopreserved in liquid nitrogen using slow freezing technique. Makler counting chamber was used to evaluate sperm concentration and motility, while the sperm viability and morphology of fresh and post-thaw sperm was determined using eosin-nigrosin staining protocol. As a result, we have successfully collected the Malayan gaur semen using EEJ technique. Sperm motility, viability and morphological changes of the post-thaw semen of Malayan gaur were found undesirable due to the complication of the cryopreservation process. On the basis of current study it can be concluded that Malayan gaur bulls semen can be obtain by EEJ with no evidence of rectal trauma. Optimization of the process of cryopreservation for Malayan gaur sperm is needed to maintain the cryoviability of the good sperm quality. The data generated in this study would be useful in conservation of genetic diversity program for Malayan gaur.

  11. CRYOPRESERVATION OF FRESHLY ISOLATED SYNAPTOSOMES PREPARED FROM THE CEREBRAL-CORTEX OF RATS

    NARCIS (Netherlands)

    GLEITZ, J; BEILE, A; WILFFERT, B; TEGTMEIER, F

    In the present study, we established a cryopreservation method for freshly isolated synaptosomes prepared from the cerebral cortex of rats. Freshly prepared synaptosomes were either shock-frozen or frozen under temperature-controlled conditions using a programmable temperature controller. Each group

  12. The Different Calcium+2 Intensity Profile and Quality of Oocyte and Goat Sperms after Cryopreservation

    Science.gov (United States)

    Ciptadi, G.; Rahayu, S.; Fatchiyah; Wahyuningsih, S.; Budiarto, A.; Nasich, M.; Putri, A. R. I.; Mudawamah, M.; Ihsan, M. N.

    2018-02-01

    This research aims were to study the effect of the oocyte and sperms cryopreservation of Indonesian local goat on the post-thawing quality and profile or characters of Calcium+2 intensity in relating with their fertility capacity. A study was conducted to test the freezing method and post-thawing viability both stock cells stored in the deep freezer and liquid nitrogen (-80°C of vs -196°C). A fertility test of sperms has been conducted through in vitro of sperm quality, while the oocytes cryopreserved test was done by in vitro maturation (IVM) rate (%). The profile of Calcium 2+ was performed and analysis by Confocal Laser Scanned Microscope (CLSM). The result showed that IVM rate of goat oocyte is considered lower when cryopreserved in -80°C than in -196°C. Meanwhile, sperm is considered having a good quality in 2 methods of cryopreservation with post-thawing motility > 40 % (SNI 2014). There is an important difference between Calcium intensity of fresh and post-thawing both for oocyte and spermatozoa. Calcium +2 profiles is varied individually on the peak of intensity, but it considered expressed the same profile of each fresh and post-thawing cell. In vitro fertilization test need to be performed to complete the viability and fertility competence of these sperm and oocyte freezing stocks.

  13. Effects of seed cryopreservation, stratification and scarification on germination for five rare species of pitcher plants.

    Science.gov (United States)

    Khanna, Sruti; Jenkins, Heather; Bucalo, Kylie; Determann, Ron O; Cruse-Sanders, Jennifer M; Pullman, Gerald S

    2014-01-01

    Habitat loss and over collection have caused North American pitcher plants to become rare, including U.S. federally endangered Sarracenia alabamensis and S. oreophila, and S. leucophylla, S. psittacina and S. purpurea spp. venosa, endangered in several states. To develop reliable seed cryopreservation protocols for endangered Sarracenia species enabling similar germination percentages before and after storage in liquid nitrogen (LN) either in vivo or using in vitro tools. Seed germination pre- and post-cryopreservation were compared following seed drying with germination in soil, aseptic environment with wet filter paper or enriched medium, and using scarification or stratification for dormancy removal. After cryostorage, germination in vitro (1/6- or 1/3-strength MS medium) increased compared to germination on peat moss. Germination pre- and post-cryopreservation was similar for S. alabamensis and S. oreophila when seeds were stratified and grown in vitro. S. leucophylla and S. psittacina also showed high germination after cryopreservation when germinated on medium following stratification. Rapid liquid nitrogen exposure and rewarming induced seed coat cracking that damaged seeds, likely allowing internal damage during acid scarification and microbial entry during germination in non-sterile environments.

  14. Shoot regeneration and embryogenesis in lily shoot tips cryopreserved by droplet vitrification

    Science.gov (United States)

    Shoot regeneration and embryogenesis were, for the first time, achieved directly in shoot tips of Lilium Oriental hybrid ‘Siberia’ following cryopreservation by droplet-vitrification. Shoot tips (2 mm in length) including 2-3 leaf primordia were excised from 4-week-old adventitious shoots directly r...

  15. The effect of Tribulus terrestris extract on motility and viability of human sperms after cryopreservation.

    Science.gov (United States)

    Asadmobini, Atefeh; Bakhtiari, Mitra; Khaleghi, Sara; Esmaeili, Farzaneh; Mostafaei, Ali

    2017-04-01

    Semen cryopreservation produces significant amounts of reactive oxygen species (ROS), which may lead to impairment of sperm morphology, function, and ultimately, male fertility. Since Tribulus terrestris has antioxidant and free-radical-scavenging properties, this study aims to reveal the effect of the Tribulus terrestris extract on motility and vitality of human sperms after cryopreservation. Semen specimens from 80 healthy volunteers were divided into eight groups: fresh control (group I), freeze control (group II), groups III, IV, and V, which had 20, 40, and 50 μg/mL doses of Tribulus terrestris extract added before cryopreservation, and groups VI, VII, and VIII, which were supplemented by these extract doses after the freeze-thaw process. To evaluate the effects of the Tribulus terrestris extract, the semen samples were incubated with the extract and evaluated with a light microscope for motility and viability. After cryopreservation, a significant improvement in spermatozoa viability was observed in group VII. In groups VII and VIII, motility, according to World Health Organization (WHO) criteria, increased considerably (p Tribulus terrestris, which improves human sperm motility and viability, may be due to its antioxidant properties. On the basis of the results, the researchers concluded that Tribulus terrestris can be used as a safe therapeutic alternative to current modalities for the management of motility dysfunction in males. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation

    Science.gov (United States)

    Cholesterol to phospholipid ratio is used as a representation for membrane fluidity, and predictor of cryopreservation success but results are not consistent across species and ignore the impact of membrane proteins. Therefore, this research explored the modulation of membrane fluidity and protein ...

  17. Cryopreserved Cadaveric Arterial Allograft for Arterial Reconstruction in Patients with Prosthetic Infection.

    Science.gov (United States)

    Lejay, Anne; Delay, Charline; Girsowicz, Elie; Chenesseau, Bettina; Bonnin, Emilie; Ghariani, Mohamed-Zied; Thaveau, Fabien; Georg, Yannick; Geny, Bernard; Chakfe, Nabil

    2017-11-01

    The aim of this study was to report outcomes of cryopreserved arterial allografts used as a vascular substitute in the setting of prosthetic material infection. A retrospective analysis of prospectively collected data was conducted including all consecutive interventions performed with cryopreserved arterial allografts used for vascular reconstruction in the setting of prosthetic material infection between January 2005 and December 2014. Five year outcomes included allograft related re-interventions, survival, primary patency, and limb salvage rates. Fifty-three procedures were performed using cryopreserved allografts for vascular prosthetic infection: 25 procedures (47%) were performed at aorto-iliac level (Group 1) and 28 procedures (53%) at peripheral level (Group 2). The mean follow-up was 52 months. Five year allograft related re-intervention was 55% in Group 1 (6 allograft ruptures and 5 allograft aneurysm degenerations) and 33% in Group 2 (2 allograft ruptures and 7 allograft aneurysm degenerations). Five year survival was 40% and 68%, primary patency was 89% and 59% and limb salvage was 100% and 89% for Group 1 and 2 respectively. Use of cryopreserved arterial allografts provides acceptable results but is tempered by suboptimal 5 year outcomes with high re-intervention rates. Copyright © 2017 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

  18. Identification of a highly successful cryopreservation method (droplet-vitrification) for petunia

    Science.gov (United States)

    Petunia (Petunia × hybrida Vilm.) is a very important crop conserved in the National Genebank of China. Petunia cultivar “Niu 2” was used to develop a droplet-vitrification protocol to cryopreserve shoot tips. Six variables (age of the in vitro plants, concentration of sucrose in the preculture solu...

  19. High-throughput optimization by statistical designs: example with rat liver slices cryopreservation.

    Science.gov (United States)

    Martin, H; Bournique, B; Blanchi, B; Lerche-Langrand, C

    2003-08-01

    The purpose of this study was to optimize cryopreservation conditions of rat liver slices in a high-throughput format, with focus on reproducibility. A statistical design of 32 experiments was performed and intracellular lactate dehydrogenase (LDHi) activity and antipyrine (AP) metabolism were evaluated as biomarkers. At freezing, modified University of Wisconsin solution was better than Williams'E medium, and pure dimethyl sulfoxide was better than a cryoprotectant mixture. The best cryoprotectant concentrations were 10% for LDHi and 20% for AP metabolism. Fetal calf serum could be used at 50 or 80%, and incubation of slices with the cryoprotectant could last 10 or 20 min. At thawing, 42 degrees C was better than 22 degrees C. After thawing, 1h was better than 3h of preculture. Cryopreservation increased the interslice variability of the biomarkers. After cryopreservation, LDHi and AP metabolism levels were up to 84 and 80% of fresh values. However, these high levels were not reproducibly achieved. Two factors involved in the day-to-day variability of LDHi were identified: the incubation time with the cryoprotectant and the preculture time. In conclusion, the statistical design was very efficient to quickly determine optimized conditions by simultaneously measuring the role of numerous factors. The cryopreservation procedure developed appears suitable for qualitative metabolic profiling studies.

  20. Potentials of short term and long term cryopreserved sperm of the ...

    African Journals Online (AJOL)

    To service the growing demand for male African giant catfish (Clarias gariepinus) broodstock for aquaculture in Nigeria, and to conserve valuable genetic resources, we improved both short-term (in deep freezer at -35°C) and long-term cryopreservation (in liquid nitrogen at -296°C) of catfish sperm. Catfish sperm ...

  1. HPMA copolymer conjugates of DOX and mitomycin C for combination therapy: physicochemical characterization, cytotoxic effects, combination index analysis, and anti-tumor efficacy

    Czech Academy of Sciences Publication Activity Database

    Kostková, Hana; Etrych, Tomáš; Říhová, Blanka; Kostka, Libor; Starovoytova, Larisa; Kovář, Marek; Ulbrich, Karel

    2013-01-01

    Roč. 13, č. 12 (2013), s. 1648-1660 ISSN 1616-5187 R&D Projects: GA ČR GAP301/11/0325; GA MŠk EE2.3.30.0029 Institutional support: RVO:61389013 ; RVO:61388971 Keywords : doxorubicin * HPMA copolymers * mitomycin C Subject RIV: CE - Biochemistry; FD - Oncology ; Hematology (MBU-M) Impact factor: 3.650, year: 2013

  2. Repairability during G 1 phase of inducting lesions of sister chromatid exchange produced by mitomycin C in salivary gland cells of mice In Vivo

    International Nuclear Information System (INIS)

    Cruz Vallejo, V.L.

    1991-01-01

    The repairability of the injuries that lead to the formation of sister chromatid exchange (SCE) produced by mitomycin C (MMC) with a dose of 2.1 mg/g in vivo, during the G 1 phase in the first cycle of cellular division (before the incorporation of BrdU [5-bromo-2 deoxyurine] to the DNA), as well as during the G 1 phase of the second cycle of cellular division (after the incorporation of BrdU) were analyzed. A 35.1% decrease in the frequency of SCE produced by Mitomycin C was observed, in the early G 1 phase of the first division, with respect to the frequency of SCE induced in the later G 1 phase. When Mitomycin C is given to cells whose DNA is substituted with BrdU in only one of the chain's filaments such decrease is not observed. The results suggest that the injuries caused by MMC, which give place to the SCE, in cells of the salivary glands of the mouse in vivo, are partially repaired only when induced in DNA which has not been substituted with BrdU. (Author)

  3. In vitro and in vivo antimutagenic effects of DIG, a herbal preparation of Berberis vulgaris, Taraxacum officinale and Arctium lappa, against mitomycin C.

    Science.gov (United States)

    Di Giorgio, C; Boyer, L; De Meo, M; Laurant, C; Elias, R; Ollivier, E

    2015-07-01

    DIG, a liquid herbal preparation made from a mixture of diluted mother tinctures of Berberis vulgaris, Taraxacum officinale and Arctium lappa, was assessed for its antimutagenic properties against mitomycin C. The micronucleus assay on Chinese hamster ovary (CHO)-K1 cells was used to evaluate the in vitro anticlastogenic activity of DIG compared to those of separately diluted mother tinctures. The micronucleus assay was performed on mouse erythrocytes and the comet assay was performed on mouse liver, kidney, lung, brain and testicles to assess the protective effects of DIG (0.2 and 2 % at libitum) against an intraperitoneal injection of mitomycin C (1 mg Kg(-1)) in mice. DIG exerted a powerful anticlastogenic activity, under both pretreatment and simultaneous treatment conditions as assessed by the micronucleus assay in CHO-K1 cells. Its protective activity was greater than that observed for each mother tincture. DIG reduced micronuclei levels in mouse erythrocytes and suppressed >80 % of DNA strand breaks in the liver, kidney, lung, brain and testicles of mice exposed to mitomycin C.

  4. Effect of mitomycin combined with Nd-YAG laser on cell proliferation and invasion as well as MEK/ERK signaling pathway in obstructive lacrimal duct model

    Directory of Open Access Journals (Sweden)

    Yu Yan

    2017-11-01

    Full Text Available Objective: To study the effect of mitomycin (MMC combined with Nd-YAG laser on cell proliferation and invasion as well as MEK/ERK signaling pathway in obstructive lacrimal duct model. Methods: New Zealand rabbits were selected as experimental animals and divided into model group, laser group and MMC + laser group; obstructive lacrimal duct model was established, then laser group were given Nd-YAG laser intervention, and MMC + laser group were given Nd-YAG laser combined with mitomycin intervention. 2 months after intervention, the expression of proliferation molecules, invasion molecules and MEK-ERK signaling molecules in lacrimal duct tissue were measured. Results: TGF-β, CTGF, PCNA, Ki-67, Col-I, Col-III, MEK, ERK1/2, MMP2 and MMP9 protein levels in lacrimal duct tissue of laser group were significantly higher than those of model group while TSG-6, Cthrc1 and TIMP1 protein levels were significantly lower than those of model group; TGF-β, CTGF, PCNA, Ki- 67, Col-I, Col-III, MEK, ERK1/2, MMP2 and MMP9 protein levels in lacrimal duct tissue of MMC + laser group were significantly lower than those of laser group while TSG-6, Cthrc1 and TIMP1 protein levels were significantly higher than those of laser group. Conclusion: Mitomycin can inhibit cell proliferation and invasion as well as MEK/ERK signaling pathway activation in obstructive lacrimal duct model after Nd-YAG laser treatment.

  5. Effect of cryopreservation and lyophilization on viability and growth of strict anaerobic human gut microbes.

    Science.gov (United States)

    Bircher, Lea; Geirnaert, Annelies; Hammes, Frederik; Lacroix, Christophe; Schwab, Clarissa

    2018-04-17

    Strict anaerobic gut microbes have been suggested as 'next-generation probiotics' for treating several intestinal disorders. The development of preservation techniques is of major importance for therapeutic application. This study investigated cryopreservation (-80°C) and lyophilization survival and storage stability (4°C for 3 months) of the strict anaerobic gut microbes Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii, Roseburia intestinalis, Anaerostipes caccae, Eubacterium hallii and Blautia obeum. To improve preservation survival, protectants sucrose and inulin (both 5% w/v) were added for lyophilization and were also combined with glycerol (15% v/v) for cryopreservation. Bacterial fitness, evaluated by maximum growth rate and lag phase, viability and membrane integrity were determined using a standardized growth assay and by flow cytometry as markers for preservation resistance. Lyophilization was more detrimental to viability and fitness than cryopreservation, but led to better storage stability. Adding sucrose and inulin enhanced viability and the proportion of intact cells during lyophilization of all strains. Viability of protectant-free B. thetaiotaomicron, A. caccae and F. prausnitzii was above 50% after cryopreservation and storage and increased to above 80% if protectants were present. The addition of glycerol, sucrose and inulin strongly enhanced the viability of B. obeum, E. hallii and R. intestinalis from 0.03-2% in protectant-free cultures to 11-37%. This is the first study that quantitatively compared the effect of cryopreservation and lyophilization and the addition of selected protectants on viability and fitness of six strict anaerobic gut microbes. Our results suggest that efficiency of protectants is process- and species-specific. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  6. In Vitro Propagarion and Cryopreservation of Important Grape Cultivars (Vitis Vinifera L. and Rootstocks

    Directory of Open Access Journals (Sweden)

    F. CELEBI TOPRAK

    2014-06-01

    Full Text Available Grape (Vitis vinifera L. is among the most important species that is cultivated almost all around the world. There are over one thousand varieties that are grown for raisin, fresh consumption and wine making purposes. The grape germplasm resources are generally maintained as whole plants under field conditions. The traditional way of germplasm preservation is very risky due to natural uncertainties. In vitro technologies can help producing healthy propagation materials free from viroids, viruses, bacteria, phytoplasmas, fungi, and nematodes. When combined with cryopreservation technologies in vitro preservation systems can allow safe protection and propagation of valuable Vitis genetic resources. In this study, 12 commercial cultivar and two rootstock materials were tested for the applicability of long term preservation by in vitro clonal propagation and cryopreservation techniques. Axillary shoot tips collected from newly emerging shoots were placed in Magenda boxes containing 30 g/l sucrose on MS medium and cultured in a growth chamber adjusted to 16 h ligth/25o C and 8 h dark/17o C. All grape genotypes tested responded well to this application and produced healthy root and shoots. Shoot explants from these in vitro stocks were subcultured in every three months for one year. Apical dome explants excised from in vitro grape plants were stored in liquid nitrogen for cryopreservation. Genotypes varied in their responses to cryopservation treatment. Five genotypes showed shoot or callus formation. Regenerated shoots continued to grow and produced normal shoots and roots, but no plants could be developed from calli. Flow cytometry analysis of regenerants from continuous subculture and cryopreservation did not show any chromosome number abnormalities. In vitro micropropagation is an excellent choice for a long-term conservation of grape germplasm, which allows access to actively growing plant materials without seasonal restriction. Such cultures are

  7. Improved quality of cryopreserved cheetah (Acinonyx jubatus) spermatozoa after centrifugation through Accudenz.

    Science.gov (United States)

    Crosier, Adrienne E; Henghali, Josephine N; Howard, Jogayle; Pukazhenthi, Budhan S; Terrell, Kimberly A; Marker, Laurie L; Wildt, David E

    2009-01-01

    Sperm cryopreservation, in combination with assisted reproductive techniques, is a valuable tool for the genetic management of endangered felids. However, the acrosome of the cheetah spermatozoon is especially sensitive to cryopreservation, with approximately 40% of spermatozoa experiencing acrosomal damage immediately after thawing and then another approximately 15% loss during the next 4 hours in vitro. Additionally, thawing causes a reduction in sperm motility by approximately 20% with another decrease of approximately 12% during subsequent incubation in vitro. We hypothesized that slow removal of glycerol from cryopreserved cheetah spermatozoa using an Accudenz gradient would improve acrosomal integrity, sperm motility longevity, and structural morphology. Accudenz was compared with traditional cheetah sperm processing methods for glycerol removal that involves washing, multistep resuspension, and swim-up processing. Electroejaculates (n = 21 total from 8 males) were washed in Ham F10 medium, and sperm pellets were resuspended in TEST-yolk buffer with 0% glycerol. Samples were cryopreserved in straws in 4% final glycerol, thawed, and assessed for percent intact acrosomes (% IA), percent motility (% M), and forward progressive status (FPS; scale, 0-5). Sperm motility index (SMI) was calculated as (% M + [FPS x 20]) / 2. In study 1, glycerol removal by centrifugation through an Accudenz gradient (4%, 10%) was compared with traditional sperm washing (control) and multistep resuspension protocols. At each time after centrifugation (hourly for 4 hours), % IA was improved (P cheetah sperm mitigates the significant loss in sperm quality that occurs after freeze-thawing. This alleviation of cellular damage resulting from cryopreservation contributes to a more than 10% improvement in overall sperm motility and, more importantly, allows retention of 40% or more of sperm with intact acrosomes.

  8. Developmental competence of oocytes isolated from surplus medulla tissue in connection with cryopreservation of ovarian tissue for fertility preservation

    DEFF Research Database (Denmark)

    Wilken-Jensen, Helle N; Kristensen, Stine G; Jeppesen, Janni V

    2014-01-01

    OBJECTIVE: Evaluating the developmental competence of immature oocytes collected from surplus medulla tissue in connection with ovarian tissue cryopreservation for fertility preservation. DESIGN: Cohort comparative study. SETTING: University laboratory in Denmark from 2011-2012. POPULATION: 69...

  9. Effects of transportation time after extraction on the magnetic cryopreservation of pulp cells of rat dental pulp

    Directory of Open Access Journals (Sweden)

    Mao-Suan Huang

    2011-03-01

    Conclusions: The freezing technique used in this animal study provided positive effects on pulp cell storage. In addition, the storage time before the freezing procedure is an important issue for cryopreserving pulp cells in intact teeth.

  10. Cryopreservation of testicular tissue before long-term testicular cell culture does not alter in vitro cell dynamics

    NARCIS (Netherlands)

    Baert, Yoni; Braye, Aude; Struijk, Robin B.; van Pelt, Ans M. M.; Goossens, Ellen

    2015-01-01

    To assess whether testicular cell dynamics are altered during long-term culture after testicular tissue cryopreservation. Experimental basic science study. Reproductive biology laboratory. Testicular tissue with normal spermatogenesis was obtained from six donors. None. Detection and comparison of

  11. Comparative proteome analysis of cryopreserved flagella and head plasma membrane proteins from sea bream spermatozoa: effect of antifreeze proteins.

    Science.gov (United States)

    Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

    2014-01-01

    Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to

  12. Sperm DNA fragmentation induced by cryopreservation: new insights and effect of a natural extract from Opuntia ficus-indica.

    Science.gov (United States)

    Meamar, Mehrdad; Zribi, Nassira; Cambi, Marta; Tamburrino, Lara; Marchiani, Sara; Filimberti, Erminio; Fino, Maria Grazia; Biggeri, Annibale; Menezo, Yves; Forti, Gianni; Baldi, Elisabetta; Muratori, Monica

    2012-08-01

    To analyze the effect of cryopreservation on sperm DNA fragmentation (SDF) in two cytometric sperm populations, PI(brighter) and PI(dimmer), and to test the effects of Opuntia ficus-indica (OFI) extracts, which contain antioxidants and flavanoids, and of resveratrol on cryopreservation of human semen. In vitro prospective study. Institutional study. Twenty-one normozoospermic men undergoing semen analysis for couple infertility. Cryopreservation using the routine method in the presence of OFI extracts or resveratrol. Measurement of SDF by TUNEL/PI flow cytometric method to evaluate sperm motility (by automated motion analysis, CASA system) and viability (by eosin/nigrosin staining) in the two populations of sperm PI(br) and PI(dim). Cryopreservation induced an increase of SDF only in the PI(br) sperm population. The increase was negatively dependent on the basal values of SDF in the same population. Addition of OFI extracts and resveratrol to the cryopreservation medium slightly but statistically significantly reduced SDF in the PI(br) population without affecting the deleterious effect of cryopreservation on sperm motion parameters or viability. The increase of SDF in the PI(br) population, which is unrelated to semen quality, suggests that caution must be taken in using cryopreserved semen, as morphologically normal and motile sperm may be damaged. The addition of substances with multifunctional properties such as OFI extracts to cryopreservation medium is only slightly effective in preventing the dramatic effects on SDF. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  13. The Effect of a GnRH Agonist Injection or Progesterone Implant at Diestrus in Cryopreserved Embryo Transferred Cows

    OpenAIRE

    KIRBAŞ, Mesut; BÜLBÜL, Bülent; KÖSE, Mehmet; DURSUN, Şükrü; ÇOLAK, Mehmet

    2014-01-01

    In this study, the effect of a single dose of GnRH on d 13 or progesterone implant for 7 days between d 13 and 20 on plasma progesterone levels and pregnancy rates on cryopreserved embryo transferred cows were investigated. Synchronized 48 Brown Swiss recipient cows were used as animal material. Seven days after estrus detection, cryopreserved cattle embryos were transferred into recipients and cows were assigned randomly into three groups. In GnRH group (n=16), cows were intramuscularly inje...

  14. Cryopreservation of chayote (Sechium edule JACQ.SW.) zygotic embryos and shoot-tips from in vitroplantlets

    OpenAIRE

    Abdelnour-Esquivel, Ana; Engelmann, Florent

    2002-01-01

    This paper presents the development of cryopreservation protocols for zygotic embryos and apices of chayote (Sechium edule Jacq. Sw.), a tropical plant species with recalcitrant seeds. Zygotic embryos of two cultivars, Ccocro negro (CN) and Claudio (Cl) could withstand cryopreservation, with survival percentages of 10 and 30 %, after desiccation to 23 and 19 % moisture content (fresh weight basis), respectively. Apices sampled on in vitro plantlets of cultivars Cl, 13 and JM we...

  15. Effects of combined cryopreservation and decellularization on the biomechanical, structural and biochemical properties of porcine pulmonary heart valves.

    Science.gov (United States)

    Theodoridis, Karolina; Müller, Janina; Ramm, Robert; Findeisen, Katja; Andrée, Birgit; Korossis, Sotirios; Haverich, Axel; Hilfiker, Andres

    2016-10-01

    Non-fixed, decellularized allogeneic heart valve scaffolds seem to be the best choice for heart valve replacement, their availability, however, is quite limited. Cryopreservation could prolong their shelf-life, allowing for their ideal match to a recipient. In this study, porcine pulmonary valves were decellularized using detergents, either prior or after cryopreservation, and analyzed. Mechanical integrity was analyzed by uniaxial tensile testing, histoarchitecture by histological staining, and composition by DNA, collagen (hydroxyproline) and GAG (chondroitin sulfate) quantification. Residual sodium dodecyl sulfate (SDS) in the scaffold was quantified by applying a methylene blue activation assay (MBAS). Cryopreserved decellularized scaffolds (DC) and scaffolds that were decellularized after cryopreservation (CD) were compared to fresh valves (F), cryopreserved native valves (C), and decellularized only scaffolds (D). The E-modulus and tensile strength of decellularized (D) tissue showed no significant difference compared to DC and CD. The decellularization resulted in an overall reduction of DNA and GAG, with DC containing the lowest amount of GAGs. The DNA content in the valvular wall of the CD group was higher than in the D and DC groups. CD valves showed slightly more residual SDS than DC valves, which might be harmful to recipient cells. In conclusion, cryopreservation after decellularization was shown to be preferable over cryopreservation before decellularization. However, in vivo testing would be necessary to determine whether these differences are significant in biocompatibility or immunogenicity of the scaffolds. Absence of adverse effects on biomechanical stability of acellular heart valve grafts by cryopreservation, neither before nor after decellularization, allows the identification of best matching patients in a less time pressure dictated process, and therefore to an optimized use of a very limited, but best-suited heart valve prosthesis

  16. Cryopreservation of Boer goat spermatozoa: Comparison of two freezing extenders based on post-thaw sperm quality and fertility rates

    Directory of Open Access Journals (Sweden)

    Fitra Aji Pamungkas

    2014-06-01

    Full Text Available Boer goat have recently been popularly used for cross breeding with local goats. However, it is currently considered a breed at very limited number with relatively high prices . In this context, the cryopreservation of spermatozoa is important because it could be conserved for a very long period of time. Egg yolk extenders are most commonly used for cryopreservation of goat sperm. The aim of this study was to compare the ability of two extenders to maintain sperm viability after cryopreservation. Semen from three male Boer goat aged about 2-3 years old was collected using artificial vagina and frozen with Tris and Triladyl extender. The results showed that percentage of motility, viability and membrane integrity of spermatozoa with Tris and Triladyl extenders at every stage of cryopreservation showed not significantly difference (P>0.05, except the percentage of sperm motility post thawing of Triladyl was higher than Tris extender (52.00±4.47% vs 47.50±2.74%, P<0.05. Cryopreserved semen in Tris extender provided the same fertility rates after cervical insemination compared to Triladyl (62.50% vs 60.00%. In conclusion, the Tris extender has the same capabilities to Triladyl in cryopreservation of Boer goat spermatozoa as to maintain sperm quality and fertility rates.

  17. Cryopreservation of human oocytes, zygotes, embryos and blastocysts: A comparison study between slow freezing and ultra rapid (vitrification methods

    Directory of Open Access Journals (Sweden)

    Tahani Al-Azawi

    2013-12-01

    Full Text Available Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans. It can also be done by preservation of ovarian tissue or entire ovary for transplantation, followed by oocyte harvesting or natural fertilization. Two basic cryopreservation techniques rule the field, slow-rate freezing, the first to be developed and vitrification which in recent years, has gained a foothold. The slow-rate freezing method previously reported had low survival and pregnancy rates, along with the high cost of cryopreservation. Although there are some recent data indicating better survival rates, cryopreservation by the slow freezing method has started to discontinue. Vitrification of human embryos, especially at early stages, became a more popular alternative to the slow rate freezing method due to reported comparable clinical and laboratory outcomes. In addition, vitrification is relatively simple, requires no expensive programmable freezing equipment, and uses a small amount of liquid nitrogen for freezing. Moreover, oocyte cryopreservation using vitrification has been proposed as a solution to maintain women’s fertility by serving and freezing their oocytes at the optimal time. The aim of this research is to compare slow freezing and vitrification in cryopreservation of oocytes, zygotes, embryos and blastocysts during the last twelve years. Therefore, due to a lot of controversies in this regard, we tried to achieve an exact idea about the subject and the best technique used.

  18. Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells.

    Science.gov (United States)

    Wang, Chuan; Xiao, Ran; Cao, Yi-Lin; Yin, Hong-Yu

    2017-09-09

    Cryopreservation provides an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs). Dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) are frequently used as cryoprotectants for this purpose. However, the use of DMSO can result in adverse effects and toxic reactions and FBS can introduce risks of viral, prion, zoonose contaminations and evoke immune responses after injection. It is therefore crucial to reduce DMSO concentrations and use serum-free solution in the cryopreservation process. Human platelet lysate (PL) is a promising candidate for use as an alternative to DMSO and FBS. Therefore, in this study, with an aim to identify a cryoprotective agent for ASC cryopreservation, we determined the viability, proliferation potential, phenotype, and differentiation potential of fresh ASCs and ASCs cryopreserved using different combinations of three cryoprotective agents: fetal bovine serum (FBS), dimethylsulfoxide (DMSO), and human platelet lysate (PL). The viability of the ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO was >80%, and the proliferation potentials, cell phenotypes, and differentiation potentials of these groups were similar to those of fresh ASCs. Together, our findings suggest that a combination of 97% PL and 3% DMSO is an ideal cryoprotective agent for the efficient cryopreservation of human ASCs. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Effects of micro-encapsulation on morphology and endocrine function of cryopreserved neonatal porcine islet-like cell clusters.

    Science.gov (United States)

    Murakami, M; Satou, H; Kimura, T; Kobayashi, T; Yamaguchi, A; Nakagawara, G; Iwata, H

    2000-10-27

    For the success of clinical islets transplantation, the development of a long-term storage method is necessary. However, the structure of digested islets is scanty for culture and cryopreservation. In this study, the effect of micro-encapsulation to cryopreserved porcine islet-like cell clusters (ICCs) was investigated. The ICCs prepared from neonatal pigs by collagenase digestion and culture technique were cryopreserved and micro-encapsulated in 5% agarose membranes. After cryopreservation, ICC cultured without encapsulation (group A) and cultured with encapsulation (group B) were assessed by comparison with no cryopreserved ICC (control) both in vitro by static incubation test and in vivo in a xenotransplantation study. Micro-encapsulation was able to maintain the fine morphology and the number of ICCs of group B after 7 days of culture. There were not significant differences in insulin secretion of group B and control on day 1 and 7 of culture (1 day:11+/-0.99, 7 days: 5.30+/-1.08 microU/ICC/hr NS versus control). On day 7 of culture, the retrieval rate of group B (105.2+/-9.8%) is obviously higher compared with group A (63.0+/-6.3%). In the xenotransplatation model, the ICCs of group B showed long survival time (7.9+/-0.4 weeks) and good transplantation effect. Our study suggests that micro-encapsulation is one of the useful method for cryopreserved ICC to maintain the fine morphology and effectively recover the endocrine function.

  20. Antioxidant and antigenotoxic role of recombinant human erythropoeitin against alkylating agents: cisplatin and mitomycin C in cultured Vero cells.

    Science.gov (United States)

    Rjiba-Touati, Karima; Ayed-Boussema, Imen; Soualeh, Nidhal; Achour, Abdellatif; Bacha, Hassen; Abid, Salwa

    2013-08-01

    Cisplatin (CDDP) and mitomycin C (MMC), two alkylating agents used against various solid tumours, are a common source of acute kidney injury. Thus, strategies for minimizing CDDP and MMC toxicity are of a clinical interest. In this study, we aimed to investigate the protective role of recombinant human erythropoietin (rhEPO) against oxidative stress and genotoxicity induced by CDDP and MMC in cultured Vero cells. Three types of treatments were performed: (i) cells were treated with rhEPO 24 h before exposure to CDDP/MMC (pre-treatment), (ii) cells were treated with rhEPO and CDDP/MMC simultaneously (co-treatment), (iii) cells were treated with rhEPO 24 h after exposure to CDDP/MMC (post-treatment). Our results showed that rhEPO decreased the reactive oxygen species levels, the malondialdehyde levels and ameliorated glutathione (reduced and oxidized glutathione) modulation induced by CDDP and MMC in cultured Vero cells. Furthermore, rhEPO administration prevented alkylating agents-induced DNA damage accessed by comet test. Altogether, our results suggested a protective role of rhEPO, against CDDP- and MMC-induced oxidative stress and genotoxicity, especially in pre-treatment condition.

  1. One-year outcomes of a bilateral randomised prospective clinical trial comparing PRK with mitomycin C and LASIK.

    Science.gov (United States)

    Wallau, A D; Campos, M

    2009-12-01

    To compare 1-year follow-up results of photorefractive keratectomy (PRK) with mitomycin C (MMC) and laser in situ keratomileusis (LASIK) for custom correction of myopia. Eighty-eight eyes of 44 patients with moderate myopia were randomised to PRK with 0.002% MMC for 1 min in one eye and LASIK in the fellow eye. The 1-year follow-up was evaluated. There were no differences between LASIK and MMC-PRK eyes preoperatively. Forty-two patients completed the 1-year follow-up. MMC-PRK eyes achieved better uncorrected visual acuity (p = 0.03) and better best-spectacle-corrected visual acuity (pPRK eyes postoperatively. Excellent vision was reported in 64% of LASIK and 74% of MMC-PRK eyes 1 year after surgery. The corneal resistance factor and corneal hysteresis (ORA, Reichert) were higher in LASIK than in MMC-PRK eyes (pPRK with 0.002% MMC was more effective than wavefront-guided LASIK for correction of moderate myopia. Further research is necessary to determine the optimal concentration, exposure time and long-term corneal side effect of MMC.

  2. Induction of protein synthesis in Escherichia coli following UV- or γ-irradiation, mitomycin C treatment or tif expression

    International Nuclear Information System (INIS)

    West, S.C.; Emmerson, P.T.

    1977-01-01

    The rate of synthesis of total cellular proteins has been studied by pulse labelling cells at various periods after irradiation with UV or γ-rays, after treatment with mitomycin C (MMC) or after expression of the temperature sensitive mutation tif. Subsequent gel electrophoresis and autoradiography reveals changes in the rate of synthesis of several proteins. The most striking change is in the protein X. Synthesis of large quantities of protein X is induced by UV, γ-rays, MMC treatment or tif expression in rec + but not recA cells. A feature of recA cells is that they break down their DNA excessively after irradiation or MMC treatment. However, if protein synthesis is prohibited by chloramphenicol, post-irradiation degradation becomes excessive in recA + cells. This inverse relationship between DNA degradation and new protein synthesis is consistent with the hypothesis that an induced protein such as X is responsible for controlling DNA degradation following irradiation. Protein X is not induced in a lexB mutant following MMC treatment. In this respect the lexB mutant behaves like lexA and recA mutants in that the ability to induce protein X can be correlated with excessive DNA degradation. Studies on the induction of proteins in inf, tif and tif sfi mutants fail to reveal any correlation between induction of protein X and either the induction of prophage lambda or septation. (orig./MG) [de

  3. Survival benefit from chemotherapy with mitomycin-c vinblastine and cisplatin in advanced non-small cell lung cancer

    International Nuclear Information System (INIS)

    Joaquin, C.F.

    1992-01-01

    Between January 1989 and May 1991 a prospective trial was conducted among the patients in the Lung Center of the Philippines who were diagnosed to have unresectable and metastatic non-small cell lung cancer (NSCLC). There were two groups of patients: those who consented to chemotherapy with the mitomycin, vinblastine and cisplatin regimen (n=31) and those who refused any form of chemotherapy or radiation (n=15). These groups were followed up and compared as to patient characteristics and duration of survival. The results show no identifiable features in the responders and non-responders to chemotherapy which could predict tumor response. The median survival of the untreated group was 15 weeks and that of the treated group was 34 weeks. This was statistically significant. No significant difference in survival between the responders and the non-responders was observed. The objective tumor response rate to the MVP regimen was 25.8%. The most common toxic effects were emesis and hematologic abnormalities. The study recommends the option of chemotherapy with the MVP regimen rather than no treatment at all after considering the risks and benefits for the patient with advanced stage NSCLC. (auth.). 19 refs.; 2 figs.; 4 tabs

  4. Effect and Mechanism of Mitomycin C Combined with Recombinant Adeno-Associated Virus Type II against Glioma

    Directory of Open Access Journals (Sweden)

    Hong Ma

    2013-12-01

    Full Text Available The effect of chemotherapy drug Mitomycin C (MMC in combination with recombinant adeno-associated virus II (rAAV2 in cancer therapy was investigated, and the mechanism of MMC affecting rAAV2’s bioactivity was also studied. The combination effect was evaluated by the level of GFP and TNF expression in a human glioma cell line, and the mechanism of MMC effects on rAAV mediated gene expression was investigated by AAV transduction related signal molecules. C57 and BALB/c nude mice were injected with rAAV-EGFP or rAAV-TNF alone, or mixed with MMC, to evaluate the effect of MMC on AAV-mediated gene expression and tumor suppression. MMC was shown to improve the infection activity of rAAV2 both in vitro and in vivo. Enhancement was found to be independent of initial rAAV2 receptor binding stage or subsequent second-strand synthesis of target DNA, but was related to cell cycle retardation followed by blocked genome degradation. In vivo injection of MMC combined with rAAV2 into the tumors of the animals resulted in significant suppression of tumor growth. It was thus demonstrated for the first time that MMC could enhance the expression level of the target gene mediated by rAAV2. The combination of rAAV2 and MMC may be a promising strategy in cancer therapy.

  5. Ocular Fluid As a Replacement for Serum in Cell Cryopreservation Media.

    Directory of Open Access Journals (Sweden)

    Vivek Phani Varma

    Full Text Available Cryostorage is of immense interest in biomedical research, especially for stem cell-based therapies and fertility preservation. Several protocols have been developed for efficient cryopreservation of cells and tissues, and a combination of dimethyl sulfoxide (DMSO and fetal bovine serum (FBS is commonly used. However, there is a need for an alternative to FBS because of ethical reasons, high cost, and risk of contamination with blood-borne diseases. The objective of the present study was to examine the possibility of using buffalo (Bubalus bubalis ocular fluid (BuOF to replace FBS in cryomedia. Frozen-thawed cells, which were cryopreserved in a cryomedia with BuOF, were assessed for viability, early and late apoptosis, and proliferation. Three cell lines (CHO, HEK, and C18-4, mouse embryonic stem (mES cells, and primary cells, such as mouse embryonic fibroblast (MEF cells, human peripheral blood mononuclear cells (hPBMCs, and mouse bone marrow cells (mBMCs, were cryopreserved in cryomedia containing 10% DMSO (D10 with 20% FBS (D10S20 or D10 with 20% BuOF (D10O20. For all three cell lines and mES cells cryopreserved in either D10S20 or D10O20, thawed cells showed no difference in cell viability or cell recovery. Western blot analysis of frozen-thawed-cultured cells revealed that the expression of Annexin V and proliferating cell nuclear antigen (PCNA proteins, and the ratio of BAX/BCL2 proteins were similar in all three cell lines, mES cells, and hPBMCs cryopreserved in D10S20 and D10O20. However, initial cell viability, cell recovery after culture, and PCNA expression were significantly lower in MEF cells, and the BAX/BCL2 protein ratio was elevated in mBMCs cryopreserved in D10O20. Biochemical and proteomic analysis of BuOF showed the presence of several components that may have roles in imparting the cryoprotective property of BuOF. These results encourage further research to develop an efficient serum-free cryomedia for several cell types

  6. INTERRELATION BETWEEN TYPE AND CULTIVAR OF BERRY-LIKE CROP AND CUTTINGS CRYOPRESERVATION RESULTS

    Directory of Open Access Journals (Sweden)

    L. V. Gorbunov

    2013-04-01

    Full Text Available Optimal cryopreservation parameters were determined for the berry-like crop cuttings: blackcurrant (Dachnica, Yuviley, Sofiivska, Kitayskaya; redcurrant (Kitaivska, Djoker, Svyatkova; gooseberries (Krasen, Malachite, Kolobok; raspberries (Novost’ Kuz’mina, Struyka, Skromnica. This procedure allowed increasing viability of deconservation samples in 2–5 times compared to the existing methods of cryopreservation. Maximal values of cutting viability were obtained after their temperature adaptation at –10 °C during 14–60 days, stepwise cooling of the samples with 0.1–0.5 °C/hr rate down to –30 °C (exposure 3–7 days, direct plunging into liquid nitrogen, storage from 1 to 30 days and thawing rate of 70 °C/min. It was found that the in the region of allowable values for maximum viability of investigated birch (control cuttings humidity values correspond 32±40%, for blackcurrant are 40±50%, and raspberries 37±40 % in affirmative temperature span and 14±28 %, 30±40 %, 20±23 % in below zero. Using dispersion analysis it has been established that the development probability of frozenthawed plant cuttings significantly depends on the selected cultivar and cryopreservation method. It is noted that the force of influence η2А, due to the individual properties of the studied cultivars of berries, is comparable to that of the force of influence η2В of cryopreservation procedures. The differences of average indexes of viability of the studied cultivars were showed within a single species. It was found that these rates are different for black currant by 92%, redcurrant — 54%, gooseberries — 48%, raspberries — 70%. Using paired Student’s t-test has reduced to 3 times the error of representativeness of the sample that enabled to improve the reliability of the significance of differences compared to the values of the P ≥0,99. Selection of the cuttings’ cultivars and cryopreservation method significantly affect the

  7. Generation of Platelet Microparticles after Cryopreservation of Apheresis Platelet Concentrates Contributes to Hemostatic Activity

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    İbrahim Eker

    2017-03-01

    Full Text Available Objective: In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs, platelet degranulation, and release of platelet-derived growth factors (PDGFs. We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation. Materials and Methods: Apheresis platelet concentrates (APCs from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing. Results: The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/μL vs. 319.9±80.5/μL, p<0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p<0.001, respectively, but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p<0.001 than those of fresh APCs. The mean endogenous thrombin potential (ETP of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p<0.001. Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014. Conclusion: Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused

  8. Alginate beads as a tool to handle, cryopreserve and culture isolated human primordial/primary follicles.

    Science.gov (United States)

    Camboni, A; Van Langendonckt, A; Donnez, J; Vanacker, J; Dolmans, M M; Amorim, C A

    2013-08-01

    One major concern of grafting cryopreserved ovarian tissue to restore fertility in cancer patients is the possibility of reintroducing tumor cells. Cryopreservation of isolated primordial/primary follicles (PFs) may circumvent this problem. The aim of our work was to compare dimethyl sulfoxide (ME2SO) and ethylene glycol (EG) as cryoprotectants (CPAs) for slow-freezing of isolated human PFs in alginate. Ovarian biopsies from four women were processed for follicle isolation. PFs were embedded in alginate (5-15 per group). Follicles were frozen-thawed using 1.4M ME2SO or 1.5M EG as CPAs. Fresh and cryopreserved isolated follicles were in vitro cultured (IVC) for 7 days. At different time periods (after isolation, cryopreservation and IVC), follicles were evaluated with live/dead assay (using fluorescent probes) and diameter measurement. Follicle viability was calculated according to the percentage of dead follicular cells and the presence of a live/dead oocyte. A total of 841 PFs were isolated, embedded in alginate and cryopreserved with ME2SO (n=424) or EG (n=259), or used as controls (n=158). After 7 days of IVC, a significant increase in follicle size was observed in the fresh and ME2SO groups, but not in the EG group. The percentage of totally viable PFs was not significantly different before or after seven days of culture in fresh (100% and 82%) or ME2SO (93.2% and 85.1%) tissue. The EG group showed significantly lower viability before (63.9%) and after IVC (66.2%) than the fresh and ME2SO groups. Our results show that 1.4M ME2SO yields better preservation of isolated PF viability after thawing and 7 days of IVC than 1.5M EG. Alginate constitutes an easy, safe hydrogel matrix to handle and cryopreserve isolated human follicles using ME2SO as a CPA. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo).

    Science.gov (United States)

    Słowińska, M; Liszewska, E; Dietrich, G J; Ciereszko, A

    2012-09-15

    This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After

  10. Cryopreservation in fish: current status and pathways to quality assurance and quality control in repository development.

    Science.gov (United States)

    Torres, Leticia; Hu, E; Tiersch, Terrence R

    2016-01-07

    Cryopreservation in aquatic species in general has been constrained to research activities for more than 60 years. Although the need for application and commercialisation pathways has become clear, the lack of comprehensive quality assurance and quality control programs has impeded the progress of the field, delaying the establishment of germplasm repositories and commercial-scale applications. In this review we focus on the opportunities for standardisation in the practices involved in the four main stages of the cryopreservation process: (1) source, housing and conditioning of fish; (2) sample collection and preparation; (3) freezing and cryogenic storage of samples; and (4) egg collection and use of thawed sperm samples. In addition, we introduce some key factors that would assist the transition to commercial-scale, high-throughput application.

  11. The Alpha consensus meeting on cryopreservation key performance indicators and benchmarks: proceedings of an expert meeting.

    Science.gov (United States)

    2012-08-01

    This proceedings report presents the outcomes from an international workshop designed to establish consensus on: definitions for key performance indicators (KPIs) for oocyte and embryo cryopreservation, using either slow freezing or vitrification; minimum performance level values for each KPI, representing basic competency; and aspirational benchmark values for each KPI, representing best practice goals. This report includes general presentations about current practice and factors for consideration in the development of KPIs. A total of 14 KPIs were recommended and benchmarks for each are presented. No recommendations were made regarding specific cryopreservation techniques or devices, or whether vitrification is 'better' than slow freezing, or vice versa, for any particular stage or application, as this was considered to be outside the scope of this workshop. Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  12. Partial protection of baboons against Schistosoma mansoni using radiation-attenuated cryopreserved schistosomula

    International Nuclear Information System (INIS)

    James, E.R.; Dobinson, A.R.; Otieno, M.; Monorei, J.; Else, J.G.

    1986-01-01

    Three groups of five baboons were vaccinated in Kenya using three doses of 10,000 viable cryopreserved schistosomula attenuated with either 10, 20 or 60 krad 60 co-irradiation. The results from perfusion indicated reductions in worm burdens in the 10, 20 and 60 krad vaccinated groups of 18%, 23% and 20% respectively, none of which was statistically significant. No stunting of adult worms could be demonstrated in any of the groups. Mean tissue egg burdens were higher in all vaccinated groups and consequently egg production per worm pair was also higher than in the challenge controls. The logistics of preparing and delivering a cryopreserved radiation-attenuated vaccine were amply demonstrated; however, in this study the levels of protection achieved were not statistically significant; possible reasons for this are discussed. (author)

  13. Live birth of twins after IVF of oocytes that were cryopreserved almost 12 years before.

    Science.gov (United States)

    Quintans, Carlos J; Donaldson, Monica J; Urquiza, M Fernanda; Carretero, Inés; Pasqualini, R Agustín; Horton, Marcos; Pasqualini, R Sergio

    2012-12-01

    A 45-year-old woman received embryos from IVF by intracytoplasmic sperm injection (ICSI) with her own oocytes that were cryopreserved (slow freezing in a low-sodium medium) 11 years and 7 and a half months before, when she was 33 years old. From seven metaphase-II oocytes thawed, five survived, four were fertilized after ICSI and two cleaving embryos were transferred on day 3. A diamniotic dichorionic term pregnancy was achieved, ending with the delivery of two healthy girls. As far as is known, this case represents, to date, the longest storage period of cryopreserved human oocytes resulting in a live birth. Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  14. Cryopreservation of spores of Dicksonia sellowiana: an endangered tree fern indigenous to South and Central America.

    Science.gov (United States)

    Rogge, G D; Viana, A M; Randi, A M

    2000-01-01

    Spores of Dicksonia sellowiana (Presl.) Hook., an endangered tree fern, were stored in liquid nitrogen. Surface sterilized spores were placed in 1 ml sterile polypropylene cryotubes and were plunged into liquid nitrogen cryo-cans for 15 minutes, 15 days, 1 month and 3 months. In all, of the treatments the percentage of germination was higher than the control (fresh spores). Germination in Dyer and MS media supplement with 10 (-7) M and 5 x 10(-7) M BA was also promoted as comparing to control. There was no difference between the germination of spores thawed rapidly in a water bath at 45 degree C during 5 minutes or slowly at room temperature. Cryopreservation seems to promote germination of some dormant spores of D. sellowiana. The pre-treatment in cryoprotective solution of dimethyl sulphoxide 15%(v/v) in 1 M glycerol inhibited the germination of cryopreserved spores

  15. Polyproline as a Minimal Antifreeze Protein Mimic That Enhances the Cryopreservation of Cell Monolayers.

    Science.gov (United States)

    Graham, Ben; Bailey, Trisha L; Healey, Joseph R J; Marcellini, Moreno; Deville, Sylvain; Gibson, Matthew I

    2017-12-11

    Tissue engineering, gene therapy, drug screening, and emerging regenerative medicine therapies are fundamentally reliant on high-quality adherent cell culture, but current methods to cryopreserve cells in this format can give low cell yields and require large volumes of solvent "antifreezes". Herein, we report polyproline as a minimum (bio)synthetic mimic of antifreeze proteins that is accessible by solution, solid-phase, and recombinant methods. We demonstrate that polyproline has ice recrystallisation inhibition activity linked to its amphipathic helix and that it enhances the DMSO cryopreservation of adherent cell lines. Polyproline may be a versatile additive in the emerging field of macromolecular cryoprotectants. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Effect of Previous Chemotherapy on the Quality of Cryopreserved Human Ovarian Tissue In Vitro.

    Directory of Open Access Journals (Sweden)

    Babak Asadi Azarbaijani

    Full Text Available Cryopreservation of ovarian tissue has been widely accepted as an option for fertility preservation among cancer patients. Some patients are exposed to chemotherapy prior to ovarian tissue cryopreservation. Consequently, assessment of the developmental capacity of human ovarian tissue after chemotherapy is of primary importance.In order to study the impact of previous chemotherapy on in vitro development and viability of ovarian follicles, quality control samples from 34 female cancer patients at median age of 15 years (range 1‒35, cryopreserved for fertility preservation before (n = 14 or after (n = 20 initiation of chemotherapy, were thawed and cultured for 7 days. The morphology and developmental stages of ovarian follicles were studied by light microscopy before and after culture. Possible associations between follicular densities, age and exposure to alkylating agents, expressed as cyclophosphamide equivalent dose (CED were tested.Exposure to chemotherapy significantly impaired the survival and development of ovarian follicles in culture. After seven days, significantly higher densities of intermediary, primary and secondary follicles and lower densities of atretic follicles was detected in the samples collected before chemotherapy. Increasing dose of alkylating agents was identified by multivariate linear regression analysis as an independent predictor of a higher density of atretic follicles, whereas increasing age of the patient predicted a better outcome with less follicle atresia and a higher density of maturing follicles.This study provides quantitative in vitro evidence of the impact of chemotherapy on developmental capacity of cryopreserved human ovarian tissue. The results indicate that fertility preservation should be carried out, if possible, before initiation of alkylating agents in order to guarantee better in vitro survival of ovarian follicles. In addition, ovarian samples from younger girls show lower viability and fewer

  17. Assisted Reproductive Technology and Newborn Size in Singletons Resulting from Fresh and Cryopreserved Embryos Transfer

    OpenAIRE

    Levi Dunietz, Galit; Holzman, Claudia; Zhang, Yujia; Talge, Nicole M.; Li, Chenxi; Todem, David; Boulet, Sheree L.; McKane, Patricia; Kissin, Dmitry M.; Copeland, Glenn; Bernson, Dana; Diamond, Michael P.

    2017-01-01

    Objectives and Study Design The aim of this study was two-fold: to investigate the association of Assisted Reproductive Technology (ART) and small newborn size, using standardized measures; and to examine within strata of fresh and cryopreserved embryos transfer, whether this association is influenced by parental infertility diagnoses. We used a population-based retrospective cohort from Michigan (2000?2009), Florida and Massachusetts (2000?2010). Our sample included 28,946 ART singletons con...

  18. ANALYSIS OF INDIVIDUAL BIOLOGICAL CHARACTERISTICS OF AMUR CARP (CYPRINUS CARPIO HAEMATOPTERUS REPRODUCED USING CRYOPRESERVED SPERM

    Directory of Open Access Journals (Sweden)

    N. Kolisnyk

    2014-12-01

    Full Text Available Purpose. To reproduce Amur carp population using cryopreserved sperm and analyze some biological and fish culture peculiarities of the reproduced fish stock. Methodology. Generally accepted methods for fish culture [1]. Experimental reproduction was carried out in pond conditions of «Carpathian vodogray» LTD (Lisnevychi village, Pustomytivsky district, Lviv region. Hydrochemical analysis was carried out classically by O. Alуokin (1970 [2], hydrobiological studies in the fatting ponds according to V. Zhadin (1956, 1960 [3, 4]. Haemoglobin concentration was determined by hemocyanin method of G. Dervis, A. Vorobiov [5]. Blood for this method was collected from fish heart with the use of Pasteur pipettes in Eppendorf tubes with heparin. Following exterior morphometric parameters were analysed: body weight (m, g, standard fish body length (l, cm, largest body height (H, cm and body circumference (O cm. Following exterior indices were calculated based on these parameters: body depth index (l/H, body circumference index (l/O and Fulton’s condition factor (Kv. The study was carried out using two groups of carp: control and experimental. The first group was reproduced from the native sperm, the second from the cryopreserved sperm. Findings. Carp reproduction and growing was carried out using native and cryopreserved sperm. This work contains the results of growing 1+ Amur carp of experimental and control groups. Hydrochemical and hydrobiological parameters of the fattening ponds were studied. Peculiarities of the exterior and some hematological parameters of the carp of different origin were characterized. Originality. For the first time we performed a comparison of some biological parameters of Amur carp reproduced using native and cryopreserved sperm. Practical Value. Considering the economic importance of Amur carp due to its use in hybridization, reproduction of its population plays an important role in the development of the stocks of the pure

  19. Thermo-mechanical stress analysis of cryopreservation in cryobags and the potential benefit of nanowarming.

    Science.gov (United States)

    Solanki, Prem K; Bischof, John C; Rabin, Yoed

    2017-06-01

    Cryopreservation by vitrification is the only promising solution for long-term organ preservation which can save tens of thousands of lives across the world every year. One of the challenges in cryopreservation of large-size tissues and organs is to prevent fracture formation due to the tendency of the material to contract with temperature. The current study focuses on a pillow-like shape of a cryobag, while exploring various strategies to reduce thermo-mechanical stress during the rewarming phase of the cryopreservation protocol, where maximum stresses are typically found. It is demonstrated in this study that while the level of stress may generally increase with the increasing amount of CPA filled in the cryobag, the ratio between width and length of the cryobag play a significant role. Counterintuitively, the overall maximum stress is not found when the bag is filled to its maximum capacity (when the filled cryobag resembles a sphere). Parametric investigation suggests that reducing the initial rewarming rate between the storage temperature and the glass transition temperature may dramatically decrease the thermo-mechanical stress. Adding a temperature hold during rewarming at the glass transition temperature may reduce the thermo-mechanical stress in some cases, but may have an adverse effect in other cases. Finally, it is demonstrated that careful incorporation of volumetric heating by means on nanoparticles in an alternating magnetic field, or nanowarming, can dramatically reduce the resulting thermo-mechanical stress. These observations display the potential benefit of a thermo-mechanical design of the cryopreservation protocols in order to prevent structural damage. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Optimized cryopreservation of mixed microbial communities for conserved functionality and diversity.

    Directory of Open Access Journals (Sweden)

    Frederiek-Maarten Kerckhof

    Full Text Available The use of mixed microbial communities (microbiomes for biotechnological applications has steadily increased over the past decades. However, these microbiomes are not readily available from public culture collections, hampering their potential for widespread use. The main reason for this lack of availability is the lack of an effective cryopreservation protocol. Due to this critical need, we evaluated the functionality as well as the community structure of three different types of microbiomes before and after cryopreservation with two cryoprotective agents (CPA. Microbiomes were selected based upon relevance towards applications: (1 a methanotrophic co-culture (MOB, with potential for mitigation of greenhouse gas emissions, environmental pollutants removal and bioplastics production; (2 an oxygen limited autotrophic nitrification/denitrification (OLAND biofilm, with enhanced economic and ecological benefits for wastewater treatment, and (3 fecal material from a human donor, with potential applications for fecal transplants and pre/probiotics research. After three months of cryopreservation at -80 °C, we found that metabolic activity, in terms of the specific activity recovery of MOB, aerobic ammonium oxidizing bacteria (AerAOB and anaerobic AOB (AnAOB, anammox in the OLAND mixed culture, resumes sooner when one of our selected CPA [dimethyl sulfoxide (DMSO and DMSO plus trehalose and tryptic soy broth (DMSO+TT] was added. However, the activity of the fecal community was not influenced by the CPA addition, although the preservation of the community structure (as determined by 16S rRNA gene sequencing was enhanced by addition of CPA. In summary, we have evaluated a cryopreservation protocol that succeeded in preserving both community structure and functionality of value-added microbiomes. This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.

  1. Cryopreserved Ultra-Thick Human Amniotic Membrane for Conjunctival Surface Reconstruction After Excision of Conjunctival Tumors.

    Science.gov (United States)

    Tanaka, Thais S; Demirci, Hakan

    2016-04-01

    Cryopreserved ultra-thick human amniotic membrane (AM) is used for glaucoma surgery. We evaluated the use of cryopreserved ultra-thick human AM for conjunctival surface reconstruction after excision of a conjunctival tumor. We retrospectively reviewed the medical records of 28 patients who underwent conjunctival surface reconstruction with cryopreserved ultra-thick human AM after excision of the tumor. The AM was secured to the surrounding conjunctiva and underlying sclera with interrupted 8-0 Vicryl sutures. Clinical data regarding demographics, diagnosis, size and location of conjunctival tumors, patient outcome, and complications were gathered. Of 28 patients, 6 (21.4%) had malignant melanoma, 4 (14.3%) had squamous cell carcinoma, 6 (21.4%) had conjunctival intraepithelial neoplasia, 1 (3.6%) had sebaceous carcinoma, 1 (3.6%) had mucoepidermoid carcinoma, 1 (3.6%) had conjunctival intraepithelial dysplasia, 5 (17.9%) had pterygium, 2 (7.1%) had compound nevus, 1 (3.6%) had a large epithelial inclusion cyst, and 1 (3.6%) patient had a granuloma. The mean area of graft size was 156 ± 120 mm2. Postoperatively, the graft was well tolerated with no failure, discomfort, or dehiscence. During the 17-month mean follow-up, symblepharon, which was clinically nonsignificant, developed in 3 (11%) patients and partial stem cell deficiency was noted in 5 (18%) patients. Cryopreserved ultra-thick human AM is a well-tolerated, effective graft material that is easy to handle. It is a viable alternative for conjunctival surface reconstruction after excision of a conjunctival tumor.

  2. Recovery from supercooling, freezing, and cryopreservation stress in larvae of the drosophilid fly, Chymomyza costata

    Czech Academy of Sciences Publication Activity Database

    Štětina, Tomáš; Hůla, Petr; Moos, Martin; Šimek, Petr; Šmilauer, P.; Košťál, Vladimír

    2018-01-01

    Roč. 8, č. 1 (2018), č. článku 4414. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GA16-06374S Institutional support: RVO:60077344 Keywords : insect * cold tolerance * cryopreservation Subject RIV: ED - Physiology OBOR OECD: Biology (theoretical, mathematical, thermal, cryobiology, biological rhythm), Evolutionary biology Impact factor: 4.259, year: 2016 http://www.nature.com/articles/s41598-018-22757-0

  3. Recent advances in the field of ovarian tissue cryopreservation and opportunities for research.

    Science.gov (United States)

    Ladanyi, Camille; Mor, Amir; Christianson, Mindy S; Dhillon, Namisha; Segars, James H

    2017-06-01

    The purpose of this study was to summarize the latest advances and successes in the field of ovarian tissue cryopreservation while identifying gaps in current knowledge that suggest opportunities for future research. A systematic review was performed according to PRISMA guidelines for all relevant full-text articles in PubMed published in English that reviewed or studied historical or current advancements in ovarian tissue cryopreservation and auto-transplantation techniques. Ovarian tissue auto-transplantation in post-pubertal women is capable of restoring fertility with over 80 live births currently reported with a corresponding pregnancy rate of 23 to 37%. The recently reported successes of live births from transplants, both in orthotopic and heterotopic locations, as well as the emerging methods of in vitro maturation (IVM), in vitro culture of primordial follicles, and possibility of in vitro activation (IVA) suggest new fertility options for many women and girls. Vitrification, as an ovarian tissue cryopreservation technique, has also demonstrated successful live births and may be a more cost-effective method to freezing with less tissue injury. Further, transplantation via the artificial ovary with an extracellular tissue matrix (ECTM) scaffolding as well as the effects of sphingosine-1-phosphate (SIP) and fibrin modified with heparin-binding peptide (HBP), heparin, and a vascular endothelial growth factor (VEGF) have demonstrated important advancements in fertility preservation. As a fertility preservation method, ovarian tissue cryopreservation and auto-transplantation are currently considered experimental, but future research may pave the way for these modalities to become a standard of care for women facing the prospect of sterility from ovarian damage.

  4. In vitro reactivity to concanavalin A of bovine lymphocytes after cryopreservation

    NARCIS (Netherlands)

    Kuil, H.

    1984-01-01

    Cryopreservation of bovine peripheral lymphocytes and its effect on the in vitro response to concanavalin A tested in a microculture system is described. Using DMSO as cryoprotectant in the medium, the cells were cooled to −30°C at 1.3°C/minute and further to −80°C at 6°C/minute and then rapidly to

  5. Characterization of cryopreserved primary human corneal endothelial cells cultured in human serum-supplemented media

    Directory of Open Access Journals (Sweden)

    Lucas Monferrari Monteiro Vianna

    2016-02-01

    Full Text Available ABSTRACT Purpose: To compare cryopreserved human corneal endothelial cells (HCECs grown in human serum-supplemented media (HS-SM with cryopreserved HCECs grown in fetal bovine serum-supplemented media (FBS-SM. Methods: Three pairs of human corneas from donors aged 8, 28, and 31 years were obtained from the eye bank. From each pair, one cornea was used to start a HCEC culture using HS-SM; the other cornea was grown in FBS-SM. On reaching confluence, the six cell populations were frozen using 10% dimethyl sulfoxidecontaining medium. Thawed cells grown in HS-SM were compared with those grown in FBS-SM with respect to morphology, growth curves, immunohistochemistry, real time-reverse transcriptase polymerase chain reaction (RT-PCR for endothelial cell markers, and detachment time. Results: No difference in morphology was observed for cells grown in the two media before or after cryopreservation. By growth curves, cell counts after thawing were similar in both media, with a slight trend toward higher cell counts in FBS-SM. Cells grown in both the media demonstrated a similar expression of endothelial cell markers when assessed by immunohistochemistry, although HCEC marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM as assessed by RT-PCR. With FBS-SM, there was a tendency of longer detachment time and lower cell passages. Conclusions: HS-SM was similar to FBS-SM for cryopreservation of cultured HCECs as assessed by analysis of cell morphology, proliferation, and protein expression, although marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM. Detachment time was longer with FBS-SM and in lower passages.

  6. Successful use of the Cryolock device for cryopreservation of scarce human ejaculate and testicular spermatozoa.

    Science.gov (United States)

    Stein, A; Shufaro, Y; Hadar, S; Fisch, B; Pinkas, H

    2015-03-01

    The existing methods for cryopreservation of very low count sperm samples are complex and sub-optimal for individual spermatozoa. Our purpose is to establish an effective simple method for cryoprotecting individual spermatozoa. Samples from patients with OTA were mixed with TYB or HEPES-buffered salt solution with glycerol + glucose and placed on a Cryolock that was plunged directly into liquid nitrogen or exposed to its vapors. Thawing was performed by direct immersion into a drop of warmed medium. The favorable method was tested on diluted samples (10-50 cells) and leftover TESE specimens from patients with azoospermia. Cryopreservation was considered successful if >30 spermatozoa, (>3 motile), or >5 spermatozoa (>1 motile) in diluted and TESE samples, were detected post-thawing. A significantly higher survival rate of seminal spermatozoa was obtained when using the Cryolock with TYB and freezing with liquid nitrogen vapor, compared to HEPES glycerol-glucose (95 vs. 35% respectively). Plunging the Cryolock into liquid nitrogen was detrimental. Cryolock combined with TYB cryoprotection and liquid nitrogen vapor freezing was highly effective for cryopreservation of individual spermatozoa in diluted and TESE samples. The Cryolock may serve for freezing very low-count sperm samples and individual spermatozoa. This method offers simplicity, efficacy, use of available materials, without requiring micromanipulation equipment or skills. © 2015 American Society of Andrology and European Academy of Andrology.

  7. Sperm quality and cryopreservation of Brazilian freshwater fish species: a review.

    Science.gov (United States)

    Viveiros, A T M; Godinho, H P

    2009-03-01

    The Brazilian freshwater fish diversity is the richest in the world. Only 0.7% of all Brazilian species have had any aspect of their sperm biology addressed up to this date. The majority of the fish species described in this review migrate during the spawning season (a phenomenon known as piracema). Urbanization, pollution, hydroelectric dams and deforestation are some of the causes of stock depletion or even local extinction of some of these species. The knowledge concerning sperm quality and minimum sperm:egg ratio is important to maximize the use of males without reducing hatching rates. Furthermore, sperm cryopreservation and gene banking can guarantee the conservation of genetic diversity and development of adequate breeding programs of native fish species. In this review, we present and evaluate the existing information on Brazilian fish species that have been subject to sperm quality and cryopreservation studies. The following parameters were evaluated: volume of extractable sperm, sperm motility, sperm concentration, freezing media, freezing methods, and post-thaw sperm quality. Although the existing protocols yield relatively high post-thaw motility and fertilization rates, the use of cryopreserved sperm in routine hatchery production is still limited in Brazil.

  8. Whole ovary cryopreservation with vascular transplantation – A future development in female oncofertility

    Directory of Open Access Journals (Sweden)

    Mats Brännström

    2010-07-01

    Full Text Available Preservation of female fertility in female cancer victims is gaining more importance in the light of the excellent survival rates today after treatment for the types of cancer, which are common in women during childhood and fertile age. In this article we discuss the risks of infertility after various cancer treatments and in what patient categories fertility preservation should be discussed. The modes of female fertility preservation used today as clinically established methods or as experimental methods that are used in the human female are reviewed. The advantages and disadvantages of the methods are also identified. Whole ovary cryopreservation has been suggested as a possible future more efficient method of fertility preservation. The research on this method in different animal models is discussed in detail and unsolved questions are identified. To date there has not been any attempt to transplant a cryopreserved whole human ovary but human ovaries have already been cryopreserved for future fertility preservation purposes, when cryobiological and microsurgical techniques have been further refined.

  9. The effect of Curcuma longa extracted (curcumin) on the quality of cryopreserved boar semen.

    Science.gov (United States)

    Chanapiwat, Panida; Kaeoket, Kampon

    2015-09-01

    The aim of this study was to determine the optimal concentration of curcumin needed for cryopreservation of boar semen. Semen samples (n = 9) were collected from nine Duroc boars which having proven fertility were used for routine artificial insemination. Semen samples were collected and divided into six groups (groups A-F) according to various concentrations of curcumin in freezing extender (i.e. 0, 0.125, 0.25, 0.50, 0.75 and 1.0 mmol/L, respectively). The semen was frozen by traditional liquid nitrogen vapor method and stored at -196°C in the liquid nitrogen tank. After storage, frozen semen samples were thawed at 50°C for 12 s and evaluated for progressive motility, viability and acrosome integrity. The present results indicated that the addition of curcumin at 0.25 (group C) or 0.50 mmol/L curcumin (group D) yielded the higher percentage of progressive motility (33.3 and 36.1%, respectively) (P curcumin during cryopreservation at a concentration of 0.25 or 0.50 mmol/L is the optimal concentration of curcumin for improving the quality (i.e. increased progressive motility and acrosome integrity) of cryopreserved boar semen. © 2015 Japanese Society of Animal Science.

  10. Noninvasive embryo assessment technique based on buoyancy and its association with embryo survival after cryopreservation.

    Science.gov (United States)

    Wessels, Cara; Penrose, Lindsay; Ahmad, Khaliq; Prien, Samuel

    2017-11-01

    Embryo cryopreservation offers many benefits by allowing genetic preservation, genetic screening, cost reduction, global embryo transport and single embryo transfer. However, freezing of embryos decreases embryo viability, as intracellular ice crystal formation often damages embryos. Success rates of frozen embryo transfer are expected to be 15-20% less than fresh embryo transfer. We have developed a noninvasive embryo assessment technique (NEAT) which enables us to predict embryo viability based on buoyancy. The purpose of this research was twofold. First was to determine if a NEAT, through a specific gravity device can detect embryo survival of cryopreservation. Second, it was to relate embryo buoyancy to embryo viability for establishing pregnancies in sheep. Blastocysts descent times were measured on one-hundred sixty-nine mice blastocysts before cryopreservation, according to standard protocol and post-thawing blastocysts descent times were measured again. There was a significant difference in blastocyst post-thaw descent times with NEAT in those blastocysts which demonstrated viability from those that did not (P embryos. Further studies on a larger scale commercial setting will evaluate the efficacy of NEAT. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Soybean lecithin-based extender preserves spermatozoa membrane integrity and fertilizing potential during goat semen cryopreservation.

    Science.gov (United States)

    Chelucci, Sara; Pasciu, Valeria; Succu, Sara; Addis, Daniela; Leoni, Giovanni G; Manca, Maria E; Naitana, Salvatore; Berlinguer, Fiammetta

    2015-04-01

    Soybean lecithin may represent a suitable alternative to egg yolk for semen cryopreservation in livestock species. However, additional studies are needed to elucidate its effects on spermatozoa functional properties. Semen collected from five Sarda bucks was cryopreserved in Tris-based extender and glycerol (4% v:v) with different supplementations. In a preliminary experiment, different soybean lecithin concentrations were tested (1%-6% wt/vol) and results in terms of viability, percentages of progressive motile and rapid spermatozoa, and DNA integrity after thawing showed that the most effective concentration was 1%. In the second experiment, semen was frozen in a Tris-based extender with no supplementation (EXT), with 1% lecithin (EXT LC), and 20% egg yolk (EXT EY). The effectiveness of these extenders was also compared with a commercial extender. The EXT EY led to the highest viability and motility parameters after freezing and thawing (P lecithin can be considered as a suitable alternative to egg yolk in goat semen cryopreservation, because it ensures higher fertilization rates and a better protection from membrane damage by cold shock. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Cryopreservation of artificial gut microbiota produced with in vitro fermentation technology.

    Science.gov (United States)

    Bircher, Lea; Schwab, Clarissa; Geirnaert, Annelies; Lacroix, Christophe

    2018-01-01

    Interest in faecal microbiota transplantation (FMT) has increased as therapy for intestinal diseases, but safety issues limit its widespread use. Intestinal fermentation technology (IFT) can produce controlled, diverse and metabolically active 'artificial' colonic microbiota as potential alternative to common FMT. However, suitable processing technology to store this artificial microbiota is lacking. In this study, we evaluated the impact of the two cryoprotectives, glycerol (15% v/v) and inulin (5% w/v) alone and in combination, in preserving short-chain fatty acid formation and recovery of major butyrate-producing bacteria in three artificial microbiota during cryopreservation for 3 months at -80°C. After 24 h anaerobic fermentation of the preserved microbiota, butyrate and propionate production were maintained when glycerol was used as cryoprotectant, while acetate and butyrate were formed more rapidly with glycerol in combination with inulin. Glycerol supported cryopreservation of the Roseburia spp./Eubacterium rectale group, while inulin improved the recovery of Faecalibacterium prausnitzii. Eubacterium hallii growth was affected minimally by cryopreservation. Our data indicate that butyrate producers, which are key organisms for gut health, can be well preserved with glycerol and inulin during frozen storage. This is of high importance if artificially produced colonic microbiota is considered for therapeutic purposes. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  13. Seedling development and evaluation of genetic stability of cryopreserved Dendrobium hybrid mature seeds.

    Science.gov (United States)

    Galdiano, Renato Fernandes; de Macedo Lemos, Eliana Gertrudes; de Faria, Ricardo Tadeu; Vendrame, Wagner Aparecido

    2014-03-01

    Vitrification, a simple, fast, and recommended cryopreservation method for orchid germplasm conservation, was evaluated for Dendrobium hybrid "Dong Yai" mature seeds. The genetic stability of regenerated seedlings was also evaluated using flow cytometry. Mature seeds from this hybrid were submitted to plant vitrification solution (PVS2) for 0, 0.5, 1, 2, 3, 4, 5, or 6 h at 0 °C. Subsequently, they were plunged into liquid nitrogen (LN) at -196 °C for 1 h and recovered in half-strength Murashige and Skoog culture medium (1/2 MS), and seed germination was evaluated after 30 days. Seeds directly submitted to LN did not germinate after cryopreservation. Seeds treated with PVS2 between 1 and 3 h presented the best germination (between 51 and 58%), although longer exposure to PVS2 returned moderated germination (39%). Germinated seeds were further subcultured in P-723 culture medium and developed whole seedlings in vitro after 180 days, with no abnormal characteristics, diseases, or nutritional deficiencies. Seedlings were successfully acclimatized under greenhouse conditions with over 80% survival. Flow cytometry analysis revealed no chromosomal changes on vitrified seedlings, as well as seedlings germinated from the control treatment (direct exposure to LN). These findings indicate that vitrification is a feasible and safe germplasm cryopreservation method for commercial Dendrobium orchid hybrid conservation.

  14. Comparison of conventional freezing and vitrification with dimethylformamide and ethylene glycol for cryopreservation of ovine embryos.

    Science.gov (United States)

    Varago, F C; Moutacas, V S; Carvalho, B C; Serapião, R V; Vieira, F; Chiarini-Garcia, H; Brandão, F Z; Camargo, L S; Henry, M; Lagares, M A

    2014-10-01

    The aim of this work was to evaluate the efficiency of the cryoprotectants dimethylformamide and ethylene glycol for cryopreservation of ovine embryos using vitrification and conventional freezing. The recovered embryos were distributed randomly in three treatment groups: Gr. 1: conventional freezing (n = 44), Gr. 2: vitrification with ethylene glycol (n = 39) and Gr. 3: vitrification with dimethylformamide (n = 38). Quality of fresh embryos in control group as well as of frozen and vitrified embryos was examined by three methodologies: staining with propidium iodide and Hoechst 33258 and evaluation under fluorescent microscopy, evaluation of re-expansion and hatching rates after culture, and determination of apoptotic index with TUNEL technique. It was established that re-expansion rate in all treatment groups was similar. In the same time, hatching rates were higher in Gr. 1 (40.5%) and Gr. 2 (35.3%) in comparison with Gr. 3 (15.5%, p conventional freezing, 10.1 ± 8.5, p conventional freezing) and fresh embryos. In conclusion, the dimethylformamide and ethylene glycol used as cryoprotectant to vitrify ovine embryos, in the concentrations and exposition time tested in this work, were not as efficient as the conventional freezing for cryopreservation of ovine embryos Thus, the conventional freezing with ethylene glycol was the most efficient method to cryopreserve ovine embryos in comparison with vitrification. © 2014 Blackwell Verlag GmbH.

  15. Should we isolate human preantral follicles before or after cryopreservation of ovarian tissue?

    Science.gov (United States)

    Vanacker, Julie; Luyckx, Valérie; Amorim, Christiani; Dolmans, Marie-Madeleine; Van Langendonckt, Anne; Donnez, Jacques; Camboni, Alessandra

    2013-04-01

    To evaluate the survival and growth potential of human preantral follicles isolated before and after cryopreservation. Pilot study. Gynecology research unit in a university hospital. Six women aged 27 to 32 years. Six ovarian biopsy samples were cut into two equal parts, half subjected to slow-freezing followed by follicle isolation (cryo-iso group) and alginate-matrigel embedding, and half immediately processed for follicle isolation and alginate-matrigel embedding followed by slow-freezing (iso-cryo group) or used as fresh controls (fresh group). Follicle number, viability, diameter, and morphology. After 1,134 preantral follicles had been isolated from fresh biopsy samples and 1,132 from frozen specimens, the three groups were compared before and after 7 days of in vitro culture (IVC) in alginate-matrigel beads. No statistically significant differences in viability were found between the three groups before or after IVC, but follicle diameter increased in all three groups after IVC. Morphology analysis revealed well-preserved follicles in both the iso-cryo and cryo-iso groups after IVC. Human preantral follicles can be successfully cryopreserved before or after isolation without impairing their ability to survive and grow in vitro. This could lead to development of new protocols for follicle cryopreservation, IVC, and grafting in clinical and research settings for fertility preservation. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  16. Behavior of lateral buds of Hancornia speciosa after cryopreservation by encapsulation-vitrification

    Directory of Open Access Journals (Sweden)

    Débora de Oliveira Prudente

    2017-05-01

    Full Text Available Hancornia speciosa is a fruitful species from Cerrado biome with high economic potential. However, the intense and disordered extractivism have caused a reduction of its population in its endemic area. In addition, seed recalcitrance negatively affects the conventional conservation of the species. Aiming to find alternatives that enable the long-term conservation of this species, the study’s objective was to assess the behavior of lateral bud’s regrowth after cryopreservation procedures by encapsulation-vitrification technique. Sodium alginate capsules containing lateral buds were pre-cultured in liquid WPM supplemented with 1.0 M glycerol, and subsequently exposed to different concentrations of sucrose (0.3; 0.75 and 1.0 M for 24 or 48 hours. The capsules were subjected to dehydration in silica gel or airflow hood for 0, 1, 2 and 3 hours before different incubation times in PVS2 (0, 15, 30, 60 and 120 minutes at 0°C. A high regeneration percentage of lateral buds was observed after cryopreservation of capsules treated with 0.75 M sucrose plus 1.0 M glycerol (24 hours, associated with dehydration in an airflow hood (1 hour and immersion in PVS2 (15 minutes. Encapsulation-vitrification allowed the long-term conservation, and provided high plant material survival rates after cryopreservation of Hancornia speciosa sensitive explants.

  17. Ovarian tissue cryopreservation by stepped vitrification and monitored by X-ray computed tomography.

    Science.gov (United States)

    Corral, Ariadna; Clavero, Macarena; Gallardo, Miguel; Balcerzyk, Marcin; Amorim, Christiani A; Parrado-Gallego, Ángel; Dolmans, Marie-Madeleine; Paulini, Fernanda; Morris, John; Risco, Ramón

    2018-04-01

    Ovarian tissue cryopreservation is, in most cases, the only fertility preservation option available for female patients soon to undergo gonadotoxic treatment. To date, cryopreservation of ovarian tissue has been carried out by both traditional slow freezing method and vitrification, but even with the best techniques, there is still a considerable loss of follicle viability. In this report, we investigated a stepped cryopreservation procedure which combines features of slow cooling and vitrification (hereafter called stepped vitrification). Bovine ovarian tissue was used as a tissue model. Stepwise increments of the Me 2 SO concentration coupled with stepwise drops-in temperature in a device specifically designed for this purpose and X-ray computed tomography were combined to investigate loading times at each step, by monitoring the attenuation of the radiation proportional to Me 2 SO permeation. Viability analysis was performed in warmed tissues by immunohistochemistry. Although further viability tests should be conducted after transplantation, preliminary results are very promising. Four protocols were explored. Two of them showed a poor permeation of the vitrification solution (P1 and P2). The other two (P3 and P4), with higher permeation, were studied in deeper detail. Out of these two protocols, P4, with a longer permeation time at -40 °C, showed the same histological integrity after warming as fresh controls. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Ice Recrystallization Inhibiting Polymers Enable Glycerol-Free Cryopreservation of Micro-organisms.

    Science.gov (United States)

    Hasan, Muhammad; Fayter, Alice E R; Gibson, Matthew I

    2018-06-22

    All modern molecular biology and microbiology is underpinned not only by the tools to handle and manipulate microorganisms, but also those to store, bank and transport them. Glycerol is the current gold-standard cryoprotectant but it is intrinsically toxic to most micro-organisms: only a fraction of cells survive freezing and the presence of glycerol can impact down-stream applications and assays. Extremophile organisms survive repeated freeze/thaw cycles by producing antifreeze proteins which are potent ice recrystallization inhibitors. Here we introduce a new concept for the storage/transport of micro-organisms by using ice recrystallization inhibiting poly(vinyl alcohol) in tandem with poly(ethylene glycol). This cryopreserving formulation is shown to result in a 4-fold increase in E. coli yield post-thaw, compared to glycerol, utilizing lower concentrations, with successful cryopreservation at just 1.1 weight percent of additive. The mechanism of protection is demonstrated to be linked to inhibiting ice recrystallization (by comparison to a recombinant antifreeze protein) but also to the significantly lower toxicity of the polymers compared to glycerol. Optimized formulations are presented and shown to be broadly applicable to the cryopreservation of a panel of Gram negative, Gram positive and Mycobacteria strains. This represents a step-change in how micro-organisms will be stored by the design of new macromolecular ice growth inhibitors; it should enable a transition from traditional solvent-based to macromolecular microbiology storage methods.

  19. Mechanical compliance and immunological compatibility of fixative-free decellularized/cryopreserved human pericardium.

    Directory of Open Access Journals (Sweden)

    Maria Cristina Vinci

    Full Text Available BACKGROUND: The pericardial tissue is commonly used to produce bio-prosthetic cardiac valves and patches in cardiac surgery. The procedures adopted to prepare this tissue consist in treatment with aldehydes, which do not prevent post-graft tissue calcification due to incomplete xeno-antigens removal. The adoption of fixative-free decellularization protocols has been therefore suggested to overcome this limitation. Although promising, the decellularized pericardium has not yet used in clinics, due to the absence of proofs indicating that the decellularization and cryopreservation procedures can effectively preserve the mechanical properties and the immunologic compatibility of the tissue. PRINCIPAL FINDINGS: The aim of the present work was to validate a procedure to prepare decellularized/cryopreserved human pericardium which may be implemented into cardiovascular homograft tissue Banks. The method employed to decellularize the tissue completely removed the cells without affecting ECM structure; furthermore, uniaxial tensile loading tests revealed an equivalent resistance of the decellularized tissue to strain, before and after the cryopreservation, in comparison with the fresh tissue. Finally, immunological compatibility, showed a minimized host immune cells invasion and low levels of systemic inflammation, as assessed by tissue transplantation into immune-competent mice. CONCLUSIONS: Our results indicate, for the first time, that fixative-free decellularized pericardium from cadaveric tissue donors can be banked according to Tissue Repository-approved procedures without compromising its mechanical properties and immunological tolerance. This tissue can be therefore treated as a safe homograft for cardiac surgery.

  20. Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood

    International Nuclear Information System (INIS)

    Lee, Myoung Woo; Moon, Young Joon; Yang, Mal Sook; Kim, Sun Kyung; Jang, In Keun; Eom, Young-woo; Park, Joon Seong; Kim, Hugh C.; Song, Kye Yong; Park, Soon Cheol; Lim, Hwan Sub; Kim, Young Jin

    2007-01-01

    Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells, with practical and ethical advantages. To date, the presence of other stem cells in UCB remains to be established. We investigated whether other stem cells are present in cryopreserved UCB. Seeded mononuclear cells formed adherent colonized cells in optimized culture conditions. Over a 4- to 6-week culture period, colonized cells gradually developed into adherent mono-layer cells, which exhibited homogeneous fibroblast-like morphology and immunophenotypes, and were highly proliferative. Isolated cells were designated 'multipotent progenitor cells (MPCs)'. Under appropriate conditions for 2 weeks, MPCs differentiated into neural tissue-specific cell types, including neuron, astrocyte, and oligodendrocyte. Differentiated cells presented their respective markers, specifically, NF-L and NSE for neurons, GFAP for astrocytes, and myelin/oligodendrocyte for oligodendrocytes. In this study, we successfully isolated MPCs from cryopreserved UCB, which differentiated into the neural tissue-specific cell types. These findings suggest that cryopreserved human UCB is a useful alternative source of neural progenitor cells, such as MPCs, for experimental and therapeutic applications

  1. Ovarian tissue cryopreservation in girls undergoing haematopoietic stem cell transplant: experience of a single centre.

    Science.gov (United States)

    Biasin, E; Salvagno, F; Berger, M; Nesi, F; Quarello, P; Vassallo, E; Evangelista, F; Marchino, G L; Revelli, A; Benedetto, C; Fagioli, F

    2015-09-01

    Fertility after childhood haemopoietic stem cell transplant (HSCT) is a major concern. Conditioning regimens before HSCT present a high risk (>80%) of ovarian failure. Since 2000, we have proposed cryopreservation of ovarian tissue to female patients undergoing HSCT at our centre, to preserve future fertility. After clinical and haematological evaluation, the patients underwent ovarian tissue collection by laparoscopy. The tissue was analysed by histologic examination to detect any tumour contamination and then frozen following the slow freezing procedure and cryopreserved in liquid nitrogen. From August 2000 to September 2013, 47 patients planned to receive HSCT, underwent ovarian tissue cryopreservation. The median age at diagnosis was 11.1 years and at the time of procedure it was 13 years, respectively. Twenty-four patients were not pubertal at the time of storage, whereas 23 patients had already experienced menarche. The median time between laparoscopy and HSCT was 25 days. Twenty-six out of 28 evaluable patients (93%) developed hypergonadotropic hypogonadism at a median time of 23.3 months after HSCT. One patient required autologous orthotopic transplantation that resulted in one live birth. Results show a very high rate of iatrogenic hypergonadotropic hypogonadism, highlighting the need for fertility preservation in these patients.

  2. Cryopreservation of adenovirus-transfected dendritic cells (DCs) for clinical use.

    Science.gov (United States)

    Gülen, D; Maas, S; Julius, H; Warkentin, P; Britton, H; Younos, I; Senesac, J; Pirruccello, Samuel M; Talmadge, J E

    2012-05-01

    In this study, we examined the effects of cryoprotectant, freezing and thawing, and adenovirus (Adv) transduction on the viability, transgene expression, phenotype, and function of human dendritic cells (DCs). DCs were differentiated from cultured peripheral blood (PB) monocytes following Elutra isolation using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6 days and then transduced using an Adv vector with an IL-12 transgene. Fresh, cryopreserved, and thawed transduced immature DCs were examined for their: 1) cellular concentration and viability; 2) antigenicity using an allogeneic mixed lymphocyte reaction (MLR); 3) phenotype (HLA-DR and CD11c) and activation (CD83); and 4) transgene expression based on IL-12 secretion. Stability studies revealed that transduced DCs could be held in cryoprotectant for as long as 75 min at 2-8°C prior to freezing with little effect on their viability and cellularity. Further, cryopreservation, storage, and thawing reduced the viability of the transduced DCs by an average of 7.7%; and had no significant impact on DC phenotype and activation. In summary, cryopreservation, storage, and thawing had no significant effect on DC viability, function, and transgene expression by Adv-transduced DCs. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Supplemental effect of varying L-cysteine concentrations on the quality of cryopreserved boar semen

    Science.gov (United States)

    Kaeoket, Kampon; Chanapiwat, Panida; Tummaruk, Padet; Techakumphu, Mongkol

    2010-01-01

    Cryopreservation is associated with the production of reactive oxygen species, which leads to lipid peroxidation of the sperm membrane and consequently a reduction in sperm motility and decreased fertility potential. The aim of this study was to determine the optimal concentration of L-cysteine needed for cryopreservation of boar semen. Twelve boars provided semen of proven motility and morphology for this study. The semen was divided into four portions in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various concentrations of L-cysteine to reach 0 mmol L−1 (group I, control), 5 mmol L−1 (group II), 10 mmol L−1 (group III) and 15 mmol L−1 (group IV). Semen suspensions were loaded in straws (0.5 mL) and placed in a controlled-rate freezer. After cryopreservation, frozen semen samples were thawed and investigated for progressive motility, viability using SYBR-14/EthD-1 staining and acrosome integrity using FITC-PNA/EthD-1 staining. There was a significantly higher (P extender for improving the quality of frozen–thawed boar semen. PMID:20601963

  4. Microencapsulation Technology: A Powerful Tool for Integrating Expansion and Cryopreservation of Human Embryonic Stem Cells

    Science.gov (United States)

    Malpique, Rita; Brito, Catarina; Jensen, Janne; Bjorquist, Petter; Carrondo, Manuel J. T.; Alves, Paula M.

    2011-01-01

    The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors. The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics. Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks. This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications. PMID:21850261

  5. Endocrine disruption screening by protein and gene expression of vitellogenin in freshly isolated and cryopreserved rainbow trout hepatocytes.

    Science.gov (United States)

    Markell, Lauren K; Mingoia, Robert T; Peterson, Heather M; Yao, Jianhong; Waters, Stephanie M; Finn, James P; Nabb, Diane L; Han, Xing

    2014-08-18

    Xenobiotics may activate the estrogen receptor, resulting in alteration of normal endocrine functions in animals and humans. Consequently, this necessitates development of assay end points capable of identifying estrogenic xenobiotics. In the present study, we screened the potential estrogenicity of chemicals via their ability to induce vitellogenin (VTG) expression in cultured primary hepatocytes from male trout. A routine method for VTG detection measures the secretion of the protein by enzyme-linked immunosorbent assay (ELISA) in freshly isolated trout hepatocytes. However, this lengthy (6 days) culturing procedure requires that hepatocyte isolation is performed each time the assay is run. We optimized this methodology by investigating the utility of cryopreserved hepatocytes, shortening the incubation time, performing a quantitative real-time PCR (qPCR) method for VTG quantification, and verifying the model system with reference chemicals 17β-estradiol, estrone, diethylstilbestrol, hexestrol, genistein, and a negative control, corticosterone. To test the performance of both freshly isolated and cryopreserved hepatocytes, mRNA was collected from hepatocytes following 24 h treatment for VTG gene expression analysis, whereas cell culture media was collected for a VTG ELISA 96 h post-treatment. EC50 values were obtained for each reference chemical except for corticosterone, which exhibited no induction of VTG gene or protein level. Our results show linear concordance between ELISA and qPCR detection methods. Although there was approximately 50% reduction in VTG inducibility following cryopreservation, linear concordance of EC50 values was found between freshly isolated and cryopreserved hepatocytes, indicating that cryopreservation does not alter the functional assessment of estrogen receptor activation and therefore VTG expression. These studies demonstrate that qPCR is a sensitive and specific method for detecting VTG gene expression that can be used together

  6. Generation of juvenile rainbow trout derived from cryopreserved whole ovaries by intraperitoneal transplantation of ovarian germ cells.

    Science.gov (United States)

    Lee, Seungki; Katayama, Naoto; Yoshizaki, Goro

    2016-09-23

    Cryopreservation of fish sperm offers the practical applications in the selective breeding and biodiversity conservation. However, because of the lack of cryopreservation methods for fish eggs and embryos, maternally inherited cytoplasmic compartments cannot be successfully preserved. We previously developed an alternative method to derive functional eggs and sperm from cryopreserved whole testis by transplanting testicular cells into female and male recipients. However, if target fish had ovaries, the previous method employing male-derived germ cells would be ineffective. Here, we aimed to generate functional gametes from cryopreserved whole ovaries by transplanting ovarian germ cells into peritoneal cavity of sterile hatchlings. Cryopreservation conditions for rainbow trout ovaries (1.0 M DMSO, 0.1 M trehalose, and 10% egg yolk) were optimized by testing several different cryoprotective agents. Ovarian germ cells from thawed ovaries were intraperitoneally transplanted into allogeneic triploid hatchlings. Transplanted germ cells migrated toward and were incorporated into recipient gonads, where they underwent gametogenesis. Transplantation efficiency of ovarian germ cells remained stable after cryopreservation period up to 1185 days. Although all triploid recipients that did not undergo transplantation were functionally sterile, 5 of 25 female recipients and 7 of 25 male recipients reached sexual maturity at 2.5 years post-transplantation. Inseminating the resultant eggs and sperm generated viable offspring displaying the donor characteristics of orange body color, green fluorescence, and chromosome numbers. This method is thus a breakthrough tool for the conservation of endangered fish species that are crucial to cryopreserve the genetic resources of female fish. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Pediatric PRK (PhotoRefractive Keratectomy) with Mitomycin C (MCC) for Persistent Anisometropic Amblyopia. A Case Report.

    Science.gov (United States)

    Crawford, Courtney M; Frazier, Travis C; Torres, Mark F; Arnold, Robert W; Mazzoli, Robert A; Raymond, William R

    2012-01-01

    To evaluate the safety and efficacy of photorefractive keratectomy (PRK) with Mitomycin C (MMC) for the treatment of severe pediatric anisometropia and amblyopia resistant to more conservative treatment modalities. A 3 year-old-child, who at 18 months old underwent unilateral diode laser treatment for threshold ROP, developed 11 diopters of anisometropic myopia and secondary dense amblyopia of the Right Eye. Only after all conservative treatment options failed was he treated with PRK and MMC. Principal outcome measures included cycloplegic refraction, the amount of refractive correction, degree of corneal haze and change in visual acuity. On presentation: BCVA: 20/CF OD; 20/30 OS. CRNS: -11.50 diopters sphere OD; -0.50 diopters sphere OS. Unilateral PRK followed by application of MMC (0.2 mg/ml) for 1 min was performed under general anesthesia. Three-month postoperative findings include: VA: 20/30 OD; 20/25 OS. CRNS: +0.25 diopters sphere OD. At one year, the BCVA remained equal at the 20/30 level despite mild myopic regression OD. CRNS OD at one year was -1.25 +050 x 116. No corneal haze was appreciated. In this child, treatment with PRK and MMC safely reduced the anisometropia thus facilitating his visual rehabilitation. While encouraging, further study is required to verify the longer term results of this single case. To evaluate the safety and efficacy of photorefractive keratectomy (PRK) with Mitomycin C (MMC) for the treatment of severe pediatric anisometropia and amblyopia resistant to more conservative treatment modalities. A 3 year-old-child, who at 18 months old underwent unilateral diode laser treatment for threshold ROP, developed 11 diopters of anisometropic myopia and secondary dense amblyopia of the Right Eye. Only after all conservative treatment options failed was he treated with PRK and MMC. Principal outcome measures included cycloplegic refraction, the amount of refractive correction, degree of corneal haze and change in visual acuity. On

  8. Persistent hypotony after primary trabeculectomy with mitomycin C Hipotonia persistente depois de trabeculectomia primária com mitomicina C

    Directory of Open Access Journals (Sweden)

    Viviane R.F. Guedes

    2000-06-01

    Full Text Available Purpose: To describe the clinical findings and treatment modalities of persistent hypotony following primary trabeculectomy with mitomycin C. Methods: We retrospectively analyzed 9 eyes with persistent hypotony, which was defined as intraocular pressure less than or equal to 5 mmHg for more than 2 months. Results: Mean hypotony duration was 7.4 months (standard deviation (SD ± 6.7 months, range 2 to 23 months. Associated findings included choroidal detachment (2 eyes and maculopathy (5 eyes. All patients who developed maculopathy were relatively young (mean age = 37 years old, SD ± 16, range 18 to 79 years. Treatments included bandaged contact lens, autologous blood injection, phacoemulsification, resuturing of the scleral flap, scleral patch graft, and Simmons' shell. After treatment, intraocular pressure (IOP raised in all patients (mean final IOP = 11.1 mmHg, SD ± 3.5. On the first day of hypotony, the mean IOP was 3 mmHg (SD ±1.7. At the last follow up, visual acuity (VA was unchanged in 3 eyes, worsened in 2 eyes (by 2 Snellen lines, and improved (by 1 to 4 Snellen lines in 4 eyes. Of those eyes whose VA improved, 3 had undergone phacoemulsification. Conclusion: Hypotony after trabeculectomy with mitomycin C can be reversed with possible improvement in vision.Objetivo: Descrever os achados clínicos e as modalidades de tratamento da hipotonia persistente após trabeculectomia primária com mitomicina C. Método: Foram retrospectivamente analisados 9 olhos com hipotonia persistente, a qual foi definida como pressão intra-ocular igual ou menor que 5 mmHg por mais que 2 meses. Resultado: Tempo médio de duração da hipotonia foi de 7.4 meses, desvio padrão ± 6.7 meses. Complicações associadas à hipotonia incluíram descolamento de coróide (2 olhos, maculopatia (5 olhos. Todos os pacientes que desen-volveram maculopatia eram relativamente novos (idade media de 37 anos, desvio padrão ±16. Tratamentos incluíram lente de contato

  9. O6-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C

    International Nuclear Information System (INIS)

    Passagne, Isabelle; Evrard, Alexandre; Depeille, Philippe; Cuq, Pierre; Cupissol, Didier; Vian, Laurence

    2006-01-01

    Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O 6 -methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC 5 values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N 7 guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism

  10. O(6)-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C.

    Science.gov (United States)

    Passagne, Isabelle; Evrard, Alexandre; Depeille, Philippe; Cuq, Pierre; Cupissol, Didier; Vian, Laurence

    2006-03-01

    Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O(6)-methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC(50) values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N(7) guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism.

  11. Mitomycin-augmented non-penetrating deep sclerectomy: preoperative gonioscopy and postoperative perimetric, tonometric and medication trends.

    Science.gov (United States)

    Sponsel, William Eric; Groth, Sylvia Linner

    2013-03-01

    Non-penetrating deep sclerectomy (NPDS) can enhance drainage of aqueous humour without disrupting the trabecular endothelial layer, reducing risks of postoperative hypotony and hyphema. This study explores associations of angle morphology with surgical efficacy in eyes with open and obstructed angles. Eighty-nine consecutive eyes undergoing successful NPDS (non-implant, with 0.4 mg/ml mitomycin C and limbus-based two-layer closure) were studied in this institutional review board-approved retrospective quality assurance study. Postoperative complication frequency, intraocular pressure (IOP), glaucoma medications required and acuity were monitored (baseline vs 3, 6, 9, 12 and 18-month postoperative levels), along with 30-2 Humphrey MD and corrected pattern standard deviation (CPSD) (baseline vs 6, 12 and 18-month postoperative values). Preoperative gonioscopy was compared with the subsequent requirement for specific postoperative interventions. IOP at all five postoperative intervals was reduced (22 ± 0.9 to 12 ± 0.5 mm Hg; p<0.0001). No hyphema were observed. Postoperative hypotony (IOP < 4 mm Hg) occurred rarely (8/445; 1.8%). Mean glaucoma medication use dropped from 3.1 ± 0.1 to 0.23 ± 0.1 at 18 months (p<0.0001). Mean 30-2 MD improved by approximately 1.4 dB at 6, 12 and 18 months (p<0.002); CPSD remained stable. Following NPDS, a sustained IOP decrease of 10 mm Hg (45%) was attained, with stable acuity, increased perimetric generalised light sensitivity and 90% reduction in medical therapy requirement. Morbidity risk was associated with narrow gonioscopic angle insertion and synechia, but not with shallow approach or trabecular pigmentation.

  12. Efficacy of A Fluoropyrimidine plus Mitomycin C in Pretreated Patients with Metastatic Colorectal Cancer Eligible for Regorafenib: A Retrospective Study

    Directory of Open Access Journals (Sweden)

    Federica Martorana

    2017-11-01

    Full Text Available Objective: In the placebo-controlled CORRECT study, individuals with metastatic colorectal cancer (mCRC receiving Regorafenib (RGR achieved significant benefits in both median overall survival (OS: 6.4 months and progression-free survival (PFS 1.9 months. Patients included in the study had previously failed all standard therapies, which must have included Fluoropyrimidines (FPDs, Oxaliplatin, Irinotecan, Bevacizumab, and Cetuximab or Panitumumab for K-RAS wild-type subjects. FPDs plus Mitomycin C (MMC represent one of the few treatment options for mCRC patients currently eligible for RGR. We wanted to investigate the therapeutic benefit of this pharmacological association in the same clinical setting defined for RGR. Methods: We retrospectively evaluated the records of mCRC patients followed in our Institutions that would have fulfilled inclusion/exclusion criteria for the CORRECT trial and received instead the combination of FPDs and MMC. We therefore collected data from 87 patients: 61 fulfilled the criteria required for this analysis. Results: Median OS was 9.3 months (95% CI 9.0–15.4, with a median PFS of 3.3 months (95% CI 2.9–3.8. One third of the patients (29.5% achieved disease control. No significant differences in OS and PFS were found between K-RAS WT and K-RAS mutant individuals. Likewise, Performance Status (PS and the primary site of disease were not associated with differences in response rates. Conclusions: These results suggest the need for a prospective study assessing RGR cost-effectiveness compared to FPDs plus MMC for mCRC patients that progress after standard treatments.

  13. LASEK for the correction of hyperopia with mitomycin C using SCHWIND AMARIS excimer laser: one-year follow-up

    Directory of Open Access Journals (Sweden)

    Khosrow Jadidi

    2015-11-01

    Full Text Available AIM: To evaluate the efficacy, safety and predictability of laser-assisted sub-epithelial keratectomy(LASEKfor the correction of hyperopia using the SCHWIND AMARIS platform.METHODS: This retrospective single-surgeon study includes 66 eyes of 33 patients with hyperopia who underwent LASEK with mitomycin C(MMC. The median age of patients was 35.42±1.12y(ranging 18 to 56y. In each patient LASEK was performed using SCHWIND AMARIS excimer laser. Postoperatively clinical outcomes were evaluated in terms of predictability, safety, efficacy, subjective and objective refractions, uncorrected visual acuity(UCVA, best spectacle-corrected visual acuity(BSCVAand adverse events. RESULTS: The mean baseline refraction was 3.2±1.6 diopters(D(ranging 0 to 7 D. The mean pre-operative and postoperative spherical equivalent(SEwere 2.34±1.76(ranging -1.25 to 7 Dand 0.30±0.84(ranging -0.2 to 0.8 Drespectively(P=0.001. The mean hyperopia was 0.63±0.84 D(ranging -1.75 to 2.76 D6 to 12mo postoperatively. Likewise, the mean astigmatism was 0.68±0.43 D(range 0 to 2 Dwith 51(77.3%and 15(22.7%eyes within ±1 and ±0.50 D respectively. The safety index and efficacy index were 1.08 and 1.6 respectively.CONCLUSION:LASEK using SCHWIND AMARIS with MMC yields good visual and refractive results for hyperopia. Moreover, there were no serious complications.

  14. Phage induction by UV and mitomycin C in Pseudomonas mori, the pathogen of bacterial blight of mulberry

    International Nuclear Information System (INIS)

    Sato, Mamoru

    1979-01-01

    Phage induction by ultraviolet radiation (UV) and mitomycin C (MMC) in some lysogenic strains of Pseudomonas mori, the pathogen of bacterial blight of mulberry, was examined. Among 5 strains tested, in the strains S 6804 and S 6805, phage was induced by both UV and MMC, and in the strain M 5, only by MMC. In the strains S 6807 and S 6808, it was not induced by both these inducers. The rate of phage production in the strain 6805 was highest when it was exposed to UV (15 W UV lamp, 40 cm) for 5 seconds, by which about 90% of the bacteria were killed, and decreased rapidly by further extending the exposure time. The bacteria suspended in 0.02 M magnesium solution were more sensitive in responding to UV than those suspended in nutrient broth, but after the UV treatment, nutrient broth was more favorable than magnesium solution for phage production. The MMC added to nutrient broth induced phage production at the concentration from 0.5 to 5 μg/ml. The strains induced by either UV or MMC their temperate phages after about 3 hours of latent period. The phage induction by UV was almost completely suppressed by 40 minute exposure to fluorescent light (a 15 W fluorescent lamp, 10 cm) or by 5 minute exposure to sunlight, given within 45 minutes after the UV treatment, i.e. within 1/4 of the latent period. Thus, the photoreversion of the UV effect on phage induction was observed in Ps. mori as well as in Ps. pyocyanea and E. coli. (Kaihara, S.)

  15. Sensitivity to mitomycin-C and radiation of cells derived from patients with familial colon polyposis: an autosomal dominant hereditary disease

    International Nuclear Information System (INIS)

    Ban, Sadayuki; Iida, Shozo; Tamura, Taizo.

    1984-04-01

    This study was undertaken to investigate the sensitivity to mitomycin-C (MMC) of skin fibroblasts derived from patients with adenomatosis coli (AC), especially familial colon polyposis. The sensitivity to X rays and ultraviolet rays of AC cells cultured at RERF was similar to that of normal human diploid cells. However, there were large individual differences in sensitivity to MMC. DNA elongation in cells sensitive to MMC was found to be inhibited after MMC treatment. Sites highly sensitive to MMC were considered to be involved in the initial stages of DNA synthesis. (author)

  16. Application of mitomycin C after endoscopic lysis of congenital laryngeal web combined with epiglottic hypoplasia in a middle-aged man.

    Science.gov (United States)

    Roh, Jong-Lyel

    2006-04-01

    Laryngeal webs and epiglottic hypoplasias are uncommon congenital anomalies. Anterior glottic web combined with epiglottic hypoplasia was found in a middle-aged man presenting with hoarseness and dyspnea on exertion. This can be considered as a unique isolated defect of the larynx during early fetal development. The laryngeal web can be successfully treated in a single stage with endoscopic lysis and topical application of mitomycin C for prevention of anterior glottic restenosis. This case and prior reports suggest that the novel approach may be effective in the treatment of laryngeal webs.

  17. Abnormal sensitivity of diploid skin fibroblasts from a family with Gardner's syndrome to the lethal effects of X-irradiation, ultraviolet light and mitomycin-C

    International Nuclear Information System (INIS)

    Little, J.B.; Nove, J.; Weichselbaum, R.R.

    1980-01-01

    Skin fibroblasts isolated from two members of the same family with the cancer-prone disease Gardner's Syndrome (intestinal polyposis, colon cancer, bone and soft tissue tumors) showed enhanced sensitivity to the lethal effects of X-irradiation, ultraviolet light and mitomycin-C. These cells showed no liquid-holding type recovery following UV-irradiation of confluent cultures, but were normal in their capacity for UV-induced unscheduled DNA synthesis. UV survival was not influenced by post-irradiation incubation with caffeine. (orig.)

  18. Knowledge, attitudes, and intentions toward fertility awareness and oocyte cryopreservation among obstetrics and gynecology resident physicians.

    Science.gov (United States)

    Yu, L; Peterson, B; Inhorn, M C; Boehm, J K; Patrizio, P

    2016-02-01

    What knowledge, attitudes and intentions do US obstetrics and gynecology (OB/GYN) residents have toward discussing age-related fertility decline and oocyte cryopreservation with their patients? Most OB/GYN residents believe that age-related fertility decline, but not oocyte cryopreservation, should be discussed during well-woman annual exams; furthermore, nearly half of residents overestimated the age at which female fertility markedly declines. Oocyte cryopreservation can be utilized to preserve fertility potential. Currently, no studies of US OB/GYN residents exist that question their knowledge, attitudes, and intentions toward discussing age-related fertility decline and oocyte cryopreservation with patients. A cross-sectional online survey was conducted during the fall of 2014 among residents in American Council for Graduate (ACOG) Medical Education-approved OB/GYN residency programs. Program directors were emailed via the ACOG Council on Resident Education in Obstetrics and Gynecology server listing and asked to solicit resident participation. Participants included 238 residents evenly distributed between post-graduate years 1-4 with varied post-residency plans; 90% of residents were women and 75% were 26-30 years old. The survey was divided into three sections: demographics, fertility awareness, and attitudes toward discussing fertility preservation options with patients. Descriptive and inferential statistics were conducted. A strong majority of residents (83%) believed an OB/GYN should initiate discussions about age-related fertility decline with patients (mean patient age 31.8), and 73% percent believed these discussions should be part of an annual exam. One third of residents overestimated the age at which there is a slight decline in female fertility, while nearly half of residents overestimated the age at which female fertility markedly declines. Over three-quarters of residents (78.4%) also overestimated the likelihood of success using assisted

  19. Production of F1 interspecies hybrid offspring with cryopreserved sperm from a live-bearing fish, the swordtail Xiphophorus helleri.

    Science.gov (United States)

    Yang, Huiping; Hazlewood, Leona; Heater, Sheila J; Guerrero, Paula A; Walter, Ronald B; Tiersch, Terrence R

    2007-03-01

    Despite study of sperm cryopreservation in more than 200 fish species, production of broods from cryopreserved sperm in live-bearing fish has not been demonstrated. This has not been due to a lack of effort, but instead is a result of the unique morphology, biology, and biochemistry of reproduction in viviparous fishes. For example, sperm of Xiphophorus helleri have a cylindrical nucleus, can swim for days after being activated, have glycolytic capabilities, and can reside in the female reproduction tract for months before fertilization. These traits are not found in fishes with external fertilization. The long-standing research use of the genus Xiphophorus has led to development of over 60 pedigreed lines among the 26 species maintained around the world. These species and lines serve as contemporary models in medical research, although they must be maintained as live populations. Previous attempts at establishing sperm cryopreservation protocols for Xiphophorus have not produced live young. To address this we have been studying the parameters surrounding cryobiology of Xiphophorus sperm and applying this information to an improved understanding of internal fertilization and reproduction. Here we report the first successful fertilization and offspring production by cryopreserved sperm in any live-bearing fish. This claim is supported by our use of artificial insemination between two species that yield distinct hybrid offspring to verify paternity via cryopreserved sperm. We provide a practical approach for preservation of valuable genetic resources from live-bearing fish species, a group that is rapidly being lost due to destruction of native habitats.

  20. Development of cryopreservation for Loxocarya cinerea---an endemic Australian plant species important for post-mining restoration.

    Science.gov (United States)

    Kaczmarczyk, Anja; Funnekotter, Bryn; Turner, Shane R; Bunn, Eric; Bryant, Gary; Hunt, Taavi E; Mancera, Ricardo L

    2013-01-01

    We report the development of a cryopreservation protocol for the endemic Western Australian plant species Loxocarya cinerea (Restionaceae). Shoot tips from two genotypes, SXH404 and SXH804, were cryopreserved using the droplet-vitrification technique. Control explants, which were cryoprotected, but not cooled, showed regeneration for both genotypes (SXH404, 22.1 +/- 5.9%; SXH804, 67.7 +/- 9.6%). Extension of incubation in PVS2 from 30 to 60 min did not lead to survival after cryopreservation. Thermal analysis using differential scanning calorimetry confirmed the beneficial effect of a loading phase but also revealed no or very little ice formation after cryoprotection of shoot tips in other treatments. Regeneration following cryopreservation was obtained for genotype SXH804 (4.3 +/- 2.1%) but not for SXH404. Regenerated explants of L. cinerea SXH804 were morphologically identical to tissue-cultured plants. As an alternative to shoot tips, callus tissues of clone SXH404 were successfully cryopreserved (> 66.7% post LN survival) using the same protocol.

  1. Second-harmonic generation microscopy used to evaluate the effect of the dimethyl sulfoxide in the cryopreservation process in collagen fibers of differentiated chondrocytes

    Science.gov (United States)

    Andreoli-Risso, M. F.; Duarte, A. S. S.; Ribeiro, T. B.; Bordeaux-Rego, P.; Luzo, A.; Baratti, M. O.; Adur, J.; de Thomaz, A. A.; Pelegati, V. B.; Carvalho, H. F.; Cesar, C. L.; Kharmadayan, P.; Costa, F. F.; Olalla-Saad, S. T.

    2012-03-01

    Cartilaginous lesions are a significant public health problem and the use of adult stem cells represents a promising therapy for this condition. Cryopreservation confers many advantages for practitioners engaged in cell-based therapies. However, conventional slow freezing has always been associated with damage and mortality due to intracellular ice formation, cryoprotectant toxicity, and dehydration. The aim of this work is to observe the effect of the usual Dimethyl Sulfoxide (DMSO) cryopreservation process on the architecture of the collagen fiber network of chondrogenic cells from mesenchymal stem cells by Second Harmonic Generation (SHG) microscopy. To perform this study we used Mesenchymal Stem Cells (MSC) derived from adipose tissue which presents the capacity to differentiate into other lineages such as osteogenic, adipogenic and chondrogenic lineages. Mesenchymal stem cells obtained after liposuction were isolated digested by collagenase type I and characterization was carried out by differentiation of mesodermic lineages, and flow cytometry using specific markers. The isolated MSCs were cryopreserved by the DMSO technique and the chondrogenic differentiation was carried out using the micromass technique. We then compared the cryopreserved vs non-cryopreserved collagen fibers which are naturally formed during the differentiation process. We observed that noncryopreserved MSCs presented a directional trend in the collagen fibers formed which was absent in the cryopreserved MSCs. We confirmed this trend quantitatively by the aspect ratio obtained by Fast Fourier Transform which was 0.76 for cryopreserved and 0.52 for non-cryopreserved MSCs, a statistical significant difference.

  2. Evaluation of Glycerol Removal Techniques, Cryoprotectants, and Insemination Methods for Cryopreserving Rooster Sperm with Implications for Breed and/or Line Regeneration

    Science.gov (United States)

    A series of experiments was designed to evaluate the quality of cryopreserved rooster sperm and its fertility so that programs needing to bank germplasm and recreate animals can do so utilizing a minimal amount of cryopreserved semen. In Experiment 1, semen from roosters was collected and cryoprese...

  3. Ovarian function after removal of an entire ovary for cryopreservation of pieces of cortex prior to gonadotoxic treatment: a follow-up study

    DEFF Research Database (Denmark)

    Rosendahl, M.; Andersen, Claus Yding; Ernst, E.

    2008-01-01

    menstruation was shown to be a good indicator of ovarian function. The cryopreservation procedure rarely complicated cancer treatment (5%) and 84% felt comforted because they had potentially secured their fertility. CONCLUSIONS: Cryopreservation of ovarian tissue should be considered in young female patients...

  4. Cell banking for regulatory T cell-based therapy: strategies to overcome the impact of cryopreservation on the Treg viability and phenotype.

    Science.gov (United States)

    Gołąb, Karolina; Grose, Randall; Placencia, Veronica; Wickrema, Amittha; Solomina, Julia; Tibudan, Martin; Konsur, Evelyn; Ciepły, Kamil; Marek-Trzonkowska, Natalia; Trzonkowski, Piotr; Millis, J Michael; Fung, John; Witkowski, Piotr

    2018-02-09

    The first clinical trials with adoptive Treg therapy have shown safety and potential efficacy. Feasibility of such therapy could be improved if cells are cryopreserved and stored until optimal timing for infusion. Herein, we report the evaluation of two cell-banking strategies for Treg therapy: 1) cryopreservation of CD4 + cells for subsequent Treg isolation/expansion and 2) cryopreservation of ex-vivo expanded Tregs (CD4 + CD25 hi CD127 lo/- cells). First, we checked how cryopreservation affects cell viability and Treg markers expression. Then, we performed Treg isolation/expansion with the final products release testing. We observed substantial decrease in cell number recovery after thawing and overnight culture. This observation might be explained by the high percentage of necrotic and apoptotic cells found just after thawing. Furthermore, we noticed fluctuations in percentage of CD4 + CD25 hi CD127 - and CD4 + FoxP3 + cells obtained from cryopreserved CD4 + as well as Treg cells. However, after re-stimulation Tregs expanded well, presented a stable phenotype and fulfilled the release criteria at the end of expansions. Cryopreservation of CD4 + cells for subsequent Treg isolation/expansion and cryopreservation of expanded Tregs with re-stimulation and expansion after thawing, are promising solutions to overcome detrimental effects of cryopreservation. Both of these cell-banking strategies for Treg therapy can be applied when designing new clinical trials.

  5. Cryopreservation, semen use and the likelihood of fatherhood in male Hodgkin lymphoma survivors: an EORTC-GELA Lymphoma Group cohort study.

    Science.gov (United States)

    van der Kaaij, M A E; van Echten-Arends, J; Heutte, N; Meijnders, P; Abeilard-Lemoisson, E; Spina, M; Moser, E C; Allgeier, A; Meulemans, B; Lugtenburg, P J; Aleman, B M P; Noordijk, E M; Fermé, C; Thomas, J; Stamatoullas, A; Fruchart, C; Eghbali, H; Brice, P; Smit, W G J M; Sebban, C; Doorduijn, J K; Roesink, J M; Gaillard, I; Coiffier, B; Lybeert, M L M; Casasnovas, O; André, M; Raemaekers, J M M; Henry-Amar, M; Kluin-Nelemans, J C

    2014-03-01

    How does the successful cryopreservation of semen affect the odds of post-treatment fatherhood among Hodgkin lymphoma (HL) survivors? Among 334 survivors who wanted to have children, the availability of cryopreserved semen doubled the odds of post-treatment fatherhood. Cryopreservation of semen is the easiest, safest and most accessible way to safeguard fertility in male patients facing cancer treatment. Little is known about what proportion of patients achieve successful semen cryopreservation. To our knowledge, neither the factors which influence the occurrence of semen cryopreservation nor the rates of fatherhood after semen has been cryopreserved have been analysed before. This is a cohort study with nested case-control analyses of consecutive Hodgkin survivors treated between 1974 and 2004 in multi-centre randomized controlled trials. A written questionnaire was developed and sent to 1849 male survivors. Nine hundred and two survivors provided analysable answers. The median age at treatment was 31 years. The median follow-up after cryopreservation was 13 years (range 5-36). Three hundred and sixty-three out of 902 men (40%) cryopreserved semen before the start of potentially gonadotoxic treatment. The likelihood of semen cryopreservation was influenced by age, treatment period, disease stage, treatment modality and education level. Seventy eight of 363 men (21%) used their cryopreserved semen. Men treated between 1994 and 2004 had significantly lower odds of cryopreserved semen use compared with those treated earlier, whereas alkylating or second-line (chemo)therapy significantly increased the odds of use; no other influencing factors were identified. We found an adjusted odds ratio of 2.03 (95% confidence interval 1.11-3.73, P = 0.02) for post-treatment fatherhood if semen cryopreservation was performed. Forty-eight out of 258 men (19%) who had children after HL treatment became a father using cryopreserved semen. Data came from questionnaires and so this

  6. Transarterial chemoperfusion with gemcitabine and mitomycin C in pancreatic carcinoma: Results in locally recurrent tumors and advanced tumor stages; Transarterielle Chemoperfusion mit Gemcitabine und Mitomycin C bei Pankreaskarzinom: Ergebnisse bei Rezidivtumoren und fortgeschrittenen Tumorstadien

    Energy Technology Data Exchange (ETDEWEB)

    Vogl, T.J.; Zangos, S.; Heller, M.; Hammerstingl, R.M.; Bauer, R.W. [Inst. fuer Diagnostische und Interventionelle Radiologie, J. W. Goethe-Univ. Frankfurt (Germany); Boecher, E. [Klinik Paradise, Medizinische Klinik, Soest (Germany); Jacob, U. [Leonardisklinik, Onkologische Fachklinik, Bad Heilbrunn (Germany)

    2007-11-15

    Purpose: The purpose of this study was to evaluate local transarterial chemoperfusion (TACP) in locally recurrent pancreatic carcinoma and advanced tumor stages which did not respond to prior systemic chemotherapy. The tumor response, survival, and pain response were retrospectively analyzed. Materials and method: Forty outpatients (median age 62 years, range 36 - 79) were treated with a minimum of 3 (mean 6, range 3 - 12) applications per patient in four-week intervals. Twenty-eight patients were in advanced tumor stages, and 12 patients had locally recurrent tumors. Gemcitabine (1,000 mg/m{sup 2}) and mitomycin C (8.5 mg/m{sup 2}) were administered within 1 hour through a celiac trunk catheter. The tumor response (diameter, volume) was measured using MRI or CT and classified according to RECIST. The pain response was defined as a reduction of pain intensity of more than 50% on a visual analog scale, or a reduction of more than 50% in analgesics consumption, or a switch to a less potent analgesic agent. Results: The treatment was tolerated well by all patients. No clinically relevant problems or grade III or IV toxicity according to CTC (Common Toxicity Criteria) were observed. Tumor-related pain was relieved in 20/32 (62.5%) cases. Radiologically, 'complete response' was found in 3/40 (7.5%), 'partial response' in 9/40 (22.5%), 'stable disease' in 16/40 (40%), and 'progressive disease' in 12/40 (30%) of the patients. The median survival period since initial diagnosis and first TACP was 16.4 months and 8.1 months, respectively. Locally recurrent tumors showed better, but still not significant results regarding tumor response (41.7% vs. 25%) as well as survival (14.4 vs. 7 months) compared to advanced tumor stages. Responders (CR + PR) showed a significant survival advantage compared to patients with tumor progression (13.0 vs. 6.0 months; p = 0.013). (orig.)

  7. The influence of family history of cancer, irradiation and anticancer medication (mitomycin C), on the occurrence of multiple primary neoplasms with breast cancer

    International Nuclear Information System (INIS)

    Yoshimoto, Masataka; Kasumi, Fujio; Fukami, Atsuo; Nishi, Mitsumasa; Kajitani, Tamaki; Sakamoto, Goi

    1985-01-01

    The influence of family history of cancer, radiation therapy and anticancer drug therapy (mitomycin C) on the occurrence of multiple primary neoplasms, following treatment of a first primary cancer of the breast, was analyzed by the person-year method in 1,359 patients, in Japan. During 14,371.8 person-years of observation, 111 multiple primary neoplasms including bilateral breast cancers were found in 109 patients. The incidence rate of multiple primary neoplasms were 0.00772 per person-year. The incidence in patients with a family history of cancer was 1.29 times greater than that in patients without such a family history, and the incidence in patients with a family history of breast cancer was about three times greater than that in those without it (p < 0.01). Radiation therapy raised the occurrence of subsequent primary neoplasms 1.28-fold (or 1.62 fold after 5 years), and mitomycin C (a total dose of 0.8 mg/kg) therapy caused no increase in the occurrence of subsequent primary cancers, after an observation of 10 years or so. (author)

  8. Mitomycin-treated undifferentiated embryonic stem cells as a safe and effective therapeutic strategy in a mouse model of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Mariana eAcquarone

    2015-04-01

    Full Text Available Parkinson’s disease (PD is an incurable progressive neurodegenerative disorder. Clinical presentation of PD stems largely from the loss of dopaminergic neurons in the nigrostriatal dopaminergic pathway, motivating experimental strategies aimed at replacing dopaminergic innervation by cellular therapy. Transplantation of dopaminergic neurons derived from embryonic stem cells significantly improves motor functions in rodent and non-human primate models of PD. However, protocols to generate dopaminergic neurons from embryonic stem cells generally meet with low efficacy and high risk of teratoma development upon transplantation. To address these issues, we have pre-treated undifferentiated mouse embryonic stem cells (mESCs with the DNA alkylating agent mitomycin C (MMC before transplantation. MMC treatment of cultures prevented tumor formation in a 12-week follow-up after mESCs were injected in nude mice. In 6-OH-dopamine-lesioned mice, intrastriatal injection of MMC-treated mESCs markedly improved motor function without tumor formation for as long as 15 months. Furthermore, we show that halting mitotic activity of undifferentiated mESCs induces a four-fold increase in dopamine release following in vitro differentiation. Our findings indicate that treating mESCs with mitomycin C prior to intrastriatal transplant is an effective strategy that could be further investigated as a novel alternative for treatment of Parkinson's disease.

  9. In vivo cell kinetic measurements in a randomized trial of continuous hyperfractionated accelerated radiotherapy with or without mitomycin C in head-and-neck cancer

    International Nuclear Information System (INIS)

    Dobrowsky, Werner; Dobrowsky, Eva; Wilson, George D.

    2003-01-01

    Purpose: Tumor cell repopulation is still considered to be a major cause of failure in radiotherapy. In this study, we investigated the influence of cell kinetic parameters on the outcome of patients treated in a randomized trial of accelerated fractionation, with or without mitomycin C, vs. conventional fractionation. Methods and Materials: Sixty-two patients were studied using administration of bromodeoxyuridine (BrdUrd), and cell kinetic parameters were measured using flow cytometry. The patients were treated with either 70 Gy for 7 weeks or 55.3 Gy for 17 continuous days (V-CHART) with or without 20 mg/m 2 mitomycin C on day 5. Results: The potential doubling time (Tpot) and labeling index (LI) failed to provide any prognostic information with regard to local control or survival. However, the duration of the S phase (Ts) revealed patients whose tumors had a long Ts had significantly worse local control (p = 0.028) and survival (p = 0.034) irrespective of treatment. A similar trend was evident within the different treatment arms particularly associated with overall survival. Conclusions: The Ts values of head-and-neck squamous cell cancers provided prognostic information that predicted clinical outcome irrespective of treatment schedule in this study. This neglected parameter of the Tpot method might provide information related to redistribution of cells during fractionated radiotherapy

  10. Use of transmission electron microscope to assess the damage to Sarcoma 180 ascites tumour cells following in vivo treatment of mitomycin-C and gamma radiation

    International Nuclear Information System (INIS)

    Majumdar, S.K.; Bhattacharya, S.; Mitra, A.; Mukherji, S.

    1991-01-01

    Five day old Sarcoma 180 tumour bearing mice were exposed to different doses of mitomycin-C (4 mg or 7 mg per kg body weight of mouse) and gamma radiation (400 R or 800 R) applied singly or in combination. Surviving populations were collected after 5 days of treatment and processed for transmission electron microscopy. The control Sarcoma 180 tumour cells has the following characteristics: profused microvilli, different sized mitochondria with poorly developed internal structure, distinct endoplasmic reticulum studded with ribosomes, the large nucleus rich in chromatin materials and distinct nucleolus containing closely interwined granular and fibrillar components with associated chromatins. Damage to treated cells were ascertained by the reduction in microvilli, swelling of mitochondria with cloudy appearance, dilation and fragmentation of endoplasmic reticulum, blebbing of nuclear membrane, condensation of heterochromatin, appearance of perichromatin granules, segregation and fragmentation of nucleolus and invagination of plasma memebrane with increased intracellular spaces. With the help of transmission electron microscope it is thus possible to assess the nature of damage to organelles effected by mitomycin C and radiation both singly and in combination. Growth inhibition and damage in the cellular ultrastructure were maximum among tumour cells which survived with concomitant treatment with 7 mg MMC and 800 R. (author). 7 refs., 4 figs., 1 tab

  11. Replication of simian virus 40 in simian virus 40-transformed hamster kidney cells induced by mitomycin C or 60Co γ irradiation

    International Nuclear Information System (INIS)

    Rakusanova, T.; Smales, W.P.; Kaplan, J.C.; Black, P.H.

    1978-01-01

    Several clones of simian virus 40 (SV40)-transformed hamster kidney cells, which are heterogeneous for induction of infectious SV40, have been studied. SV40 yields are low after induction with 60 Co γ irradiation or mitomycin C. In order to clarify the mechanism(s) by which virus is produced in induced cells, we analyzed the replication of viral DNA and production of virion (V) antigen and infectious virus after induction in various clones as well as in lytically infected permissive cells. Cells replicating SV40 DNA or synthesizing V antigen were visualized by in situ hybridization and immunofluorescence techniques, respectively. Only some cells in induced cultures were found to produce SV40 and those which did were less efficient than lytically infected monkey cells. Mitomycin C or 60 Co γ irradiation acted by inducing more cells to replicate virus rather than by increasing the amount of SV40 released from individual cells. A greater proportion of cells could be induced to replicate SV40 DNA than to synthesize V antigen in all induced clones studied. Also, SV40 DNA replication was induced at lower doses of γ irradiation than the production of either V antigen or infectious virus suggesting that synthesis of late virus protein is more restricted in induced cells than is replication of SV40 DNA. These findings indicate that one of the effects of induction treatments on SV40-transformed hamster cells is an enhancement of the cells' capacity to support SV40 replication

  12. [Correlation of single-cell gel electrophoresis and mitomycin C-induced chromosomal breakage for chromosomal instabiligy in children with Fanconi anemia].

    Science.gov (United States)

    Zhang, Li; Liu, Qiang; Zou, Yao; Liu, Xiao-ming; Zhang, Jia-yuan; Wang, Shu-chun; Chen, Xiao-juan; Guo, Ye; Yang, Wen-yu; Ruan, Min; Liu, Tian-feng; Liu, Fang; Cai, Xiao-jin; Chen, Yu-mei; Zhu, Xiao-fan

    2013-02-01

    Fanconi anemia (FA) is characterized by bone marrow failure, congenital abnormalities and predisposition to neoplasia. Hypersensitivity of FA cells to the clastogenic effect of mitomycin C (MMC) provides a unique marker for the diagnosis before the beginning of hematological manifestations. The aim of this study was to evaluate the relationship between Single-Cell Gel Electrophoresis (SCGE) and mitomycin C-induced chromosomal breakage in children with FA. Between January 2007 and June 2011, 248 children (results of the two methods and compared with each other. The receiver operating characteristic (ROC) curve was used to evaluate the parameters in SCGE. Seventeen patients were diagnosed as FA and 231 as non-FA. Chromosomal breakage was found to be significantly higher in FA patients [(32.2 ± 4.8)%] than non-FA [(19.9 ± 3.0)%] and controls[(21.6 ± 4.8)%] when induced by MMC 80 ng/ml. The parameters of SCGE were significantly different between FA patients and non-FA or controls. All the parameters were rectilinearly correlated with MMC (P = 0.000). The most closely correlated parameter was the rate of comet cell (r = 0.848, P = 0.000). The results of ROC curves suggested the comet cell rate (0.999) was more important. SCGE might be used to discriminate between FA and non-FA individuals. The relationship between SCGE and MMC-induced chromosomal breakage was significant. The rate of comet cell was the important parameter.

  13. Vitality of oligozoospermic semen samples is improved by both swim-up and density gradient centrifugation before cryopreservation.

    Science.gov (United States)

    Counsel, Madeleine; Bellinge, Rhys; Burton, Peter

    2004-05-01

    To ascertain whether washing sperm from oligozoospermic and normozoospermic samples before cryopreservation improves post-thaw vitality. Normozoospermic (n = 18) and oligozoospermic (n = 16) samples were divided into three aliquots. The first aliquot remained untreated and the second and third aliquots were subjected to the swim-up and discontinuous density gradient sperm washing techniques respectively. Vitality staining was performed, samples mixed with cryopreservation media and frozen. Spermatozoa were thawed, stained, and vitality quantified and expressed as the percentage of live spermatozoa present. Post-thaw vitality in untreated aliquots from normozoospermic samples (24.9% +/- 2.3; mean +/- SEM) was significantly higher (unpaired t-tests; P vitality was significantly higher after swim-up in normozoospermic samples (35.6% +/- 2.1; P vitality in oligozoospermic (22.4% +/- 1.0; P vitality in cryopreserved oligozoospermic samples was improved by both the swim-up and density gradient centrifugation washing techniques prior to freezing.

  14. [Ultrastructural changes in the MP3 neuron of the mollusk Lymnaea stagnalis after cryopreservation of the isolated brain].

    Science.gov (United States)

    Dmitrieva, E V; Moshkov, D A; Gakhova, E N

    2006-01-01

    Investigation of a possibility of long-term storage of frozen (-196 degrees C) viable neurons and nervous tissue is one of the central present day problems. In this study ultrastructural changes in neurons of frozen-thawed snail brain were examined as a function of time. We studied the influence of cryopreservation, cryoprotectant (Me2SO), cooling to 4-6 degrees C, and a prolonged incubation in physiological solution at 4-6 degrees C on dictyosomes of Golgi apparatus, endoplasmic reticulum (ER) cisternae and mitochondria. It has been found that responses of these intracellular structures of cryopreserved neurons to the above influences are similar: dissociation of Golgi dictyosomes, swelling of endoplasmic reticulum cisternae and mitochondrial cristae. Both freezing-thawing and cryoprotectant were seen to cause an increase in the number of lysosomes, liposomes, myelin-like structures, and to form large vacuoles. The structural changes in molluscan neurons caused by cryopreservation with Me2SO (2 M) were reversible.

  15. Effect of cryopreservation on the pre-hatching behavior in the Mexican fruit fly Anastrepha ludens Loew (Diptera, Tephritidae).

    Science.gov (United States)

    Rajamohan, Arun; Rinehart, Joseph P; Leopold, Roger A

    2018-02-01

    In a sampling of untreated embryos of the economically important fruit pest species, Anastrepha ludens, the cumulative hatch percentage in the lab was noted to be ∼85%. Approximately 70% of the larvae had eclosed through the posterior pole of the egg. This process is effected by the act of Pole Reversal (PR) of the fully developed pre-hatch larva from the wider anterior to the narrower posterior pole of the egg. Investigation of the effects of cryopreservation and various pretreatments prior to cryostorage on the PR behavior was prompted by the observation of significantly lower proportion of cryopreserved embryos exhibiting the PR behavior. Pretreatments (dechorionation and permeabilization) followed by vitrification resulted in delayed hatching, reflecting a slower embryonic development rate of ∼10 h. A smaller proportion of the treated embryos either eclosed from the anterior end of the egg or did not eclose at all despite complete development and prehatch gnawing activity. In the untreated controls, 24.0% of the embryos eclosed from the anterior pole. After permeabilization and cryopreservation, 83% and 55% (adjusted hatch) of the embryos were noted to hatch this way, respectively. An analysis of the hatch count after the treatments shows that factors contributing to the embryos' inability to properly invert polarity is not solely due to cryopreservation but also due to the pretreatment procedures including dechorionation and permeabilization. In fact, the permeabilization pre-treatment contributed the highest to this phenomenon lending support to the view that chemical toxicity rather than physical effects of cryopreservation play a major role in post-cryopreservation effects. Published by Elsevier Inc.

  16. Predictive value of early postoperative IOP and bleb morphology in Mitomycin-C augmented trabeculectomy [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Hamed Esfandiari

    2017-12-01

    Full Text Available Background: To determine the predictive value of postoperative bleb morphological features and intraocular pressure (IOP on the success rate of trabeculectomy. Methods: In this prospective interventional case series, we analyzed for one year 80 consecutive primary open angle glaucoma patients who underwent mitomycin-augmented trabeculectomy. Bleb morphology was scored using the Indiana bleb appearance grading scale (IBAGS. Success was defined as IOP ≤15 mmHg at 12 months. We applied a multivariable regression analysis and determined the area under the receiver operating characteristic curve (AUC. Results: The mean age of participants was 62±12.3 years in the success and 63.2±16.3 years in the failure group (P= 0.430 with equal gender distribution (P=0.911. IOPs on day 1, 7 and 30 were similar in both (P= 0.193, 0.639, and 0.238, respectively. The AUC of IOP at day 1, day 7 and 30 for predicting a successful outcome was 0.355, 0.452, and 0.80, respectively. The AUC for bleb morphology parameters of bleb height, extension, and vascularization, on day 14 were 0.368, 0.408, and 0.549, respectively. Values for day 30 were 0.428, 0.563, and 0.654. IOP change from day 1 to day 30 was a good predictor of failure (AUC=0.838, 95% CI: 0.704 to 0.971 with a change of more than 3 mmHg predicting failure with a sensitivity of 82.5% (95% CI: 68 to 91% and a specificity of 87.5% (95% CI: 53 to 98%. Conclusions: IOP on day 30 had a fair to good accuracy while bleb features failed to predict success except bleb vascularity that had a poor to fair accuracy.  An IOP increase more than 3 mmHg during the first 30 days was a good predictor of failure.

  17. Internal Urethrotomy With Intralesional Mitomycin C: An Effective Option for Endoscopic Management of Recurrent Bulbar and Bulbomembranous Urethral Strictures.

    Science.gov (United States)

    Farrell, M Ryan; Lawrenz, Cedric W; Levine, Laurence A

    2017-12-01

    To describe our experience with direct visual internal urethrotomy (DVIU) and mitomycin C (MMC) for recurrent bulbar and bulbomembranous urethral strictures of radiation and non-radiation-induced etiologies. We reviewed our database of consecutive patients presenting to our tertiary care institution with recurrent bulbar and bulbomembranous urethral strictures who underwent DVIU with MMC from 2011 to 2016. Patients were stratified by radiation-induced strictures (RIS) vs non-RIS. Cold-knife incisions were made at 12-, 3-, and 9-o'clock positions followed by intralesional injection of 10 mL MMC (0.4 mg/mL) in 0.2-0.4 mL aliquots and 1 month of postoperative daily clean intermittent catheterization (CIC). All 44 patients (RIS n = 18, non-RIS n = 26) failed prior endoscopic management or urethroplasty. Median stricture length was 2.0 cm (interquartile range [IQR] 1.0-2.5). Over a median follow-up of 25.8 months (IQR 12.9-47.2), 75.0% of patients (33/44) required no additional surgical intervention (RIS 12/18, 66.7%; non-RIS 21/26, 80.8%). Median time to stricture recurrence among those who recurred was 10.7 months (IQR 3.9-17.6; RIS 9.4 months, IQR 3.5-17.6; non-RIS 11.2 months, IQR 8.0-25.6). Four patients (RIS n = 2, non-RIS n = 2) elected to undergo urethroplasty for recurrence. A second DVIU with MMC was performed in the remaining recurrences (n = 7) with no further surgical intervention required in 37 of 40 of patients (92.5%) overall (RIS 14/16, 87.5%; non-RIS 23/24, 95.8%). No long-term complications were attributable to MMC. DVIU with MMC and short-term CIC for recurrent, short, bulbar and bulbomembranous urethral strictures is a safe endoscopic modality with promising early results. This approach may be useful for patients who are suboptimal candidates for open reconstruction. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Development of soya milk extender for semen cryopreservation of Karan Fries (crossbreed cattle).

    Science.gov (United States)

    Singh, V K; Singh, A K; Kumar, R; Atreja, S K

    2013-01-01

    Egg yolk based semen extenders are used widely, with the potential risk of xenobiotic contamination. This study was designed to develop a soya milk based extender to substitute egg yolk based extender for bovine semen cryopreservation. In the first experiment soya milk was prepared from fresh soya bean (Glycine max). Concentration of soya milk in tris based extender was standardized based on quality parameters of spermatozoa during liquid preservation at 5°C up to 72 h and compared with egg yolk tris (EYT) extender. Sperm in soya milk tris (SMT) extender with 25 percent soya milk showed no significant (P > 0.05) differences in all the quality parameters like motility, viability, membrane integrity and acrosome integrity, as compared to sperm in EYT extender up to 72h in liquid dilution. In the second experiment the Karan Fries semen was cryopreserved in SMT extender with 25 percent soya milk (selected from the first experiment) using different concentration of glycerol, as cryoprotectant, ranging from 6-7 percent with a difference of 0.2 percent to standardize optimum concentration based on post thaw motility of spermatozoa. Glycerol at a final concentration of 6.4 percent was found to be the best among all. Further, semen samples were split and cryopreserved in newly developed SMT extender containing 6.4 percent glycerol and compared with conventional EYT extender for post thaw sperm quality parameters and degree of cryocapacitation. There were no significant (P > 0.05) differences between sperm in EYT extender and SMT extender for post thaw motility, viability, membrane integrity, acrosome integrity and cryocapacitation. In conclusion, the newly developed SMT extender maintained comparable semen quality as compared to EYT extender hence it can.

  19. Antioxidant activity of aqueous extract of noni in dilutent for ram semen cryopreservation

    Directory of Open Access Journals (Sweden)

    Ana Lauren Costa Nascimento

    2016-03-01

    Full Text Available Noni (Morinda citrifolia L. is a fruit consumed worldwide because of its nutritional and therapeutic properties resulting from the large amount of phenolic compounds, which has aroused interest of the scientific community. In order to identify new natural sources of antioxidants, the objective of this study was to evaluate the performance of noni in diluent for ram semen cryopreservation. A completely randomized design consisting of four treatments and three repetitions per treatment was used. The treatments differed in terms of the concentration of the aqueous extract of noni added to the diluent: control, no addition of the extract, and three concentrations (24, 72, and 120 µg/mL. The physical and chemical variables of the mature fruit were evaluated: total acidity (8.78, pH (4.12, and soluble solids (8.18%. The vitamin C content was 309.42 mg per 100 g fresh matter. The aqueous extract of noni was also evaluated regarding the quantity of total phenolic compounds, antioxidant activity, and lipid peroxidation inhibition capacity. The aqueous extract contained a moderate amount of phenolic compounds (47.96 ± 1.95 mg gallic acid equivalent/100 g extract. The concentrations of the aqueous extract of 72 and 120 µg/mL in diluent used for semen cryopreservation inhibited lipid peroxidation by 21.75% and 51.32%, respectively. There was no positive effect of the lowest concentration (24 µg/mL. The antioxidant activity index of noni was 33.33, corresponding to very strong antioxidant activity. The aqueous extract of noni exhibits very strong antioxidant activity and its addition to the diluent for semen cryopreservation at a concentration of 72 µg/mL is able to inhibit lipid peroxidation.

  20. Thermomechanical Stress in Cryopreservation Via Vitrification With Nanoparticle Heating as a Stress-Moderating Effect.

    Science.gov (United States)

    Eisenberg, David P; Bischof, John C; Rabin, Yoed

    2016-01-01

    This study focuses on thermomechanical effects in cryopreservation associated with a novel approach of volumetric heating by means on nanoparticles in an alternating electromagnetic field. This approach is studied for the application of cryopreservation by vitrification, where the crystalline phase is completely avoided-the cornerstone of cryoinjury. Vitrification can be achieved by quickly cooling the material to cryogenic storage, where ice cannot form. Vitrification can be maintained at the end of the cryogenic protocol by quickly rewarming the material back to room temperature. The magnitude of the rewarming rates necessary to maintain vitrification is much higher than the magnitude of the cooling rates that are required to achieve it in the first place. The most common approach to achieve the required cooling and rewarming rates is by exposing the specimen's surface to a temperature-controlled environment. Due to the underlying principles of heat transfer, there is a size limit in the case of surface heating beyond which crystallization cannot be prevented at the center of the specimen. Furthermore, due to the underlying principles of solid mechanics, there is a size limit beyond which thermal expansion in the specimen can lead to structural damage and fractures. Volumetric heating during the rewarming phase of the cryogenic protocol can alleviate these size limitations. This study suggests that volumetric heating can reduce thermomechanical stress, when combined with an appropriate design of the thermal protocol. Without such design, this study suggests that the level of stress may still lead to structural damage even when volumetric heating is applied. This study proposes strategies to harness nanoparticles heating in order to reduce thermomechanical stress in cryopreservation by vitrification.

  1. Prevent the degradation of algicidal ability in Scenedesmus-lysing bacteria using optimized cryopreservation.

    Science.gov (United States)

    Liao, Chunli; Liu, Xiaobo

    2016-03-01

    With the anthropogenic nutrient loading increasing, the frequency and impacts of harmful algal blooms (HABs) have intensified in recent years. To biocontrol HABs, many corresponding algal-lysing bacteria have been exploited successively. However, there are few studies on an effective algal-lysing culture collection to prevent cells from death and particularly the degradation of algicidal ability to their hosts. An optimized cryopreservation was developed and experiments on the validation of this method on preventing algicidal degradation and effects of this optimized cryopreservation on the survival rate of Scenedesmus-lysing bacterium, Enterobacter NP23, isolated from Scenedesmus sp. community, China, on the algicidal dynamic of Scenedesmus wuhanensis was investigated. The optimized cryoprotectant composition consists of 30.0 g/L gelatin, 48.5 g/L sucrose, and 28.4 g/L glycerol, respectively. Using this approach, the survival rate of NP23 cells can still maintain above 90 % and the algal-lysing rate only decline 4 % after the 18-month cryoprotection. Moreover, the 16 generations' passage experiment showed a significant (p < 0.05) genetic stability of algicidal capacity after 18 months. The growth dynamic of S. wuhanensis was investigated in a 5-L bioreactor during 132 h in the absence or presence of NP23. As a result, NP23 has a significant (p < 0.05) inhibition to S. wuhanensis growth when injected into algal culture in the exponential phase at 60th hour. In addition, S. wuhanensis culture initially with NP23 exhibited a slow growth, performing a prolonged lag phase without a clear stationary phase and then rapidly decreased. Our findings, combined with the capacity of preventing the degradation of algicidal ability collectively suggest that the use of this opitimized cryopreservation may be a promising strategy for maintaining algicidal cells.

  2. Optimization of a protocol for cryopreservation of mouse spermatozoa using cryotubes.

    Science.gov (United States)

    Hasegawa, Ayumi; Yonezawa, Kazuya; Ohta, Akihiko; Mochida, Keiji; Ogura, Atsuo

    2012-01-01

    The rapid increase in the number of genetically modified mouse strains has produced a high demand for their frozen spermatozoa from laboratories and mouse banking facilities. Historically, plastic straws have been used preferentially as containers for frozen mammalian spermatozoa because spermatozoa frozen in plastic straws have a high survival rate after thawing. However, plastic straws are more fragile and are used less often than the cryotubes used for conventional cell freezing. In this study, we sought to develop a new protocol for sperm freezing using cryotubes as the container to increase the accessibility of mouse sperm cryopreservation. Epididymal spermatozoa were collected from mature ICR or C57BL/6J (B6) males and were suspended in 18% raffinose and 3% skim milk solution. We then optimized the following conditions using the sperm survival rate as an index: 1) distance of cryotubes from the surface of the liquid nitrogen at freezing, 2) volume of the sperm suspension in the cryotube and 3) temperature of warming sperm during thawing. The best result was obtained when cryotubes containing 10 µl of sperm suspension were immersed 1 cm below the surface of the liquid nitrogen and then thawed at 50 C. The fertilization rates using spermatozoa frozen and thawed using this method were 63.1% in ICR mice and 28.2% in B6 mice. The latter rate was increased to 62.3% by adding reduced glutathione to the fertilization medium. After embryo transfer, 68% and 62% of the fertilized oocytes developed into normal offspring in the ICR and B6 strains, respectively. These results show that cryotubes can be used for cryopreservation of mouse spermatozoa under optimized conditions. This protocol is easy and reproducible, and it may be used in laboratories that do not specialize in sperm cryopreservation.

  3. Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender.

    Science.gov (United States)

    Pojprasath, T; Lohachit, C; Techakumphu, M; Stout, T; Tharasanit, T

    2011-06-01

    Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integrity, plasma membrane functionality and sperm longevity. The fertility of semen frozen in the presence of sorbitol was also tested by artificial insemination. Sperm quality was significantly decreased following freezing and thawing (P sorbitol and glucose (P sorbitol-based extender when examined at 2 and 4 h post-thaw, all of these parameters plus plasma membrane functionality were improved for sperm frozen in the sorbitol extender than in the glucose extender when examined 10 min post-thaw. Two of four mares (50%) inseminated with semen frozen with a sorbitol-containing freezing extender became pregnant. It is concluded that different sugars have different abilities to protect against cryoinjury during freezing and thawing of stallion sperm. This study demonstrated that an extender containing sorbitol as primary sugar can be used to successfully cryopreserve equine sperm; moreover, the quality of frozen-thawed sperm appeared to be better than when glucose or fructose was the principle sugar in the freezing extender. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Ejaculate traits and sperm cryopreservation in the endangered Baird's tapir (Tapirus bairdii).

    Science.gov (United States)

    Pukazhenthi, Budhan S; Togna, Gina Della; Padilla, Luis; Smith, Diorene; Sanchez, Carlos; Pelican, Katey; Sanjur, Oris I

    2011-01-01

    There is little information on the reproductive biology of the male Baird's tapir (Tapirus bairdii). In this study, we characterized the ejaculate traits and evaluated the efficacy of 2 cryodiluents on sperm cryosurvival. Ejaculates were assessed for volume, pH, sperm motility, forward progression, osmolality, sperm concentration, sperm morphology, and acrosomal integrity. For cryopreservation, ejaculates with >50% total sperm motility were washed, and sperm pellets were resuspended in either Botu-Crio (CryoVital, Grandau, Germany) or INRA 96 containing 2% egg yolk and 2.5% each of methyl- and dimethylformamide (INRA 96), and they were cryopreserved over liquid nitrogen vapor. Thawed samples were incubated in vitro (25 °C) and evaluated for percent total sperm motility, forward progression, and acrosomal integrity at hourly intervals for 4 hours. Spermic ejaculates were obtained from all males, and the mean seminal volume, sperm concentration per milliliter, percent sperm motility, progressive status, and percent morphologically normal cells were 20.4 ± 4.3 mL, 101.2 ± 24.0 × 10(6)/mL, 46.1% ± 5.0%, 2.9 ± 0.1, and 6.9% ± 1.4%, respectively. There was a positive significant correlation between percent normal sperm and animal age (r = 0.66; P tapir; demonstrate that tapir spermatozoa can be cryopreserved in diluents containing amides alone or in combination with glycerol; and provide fundamental information critical for development of assisted reproductive technologies for the Baird's tapir.

  5. Consequences of metaphase II oocyte cryopreservation on mRNA content.

    Science.gov (United States)

    Chamayou, S; Bonaventura, G; Alecci, C; Tibullo, D; Di Raimondo, F; Guglielmino, A; Barcellona, M L

    2011-04-01

    We studied the consequences of freezing/thawing processes on mRNA contents in MII oocytes after slow-freezing/rapid thawing (SF/RT) and vitrification/warming (V/W) protocols, and compared the results to fresh MII oocytes. We quantified the nuclear transcript mRNA responsible for the translation of proteins belonging either to trans-regulatory protein family or to functional structural proteins such as proteins involved in DNA structural organization (NAP1L1, TOP1, H1F0H1), chromosomal structure maintenance (SMC, SCC3, RAD21, SMC1A, SMC1B, STAG3, REC8), mitochondrial energetic pathways (ATP5GJ, SDHC), cell cycle regulation and processes (CLTA, MAPK6, CKS2) and staminal cell potency-development competence stage (DPPA3, OCT4, FOXJ2). Surplus MII oocytes were donated from patients in IVF cycles and divided in three groups of 15 oocytes. Group 1 was comprised of non-cryopreserved oocytes and Groups 2 and 3 underwent SF/RT and V/W procedures, respectively. There was an overall decrease of mRNA extracted from cryopreserved oocytes compared to control group. Only 39.4% of mRNA content were preserved after SF/RT while 63.3% of mRNA content were maintained after V/W. Oocyte cryopreservation is associated with molecular injury associated with the decrease of stored mRNA. However the V/W protocol is more conservative than SF/RT resulting in a level of mRNA sufficient to maintain biologic functions in the subsequent fertilized oocyte. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. The Effect of Cryopreserved Human Placental Tissues on Biofilm Formation of Wound-Associated Pathogens.

    Science.gov (United States)

    Mao, Yong; Singh-Varma, Anya; Hoffman, Tyler; Dhall, Sandeep; Danilkovitch, Alla; Kohn, Joachim

    2018-01-08

    Biofilm, a community of bacteria, is tolerant to antimicrobial agents and ubiquitous in chronic wounds. In a chronic DFU (Diabetic Foot Ulcers) clinical trial, the use of a human cryopreserved viable amniotic membrane (CVAM) resulted in a high rate of wound closure and reduction of wound-related infections. Our previous study demonstrated that CVAM possesses intrinsic antimicrobial activity against a spectrum of wound-associated bacteria under planktonic culture conditions. In this study, we evaluated the effect of CVAM and cryopreserved viable umbilical tissue (CVUT) on biofilm formation of S. aureus and P. aeruginosa , the two most prominent pathogens associated with chronic wounds. Firstly, we showed that, like CVAM, CVUT released antibacterial activity against multiple bacterial pathogens and the devitalization of CVUT reduced its antibacterial activity. The biofilm formation was then measured using a high throughput method and an ex vivo porcine dermal tissue model. We demonstrate that the formation of biofilm was significantly reduced in the presence of CVAM- or CVUT-derived conditioned media compared to control assay medium. The formation of P. aeruginosa biofilm on CVAM-conditioned medium saturated porcine dermal tissues was reduced 97% compared with the biofilm formation on the control medium saturated dermal tissues. The formation of S. auerus biofilm on CVUT-conditioned medium saturated dermal tissues was reduced 72% compared with the biofilm formation on the control tissues. This study is the first to show that human cryopreserved viable placental tissues release factors that inhibit biofilm formation. Our results provide an explanation for the in vivo observation of their ability to support wound healing.

  7. The Effect of Cryopreserved Human Placental Tissues on Biofilm Formation of Wound-Associated Pathogens

    Directory of Open Access Journals (Sweden)

    Yong Mao

    2018-01-01

    Full Text Available Biofilm, a community of bacteria, is tolerant to antimicrobial agents and ubiquitous in chronic wounds. In a chronic DFU (Diabetic Foot Ulcers clinical trial, the use of a human cryopreserved viable amniotic membrane (CVAM resulted in a high rate of wound closure and reduction of wound-related infections. Our previous study demonstrated that CVAM possesses intrinsic antimicrobial activity against a spectrum of wound-associated bacteria under planktonic culture conditions. In this study, we evaluated the effect of CVAM and cryopreserved viable umbilical tissue (CVUT on biofilm formation of S. aureus and P. aeruginosa, the two most prominent pathogens associated with chronic wounds. Firstly, we showed that, like CVAM, CVUT released antibacterial activity against multiple bacterial pathogens and the devitalization of CVUT reduced its antibacterial activity. The biofilm formation was then measured using a high throughput method and an ex vivo porcine dermal tissue model. We demonstrate that the formation of biofilm was significantly reduced in the presence of CVAM- or CVUT-derived conditioned media compared to control assay medium. The formation of P. aeruginosa biofilm on CVAM-conditioned medium saturated porcine dermal tissues was reduced 97% compared with the biofilm formation on the control medium saturated dermal tissues. The formation of S. auerus biofilm on CVUT-conditioned medium saturated dermal tissues was reduced 72% compared with the biofilm formation on the control tissues. This study is the first to show that human cryopreserved viable placental tissues release factors that inhibit biofilm formation. Our results provide an explanation for the in vivo observation of their ability to support wound healing.

  8. Comparison of the Viability of Cryopreserved Fat Tissue in Accordance with the Thawing Temperature

    Directory of Open Access Journals (Sweden)

    So-Min Hwang

    2015-03-01

    Full Text Available BackgroundAdipose tissue damage of cryopreserved fat after autologous fat transfer is inevitable in several processes of re-transplantation. This study aims to compare and analyze the survivability of adipocytes after thawing fat cryopreserved at -20℃ by using thawing methods used in clinics.MethodsThe survival rates of adipocytes in the following thawing groups were measured: natural thawing at 25℃ for 15 minutes; natural thawing at 25℃ for 5 minutes, followed by rapid thawing at 37℃ in a water bath for 5 minutes; and rapid thawing at 37℃ for 10 minutes in a water bath. The survival rates of adipocytes were assessed by measuring the volume of the fat layer in the top layers separated after centrifugation, counting the number of live adipocytes after staining with trypan blue, and measuring the activity of mitochondria in the adipocytes.ResultsIn the group with rapid thawing for 10 minutes in a water bath, it was observed that the cell count of live adipocytes and the activity of the adipocyte mitochondria were significantly higher than in the other two groups (P<0.05. The volume of the fat layer separated by centrifugation was also measured to be higher, which was, however, not statistically significant.ConclusionsIt was shown that the survival rate of adipocytes was higher when the frozen fat tissue was thawed rapidly at 37℃. It can thus be concluded that if fats thawed with this method are re-transplanted, the survival rate of cryopreserved fats in transplantation will be improved, and thus, the effect of autologous fat transfer will increase.

  9. Effect of mitomycin-c on biodistribution of 99 mTc-sodium pyrophosphate radiopharmaceuticals in mice; Efeito da mitomicina-c na biodistribuicao do radiofarmaco pirofosfato de sodio marcado com Tecnecio-99m em camundongos

    Energy Technology Data Exchange (ETDEWEB)

    Gomes, Maria L.; Braga, Ana C.S.; Gutfilen, Bianca; Bernardo-Filho, Mario [Instituto Nacional do Cancer, Rio de Janeiro, RJ (Brazil)

    1997-12-01

    The desirable characteristics of technetium-99 have stimulated the development of labeling techniques for different molecular and cellular. It is generally acceptable that a variety of factors other than disease can alter the biodistribution of radiopharmaceutical and one such factor is the drug therapy. The absence of knowledge of these factors may result in an unexpected behavior of the radiopharmaceutical. Since patients on chemotherapeutic treatment ca be submitted, for different reasons, to a nuclear medicine procedure, we have studied in mice, the effect of mitomycin-C on the biodistribution of the radiopharmaceutical {sup 99m} Tc-pyrophosphate ({sup 99m} Tc-PYP). Mitomycin-C is an antineoplastic agent obtained from Streptomyces caesptosus. It was administered 0,15 mg of mitomycin-C in Balb/c in Balb/c female mice with an interval of 72 hours. After one hour of the last dose, 0.3 ml of {sup 99m} Tc-PYP (7.4 MBq) were injected after 0.5 h the animals were sacrificed. The organs were isolated, weighted and counted in a well counter. The percentages of radioactivity per gran of organs (% rad/g) were calculated and statistics analyses were performed (wilcoxon). The results have shown that the % rad/g has increased in pancreas, stomach, bone and lungs. These increased of radioactivity can be justified by the metabolic process or the therapeutic effect of mitomycin-C. (author). 8 refs., 2 tabs.

  10. Experimental study on active specific immunotherapy utilizing the immune reaction of low-dose irradiated tumor tissue, 9

    International Nuclear Information System (INIS)

    Ogawa, Yasuhiro; Maeda, Tomoho; Yoshida, Shoji

    1983-01-01

    We have already reported the remarkable effect of an active specific immunotherapy using cryopreserved tumor cells and infiltrating mononuclear cells prepared from a low-dose irradiated tumor tissue after cytoreductive radiotherapy. In the present study, the effect of a biological response modifier, OK-432 combined with the active specific immunotherapy was investigated. Twelve-week-aged female C3H/He mice transplanted with MM 46 tumor cells were received local radiotherapy with a dose of 3,000 rads by high energy electron beams on the fifth day after inoculation. The tumor cells and infiltrating mononuclear cells cryopreserved for two months were thawed and treated with mitomycin-C at concentration of 20 μg/10 7 cells at 37 0 C for 30 min. Then, these cells were injected subcutaneously into the left hind paws as a mitomycin C-treated, cryopreserved active specific immunotherapy on the thirteenth day, and daily dose of 1 KE of OK-432 was injected intraperitoneally from the sixth to the tenth days. The inhibition of the tumor growth was similarly observed in the group which received this active specific immunotherapy combined with a biological response modifier, OK-432. (author)

  11. A new portable device for automatic controlled-gradient cryopreservation of blood mononuclear cells

    DEFF Research Database (Denmark)

    Hviid, L; Albeck, G; Hansen, B

    1993-01-01

    Protection of the functional integrity of mononuclear cells stored in liquid N2 requires careful control of the freezing procedure. Consequently, optimal quality of cryopreserved cells is usually assured by freezing according to a specified time-temperature gradient generated by computer......-controlled freezing devices. While such equipment offers large capacity and secures maximum survival and functional integrity of the lymphocytes upon thawing, it is quite costly and strictly stationary. We have previously developed and tested an alternative, manual device for controlled-gradient lymphocyte freezing...

  12. Cryopreservation of ovarian tissue for fertility preservation in young female oncological patients

    DEFF Research Database (Denmark)

    Andersen, Claus Yding; Kristensen, Stine Gry; Greve, Tine

    2012-01-01

    Girls and women suffering from a cancer that requires treatment with gonadotoxic drugs may experience cessation of reproductive function as a side effect due to obliteration of the ovarian pool of follicles. Techniques are now available for fertility preservation, such as cryopreservation of mature...... and growth of follicles, giving rise to menstrual cycles and hormone production for several years. Worldwide, the procedure has resulted in the birth of 15 healthy children. Many cancer patients including girls and young women want fertility preservation, and the techniques are now being further developed...

  13. Cholesterol addition aids the cryopreservation of dromedary camel (Camelus dromedarius) spermatozoa.

    Science.gov (United States)

    Crichton, Elizabeth G; Pukazhenthi, Budhan S; Billah, M; Skidmore, Julian A

    2015-01-15

    The cryopreservation of dromedary camel (Camelus dromedarius) sperm has proved challenging with little success reported. The routine application of artificial insemination with frozen semen would assist the flow of valuable genetic material nationally and internationally. The current study sought to examine the effects of cholesterol (cholesterol-loaded cyclodextrin [CLC]) preloading on camel sperm cryosurvival. Ejaculates (n = 3 males; 3 ejaculates per male) were collected using an artificial vagina during the breeding season and extended in HEPES-buffered Tyrode's albumin lactate pyruvate (TALP) and allowed to liquefy in the presence of papain (0.1 mg/mL) before removal of the seminal plasma by centrifugation. Sperm pellets were resuspended (120 million/mL) in fresh TALP and incubated (15 minutes; 37 °C) with 0, 1.5, or 4.5 mg CLC/mL. Sperm suspensions were then centrifuged and reconstituted in INRA-96 containing 20% (v:v) egg yolk and 2.5% (v:v) methylformamide, loaded in 0.5-mL plastic straws, sealed, and cooled for 20 minutes at 4 °C. Straws were frozen over liquid nitrogen (4 cm above liquid; 15 minutes), plunged, and stored. Sperm motility, forward progressive status, and acrosomal integrity were recorded at 0 and 3 hours after thawing and compared with these same parameters before freezing. Aliquots also were stained with chlortetracycline hydrochloride to assess spontaneous sperm capacitation status before freezing and post-thaw. Pretreatment with CLC (1.5 and 4.5 mg/mL) enhanced cryosurvival. Post-thaw sperm motility was highest (P < 0.05) in 1.5 mg CLC/mL immediately after thawing (44%) and after 3 hours incubation at room temperature (34%). Highest post-thaw sperm progressive status was also achieved in the presence of 1.5 CLC. Greater proportions of spermatozoa retained acrosomal membrane integrity when cryopreserved in the presence of CLC, but there was no difference between 1.5 and 4.5 CLC. Although thawed spermatozoa underwent spontaneous

  14. Novel glyceryl glucoside is a low toxic alternative for cryopreservation agent

    Energy Technology Data Exchange (ETDEWEB)

    Su, Cathy; Allum, Allison J. [Department of Environmental & Radiological Health Sciences, Colorado State University, 1618 Campus Delivery, Fort Collins, CO 80523 (United States); Aizawa, Yasushi [Research and Development Group, Toyo Sugar Refining Co. Ltd., Tokyo 103-0046 (Japan); Kato, Takamitsu A., E-mail: Takamitsu.Kato@Colostate.edu [Department of Environmental & Radiological Health Sciences, Colorado State University, 1618 Campus Delivery, Fort Collins, CO 80523 (United States)

    2016-08-05

    Glyceryl glucoside (GG, α-D-glucosyglycerol) is a natural glycerol derivative found in alcoholic drinks. Recently GG has been used as an alternative for glycerol in cosmetic products. However, the safety of using GG is still unclear. Currently, dimethyl sulfoxide (DMSO) and glycerol are wildly used in cryopreservation. Despite GG being a derivative of glycerol, the ability of GG in cryopreservation is still unknown. By using a system of Chinese Hamster Ovary cells (CHO), A549 cells and AG1522 cells, the study examined the cryoprotective effects of DMSO, glycerol and GG. Cytotoxic and genotoxic responses induced by the three chemicals were also investigated with CHO to determine the safety of GG for cosmetic products. Our data suggests that GG has great cryopresearvation ability in the concentration of 30%–40% (v/v). For cytotoxic studies, DMSO showed the highest cytotoxicity above 3% (v/v) in cell doubling time delay among three chemicals. For the acute cytotoxicity with trypan blue dye exclusion assay, GG showed stronger cell killing effect within 24 h above 4% (v/v). For the continuous cytotoxicity with colony formation assay for 7 days, DMSO showed significantly reduced clonogenic ability above 2%. In genotoxicity studies, CHO treated with glycerol at 2% concentration induced three times higher frequencies of sister chromatid exchange (SCE) than background levels. GG did not induce significant amounts of SCE compared to background. Micronuclei formation was equally observed in the 2% and above concentrations of glycerol and GG. Our data showed that GG has significant effects on cryopreservation compared to DMSO. Glycerol and GG have similar cytotoxicity effects to CHO, but glycerol induced genotoxic responses in the same concentration. Therefore, we conclude that GG may be a safer alternative compound to glycerol in cosmetic products and safer alternative to DMSO in cryopreservation. -- Highlights: •Glyceryl Glucoside is low cytotoxicity and genotoxicity

  15. Thixotropic injectable hydrogel using a polyampholyte and nanosilicate prepared directly after cryopreservation

    Energy Technology Data Exchange (ETDEWEB)

    Jain, Minkle; Matsumura, Kazuaki, E-mail: mkazuaki@jaist.ac.jp

    2016-12-01

    Success of tissue engineering applications in regenerative medicine requires the preservation of tissue-engineered products at a low temperature. This can be successfully achieved by the use of cryoprotective agent (CPA). In this study, we formulated a unique injectable hydrogel for the purpose of cell delivery after cryopreservation by using polyampholyte CPA. The polyampholyte showed excellent post-thaw cell survival, and after thawing, the polymeric CPA did not have to be removed because of its low cytotoxicity. The polyampholyte could be transformed into a hydrogel by mixing with nanosilicates. Previously, nanosilicates were used to improve mechanical properties, but this is the first report of the use of a nanosilicate together with CPA to formulate hydrogels. Inclusion of the nanosilicate led to the formation of thixotropic hydrogels, which can be injected using fine needles. These gels with tunable mechanical properties can be injected into defect sites to form scaffolds for cell growth and tissue repair, and they do not require any separate seeding of cells before injection, thus eliminating the need for cell harvesting and cell maintenance. This is a distinct system in which cells can be cryopreserved until before usage; when required, the cells in the polyampholyte can be revived to their original state and the thixotropic hydrogel can be formed. The combination of thixotropy and cytocompatibility of the gels could enable a wide range of biomedical applications such as cell delivery and orthopedic repair. - Graphical abstract: A novel thixotropic, cytocompatible and injectable nanocomposite hydrogel with tunable mechanical properties directly after cryopreservation was formulated using an efficient polymer cryoprotectant and laponite. This is an efficient system in which cells remain viable and can proliferate even after one week. The combined use of thixotropy and cytocompatibility enables this system to be used for cell delivery applications

  16. Reconstruction of Chest Wall by Cryopreserved Sternal Allograft after Resection of Aneurysmal Bone Cyst of Sternum

    Directory of Open Access Journals (Sweden)

    Kambiz Sheikhy

    2017-01-01

    Full Text Available A 20-year-old female was referred to our hospital due to deformity and bulging in anterior aspect of chest wall in sternal area. Chest X-ray and CT scan confirmed a large mass with destruction of sternum. Pathologic diagnosis after incisional biopsy was compatible with aneurysmal bone cyst. We resected sternum completely and reconstructed large anterior defect by a cryopreserved sternal allograft. In follow-up of patient there was no unstability of chest wall with good cosmetic result.

  17. Simultaneous Integrated Boost–Intensity Modulated Radiation Therapy With Concomitant Capecitabine and Mitomycin C for Locally Advanced Anal Carcinoma: A Phase 1 Study

    Energy Technology Data Exchange (ETDEWEB)

    Deenen, Maarten J. [Division of Clinical Pharmacology, Department of Medical Oncology, The Netherlands Cancer Institute, Amsterdam (Netherlands); Dewit, Luc [Department of Radiotherapy, The Netherlands Cancer Institute, Amsterdam (Netherlands); Boot, Henk [Division of Gastroenterology and Hepatology, Department of Medical Oncology, The Netherlands Cancer Institute, Amsterdam (Netherlands); Beijnen, Jos H. [Department of Pharmacy and Pharmacology, Slotervaart Hospital, Amsterdam (Netherlands); Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmaco-epidemiology and Clinical Pharmacology, Utrecht University, Utrecht (Netherlands); Schellens, Jan H.M. [Division of Clinical Pharmacology, Department of Medical Oncology, The Netherlands Cancer Institute, Amsterdam (Netherlands); Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmaco-epidemiology and Clinical Pharmacology, Utrecht University, Utrecht (Netherlands); Cats, Annemieke, E-mail: a.cats@nki.nl [Division of Gastroenterology and Hepatology, Department of Medical Oncology, The Netherlands Cancer Institute, Amsterdam (Netherlands)

    2013-04-01

    Purpose: Newer radiation techniques, and the application of continuous 5-FU exposure during radiation therapy using oral capecitabine may improve the treatment of anal cancer. This phase 1, dose-finding study assessed the feasibility and efficacy of simultaneous integrated boost–intensity modulated radiation therapy (SIB-IMRT) with concomitant capecitabine and mitomycin C in locally advanced anal cancer, including pharmacokinetic and pharmacogenetic analyses. Methods and Materials: Patients with locally advanced anal carcinoma were treated with SIB-IMRT in 33 daily fractions of 1.8 Gy to the primary tumor and macroscopically involved lymph nodes and 33 fractions of 1.5 Gy electively to the bilateral iliac and inguinal lymph node areas. Patients received a sequential radiation boost dose of 3 × 1.8 Gy on macroscopic residual tumor if this was still present in week 5 of treatment. Mitomycin C 10 mg/m{sup 2} (maximum 15 mg) was administered intravenously on day 1, and capecitabine was given orally in a dose-escalated fashion (500-825 mg/m{sup 2} b.i.d.) on irradiation days, until dose-limiting toxicity emerged in ≥2 of maximally 6 patients. An additional 8 patients were treated at the maximum tolerated dose (MTD). Results: A total of 18 patients were included. The MTD of capecitabine was determined to be 825 mg/m{sup 2} b.i.d. The predominant acute grade ≥3 toxicities included radiation dermatitis (50%), fatigue (22%), and pain (6%). Fifteen patients (83% [95%-CI: 66%-101%]) achieved a complete response, and 3 (17%) patients a partial response. With a median follow-up of 28 months, none of the complete responders, and 2 partial responders had relapsed. Conclusions: SIB-IMRT with concomitant single dose mitomycin C and capecitabine 825 mg/m{sup 2} b.i.d. on irradiation days resulted in an acceptable safety profile, and proved to be a tolerable and effective treatment regimen for locally advanced anal cancer.

  18. Resultados refractivos en pacientes operados por LASIK versus LASEK con mitomicina C Refractive results in patients operated by LASIK versus LASEK with Mitomycin C

    Directory of Open Access Journals (Sweden)

    Abel Cabrera Martínez

    2009-06-01

    Full Text Available OBJETIVO: Evaluar comparativamente los resultados refractivos obtenidos en ojos operados con el uso de la keratomileusis in situ asistida con láser (LASIK contra la keratomileusis subepitelial asistida con láser combinándosele a esta última el uso de la mitomicina C (LASEK+MC. MÉTODOS: Se realizó un estudio prospectivo, experimental, comparativo en un total de 210 ojos de pacientes que acudieron a consulta para ser operados por algún defecto refractivo. Un total de 104 ojos fueron intervenidos por LASIK y 106 por LASEK+MC. Para comparar los resultados del estudio se realizaron varios exámenes antes y después de operados los pacientes. RESULTADOS: Los defectos refractivos fueron superiores con significación estadística (pOBJECTIVE: Evaluating comparatively the refractive results obtained in eyes operated by keratomileusis in situ assisted with laser (LASIK versus keratomileusis subepithelial assisted with laser plus Mitomycin C (LASEK + MC. METHODS: A prospective, experimental and comparative study was performed in 210 eyes of patients who went to see the specialist to be operated from some refractive defect. A total of 104 eyes were operated by LASIK and 106 by LASEK + MC. In order to compare the results of study, several tests were made before and after surgery. RESULTS: The refractive defects were greater with statistical significance (p< 0,05, for Student's t for the eyes operated by LASEK + MC. The visual result of the LASEK with MC surgery was better than that of LASIK. Haze was a problem resolved for the LASEK when included Mitomycin C in patients with high myopia, in addition to improvement of quality of life of the patients. CONCLUSIONS: The corneal surgery with laser through the LASEK technique plus use of Mitomycin C demonstrated being as reliable as that with LASIK for ametropy correction, since it provides additional advantages that increases the quality of life of patient, with less postoperative risks.

  19. Single-step transepithelial ASLA (SCHWIND with mitomycin-C for the correction of high myopia: long term follow-up

    Directory of Open Access Journals (Sweden)

    Aslanides IM

    2014-12-01

    Full Text Available Ioannis M Aslanides, Panagiotis N Georgoudis, Vasilis D Selimis, Achyut N Mukherjee Emmetropia Mediterranean Eye Institute, Heraklion, Crete, Greece Purpose: We wanted to compare the outcomes of single-step modified transepithelial photorefractive keratectomy (tPRK termed a SCHWIND all surface laser ablation (ASLA versus conventional alcohol-assisted photorefractive keratectomy (PRK and laser-assisted in situ keratomileusis (LASIK for the correction of higher myopia of 6.00 diopters (D or more, in an area with high risk of haze due to high intensity of sunlight.Methods: We used a prospective interventional cohort with matched retrospective control groups. Patients with >6 D myopia and <3.5 D of astigmatism were included. All treatments were performed with the SCHWIND Amaris system using aspheric ablation profiles. Mitomycin C was used in all PRK and ASLA cases. Outcomes were postoperative refraction, visual acuity, stability, and complications. The follow-up period was up to 12 months.Results: In total, 101 eyes were included after exclusions. Mean preoperative spherical equivalent refraction was −7.9 D, −8.2 D, and −7.4 D in the ASLA (n=41, PRK (n=29, and LASIK (n=31 groups. Mean postoperative spherical equivalent at 12 months postoperatively was −0.1 (standard deviation [SD]: 0.34, −0.2 (SD: 0.59, and −0.08 (SD: 0.36 in the ASLA, PRK, and LASIK groups, with 91.4%, 85.7%, and 83.9% within 0.5 D of target, respectively. Refractive outcomes and regression at 12 months did not vary among groups (P>0.05. Mean logMAR (logarithm of the minimum angle of resolution uncorrected distance visual acuity at 12 months was 0.00 (SD: 0.05, 0.06 (SD: 0.1, and 0.05 (SD: 0.09 in the ASLA, PRK, and LASIK groups, with significantly better vision in the tPRK group versus LASIK (P=0.01 and PRK (P=0.01 groups.Conclusion: ASLA (SCHWIND tPRK with mitomycin C for high myopia demonstrates comparable refractive outcomes to LASIK and PRK, with relatively

  20. Eradication of Biofilm-like Microcolony Structures of Borrelia burgdorferi by Daunomycin and Daptomycin but not Mitomycin C in Combination with Doxycycline and Cefuroxime

    Directory of Open Access Journals (Sweden)

    Jie eFeng

    2016-02-01

    Full Text Available Lyme disease, caused by Borrelia burgdorferi, is the most common vector-borne disease in the United States and Europe. While the majority of Lyme disease patients can resolve their symptoms if treated promptly, 10-20% of patients suffer from prolonged symptoms called post-treatment Lyme disease syndrome (PTLDS. Although the cause for PTLDS is unclear, one possibility is the presence of bacterial persisters not effectively cleared by the current Lyme antibiotics. Recent studies identified several drug candidates including daptomycin, daunomycin, doxorubicin, and mitomycin C that had good activity against B. burgdorferi persisters. However, their relative activities against B. burgdorferi persisters have not been evaluated under the same conditions. In this study, we tested the anti-persister activities of these drugs against both 7-day and 15-day old stationary phase cultures of B. burgdorferi individually as well as in combination with Lyme antibiotics doxycycline and cefuroxime (Ceftin. Our findings demonstrate daunomycin and daptomycin were more active than mitomycin C in single drug comparison at 10 and 20 µM, as well as in drug combinations with doxycycline and cefuroxime. In addition, daunomycin was more active than doxorubicin which correlated with their ability to stain and accumulate in B. burgdorferi. The two drug combination of doxycycline and cefuroxime was unable to eradicate biofilm-like microcolonies of B. burgdorferi persisters. However, the addition of either daunomycin or daptomycin to the doxycycline + cefuroxime combination completely eradicated the biofilm-like structures and produced no visible bacterial regrowth after 7 days and 21 days, while the addition of doxorubicin was unable to prevent regrowth at either 7 day or 21 day subculture. Mitomycin C in combination with doxycycline and cefuroxime caused no regrowth at 7 days but visible spirochetal regrowth occurred after 21 day subculture. Furthermore, we found that

  1. Simultaneous Integrated Boost–Intensity Modulated Radiation Therapy With Concomitant Capecitabine and Mitomycin C for Locally Advanced Anal Carcinoma: A Phase 1 Study

    International Nuclear Information System (INIS)

    Deenen, Maarten J.; Dewit, Luc; Boot, Henk; Beijnen, Jos H.; Schellens, Jan H.M.; Cats, Annemieke

    2013-01-01

    Purpose: Newer radiation techniques, and the application of continuous 5-FU exposure during radiation therapy using oral capecitabine may improve the treatment of anal cancer. This phase 1, dose-finding study assessed the feasibility and efficacy of simultaneous integrated boost–intensity modulated radiation therapy (SIB-IMRT) with concomitant capecitabine and mitomycin C in locally advanced anal cancer, including pharmacokinetic and pharmacogenetic analyses. Methods and Materials: Patients with locally advanced anal carcinoma were treated with SIB-IMRT in 33 daily fractions of 1.8 Gy to the primary tumor and macroscopically involved lymph nodes and 33 fractions of 1.5 Gy electively to the bilateral iliac and inguinal lymph node areas. Patients received a sequential radiation boost dose of 3 × 1.8 Gy on macroscopic residual tumor if this was still present in week 5 of treatment. Mitomycin C 10 mg/m 2 (maximum 15 mg) was administered intravenously on day 1, and capecitabine was given orally in a dose-escalated fashion (500-825 mg/m 2 b.i.d.) on irradiation days, until dose-limiting toxicity emerged in ≥2 of maximally 6 patients. An additional 8 patients were treated at the maximum tolerated dose (MTD). Results: A total of 18 patients were included. The MTD of capecitabine was determined to be 825 mg/m 2 b.i.d. The predominant acute grade ≥3 toxicities included radiation dermatitis (50%), fatigue (22%), and pain (6%). Fifteen patients (83% [95%-CI: 66%-101%]) achieved a complete response, and 3 (17%) patients a partial response. With a median follow-up of 28 months, none of the complete responders, and 2 partial responders had relapsed. Conclusions: SIB-IMRT with concomitant single dose mitomycin C and capecitabine 825 mg/m 2 b.i.d. on irradiation days resulted in an acceptable safety profile, and proved to be a tolerable and effective treatment regimen for locally advanced anal cancer

  2. Cryopreservation of adult unrelated donor products in hematopoietic cell transplantation: the OneMatch experience and systematic review of the literature.

    Science.gov (United States)

    Aziz, Joseph; Morris, Gail; Rizk, Mina; Shorr, Risa; Mercer, Dena; Young, Kimberly; Allan, David

    2017-11-01

    The frequency of cryopreserving blood stem or progenitor products from unrelated donors is not known and the underlying reasons are poorly documented. Greater insight is needed to develop policies on cryopreservation that balance donor safety with patient needs. Cryopreservation requests between January 1, 2014, and May 31, 2016, at the OneMatch Stem Cell and Marrow Network at Canadian Blood Services were reviewed and a systematic review of the literature was performed. Thirty products of 719 (4.2%) unrelated donor collections facilitated by OneMatch were cryopreserved. Patient-related reasons were most common and included the need to delay transplant for continued antimicrobial treatment (six patients), patient too deconditioned to proceed with scheduled transplant (five patients), and/or need for more treatment for relapsed disease (three patients). Donor-related issues leading to cryopreservation requests were less common (five cases), mainly due to lack of donor availability after attempting to reschedule. Cryopreservation of a product that was never infused occurred infrequently (two cases, 7%). In our systematic review of the literature, 993 cases were identified in 32 published reports. Both patient-related and donor-related reasons were cited but not specifically reported, precluding quantitative insight regarding the relative frequency of causes. The impact of cryopreservation on hematopoietic engraftment appears negligible when compared to controls in a subset of studies; however, reporting of outcomes was inconsistent. Future studies with standard outcome measures are needed to clarify the impact of cryopreservation on engraftment and other transplant outcomes. International guidelines that consider the ethical framework surrounding requests for donor product cryopreservation are needed. © 2017 AABB.

  3. Analysis of the impact of cryopreservation and theophylline on motility of sperm

    Directory of Open Access Journals (Sweden)

    Elaheh Gorji

    2018-06-01

    Full Text Available Objective: Sperm parameters, particularly motility, decrease during cryopreservation. Theophylline generally enhances sperm motility. We analyzed effects of theophylline and freezing on sperm motility.Design: Experimental study.Setting: Private IVF lab.Setting: IVF lab of Mehrgan Hospital. Method: 22–55 year-old men participated in this study (30 fresh ejaculation and 8 TESE samples. After sperm analysis, we added theophylline (40 mM to half of our samples as case group to compare motility with the remaining samples as control group. Cryopreservation was performed in two groups. After thawing, motility of both groups was recorded. Furthermore, theophylline (40 mM was applied to both groups after thawing again. Result: After adding theophylline, sperm motility improved significantly in all samples. Sperm motility reduced in control group more than the study group after freeze-thaw procedure (P < 0.002, normal morphology <5%. Sperm motility was not enhanced significantly by re-adding of theophylline to the two groups. Interactions between stages and groups were statistically significant in semen and biopsy samples (p < 0.001. Conclusion: Adding theophylline before freezing can preserve motility of sperms in samples with different parameters and even sperms extracted in testicular biopsy. Theophylline may have protective impact on sperms in freezing procedure. Keywords: Sperm motility, Theophylline, Freezing, Morphology, Biopsy

  4. Initial 10-year Experience of Sperm Cryopreservation Services for Cancer Patients

    Directory of Open Access Journals (Sweden)

    Hong-Chiang Chang

    2006-01-01

    Full Text Available Offering sperm cryopreservation to preserve the fertility of male cancer patients is a relatively recent service in Asia. This study analyzed the types of cancer, timing of collection, sperm quality, and utilization for reproductive services by patients during a 10-year period at a medical center in Taiwan. A total of 75 oncology patients elected to freeze sperm for fertility preservation at our medical center during the initial 10 years of the availability of this service. The mean age of the patients was 25.7 years. Storage was discontinued in 13 (17% patients and their survival duration was 13.1 ±11.1 months. The utilization rate of sperm cryo-preservation was 2.8% (75/2642. The types of cancer varied, with leukemia (35%, lymphoma (25%, and testicular cancer (13% comprising the largest groups. A significantly lower sperm count was found in patients with chronic myelogenous leukemia, suggesting the need for earlier sperm collection after initiation of cancer treatment. Only three (4% patients utilized their specimens for reproductive purposes. There was no clinical pregnancy during the study period, although one biochemical pregnancy was achieved. The low rates of sperm cryostorage for fertility preservation in cancer patients in this study suggest that there is a need for greater emphasis of this option for male oncology patients whose fertility is likely to be affected by chemotherapeutic treatment.

  5. Biomechanical and ultrastructural comparison of cryopreservation and a novel cellular extraction of porcine aortic valve leaflets.

    Science.gov (United States)

    Courtman, D W; Pereira, C A; Omar, S; Langdon, S E; Lee, J M; Wilson, G J

    1995-12-01

    Heart valve substitutes of biological origin often fail by degenerative mechanisms. Many authors have hypothesized that mechanical fatigue and structural degradation are instrumental to in vivo failure. Since the properties of the structural matrix at implantation may predetermine failure, we have examined the ultrastructure, fracture, mechanics, and uniaxial high-strain-rate viscoelastic properties of: (1) fresh, (2) cryopreserved, and (3) cellular extracted porcine aortic valve leaflets. The cellular extraction process is being developed in order to reduce immunological attack and calcification. Cryopreservation causes cellular disruption and necrotic changes throughout the tissue, whereas extraction removes all cells and lipid membranes. Both processes leave an intact collagen and elastin structural matrix and preserve the high-strain-rate viscoelastic characteristics of the fresh leaflets. Extraction does cause a 20% reduction in the fracture tension and increases tissue extensibility, with the percent strain at fracture rising to 45.3 +/- 4 (mean +/- SEM) from 31.5 +/- 3 for fresh leaflets. However, extraction does preserve matrix structure and mechanics over the physiological loading range. Glutaraldehyde fixation produces increased extensibility, increased elastic behavior, and, when applied to extracted leaflets, it causes a marked drop in fracture tension, to 50% of that for fresh leaflets. The combination of extraction and fixation may lead to early degenerative failure. The cellular extraction technique alone may be a useful alternative to glutaraldehyde fixation in preparing bioprosthetic heart valves.

  6. EFFECT OF DIFFERENT CRYOPROTECTIVE AGENTS ON SKIM MILK AND DIMITROPOULUS EXTENDER FOR STALLION SEMEN CRYOPRESERVATION

    Directory of Open Access Journals (Sweden)

    R.I. Arifiantini

    2014-10-01

    Full Text Available s to assess different CPAs on stallion semen cryopreservation. Skim milk (SM and Dimitropoulos(DV were the extenders used in this study; each was added by glycerol (Gly, combination of ethyleneglycol-glycerol (EG+Gly or dimethilformamide (DMF. Each semen sample was evaluated and dividedequally into six tubes; semen in the three tubes was diluted 1:1 with (SM, while in the remaining tubesthe semen was diluted 1:1 by DV. After being diluted, all tubes were centrifuged at 1006xg for 10minutes. The supernatan discarded, the pellet was rediluted by SM trehalosa or DV trehalose, and addedby G, EG+Gly, or DMF to reach the final sperm concentration of 200x106/ml. The extended semen wasindividually packed in 0.3 ml minitube, equilibrated at 4oC for 2 hours, frozen in liquid nitrogen vaporfor 10 minutes, and then was stored in liquid nitrogen container at -196 oC. After 24 hours, the semenwas thawed at 37 oC for 30 second. There were no significantly different (p>0.05 on the percentages ofmotile and viable sperm in SMT (21.7% and 43.4%, respectively compared with those extended withDV T extender (26.9% and 50.8%, respectively. DMF demonstrated better results as CPA compared tothe others; and DVTDMF combination had the best protection during cryopreservation in this study.

  7. Establishment and cryopreservation of a giant panda skeletal muscle-derived cell line.

    Science.gov (United States)

    Yu, Fang-Jian; Zeng, Chang-Jun; Zhang, Yan; Wang, Cheng-Dong; Xiong, Tie-Yi; Fang, Sheng-Guo; Zhang, He-Min

    2015-06-01

    The giant panda Ailuropoda melanoleuca is an endangered species and is a symbol for wildlife conservation. Although efforts have been made to protect this rare and endangered species through breeding and conservative biology, the long-term preservation of giant panda genome resources (gametes, tissues, organs, genomic libraries, etc.) is still a practical option. In this study, the giant panda skeletal muscle-derived cell line was successfully established via primary explants culture and cryopreservation techniques. The population doubling time of giant panda skeletal cells was approximately 33.8 h, and this population maintained a high cell viability before and after cryopreservation (95.6% and 90.7%, respectively). The two skeletal muscle-specific genes SMYD1 and MYF6 were expressed and detected by RT-PCR in the giant panda skeletal muscle-derived cell line. Karyotyping analysis revealed that the frequencies of giant panda skeletal muscle cells showing a chromosome number of 2n=42 ranged from 90.6∼94.2%. Thus, the giant panda skeletal muscle-derived cell line provides a vital resource and material platform for further studies and is likely to be useful for the protection of this rare and endangered species.

  8. Random-start ovarian stimulation in women desiring elective cryopreservation of oocytes.

    Science.gov (United States)

    Pereira, Nigel; Voskuilen-Gonzalez, Anna; Hancock, Kolbe; Lekovich, Jovana P; Schattman, Glenn L; Rosenwaks, Zev

    2017-10-01

    The current study investigates the utility of random-start ovarian stimulation in women desiring elective oocyte cryopreservation. Women in the study cohort underwent random-start ovarian stimulation, and were subdivided based on the phase of the menstrual cycle that ovarian stimulation began, i.e. early follicular, late follicular or luteal phase. Women undergoing conventional cycle day (CD) 2/3 ovarian stimulation start were controls. A total of 1302 women were included - 859 (66.0%) conventional CD 2/3, 342 (26.3%) early follicular, 42 (3.2%) late follicular and 59 (4.5%) luteal ovarian stimulation starts. There was no difference in the demographics or baseline ovarian stimulation characteristics. The duration of ovarian stimulation (11 versus 9 days; P start group. The number of total and MII oocytes in the control and random-start groups was similar. A non-significant trend towards increased cycle cancellation was noted in the late follicular start group (7.1%). Study findings indicate the number of total and MII oocytes derived from random-start protocols initiated during any phase of the menstrual cycle is similar to conventional CD 2/3 ovarian stimulation start protocols in women desiring elective oocyte cryopreservation. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  9. Effects of some cryopreservation procedures on recalcitrant zygotic embryos of Ammocharis coranica.

    Science.gov (United States)

    Nomali, Z; Ngobese; Sershen; Berjak, P; Pammenter, N W

    2014-01-01

    Cryopreservation, the most promising method for the long-term conservation of recalcitrant (desiccation-sensitive) seed germplasm, is often associated with high viability losses. Cryo-procedures involve a sequence of steps which must be optimised to reduce the impact of the stresses. This study reports on the effects of some of the steps of cryopreservation on the recalcitrant zygotic embryos of the amaryllid, Ammocharis coranica. Embryos were subjected to cryoprotection with glycerol and/or DMSO, rapid (flash) drying, and rapid (>100 degree C s(-1)) or slow (1 degree C s(-1)) cooling. Rapid dehydration (from c. 2.7 to 0.9 g g(-1) over 60 min) and cooling had a detrimental effect on the viability of the embryos, which was exacerbated when these steps were applied sequentially. After cooling, seedling production (30%) was obtained only from embryos that had been cryoprotected with glycerol prior to drying and rapid cooling, while 30% of non-treated embryos and 70% of those that had undergone cathodic protection during flash drying produced callus. Noting that no post-cryo survival of A. coranica embryos had previously been obtained, this study identified cryoprotection with glycerol and the incorporation of cathodic protection during flash drying as promising intervention points for future studies.

  10. In vitro assessment of soybean lecithin and egg yolk based diluents for cryopreservation of goat semen.

    Science.gov (United States)

    Salmani, Hossein; Towhidi, Armin; Zhandi, Mahdi; Bahreini, Majid; Sharafi, Mohsen

    2014-04-01

    Soybean lecithin is a suitable plant-based cryoprotectant for freezing ruminant sperm. Optimum level of lecithin was not clear for goat semen cryopreservation. The objective of this study was to investigate the effects of different levels of soybean lecithin in semen extender on post-thaw sperm quality including CASA-motion parameters, viability, plasma membrane integrity and lipid peroxidation. Semen samples were collected from 4 Mahabadi bucks using an artificial vagina. Different concentrations of soy lecithin (SL, 0.5%, 1%, 1.5%, 2% and 2.5% w/v) were compared to 15% (v/v) egg yolk-based extender (TR-EY). No significant difference was observed for sperm progressive motility, viability or plasma membrane integrity in 1.5% SL media (33.8%, 66%, and 62.7%, respectively) and TR-EY medium (35.4%, 67.2%, and 64.9%, respectively). Sperm motion characteristics (VAP, VSL, VCL, ALH and LIN) and rapid spermatozoa were improved with extender containing 1% and 1.5% SL, compared to TR-EY extender. Furthermore, egg yolk produced significantly higher malondialdehyde (4.02±0.21) than other groups. Results suggest that the optimal lecithin concentration in the semen extender was 1.5% and also soy lecithin can substitute for egg yolk during cryopreservation for caprine sperm. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Application of Pectin From Rauvolfia serpentina (L.) Benth to the Cryopreservation of Human Leucocyte Cell Suspensions.

    Science.gov (United States)

    Zaitseva, O O; PoleZhaeva, T V; Khudvakov, A N; Solomina, O N; Golovchenko, V V

      BACKGROUND: Due to their valuable medicinal properties and high physiological activity, plant polysaccharides are currently being extensively studied. The present study aims to investigate rauwolfian (pectin for Rauvolfia serpentina) supplementation on human leukocytes cryopreservation. We determined the сharacteristics of leukocytes undergoing freezing with pectin at different temperatures. Donor leukocytes were frozen under the protection of comprehensive cryoprotectant solution and stored in electrical freezers (-20C, -40C, -80C). A regular decrease of all values starting from a higher temperature (-20С) through to the lower temperature (-80С) was identified. The study showed that pectin rauwolfian stimulated both the oxygen-independent and the oxygen-dependent killer response. We also found that the oxygen-dependent neutrophil killer effect was reduced as the storage temperature was lowered. It was determined that the LPO levels in the cells with added pectin-containing solutions remained the same before freezing, while their antioxidant activity positively increased, which is beneficial for neutrophils for their further freezing to -20C, -40C and -80C. The results of the study make it possible to assume that rauwolfian, a pectin extracted from Rauvolfia serpentina, has an exocellular protectant effect as part of cryopreservative solution on human white blood cells stored at different low temperatures. The versatility of the substance is probably due to the degree of the macromolecule branching, in particular, the structure of carbohydrate side chains, which contain a large number of hydroxyl groups.

  12. Effect of cryopreservation on mitochondrial activity in buffalo sperm Bubalus bubalis

    Directory of Open Access Journals (Sweden)

    O. Kandil

    2010-02-01

    Full Text Available Sperm mitochondrial activity is investigated and used as “in vitro” spermatozoa vitality indicator and about quality effectiveness of different sperm diluents. It was studied the cytochemically activity of NADPH-diaphorase and LDH-C4 in cryopreserved buffalo sperm. Low intensity of the enzyme reaction was established in all examined sperm samples in both enzymes, regardless from the used cryoprotectors. The main part of the enzyme reaction was localized in mitochondrial sheath and in a very small degree in the head base of spermatozoa. No increase of the enzymes activities or the spermatozoa motility has been found after the incubating with Sp-TALP medium although the established caffeine stimulating effect on the glycolysis and fresh spermatozoa motility. Established by us low sperm motility after cryopreservation may be due to low LDH and NADPH-diaphorase activity due to glycolisis disturbances and ATP synthesis. This method allows to estimate quality of buffalo semen and to find some different disturbances in mitochondrial sheath, which could not be found by routine morphological studies and could be used in practice ejaculates with high number of metabolic active sperm cells.

  13. Value of lymphocyte cryo-preservation after a radiological or nuclear accident

    International Nuclear Information System (INIS)

    Laroche, P.; Lataillade, J.J.; Chambrette, V.; Voisin, Ph.

    1997-01-01

    The conventional cytogenetic method in biological dosimetry is most useful for the estimation of the received radiation dose. It shows resulting unstable chromosomal aberrations (dicentrics, centric rings and fragments) in peripheral blood lymphocytes. This method has been used over the past 30 years and is used in forensic medicine. Nevertheless, it is long and fastidious. Accordingly, the number of simultaneous analyses of blood samples is limited and depends on the capacity of specialized laboratories. This capacity may be insufficient in the case of large scale radiological or nuclear accidents. Cryo-preservation is the usual method to store cells before analysis or use, for instance for biological dosimetry purposes. Some investigations have shown that thawing following freezing may induce cell injury but few studies have been made on the effect of cryo-preservation on cells containing radiation-induced unstable chromosomal aberrations. In this work, lymphocytes were irradiated with 1 to 4 Gy gamma rays and stored in liquid nitrogen. The dicentric and centric ring yields were analysed after storage periods of 1 week, 1 month, 3 months, and 1 year. No difference in aberration frequency from control, unfrozen samples was observed over this period. Lymphocytes stored at -196 deg C for up to least 1 year may therefore be used for chromosome aberration scoring when overexposure to ionizing radiation is suspected. (author)

  14. B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody

    Directory of Open Access Journals (Sweden)

    Philipp Fecher

    2018-05-01

    Full Text Available In individuals who have once developed humoral immunity to an infectious/foreign antigen, the antibodies present in their body can mediate instant protection when the antigen re-enters. Such antigen-specific antibodies can be readily detected in the serum. Long term humoral immunity is, however, also critically dependent on the ability of memory B cells to engage in a secondary antibody response upon re-exposure to the antigen. Antibody molecules in the body are short lived, having a half-life of weeks, while memory B cells have a life span of decades. Therefore, the presence of serum antibodies is not always a reliable indicator of B cell memory and comprehensive monitoring of humoral immunity requires that both serum antibodies and memory B cells be assessed. The prevailing view is that resting memory B cells and B cell blasts in peripheral blood mononuclear cells (PBMC cannot be cryopreserved without losing their antibody secreting function, and regulated high throughput immune monitoring of B cell immunity is therefore confined to—and largely limited by—the need to test freshly isolated PBMC. Using optimized protocols for freezing and thawing of PBMC, and four color ImmunoSpot® analysis for the simultaneous detection of all immunoglobulin classes/subclasses we show here that both resting memory B cells and B cell blasts retain their ability to secrete antibody after thawing, and thus demonstrate the feasibility of B cell immune monitoring using cryopreserved PBMC.

  15. Cryopreservation of yamú (Brycon amazonicus) sperm for large scale fertilization

    DEFF Research Database (Denmark)

    Velasco-Santamaría, Yohana M.; Medina-Robles, Mauricio; Cruz-Casallas, Pablo E.

    2006-01-01

      To determine the effect of straw size and thawing temperature on cryopreserved sperm quality of yamú (Brycon amazonicus), ovulation and spermiation were induced in sexually mature broodstock using Carp Pituitary Extract. Sperm quality was evaluated by motility, activation time and fertility...... assays consisted of 40 g eggs inseminated with approximately 5.0 mL (ca. 75,000 motile spermatozoa/egg) of cryopreserved sperm in large straws thawed at 35 °C. The fertilization rate was estimated 6 h post-insemination. In all straws, postthaw motility was significantly lower than for fresh sperm (pb0.......05) to sperm frozen in 0.5-mL straws (48±2%, 51±2%, 52±2% and 54±3%, respectively). In large scale fertilization trials, fresh sperm showed a higher (pb0.05) fertilization rate (83±1%) than frozen-thawed sperm (68±1%). Although the fertility percentage with fresh sperm was significantly higher than with frozen...

  16. Use of commercial extenders and alternatives to prevent sperm agglutination for cryopreservation of brown bear semen.

    Science.gov (United States)

    Gomes-Alves, S; Alvarez, M; Nicolas, M; Lopez-Urueña, E; Martínez-Rodríguez, C; Borragan, S; de Paz, P; Anel, L

    2014-08-01

    The objective of this study was to evaluate different bovine and canine commercial semen extenders for cryopreservation of brown bear ejaculates and the effect of semen collection directly into extender on sperm agglutination. Semen samples were obtained by electroejaculation from 13 adult males. In experiment 1, eleven ejaculates from eight bears were used to evaluate Bioxcell and Andromed as extenders, whereas in experiment 2, nine ejaculates from six bears were used to evaluate Triladyl canine, CaniPro, and Extender 2 as extenders. An extender specifically developed for brown bears (Test-Tris-fructose-egg yolk-glycerol, TTF-ULE/bear) served as a control extender in both experiments. After thawing, total and progressive sperm motility and sperm viability were greater (P bear and Andromed extenders than for Bioxcell in experiment 1 and greater (P bear extender than for Triladyl Canine, CaniPro, and Extender 2 in experiment 2. In experiment 3, addition of handling extender (TTF-H) to the semen collection tube for eight ejaculates from seven bears resulted in less (P bear is the most suitable extender for brown bear semen cryopreservation, but comparable results can be obtained with the commercial extender Andromed. In addition, collection of ejaculates directly in TTF-H extender decreases sperm agglutination in fresh samples. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Optimization of conditions for long-term prefreezing storage of brown bear sperm before cryopreservation.

    Science.gov (United States)

    López-Urueña, E; Alvarez, M; Gomes-Alves, S; Anel-López, L; Martínez-Rodríguez, C; Manrique, P; Borragan, S; Anel, L; de Paz, P

    2015-10-15

    Brown bear ejaculates are usually collected in field conditions and may need to be shipped to a laboratory for the application of reproductive biotechnologies before cryopreservation. The aim of this study was to extend the prefreezing step to 48 hours (1 hour vs. long-term storage [LS] to 24 and 48 hours) to enable the sample to be transported. The effects of storage temperature (experiment 1), glycerol concentration (experiment 2), and dilution rate (experiment 3) on sperm were evaluated. Electroejaculates from brown bears were stored under different experimental conditions and cryopreserved. The sperm motility and viability, apoptotic status, and acrosomal status of sperm were assessed before freezing (prefreezing), after thawing, and after 2-hour incubation at 37 °C (thermal stress test). In all experiments, one control sample was frozen using a standard protocol (control). In experiment 1, three temperatures during LS with 6% glycerol were tested: 5 °C (T5), 15 °C (T15), and room temperature (RT). The LS-T5 sample yielded the highest postthawing results for viability (42.4%), progressive motility (15.6%), and intact acrosome (83.1%) after 24 hours in comparison with the other temperatures (P bear electroejaculates are at 5 °C, at a concentration of 100 × 10(6) sperm/mL, and with 6% glycerol. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Development of a vitrification-based cryopreservation protocol for the storage of saltcedar (Tamarix boveana Bunge).

    Science.gov (United States)

    Cano-Castillo, M; Casas, J L

    2012-01-01

    We cryopreserved in vitro shoot tips of saltcedar (Tamarix boveana Bunge) using the vitrification technique. The success of the cryopreservation protocol was strongly affected by preculture, loading duration, dehydration duration in plant vitrification solution 2 (PVS2), and medium composition during post-warming regrowth. The highest explant regrowth (50 percent) occurred when the following conditions were employed: preculture in 0.4 M glycerol; treatment with a loading solution (LS) consisting of 2 M glycerol + 0.4 M sucrose in culture medium for 40 min at room temperature; and dehydration in PVS2 at 0 degree C for 45 min before rapid immersion in liquid nitrogen (LN). Rewarming was performed in a water-bath at 40 degree C for 2 min. Explants were then immersed in unloading solution for 10 min before plating on recovery medium supplemented with 0.01 mg per liter thidiazuron (TDZ). TDZ was progressively eliminated from the medium over a period of 6 weeks. Plantlets were transferred to a double-layer medium to enhance rooting. This protocol was successfully applied to three individuals of T. boveana harvested from the wild.

  19. Lipid content and composition of oocytes from five coral species: potential implications for future cryopreservation efforts.

    Directory of Open Access Journals (Sweden)

    Chiahsin Lin

    Full Text Available Given the previously documented importance of lipid concentration and composition in the successful cryopreservation of gorgonian corals, these parameters were assessed in oocytes of five species of scleractinian coral; Platygyra daedalea, Echinopora gemmacea, Echinophyllia aspera, Oxypora lacera and Astreopora expansa. Wax esters, phosphatidylethanolamine, phosphatidylcholine, and fatty acids were all measured at detectable levels, and the latter were produced at significantly elevated quantities in E. gemmacea, E. aspera, and O. lacera. On the other hand, phosphatidylethanolamine, phosphatidylcholine, and wax ester were found at significantly higher concentrations in A. expansa oocytes. Triacylglycerol was not present in any species. Interestingly, the total lipid content of oocytes from all five scleractinians was significantly lower than that of oocytes of two gorgonian species, Junceella juncea and Junceella fragilis. As higher total lipid concentrations may be correlated with greater degrees of cellular membrane fluidity at lower temperatures, it stands to reason that gorgonian coral oocytes may be more likely to survive the cryopreservation process than oocytes of scleractinian corals.

  20. B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody.

    Science.gov (United States)

    Fecher, Philipp; Caspell, Richard; Naeem, Villian; Karulin, Alexey Y; Kuerten, Stefanie; Lehmann, Paul V

    2018-05-31

    In individuals who have once developed humoral immunity to an infectious/foreign antigen, the antibodies present in their body can mediate instant protection when the antigen re-enters. Such antigen-specific antibodies can be readily detected in the serum. Long term humoral immunity is, however, also critically dependent on the ability of memory B cells to engage in a secondary antibody response upon re-exposure to the antigen. Antibody molecules in the body are short lived, having a half-life of weeks, while memory B cells have a life span of decades. Therefore, the presence of serum antibodies is not always a reliable indicator of B cell memory and comprehensive monitoring of humoral immunity requires that both serum antibodies and memory B cells be assessed. The prevailing view is that resting memory B cells and B cell blasts in peripheral blood mononuclear cells (PBMC) cannot be cryopreserved without losing their antibody secreting function, and regulated high throughput immune monitoring of B cell immunity is therefore confined to-and largely limited by-the need to test freshly isolated PBMC. Using optimized protocols for freezing and thawing of PBMC, and four color ImmunoSpot ® analysis for the simultaneous detection of all immunoglobulin classes/subclasses we show here that both resting memory B cells and B cell blasts retain their ability to secrete antibody after thawing, and thus demonstrate the feasibility of B cell immune monitoring using cryopreserved PBMC.

  1. A method for generation of bone marrow-derived macrophages from cryopreserved mouse bone marrow cells.

    Directory of Open Access Journals (Sweden)

    Fernanda M Marim

    Full Text Available The broad use of transgenic and gene-targeted mice has established bone marrow-derived macrophages (BMDM as important mammalian host cells for investigation of the macrophages biology. Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM. Cryopreserved murine BM cells maintain their potential for BMDM differentiation for more than 6 years. We compared BMDM obtained from fresh and frozen BM cells and found that both are similarly able to trigger the expression of CD80 and CD86 in response to LPS or infection with the intracellular bacteria Legionella pneumophila. Additionally, BMDM obtained from fresh or frozen BM cells equally restrict or support the intracellular multiplication of pathogens such as L. pneumophila and the protozoan parasite Leishmania (L. amazonensis. Although further investigation are required to support the use of the method for generation of dendritic cells, preliminary experiments indicate that bone marrow-derived dendritic cells can also be generated from cryopreserved BM cells. Overall, the method described and validated herein represents a technical advance as it allows ready and easy generation of BMDM from a stock of frozen BM cells.

  2. Cryopreservation of human insulin expressing cells macro-encapsulated in a durable therapeutic immunoisolating device theracyte.

    Science.gov (United States)

    Yakhnenko, Ilya; Wong, Wallace K; Katkov, Igor I; Itkin-Ansari, Pamela

    2012-01-01

    Encapsulating insulin producing cells (INPCs) in an immunoisolation device have been shown to cure diabetes in rodents without the need for immunosuppression. However, micro-encapsulation in semi-solid gels raises longevity and safety concerns for future use of stem cell derived INPCs. We have focused on a durable and retrievable macro-encapsulation (> 10(6) cells) device (TheraCyte). Cryopreservation (CP) of cells preloaded into the device is highly desirable but may require prolonged exposure to cryoprotectants during loading and post-thaw manipulations. Here, we are reporting survival and function of a human islet cell line frozen as single cells or as islet-like cell clusters. The non-clusterized cells exhibited high cryosurvival after prolonged pre-freeze or post-thaw exposure to 10 percent DMSO. However, both clusterization and especially loading INPCs into the device reduced viable yield even without CP. The survived cryopreserved macro-encapsulated INPCs remained fully functional suggesting that CP of macro-encapsulated cells is a promising tool for cell based therapies.

  3. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification.

    Science.gov (United States)

    Sakai, A; Kobayashi, S; Oiyama, I

    1990-06-01

    The nucellar cells of navel orange(Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved by vitrification. In this method, cells were sufficiently dehydrated with highly concentrated cryoprotective solution(PVS2) prior to direct plunge in liquid nitrogen. The PVS2 contains(w/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in Murashige-Tucker medium(MT) containing 0.15 M sucrose. Cells were treated with 60% PVS2 at 25°C for 5 min and then chilled PVS2 at 0°C for 3 min. The cell suspension of about 0.1 ml was loaded in a 0.5 ml transparent plastic straw and directly plunged in liquid nitrogen for 30 min. After rapid warming, the cell suspension was expelled in 2 ml of MT medium containing 1.2 M sucrose. The average rate of survival was about 80%. The vitrified cells regenerated plantlets. This method is very simple and the time required for cryopreservation is only about 10 min.

  4. The Development of Optimal Cryopreservation Media For Longspine Scraper (Capoeta trutta Sperm

    Directory of Open Access Journals (Sweden)

    Erdinç Şahinöz

    2018-03-01

    Full Text Available This study is performed to determine some of sperm quality after applying freezing / thawing process. Thus, the aim of this study is to examine different cryprotective agents with additives in terms of their effects at different pH on the cryopreservation process of longspine scraper (Capoeta trutta. The present study, twelve media were prepared by mixing three different cryoprotectants (dimethyl sulfoxide (DMSO, (CH32SO; methanol (CH3OH; methyl glycol (MG, CH3O (CH22OH with an extenders (glucose at four different pH (7.2, 7.6, 8.0 and 8.4 for longspine scraper semen. Considering the findings from the examination (The motility rate after thawing process and duration of motility obtained in DMSO as 81% and 20 min, in methanol as 73% and 12 min, in methyl glycol as 60% and 15 min., we can conclude that the DMSO is the best freezing media in order to create new essays in cryopreservation for sperm of Capoeta trutta in the future.

  5. Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation.

    Science.gov (United States)

    Fonseca, Fernanda; Meneghel, Julie; Cenard, Stéphanie; Passot, Stéphanie; Morris, G John

    2016-01-01

    During cryopreservation ice nucleation and crystal growth may occur within cells or the intracellular compartment may vitrify. Whilst previous literature describes intracellular vitrification in a qualitative manner, here we measure the intracellular vitrification temperature of bacteria and yeasts under conditions relevant to cryopreservation, including the addition of high levels of permeating and nonpermeating additives and the application of rapid rates of cooling. The effects of growth conditions that are known to modify cellular freezing resistance on the intracellular vitrification temperature are also examined. For bacteria a plot of the activity on thawing against intracellular glass transition of the maximally freeze-concentrated matrix (Tg') shows that cells with the lowest value of intracellular Tg' survive the freezing process better than cells with a higher intracellular Tg'. This paper demonstrates the role of the physical state of the intracellular environment in determining the response of microbial cells to preservation and could be a powerful tool to be manipulated to allow the optimization of methods for the preservation of microorganisms.

  6. Schistosoma mansoni: vaccination of mice with 10-krad-irradiated, cryopreserved schistosomules

    International Nuclear Information System (INIS)

    Lewis, F.A.; Stirewalt, M.A.; Leef, J.L.

    1984-01-01

    Protection against a Schistosoma mansoni cercarial challenge was evaluated in mice immunized with a vaccine composed of 10-krad-irradiated, cryopreserved schistosomules. The level of resistance induced in C57B1/6 or NMRI (CV) mice increased with the number of schistosomules injected. Up to 83% reduction in challenge worm burden was achieved when 5000 schistosomules were injected per mouse. Intramuscular injection of the vaccine was superior to subcutaneous. Multiple immunizations, up to 3 at 4-week intervals, did not increase the resistance induced by a single immunization. A high level of protection developed in as little as 2 weeks and was maintained through at least 12 weeks postimmunization. The vaccine irradiated with 10 krad from either a 60-cobalt or 137-cesium source induced equivalent levels of resistance, and no differences were found in the immunogenicity of vaccines comprised of organisms irradiated as cercariae or as 1- to 3-hr-old schistosomules. These findings are basic to the development of a cryopreserved, live vaccine against schistosomiasis of humans or domestic animals

  7. Effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation.

    Science.gov (United States)

    Gómez-Fernández, José; Gómez-Izquierdo, Emilio; Tomás, Cristina; Mocé, Eva; de Mercado, Eduardo

    2012-07-01

    The aim of the present study was to evaluate the cryoprotectant effect of different non-permeating sugars for boar sperm. Pooled semen from three boars was used for the experiments. In the first experiment, the sperm quality of boar sperm cryopreserved with an egg-yolk based extender supplemented with different monosaccharides (glucose, galactose or fructose) was compared to a control cryopreserved in lactose-egg yolk extender. In the second experiment, the effect of five disaccharides (lactose, sucrose, lactulose, trehalose or melibiose) on boar sperm cryosurvival was studied. Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37°C after thawing: percentages of sperm with intact plasma membrane (SIPM), sperm presenting high plasma membrane fluidity (HPMF), sperm with intracellular reactive oxygen substances production (IROSP) and apoptotic sperm (AS). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. Freezing extenders supplemented with each of the monosaccharide presented smaller cryoprotective effect than the control extender supplemented with lactose (Pextender supplemented with lactulose exhibited in general the lowest sperm quality, except for the percentage of capacitated sperm, which was highest (Pextender. Our results suggest that disaccharides have higher cryoprotective effect than monosaccharides, although the monosaccharide composition of the disaccharides is also important, since the best results were obtained with those disaccharides presenting glucose in their composition. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Boar spermatozoa cryopreservation in low glycerol/trehalose enriched freezing media improves cellular integrity.

    Science.gov (United States)

    Gutiérrez-Pérez, Oscar; Juárez-Mosqueda, María de Lourdes; Carvajal, Salvador Uribe; Ortega, María Elena Trujillo

    2009-06-01

    The use of glycerol for boar semen cryopreservation results in low fertility, possibly due to toxicity. This has led to recommend the use of solutions with less than 4% glycerol. Trehalose is a disaccharide known to stabilize proteins and biologic membranes during processes such as cryopreservation. Thus, it was decided to evaluate the cryoprotective effect of glycerol/trehalose mixtures. Effects on motility (M), viability (Vb) and acrosomal integrity (nA) were evaluated. Sperm samples were frozen in three different extenders: G4 contained 4% glycerol; T1 contained 1% glycerol plus 250 mM trehalose and T0.5 was constituted by 0.5% glycerol plus 250 mM trehalose. All extenders yielded similar post-freezing/thawing motility rates. Viability was diminished in T0.5 as compared to the others. In regard to acrosome integrity, it was twice as high (Pextender. Thus, T1 twice as many spermatozoa were alive, motile and intact, than in either T0.5 or G4, i.e. during freeze/thawing the use of T1 resulted in twice as many fertile cells as when using the other extenders. During our study, we noted that there were wide individual variations both in sperm viability and in motility.

  9. Dimethylformamide is not better than glycerol for cryopreservation of boar semen.

    Science.gov (United States)

    Malo, C; Gil, L; Cano, R; Martínez, F; García, A; Jerez, R A

    2012-05-01

    To improve the boar sperm cryopreservation process, the influence of the sugar (lactose, trehalose) source and the cryoprotectant [glycerol, dimethylformamide (DMF)] on the success of freezing was investigated. Sperm samples were frozen in one of six extenders: lactose plus 3% glycerol (LG); lactose plus 1.5% glycerol and 1.5% DMF (LGD); lactose plus 3% DMF (LD); trehalose plus 3% glycerol (TG); trehalose plus 1.5% glycerol and 1.5% DMF (TGD); trehalose plus 3% DMF (TD). Effects on motility, viability, acrosome integrity and hypoosmotic test (HOST) were measured. The results showed that extender containing 3% glycerol retained the highest motility percentages. In regard to viability and acrosome integrity, all extenders yielded similar rates except for the decreasing values of TD. Endosmosis was diminished in TD and LD at 2 h (P = 0.0018), as compared with the others. The results of the study demonstrated that the use of DMF as a cryoprotectant adversely affected boar sperm quality after cryopreservation. © 2011 Blackwell Verlag GmbH.

  10. On the Effects of Thermal History on the Development and Relaxation of Thermo-Mechanical Stress in Cryopreservation.

    Science.gov (United States)

    Eisenberg, David P; Steif, Paul S; Rabin, Yoed

    2014-01-01

    This study investigates the effects of the thermal protocol on the development and relaxation of thermo-mechanical stress in cryopreservation by means of glass formation, also known as vitrification. The cryopreserved medium is modeled as a homogeneous viscoelastic domain, constrained within either a stiff cylindrical container or a highly compliant bag. Annealing effects during the cooling phase of the cryopreservation protocol are analyzed. Results demonstrate that an intermediate temperature-hold period can significantly reduce the maximum tensile stress, thereby decreasing the potential for structural damage. It is also demonstrated that annealing at temperatures close to glass transition significantly weakens the dependency of thermo-mechanical stress on the cooling rate. Furthermore, a slower initial rewarming rate after cryogenic storage may drastically reduce the maximum tensile stress in the material, which supports previous experimental observations on the likelihood of fracture at this stage. This study discusses the dependency of the various stress components on the storage temperature. Finally, it is demonstrated that the stiffness of the container wall can affect the location of maximum stress, with implications on the development of cryopreservation protocols.

  11. Exploring the Possibility of Cryopreservation of Feline and Canine Erythrocytes by Rapid Freezing with Penetrating and Non-Penetrating Cryoprotectants.

    Directory of Open Access Journals (Sweden)

    Denys Pogozhykh

    Full Text Available Efficient application of veterinary blood transfusion approaches for small companion animals requires readily available supply of the donor material. This can be achieved by developing of effective biobanking technologies allowing long-term storage of donor blood components via cryopreservation. Transfusion of an erythrocyte concentrate allows the successful correction of various hematological pathologies, severe bleeding, and etc. While in the past there were several approaches to cryopreserve red blood cells of dogs, to our knowledge t