Sample records for subcutaneously transplanted cryopreserved

  1. Extensive Hair Shaft Growth after Mouse Whisker Follicle Isolation, Cryopreservation and Transplantation in Nude Mice. (United States)

    Cao, Wenluo; Li, Lingna; Tran, Benjamin; Kajiura, Satoshi; Amoh, Yasuyuki; Liu, Fang; Hoffman, Robert M


    We previously demonstrated that whole hair follicles could be cryopreserved to maintain their stem-cells differentation potential. In the present study, we demonstrated that cryopreserved mouse whisker hair follicles maintain their hair growth potential. DMSO better cryopreserved mouse whisker follicles compared to glycerol. Cryopreserved hair follicles also maintained the hair follicle-associated-pluripotent (HAP) stem cells, evidenced by P75NTR expression. Subcutaneous transplantation of DMSO-cryopreserved hair follicles in nude mice resulted in extensive hair fiber growth over 8 weeks, indicating the functional recovery of hair shaft growth of cryopreserved hair follicles.

  2. Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation. (United States)

    Jitraruch, Suttiruk; Dhawan, Anil; Hughes, Robin D; Filippi, Celine; Lehec, Sharon C; Glover, Leanne; Mitry, Ragai R


    Intraperitoneal transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. Establishment of banked cryopreserved hepatocyte microbeads is important for emergency use. The aim of this study was to develop an optimized protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated. Human and rat hepatocytes cryopreserved with University of Wisconsin (UW)/10% dimethyl sulfoxide (DMSO)/5% (300 mM) glucose and CryoStor CS10 showed better postthawing cell viability, attachment, and hepatocyte functions than with histidine-tryptophan-ketoglutarate/10% DMSO/5% glucose and Bambanker. The 2 freezing solutions that gave better results were studied with human and rat hepatocytes microbeads. Similar effects on cryopreserved microbead morphology (external and ultrastructural), viability, and hepatocyte-functions post thawing were observed over 7 d in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone [ZVAD]), an antioxidant (desferoxamine [DFO]), and a buffering and mechanical protectant (human serum albumin [HSA]) on RMBs. ZVAD (60 µM) had a beneficial effect on cell viability that was greater than with DFO (1 mM), HSA (2%), and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes and a lower degree of cell apoptosis were observed with all 3 cytoprotectants, with ZVAD tending to provide the greatest effect. Cytochrome P450 activity was significantly higher in the 3 cytoprotectant groups than with fresh microbeads. In conclusion, developing an optimized cryopreservation protocol by adding

  3. The safety of transplanting cryopreserved ovarian tissue in cancer patients

    DEFF Research Database (Denmark)

    Rosendahl, Mikkel; Greve, Tine; Andersen, Claus Yding


    Transplantation of frozen/thawed ovarian tissue from patients with a malignant condition is associated with a risk of re-introduction of the disease as the tissue usually is removed before anti-cancer therapy and may thus contain malignant cells. We review studies investigating the presence...... of malignant cells in cryopreserved ovarian tissue from patients with malignant disease and based on the strength of the evidence, recommendations for transplantations are proposed....

  4. Rat Hindlimb Cryopreservation and Transplantation: A Step Toward "Organ Banking". (United States)

    Arav, A; Friedman, O; Natan, Y; Gur, E; Shani, N


    In 2016, over 5 million reconstructive procedures were performed in the United States. The recent successes of clinical vascularized composite allotransplantations, hand and face transplantations included, established the tremendous potential of these life-enhancing reconstructions. Nevertheless, due to limited availability and lifelong immunosuppression, application is limited. Long-term banking of composite transplants may increase the availability of esthetically compatible parts with partial or complete HLA matching, reducing the risk of rejection and the immunosuppressive burden. The study purpose was to develop efficient protocols for the cryopreservation and transplantation of a complete rodent limb. Directional freezing is a method in which a sample is cooled at a constant-velocity linear temperature gradient, enabling precise control of the process and ice crystal formation. Vitrification is an alternative cryopreservation method in which the sample solidifies without the formation of ice crystals. Testing both methods on a rat hindlimb composite tissue transplantation model, we found reliable, reproducible, and stable ways to preserve composite tissue. We believe that with further research and development, cryopreservation may lead to composite tissue "banks." This may lead to a paradigm shift from few and far apart emergent surgeries to wide-scale, well-planned, and better-controlled elective surgeries. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

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

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


    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. Optimizing outcomes from ovarian tissue cryopreservation and transplantation; activation versus preservation

    DEFF Research Database (Denmark)

    Meirow, Dror; Roness, Hadassa; Kristensen, Stine Gry


    Ovarian tissue cryopreservation and transplantation (OTCP) is gaining increasing traction in the field of fertility preservation as a result of accumulated successes. We now have a decade of experience with the technique, with tens of live births and greater than 90% return of ovarian function...... be generated in the immediate short term after transplantation. By contrast, conventional OTCP seeks to maintain dormancy and thus preserve the follicle reserve in the graft with the aim of maximizing graft lifespan. This opinion paper will compare the two methods of OTCP, highlighting their respective...

  7. Effects of cryopreservation on the immunogenicity of porcine arterial allografts in early stages of transplant vasculopathy. (United States)

    Solanes, Núria; Rigol, Montserrat; Khabiri, Ebrahim; Castellà, Manuel; Ramírez, José; Roqué, Mercè; Agustí, Elba; Roig, Eulàlia; Pérez-Villa, Fèlix; Segalés, Joaquim; Pomar, José Luís; Engel, Pablo; Massaguer, Anna; Martorell, Jaume; Rodríguez, Jose Antonio; Sanz, Ginés; Heras, Magda


    The number of revascularization procedures including coronary and lower extremity bypass, have increased greatly in the last decade. It suggests a growing need for vascular grafts. Cryopreserved allografts could represent a viable alternative but their immunologic reactivity remains controversial. 71 pigs (40 recipients and 31 donors) were used. Two femoral grafts per recipient animal were implanted for 3, 7, and 30 days. Types of grafts: fresh autograft as a control graft (n=19), fresh allograft (n=31) and cryopreserved allograft (n=30). Histological and immunohistochemical studies were performed. Fresh allografts compared to autografts showed intimal inflammatory infiltration at 3 days (328 vs. 0 macrophages/mm2; P<0.05) and 7 days (962 vs. 139 T lymphocytes/mm2; P<0.05) post-transplantation. At 30 days, there was a loss of endothelial cells, presence of luminal thrombus and aneurismal lesions (total area=15.8 vs. 8.4 mm2; P<0.05). Cryopreservation did not reduce these lesions nor modify endothelial nitric oxide synthase (eNOS) expression nor modify the number of animals that developed anti-SLA antibodies. Moreover, at 7 days, cryopreserved allografts compared to fresh allografts showed a higher expression of P-selectin (5 out of 5 vs. 1 out of 5; P<0.05) and, at 30 days, a greater inflammatory reactivity (2692 vs. 1107 T lymphocytes/mm2 in media; P<0.05) with a trend towards a higher presence of multinucleated giant cells than in the fresh ones. The cryopreservation method used maintained immunogenicity of allografts and increased the inflammatory reactivity found in fresh allografts up to 30 days of vascular transplantation.

  8. Cryopreservation and transplantation of ovarian tissue exclusively to postpone menopause: technically possible but endocrinologically doubtful. (United States)

    von Wolff, Michael; Stute, Petra


    Transplantation of cryopreserved ovarian tissue has been shown to induce pregnancies and puberty successfully. Therefore, using cryopreserved ovarian tissue to postpone menopause (tissue hormone therapy [THT]) seems to be an interesting option to avoid conventional menopause hormone therapy (MHT). Pregnancy induction and replacing MHT by THT, however, are completely different topics as different requirements need to be met. First, MHT requires long-lasting and continuous hormone production. It still needs to be proven if the transplanted tissue is active for at least 5 years with a continuous follicle growth to avoid phases with low oestrogen production, which would otherwise cause menopausal symptoms and could reduce the postulated benefit for women's health. Second, the advantage of a physiological hormone production over a non-physiological MHT is still hypothetical. Third, women who have undergone hysterectomies who do not need progesterone for endometrial protection would only require oestrogens, imposing more health benefits (cardiovascular system, mammary gland) than oestrogen and progesterone production or replacement. Therefore, transplanting ovarian tissue exclusively to postpone menopause is endocrinologically doubtful and should only be carried out within clinical trials. Copyright © 2015. Published by Elsevier Ltd.

  9. Whole ovary cryopreservation with vascular transplantation – A future development in female oncofertility

    Directory of Open Access Journals (Sweden)

    Mats Brännström


    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.

  10. Safety assessment of ovarian cryopreservation and transplantation in nude mice bearing human epithelial ovarian cancer. (United States)

    Zhu, Gen-Hai; Wang, Sheng-Tan; Yang, Zhao-Xin; Cai, Jun-Hong; Chen, Chun-Ying; Yao, Mao-Zhong; Hong, Lan; He, Guo-Li; Yang, Shu-Ying


    Nude mice with orthotopic transplantation of human ovarian epithelial cancer were used to investigate screening criteria for paraneoplastic normal ovarian tissue and the security of the freezing and thawing for ovarian tissue transplantation. Expression of CK-7, CA125, P53, survivin, MMP-2/TIMP- 2 in paraneoplastic normal ovarian tissues were detected by RT-PCR as well as immunohistochemistry. The tissues of the groups with all negative indicators of RT-PCR, all negative indicators of immunohistochemistry, negative expression of CK-7, CA125 and survivin, positive expression of CK-7, CA125 and survivin, cancer tissues and normal ovarian tissues of nude mice were used for freezing and thawing transplantation, to analyze overt and occult carcinogenesis rates after transplantation. When all indicators or the main indicators, CK-7, CA125 and survivin, were negative, tumorigenesis did not occur after transplantation. In addition the occult carcinogenesis rate was lower than in the group with positive expression of CK-7, CA125 and survivin (Povarian tissues, rates did not change (P>0.05). There was no statistical significance among rates after transplantation of ovarian tissues which were obtained under different severity conditions (P>0.05). Negative expression of CK-7, CA125 and survivin can be treated as screening criteria for security of ovarian tissues for transplantation. Immunohistochemical methods can be used as the primary detection approach. Both subcutaneous and orthotopic transplantation are safe. The initial severity does not affect the carcinogenesis rate after tissue transplantation. Freezing and thawing ovarian tissue transplantation in nude mice with human epithelial ovarian carcinoma is feasible and safe.

  11. Effects of three different types of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation.

    Directory of Open Access Journals (Sweden)

    Jaewang Lee

    Full Text Available Ovarian tissue (OT cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, the damage incurred at different steps during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function.This study was designed to investigate the beneficial effects of different antifreeze proteins (AFPs on mouse ovarian tissue cryopreservation and transplantation.Ovaries were obtained from 5-week-old B6D2F1 mice, and each ovary was cryopreserved using two-step vitrification and four-step warming procedures. In Experiment I, ovaries were randomly allocated into fresh, vitrification control, and nine experimental groups according to the AFP type (FfIBP, LeIBP, type III and concentration (0.1, 1, 10 mg/mL used. After vitrification and warming, 5,790 ovarian follicles were evaluated using histology and TUNEL assays, and immunofluorescence for τH2AX and Rad51 was used to detect DNA double-strand breaks (DSBs and repair (DDR, respectively. In Experiment II, 20 mice were randomly divided into two groups: one where the vitrification and warming media were supplemented with 10 mg/mL LeIBP, and the other where media alone were used (control. Ovaries were then autotransplanted under both kidney capsules 7 days after vitrification together with the addition of 10 mg/mL LeIBP in the vitrification-warming media. After transplantation, the ovarian follicles, the percentage of apoptotic follicles, the extent of the CD31-positive area, and the serum FSH levels of the transplanted groups were compared.In Experiment I, the percentage of total grade 1 follicles was significantly higher in the 10 mg/mL LeIBP group than in the vitrification control, while all AFP-treated groups had significantly improved grade 1 primordial follicle numbers compared with those of the vitrification control. The number of apoptotic (TUNEL

  12. Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells

    Directory of Open Access Journals (Sweden)

    M.G. Fukushima


    Full Text Available The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (10(6 cells obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5 were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs were also labeled with tetramethylrhodamine-labeled alpha-bungarotoxin. The development of ectopic muscles was successful in 86% of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4, resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. The presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 ± 1.0 µm; N = 213, the diameter of ectopic fibers (59.1 µm; N = 213 did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.

  13. Cryopreserved neonatal hepatocytes may be a source for transplantation: Evaluation of functionality toward clinical use. (United States)

    Lee, Charlotte A; Dhawan, Anil; Iansante, Valeria; Lehec, Sharon; Khorsandi, Shirin E; Filippi, Celine; Walker, Simon; Fernandez-Dacosta, Raquel; Heaton, Nigel; Bansal, Sanjay; Mitry, Ragai R; Fitzpatrick, Emer


    Neonatal livers are a potential source of good-quality hepatocytes for clinical transplantation. We compared viability and function of neonatal hepatocytes (NHs) and adult hepatocytes (AHs) and report their clinical use both intraportally and in alginate microbeads. Following isolation from donor livers, hepatocyte function was assessed using albumin, alpha-1-antitrypsin, and factor VII. Metabolic function was investigated by measuring resorufin conjugation, ammonia metabolism, uridine diphosphate glucuronosyltransferase enzyme activity, and cytochrome P450 (CYP) function following induction. Activation of the instant blood-mediated inflammatory reaction by NHs and AHs was investigated using an in vitro blood perfusion model, and tissue factor expression was analyzed using real-time polymerase chain reaction (RT-PCR). Clinical hepatocyte transplantation (HT) was undertaken using standard protocols. Hepatocytes were isolated from 14 neonatal livers, with an average viability of 89.4% ± 1.8% (mean ± standard error of the mean) and average yield of 9.3 × 10 6 ± 2.0 × 10 6 cells/g. Hepatocytes were isolated from 14 adult livers with an average viability of 78.6% ± 2.4% and yield 2.2 × 10 6 ± 0.5 × 10 5 cells/g. NHs had significantly higher viability after cryopreservation than AHs, with better attachment efficiency and less plasma membrane leakage. There were no differences in albumin, alpha-1-antitrypsin, and factor VII synthesis between NHs and AHs (P > 0.05). Neonatal cells had inducible phase 1 enzymes as assessed by CYP function and functional phase 2 enzymes, in which activity was comparable to AHs. In an in vitro blood perfusion model, AHs elicited increased thrombus formation with a greater consumption of platelets and white cells compared with NHs (28.3 × 10 9 versus 118.7 × 10 9 and 3.3 × 10 9 versus 6.6 × 10 9 ; P < 0.01). Intraportal transplantation and intraperitoneal transplantation of alginate encapsulated hepatocytes was safe, and

  14. Cryopreservation of adult unrelated donor products in hematopoietic cell transplantation: the OneMatch experience and systematic review of the literature. (United States)

    Aziz, Joseph; Morris, Gail; Rizk, Mina; Shorr, Risa; Mercer, Dena; Young, Kimberly; Allan, David


    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.

  15. Safety considerations for transplanting cryopreserved ovarian tissue to restore fertility in female patients who have recovered from Ewing's sarcoma

    DEFF Research Database (Denmark)

    Sørensen, Stine D; Greve, Tine; Wielenga, Vera Timmermans


    Ewing's sarcoma (EWS) is a highly malignant cancer in children, adolescents and young adults. The chemotherapy required to treat female EWS patients may cause primary ovarian insufficiency and infertility as a side effect. Cryopreservation of ovarian tissue before the start of chemotherapy can...... ovarian tissue, potentially causing recrudescence of the original cancer after transplantation. The risk of EWS metastasizing to the ovary is probably low but has not been studied in great detail. This review describes the available evidence on the risk of malignant cell contamination in the ovaries...... of EWS patients and presents a new case of malignant cells in an ovarian biopsy from a girl with EWS....

  16. Production of live offspring from testicular tissue cryopreserved by vitrification procedures in Japanese quail (Coturnix japonica). (United States)

    Liu, Jianan; Cheng, Kimberly M; Silversides, Frederick G


    Cryopreservation of testicular tissue can be used for ex situ conservation of male germplasm of avian species. The possibility of using vitrification and transplantation of testicular tissue for fertility preservation and recovery was tested in Japanese quail. Testes were removed from 1-wk-old Japanese quail; transfixed on acupuncture needles; equilibrated with dimethyl sulphoxide, ethylene glycol, and sucrose; plunged into liquid nitrogen; and stored in 2-ml straws. Cryopreserved tissue was warmed in sucrose solution at room temperature or at 40°C. Fresh and cryopreserved tissue were transplanted subcutaneously into castrated, 1-wk-old recipients. Twenty of 21 recipients survived the surgery, and 18 had viable transplants at maturity, with no difference in transplantation success between fresh and cryopreserved tissue. Fluid extrusion from 11 of the transplants was collected and inseminated surgically into the magnum of 22 quail hens, and 10 inseminations included foam from the proctodeal gland of the same recipients. Egg production in the 2 wk after insemination was reduced, and none of the hens inseminated with foam produced fertile eggs. Five hens inseminated without foam produced a total of eight live offspring; four of these hens had been inseminated with fluid extrusion from cryopreserved tissue. Histological examination showed spermatogenesis in the transplants, and the tubules, lumens, and epithelium of the seminiferous tubules were of comparable size to those of testicular tissue from intact males. These results demonstrate that testicular tissue of Japanese quail can be preserved using vitrification procedures and recovered through transplantation.

  17. A Retrievable, Efficacious Polymeric Scaffold for Subcutaneous Transplantation of Rat Pancreatic Islets. (United States)

    Smink, Alexandra M; Hertsig, Don T; Schwab, Leendert; van Apeldoorn, Aart A; de Koning, Eelco; Faas, Marijke M; de Haan, Bart J; de Vos, Paul


    We aim on developing a polymeric ectopic scaffold in a readily accessible site under the skin. The liver as transplantation site for pancreatic islets is associated with significant loss of islets. Several extrahepatic sites were tested in experimental animals, but many have practical limitations in the clinical setting and do not have the benefit of easy accessibility. Functional survival of rat islets was tested during 7 days of culture in the presence of poly(D,L-lactide-co-ε-caprolactone) (PDLLCL), poly(ethylene oxide terephthalate)/polybutylene terephthalate (PEOT/PBT) block copolymer, and polysulfone. Tissue responses were studied in vivo after subcutaneous implantation in rats. Culture on PEOT/PBT and polysulfone profoundly disturbed function of islets, and induced severe tissue responses in vivo. Modification of their hydrophilicity did not change the suitability of the polymers. PDLLCL was the only polymer that promoted functional survival of rat islets in vitro and was associated with minor tissue reactions after 28 days. Rat islets were transplanted in the PDLLCL scaffold in a diabetic rat model. Before islet seeding, the scaffold was allowed to engraft for 28 days to allow the tissue response to dampen and to allow blood vessel growth into the device. Islet transplantation into the scaffold resulted in normoglycemia within 3 days and for the duration of the study period of 16 weeks. In conclusion, we found that some polymers such as PEOT/PBT and polysulfone interfere with islet function. PDLLCL is a suitable polymer to create an artificial islet transplantation site under the skin and supports islet survival.

  18. Cryopreservation of Hair-Follicle Associated Pluripotent (HAP) Stem Cells Maintains Differentiation and Hair-Growth Potential. (United States)

    Hoffman, Robert M; Kajiura, Satoshi; Cao, Wenluo; Liu, Fang; Amoh, Yasuyuki


    Hair follicles contain nestin-expressing pluripotent stem cells which originate above the bulge area of the follicle, below the sebaceous gland. We have termed these cells hair follicle-associated pluripotent (HAP) stem cells. We have established efficient cryopreservation methods of the hair follicle that maintain the pluripotency of HAP stem cells as well as hair growth. We cryopreserved the whole hair follicle by slow-rate cooling in TC-Protector medium or in DMSO-containing medium and storage in liquid nitrogen or at -80 °C. After thawing and culture of the cryopreserved whisker follicles, growing HAP stem cells formed hair spheres. The hair spheres contained cells that differentiated to neurons, glial cells, and other cell types. The hair spheres derived from slow-cooling cryopreserved hair follicles were as pluripotent as hair spheres from fresh hair follicles. We have also previously demonstrated that cryopreserved mouse whisker hair follicles maintain their hair-growth potential. DMSO better cryopreserved mouse whisker follicles compared to glycerol. DMSO-cryopreserved hair follicles also maintained the HAP stem cells, evidenced by P75 ntr expression. Subcutaneous transplantation of DMSO-cryopreserved hair follicles in nude mice resulted in extensive hair fiber growth over 8 weeks, indicating the functional recovery of hair-shaft growth of cryopreserved hair follicles. HAP stem cells can be used for nerve and spinal-cord repair. This biobanking of hair follicles can allow each patient the potential for their own stem cell use for regenerative medicine or hair transplantation.

  19. Safeguarding Fertility With Whole Ovary Cryopreservation and Microvascular Transplantation: Higher Follicular Survival With Vitrification Than With Slow Freezing in a Ewe Model. (United States)

    Torre, Antoine; Vertu-Ciolino, Delphine; Mazoyer, Claire; Selva, Jacqueline; Lornage, Jacqueline; Salle, Bruno


    In young women, ovarian cortex cryopreservation before gonadotoxic chemotherapy and its avascular grafting after cancer healing permitted fertility restoration. However, ischemia reduced the grafts' lifespan. Microvascular transplantation of cryopreserved whole ovary may allow immediate revascularization, ensuring better fertility preservation, but the best cryopreservation method is unknown. We aimed to compare slow freezing and vitrification of whole ovary for fertility preservation purposes, in an ewe model. Twelve ewes were allocated at random to slow freezing (n = 6) or vitrification group (n = 6). Ewes' left ovary was removed and cryopreserved. Dimethyl sulfoxide 2 M was used as cryoprotector for slow freezing. Vitrification was obtained using increasing concentrations of a vitrification solution of the latest generation (VM3) and gradual temperature lowering to minimize toxicity. After a month, the right ovary was removed, the left ovary was thawed/warmed, and its vessels were anastomosed to the right pedicle. Fertility and ovarian function were assessed for 3 years. Ovarian follicles in native and transplanted ovaries were counted and compared at study completion. Hormonal secretion resumed in all ewes of both groups. One ewe of the slow-freezing group delivered healthy twins 1 year 9 months and 12 days after transplantation. Estimated whole follicle survival was very low in both groups but significantly higher after vitrification than after slow freezing (0.3% ± 0.5% vs 0.017% ± 0.019%, respectively; p < 0.05). Further progress is needed before whole-ovary cryopreservation can be considered an option for safeguarding fertility. Whole ovary vitrification provides better follicular survival compared to slow freezing and may be a valuable cryopreservation option.

  20. 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


    INTRODUCTION: The aim of this study is to report the current status of ovarian tissue cryopreservation among alternatives for fertility preservation in the Nordic countries. MATERIAL AND METHODS: A questionnaire was sent to 14 Nordic academic reproductive centers with established fertility preser...

  1. The expression of TP53 pathway-related proteins in ovarian carcinoma transplanted subcutaneously in nude mice. (United States)

    Zhang, S-R; Li, D-B; Xue, J-W


    Given the important functions of TP53 pathway in various biological processes, this study aimed to investigate the expression of TP53 pathway-related proteins in ovarian carcinoma transplanted subcutaneously in nude mice with and without the presence of p53 inhibitor and to explore possible roles of p53 in the development of ovarian cancer. Thirty BALB/c-nu female nude mice were randomly divided into model group, control group and p53 inhibitor group (Pftα group). There were 10 rats in each group. The nude mice were subcutaneously inoculated with human ovarian cancer cell line SKOV3, and the tumor growth was observed. Morphological changes of tumor tissue were observed by hematoxylin and eosin (HE) staining. The mRNA and protein levels of TP53 pathway related factors-p53, p21 and mouse double minute 2 homolog (MDM2) were detected by RT-PCR and Western blot. p53 inhibitor can increase the growth rate of subcutaneously transplanted tumor in nude mice. p53 inhibitor could decrease the expression of p53 and p21 at both mRNA and protein levels and increase the expression of MDM2 at both mRNA and protein levels in ovarian carcinoma transplanted subcutaneously in nude mice. TP53 pathway may play pivotal roles in the development of ovarian cancer and TP53 pathway may be a new target for the treatment of ovarian cancer.

  2. Restoration of ovarian activity and pregnancy after transplantation of cryopreserved ovarian tissue

    DEFF Research Database (Denmark)

    Donnez, Jacques; Dolmans, Marie-Madeleine; Pellicer, Antonio


    Aggressive chemotherapy/radiotherapy and bone marrow transplantation can cure >90% of girls and young women affected by disorders requiring such treatment. However, the ovaries are very sensitive to cytotoxic drugs, especially to alkylating agents. Several options are currently available...

  3. Legal termination of a pregnancy resulting from transplanted cryopreserved ovarian tissue due to cancer recurrence

    DEFF Research Database (Denmark)

    Ernst, Emil Hagen; Offersen, Birgitte Vrou; Andersen, Claus Yding


    To report on a woman who conceived naturally and had a normal intrauterine pregnancy following transplantation of frozen/thawed ovarian tissue but decided to have an early abortion due to recurrence of breast cancer.......To report on a woman who conceived naturally and had a normal intrauterine pregnancy following transplantation of frozen/thawed ovarian tissue but decided to have an early abortion due to recurrence of breast cancer....

  4. Outcomes of transplantations of cryopreserved ovarian tissue to 41 women in Denmark

    DEFF Research Database (Denmark)

    Jensen, A K; Kristensen, S G; Macklon, K T


    with a pregnancy-wish. WHAT IS KNOWN ALREADY: Cryopreservation of ovarian tissue is now gaining ground as a valid method for fertility preservation. More than 36 children worldwide have now been born following this procedure. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study of 41 women who had...... by monitoring relapse in cancer survivors. MAIN RESULTS, AND THE ROLE OF CHANCE: Among 32 women with a pregnancy-wish, 10 (31%) had a child/children (14 children in total); this included 1 woman with a third trimester on-going pregnancy. In addition, two legal abortions and one second trimester miscarriage...... this study. They had no role in the study design, collection and analysis of data, data interpretation or writing of the report. The authors have no conflict of interest to disclose....

  5. 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


    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

  6. A Retrievable, Efficacious Polymeric Scaffold for Subcutaneous Transplantation of Rat Pancreatic Islets

    NARCIS (Netherlands)

    Smink, Alexandra M; Hertsig, Don T; Schwab, Leendert; van Apeldoorn, Aart A; de Koning, Eelco; Faas, Marijke M; de Haan, Bart J; de Vos, Paul


    OBJECTIVE: We aim on developing a polymeric ectopic scaffold in a readily accessible site under the skin. SUMMARY BACKGROUND DATA: The liver as transplantation site for pancreatic islets is associated with significant loss of islets. Several extrahepatic sites were tested in experimental animals,

  7. A Retrievable, Efficacious Polymeric Scaffold for Subcutaneous Transplantation of Rat Pancreatic Islets

    NARCIS (Netherlands)

    Smink, Alexandra M; Hertsig, Don T; Schwab, Leendert; van Apeldoorn, Aart A; de Koning, Eelco; Faas, Marijke M; de Haan, Bart J; de Vos, Paul

    OBJECTIVE: We aim on developing a polymeric ectopic scaffold in a readily accessible site under the skin. SUMMARY BACKGROUND DATA: The liver as transplantation site for pancreatic islets is associated with significant loss of islets. Several extrahepatic sites were tested in experimental animals,

  8. Myeloid Sarcoma Presenting With Multiple Skin and Subcutaneous Mass Without Leukemic Manifestations After Renal Transplantation. (United States)

    Shen, J; Du, X; Diao, X; Zhao, L; Wang, W; Yu, Y; Luo, H


    Myeloid sarcoma is well described and known in clinical practice, however, it is a rare condition after receiving renal transplantation. Immunosuppressive therapy is thought to be the main cause in these cases. A 45-year-old woman accepted a right kidney transplantation because of her chronic renal insufficiency and uremia in May 2011. She had to receive a left kidney transplantation again in February 2012 because she had renal failure again after receiving the right kidney transplantation. She received immune inhibitors treatment. After the latter operation, her renal function was normal. The third operation was done to remove the right transplanted kidney in July 2012. The diagnosis of the kidney was myeloid sarcoma. The blood and bone marrow biopsy had no evidence of leukemia. She then received chemotherapy. There was a small skin nodule on the left arm of approximately 0.5 cm in August 2012; after that its diameter enlarged progressively to about 5 cm and more nodules and masses gradually appeared on her face, arms, trunk, lower limbs, and feet over the course of 1 year. The skin biopsy specimen obtained from her left arm showed myeloid sarcoma too. She was admitted to the Orthopedics Department for severe pain and swelling in the left foot in September 2014 and underwent an operation for resecting the mass in the left foot. Pain was apparently alleviated and the incision healed well. The patient is still alive with no evidence of leukemia after a 30-month follow-up. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Subcutaneous phaeohyphomycosis due to Alternaria infectoria in a renal transplant patient: surgical treatment with no long-term relapse. (United States)

    Salido-Vallejo, Rafael; Linares-Sicilia, María José; Garnacho-Saucedo, Gloria; Sánchez-Frías, Marina; Solís-Cuesta, Francisco; Gené, Josepa; Moreno-Giménez, José Carlos


    Phaeohyphomycosis can be caused by a number of different species, being the most common Alternaria alternata and Alternaria infectoria. The biggest risk factor for the development of the infection is immunosuppression. We present the case of a 64-year-old male renal transplant patient who came to hospital for presenting a tumour in the Achilles region which had been gradually growing in size. A skin biopsy was taken for histological study and culture of fungi and mycobacteria. Blood tests and imaging studies were performed. Histopathology study and cultures identified A. infectoria as the causal agent. Imaging studies ruled out internal foci of infection. The lesion was surgically removed with no signs of recurrence after 24 months of follow-up. There are no treatment guidelines at present for cutaneous and subcutaneous Alternaria spp. infections. Various systemic antifungals have been used, either in combination with surgical removal or alone, with varying results. Surgery alone could be useful in the treatment of solitary, localised lesions in transplant patients in whom there are difficulties in controlling immunosuppression. Copyright © 2012 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

  10. Effect of cryopreservation on proliferation and differentiation of periodontal ligament stem cell sheets. (United States)

    Li, Mengying; Feng, Cheng; Gu, Xiuge; He, Qin; Wei, Fulan


    Cryopreservation has been extensively applied to the long-term storage of a diverse range of biological materials. However, no comprehensive study is currently available on the cryopreservation of periodontal ligament stem cell (PDLSC) sheets which have been suggested as excellent transplant materials for periodontal tissue regeneration. The aim of this study is to investigate the effect of cryopreservation on the structural integrity and functional viability of PDLSC sheets. PDLSC sheets prepared from extracted human molars were divided into two groups: the cryopreservation group (cPDLSC sheets) and the freshly prepared control group (fPDLSC sheets). The cPDLSC sheets were cryopreserved in a solution consisting of 90% fetal bovine serum and 10% dimethyl sulfoxide for 3 months. Cell viability and cell proliferation rates of PDLSCs in both groups were evaluated by cell viability assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. The multilineage differentiation potentials of the cells were assessed by von Kossa staining and Oil Red O staining. The chromosomal stability was examined by karyotype analysis. Moreover, the cell sheets in each group were transplanted subcutaneously into the dorsal site of nude mice, after which Sirius Red staining was performed to analyze the efficiency of tissue regeneration. The PDLSCs derived from both groups of cell sheets showed no significant difference in their viability, proliferative capacities, and multilineage differentiation potentials, as well as chromosomal stability. Furthermore, transplantation experiments based on a mouse model demonstrated that the cPDLSC sheets were equally effective in generating viable osteoid tissues in vivo as their freshly prepared counterparts. In both cases, the regenerated tissues showed similar network patterns of bone-like matrix. Our results offer convincing evidence that cryopreservation does not alter the biological properties of PDLSC sheets


    Directory of Open Access Journals (Sweden)

    D. V. Grizay


    Full Text Available Aim. To study the therapeutic potential of cryopreserved fetal liver cells seeded into macroporous alginategelatin scaffolds after implantation to omentum of rats with hepatic failure.Materials and methods.Hepatic failure was simulated by administration of 2-acetyl aminofl uorene followed partial hepatectomy. Macroporous alginate-gelatin scaffolds, seeded with allogenic cryopreserved fetal liver cells (FLCs were implanted into rat omentum. To prevent from colonization of host cells scaffolds were coated with alginate gel shell. Serum transaminase activity, levels of albumin and bilirubin as markers of hepatic function were determined during 4 weeks after failure model formation and scaffold implantation. Morphology of liver and scaffolds after implantation were examined histologically. Results. Macroporous alginate-gelatin scaffolds after implantation to healthy rats were colonized by host cells. Additional formation of alginate gel shell around scaffolds prevented the colonization. Implantation of macroporous scaffolds seeded with cryopreserved rat FLCs and additionally coated with alginate gel shell into omentum of rats with hepatic failure resulted in signifi cant improvement of hepatospecifi c parameters of the blood serum and positive changes of liver morphology. The presence of cells with their extracellular matrix within the scaffolds was confi rmed after 4 weeks post implantation.Conclusion. The data above indicate that macroporous alginate-gelatin scaffolds coated with alginate gel shell are promising cell carriers for the development of bioengineered liver equivalents.

  12. Orchid cryopreservation

    Directory of Open Access Journals (Sweden)

    Wagner Vendrame


    Full Text Available Orchids are lush and highly valuable plants due to their diversity and the beauty of their flowers, which increases their commercialization. The family Orchidaceae comprises approximately 35,000 species, distributed among more than 1,000 distinct genera and 100,000 hybrids, totaling approximately 8% to 10% of all flowering plants. With the advance of agriculture and the constant destruction of their natural habitat, orchid species are collected in an indiscriminate manner by collectors and vendors, and this extractive activity threatens many species with extinction, drastically reducing their genetic variability in nature. Therefore, it is essential to seek alternatives that make the preservation of such species feasible using techniques with low maintenance costs that provide greater storage time and that enable good phytosanitary conditions for the plant material for commercial use. Cryopreservation involves the conservation of biological materials at ultra-low temperatures, generally in liquid nitrogen at -196 ºC or in its vapor phase at -150 ºC. This is the only technique currently available for the long-term preservation of the germplasm of plant species that are vegetatively propagated or that have unviable, recalcitrant or intermediate seeds. The objective of this bibliographic review is to report on the importance, methods and application of cryopreservation for orchids. According to the studies reviewed, this is an incipient, developing and relevant field that generates a lot of discussion and requires further research relative to the type of treatment to use for cryopreservation and the methodology to be applied according to the species. The types of methods that are used for cryopreservation and the large variation in the responses of orchids to the cryopreservation methods observed in this study emphasize the need for the development of more appropriate protocols for the preservation of orchids.


    African Journals Online (AJOL)

    stage kidney disease. There is good evidence that transplantation improves both the quality and quantity of life in renal transplant recipients when compared with dialysis.1,2. Living donor kidney transplantation has gained popularity, not only owing ...

  14. A new strategy and system for the ex vivo ovary perfusion and cryopreservation: An innovation. (United States)

    Ali Mohamed, Mohamed Shehata


    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.

  15. The Efficacy of a Prevascularized, Retrievable Poly(D,L,-lactide-co-epsilon-caprolactone) Subcutaneous Scaffold as Transplantation Site for Pancreatic Islets

    NARCIS (Netherlands)

    Smink, Alexandra M; Li, Shiri; Hertsig, Don T; de Haan, Bart J; Schwab, Leendert; van Apeldoorn, Aart A; de Koning, Eelco; Faas, Marijke M; Lakey, Jonathan R T; de Vos, Paul

    Background. The liver as transplantation site for human pancreatic islets is a harsh microenvironment for islets and it lacks the ability to retrieve the graft. A retrievable, extrahepatic transplantation site that mimics the pancreatic environment is desired. Ideally, this transplantation site

  16. Cryopreservation of Living Organs (United States)

    Tanasawa, Ichiro; Nagata, Shinichi; Kimura, Naohiro

    Cryopreservation is considered to be the most promising way of preserving living organs or tissues for a long period of time without casuing any damage to their biological functions. However, cryopreservation has been succeeded only for simple and small-size tissues such as spermatozoon, ovum, erythrocyte, bone marrow and cornea. Cryopreservation of more complex and large-scale organs are not yet succssful. The authors have attempted to establish a technique for cryopreservation of larger living organs. An experiment was carried out using daphnia (water flea). The optimum rates of freezing and thawing were determined together with the optimum selection of cryoprotectant. High recovery rate was achieved under these conditions.

  17. Biological Properties and Therapeutic Value of Cryopreserved Fat Tissue. (United States)

    Mashiko, Takanobu; Wu, Szu-Hsien; Kanayama, Koji; Asahi, Rintaro; Shirado, Takako; Mori, Masanori; Sunaga, Ataru; Sarukawa, Shunji; Uda, Hirokazu; Yoshimura, Kotaro


    Fat grafting frequently requires multiple treatments and thus repeated liposuction to achieve treatment goals. The purpose of this study was to evaluate whether cryopreservation of adipose tissue may facilitate future fat grafting. Lipoaspirates were harvested from six women and preserved using two cryopreservation methods: (1) simple cooling to -80°C (cryo-1); or (2) programmed cooling to -196°C (cryo-2). Fresh fat, cryo-1 fat, and cryo-2 fat were analyzed both in vitro and in vivo. Immunohistochemistry of both types of cryopreserved adipose tissue revealed that most adipocytes were necrotic. The cell number and viability of stromal vascular fraction cells were significantly decreased in cryo-1 fat (1.7 × 10 cells, 42.6 percent viable) and cryo-2 fat (2.0 × 10 cells, 55.4 percent viable), compared with fresh fat (3.9 × 10 cells, 90.6 percent viable). Although adipose-derived stem cells were cultured successfully from all fats, functional adipose-derived stem cells from cryopreserved fats were much fewer, with comparable multilineage differentiating capacity. In vivo studies using human fat grafted into immunocompromised mice revealed that, 3 months after transplantation, all of the cryopreserved fats maintained their volume to some extent; however, the cryopreserved fats were mostly filled with dead tissue and produced significantly lower engraftment scores than fresh fat. Most adipocytes were killed in the process of cryopreservation and thawing. Adipose-derived stem cells were isolated from cryopreserved fat, but the number of functional adipose-derived stem cells was very limited in both cryopreservation methods. After grafting, cryopreserved fat was retained as dead and fibrous tissue, suggesting a risk of clinical complications such as oil cysts.

  18. Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy (United States)


    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. PMID:28122047

  19. 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.

  20. Cryopreservation of crane semen (United States)

    Gee, G.F.; Harris, James


    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.

  1. 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.

  2. Cryopreservation of Human Mucosal Leukocytes. (United States)

    Hughes, Sean M; Shu, Zhiquan; Levy, Claire N; Ferre, April L; Hartig, Heather; Fang, Cifeng; Lentz, Gretchen; Fialkow, Michael; Kirby, Anna C; Adams Waldorf, Kristina M; Veazey, Ronald S; Germann, Anja; von Briesen, Hagen; McElrath, M Juliana; Dezzutti, Charlene S; Sinclair, Elizabeth; Baker, Chris A R; Shacklett, Barbara L; Gao, Dayong; Hladik, Florian


    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.

  3. Cryopreservation of oocytes

    International Nuclear Information System (INIS)

    Jadoon, S.


    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)

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

    DEFF Research Database (Denmark)

    Rosendahl, Mikkel; Schmidt, Kirsten Tryde; Andersen, Anders Nyboe


    This paper presents the Danish 10-year experience (1999-2009) with cryopreservation (n=386) and autotransplantation of ovarian tissue (n=18). Before applying the technique to humans, the method was thoroughly tested and validated. The cryoprotectant solution was chosen after histological evaluation...... validated. Overnight transport of fresh ovarian tissue prior to cryopreservation was evaluated when human ovarian tissue was kept on ice for 20h and then cryopreserved. The thawed ovarian tissue was transplanted to an oophorectomized Nude mouse and histology confirmed viability. In Denmark 12 women have...... received a total of 18 autotransplantations of ovarian tissue. All women resumed ovarian function and three healthy babies were born to two women. In both women, the tissue was transported on ice for 4-5h prior to cryopreservation. Ovarian tissue cryopreservation is an important method for fertility...

  5. Decellularized scaffold of cryopreserved rat kidney retains its recellularization potential.

    Directory of Open Access Journals (Sweden)

    Baldeep Chani

    Full Text Available The multi-cellular nature of renal tissue makes it the most challenging organ for regeneration. Therefore, till date whole organ transplantations remain the definitive treatment for the end stage renal disease (ESRD. The shortage of available organs for the transplantation has, thus, remained a major concern as well as an unsolved problem. In this regard generation of whole organ scaffold through decellularization followed by regeneration of the whole organ by recellularization is being viewed as a potential alternative for generating functional tissues. Despite its growing interest, the optimal processing to achieve functional organ still remains unsolved. The biggest challenge remains is the time line for obtaining kidney. Keeping these facts in mind, we have assessed the effects of cryostorage (3 months on renal tissue architecture and its potential for decellularization and recellularization in comparison to the freshly isolated kidneys. The light microscopy exploiting different microscopic stains as well as immuno-histochemistry and Scanning electron microscopy (SEM demonstrated that ECM framework is well retained following kidney cryopreservation. The strength of these structures was reinforced by calculating mechanical stress which confirmed the similarity between the freshly isolated and cryopreserved tissue. The recellularization of these bio-scaffolds, with mesenchymal stem cells quickly repopulated the decellularized structures irrespective of the kidneys status, i.e. freshly isolated or the cryopreserved. The growth pattern employing mesenchymal stem cells demonstrated their equivalent recellularization potential. Based on these observations, it may be concluded that cryopreserved kidneys can be exploited as scaffolds for future development of functional organ.

  6. Decellularized scaffold of cryopreserved rat kidney retains its recellularization potential. (United States)

    Chani, Baldeep; Puri, Veena; Sobti, Ranbir C; Jha, Vivekanand; Puri, Sanjeev


    The multi-cellular nature of renal tissue makes it the most challenging organ for regeneration. Therefore, till date whole organ transplantations remain the definitive treatment for the end stage renal disease (ESRD). The shortage of available organs for the transplantation has, thus, remained a major concern as well as an unsolved problem. In this regard generation of whole organ scaffold through decellularization followed by regeneration of the whole organ by recellularization is being viewed as a potential alternative for generating functional tissues. Despite its growing interest, the optimal processing to achieve functional organ still remains unsolved. The biggest challenge remains is the time line for obtaining kidney. Keeping these facts in mind, we have assessed the effects of cryostorage (3 months) on renal tissue architecture and its potential for decellularization and recellularization in comparison to the freshly isolated kidneys. The light microscopy exploiting different microscopic stains as well as immuno-histochemistry and Scanning electron microscopy (SEM) demonstrated that ECM framework is well retained following kidney cryopreservation. The strength of these structures was reinforced by calculating mechanical stress which confirmed the similarity between the freshly isolated and cryopreserved tissue. The recellularization of these bio-scaffolds, with mesenchymal stem cells quickly repopulated the decellularized structures irrespective of the kidneys status, i.e. freshly isolated or the cryopreserved. The growth pattern employing mesenchymal stem cells demonstrated their equivalent recellularization potential. Based on these observations, it may be concluded that cryopreserved kidneys can be exploited as scaffolds for future development of functional organ.

  7. Cryopreservation of Fish Sperm (United States)

    Kurokura, Hisashi

    Present status of research activities in cryopreservation of fish gamete in aquaculture field was introduced. More than 59 fish species have been reported in the research histories and nearly half of them were studied during recent 10 years. This means that the research activities are increasing, though commercial profit have not obtained yet. Fish species of which sperm can successfully cryopreserved is still limited comparing to numerous species in telost. One of the major obstacle for improvement of the technique is existence of wide specie specific variance in the freezing tolerance of fish sperm. The varianc can possibly be explaind thorugh the informations obtained by the studies in comparative spermatology, which is recently activated field in fish biology.

  8. Cryopreservation of Parathyroid Glands

    Directory of Open Access Journals (Sweden)

    Marlon A. Guerrero


    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.

  9. Effect of Antioxidants and Apoptosis Inhibitors on Cryopreservation of Murine Germ Cells Enriched for Spermatogonial Stem Cells (United States)

    Lee, Yong-An; Kim, Yong-Hee; Kim, Bang-Jin; Jung, Sang-Eun; Pang, Myeong-Geol; Ryu, Buom-Yong


    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. PMID:27548381

  10. Widespread applications of citrus cryopreservation (United States)

    Citrus genetic resources can now be successfully cryopreserved, which means that they can be placed into long-term storage at liquid nitrogen temperatures. This cryopreservation technology was specifically developed to address the immediate need to have secure long-term back-up storage for citrus co...

  11. Cryopreservation and autotransplantation of human ovarian tissue prior to cytotoxic therapy--a technique in its infancy but already successful in fertility preservation

    DEFF Research Database (Denmark)

    von Wolff, Michael; Donnez, Jacques; Hovatta, Outi


    of reproductive medicine and transplanted in case the women experience premature ovarian failure (POF). This review summarises the European expertise on cryopreservation and transplantation of ovarian tissue, following around 30 reported transplantations globally, resulting in six live births and several ongoing...

  12. Cryopreservation of adult cervid testes. (United States)

    Pothana, Lavanya; Devi, Lalitha; Goel, Sandeep


    Several species of cervids are currently classified as threatened or endangered due to a rapid decline in their populations. Sperm cryopreservation, in association with assisted reproductive technologies, can find application for the conservation of endangered cervids. In cases of unsuccessful sperm retrieval through other means prior to the death of the animal, adult testis is the only source of sperm. Recovery of viable sperm from adult testes depends on the effective preservation of testicular tissues through optimization of cryopreservation protocols. The present study evaluated combinations of 10% dimethyl sulfoxide (DMSO) with 0% or 80% fetal bovine serum (FBS) and 20% DMSO with 0 or 20% FBS for the cryopreservation of testicular tissues of three adult cervids using uncontrolled slow freezing protocol. The cryopreserved testis was compared to chilled tissue without cryoprotectants. Results revealed that testicular tissues of barking deer cryopreserved in 20% DMSO (D20) had all the analyzed 7 parameters (number of TNP1-, PRM2 and acrosin-expressing cells/tubule and, the number of viable, morphologically normal, acrosome intact, Annexin V-negative sperm) comparable to the chilled testis. However, testicular tissues of sambhar and hog deer cryopreserved only in D20S20 had 5 of 7 parameters comparable to the chilled testis. In conclusion, D20 is acceptable for cryopreservation of barking deer and D20S20 for sambar and hog deer testes. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Sensing and Sensibility: Single-Islet-based Quality Control Assay of Cryopreserved Pancreatic Islets with Functionalized Hydrogel Microcapsules. (United States)

    Chen, Wanyu; Shu, Zhiquan; Gao, Dayong; Shen, Amy Q


    Despite decades of research and clinical studies of islet transplantations, finding simple yet reliable islet quality assays that correlate accurately with in vivo potency is still a major challenge, especially for real-time and single-islet-based quality assessment. Herein, proof-of-concept studies of a cryopreserved microcapsule-based quality control assays are presented for single islets. Individual rat pancreatic islets and fluorescent oxygen-sensitive dye (FOSD) are encapsulated in alginate hydrogel microcapsules via a microfluidic device. To test the susceptibility of the microcapsules and the FOSD to cryopreservation, the islet microcapsules containing FOSD are cryopreserved and the islet functionalities (adenosine triphosphate, static insulin release measurement, and oxygen consumption rate) are assessed after freezing and thawing steps. The cryopreserved islet capsules with FOSD remain functional after encapsulation and freezing/thawing procedures, validating a simple yet reliable individual-islet-based quality control method for the entire islet processing procedure prior to transplantation. This work also demonstrates that the functionality of cryopreserved islets can be improved by introducing trehalose into the routinely used cryoprotectant dimethyl sulfoxide. The functionalized alginate hydrogel microcapsules with embedded FOSD and optimized cryopreservation protocol presented in this work serve as a versatile islet quality assay and offer tremendous promise for tackling existing challenges in islet transplantation procedures. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Effects of human adipose-derived stem cells and stromal vascular fraction on cryopreserved fat transfer. (United States)

    Bae, Yong Chan; Song, Ji Sun; Bae, Seong Hwan; Kim, Joo Hyoung


    The use of cryopreserved adipose tissue for soft tissue augmentation is common, but unpredictability of fat graft viability remains a limitation. Adipose-derived stem cell (ADSC) and stromal vascular fraction (SVF) have been introduced to enhance viability and improve the survival of transplanted fat tissue. To investigate whether supplementation with ADSC or SVF improved the survival of cryopreserved fat grafts. The cryopreserved fat grafts were treated with ADSC, SVF, or normal saline in 30 six-week-old male nude mice to test whether ADSC and SVF could improve the survival of the transplanted fat tissue. The authors examined the weight, volume, and histological features of each group (n = 10) at 8 weeks after transplantation to evaluate the survival of the fat tissue. There was no difference between the control and SVF groups with respect to weight, volume, and histological findings. However, the ADSC group showed a significant increase in weight and volume compared with the control and SVF groups. Histological examination showed that the ADSC supplementation improved the quality of the transplanted fat grafts. Taken together, these results suggest a potential clinical utility of ADSC but no advantage of SVF in facilitating cryopreserved fat transfer.

  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.


    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. Expansion and cryopreservation of porcine and human corneal endothelial cells. (United States)

    Marquez-Curtis, Leah A; McGann, Locksley E; Elliott, Janet A W


    Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me 2 SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me 2 SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Stemness of spermatogonial stem cells encapsulated in alginate hydrogel during cryopreservation. (United States)

    Pirnia, A; Parivar, K; Hemadi, M; Yaghmaei, P; Gholami, M


    This study investigated the effect of spermatogonial stem cell encapsulated in alginate hydrogel during cryopreservation, as cells were protected against damage during cryopreservation within the hydrogel. Spermatogonial stem cells were isolated from the testes of Balb/c mice pups (6 days old), purified in laminin-coated dishes and CD90.1 microbeads, encapsulated in alginate hydrogel and then cryopreserved. After thawing, cell viability and Spermatogonial stem cell (SSC) colony diameter were evaluated. After RNA was isolated and cDNA was synthesised, the expression of stemness genes was considered using RT real-time PCR. Finally, spermatogonial stem cells labelled with BrdU were transplanted to busulfan azoospermic mouse models. Lin28a and Sall4 genes were significantly upregulated after cryopreservation in alginate hydrogel. However, cell viability was significantly decreased. The diameter of colonies consisting of spermatogonial stem cells freeze-thawed in alginate microbeads showed no significant difference with fresh spermatogonial stem cells and the control group. The injection of freeze-thawed spermatogonial stem cells encapsulated in alginate hydrogel resulted in spermatogenesis recovery. Alginate mimics the extracellular matrices (ECM) for spermatogonial stem cells; therefore, it can support stemness potential during the cell cryopreservation process and restart spermatogenesis after transplantation. © 2016 Blackwell Verlag GmbH.

  18. Embryo cryopreservation and preeclampsia risk. (United States)

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


    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.

  19. Hematopoietic stem cell transplantation in Algeria. (United States)

    Bekadja, Mohamed Amine; Brahimi, Mohamed; Osmani, Soufi; Yafour, Nabil; Krim, Amina; Serradj, Faiza; Talhi, Souad; Amani, Kamila; Bouhass, Rachid Amar


    Algeria is a country of 40.4 million inhabitants and half of which is under 30years. In Algeria, Health-care insurance covered, 90% of the population. Health care is free and it is supported by the Ministry of Health. 16 university hospitals exist in Algeria and only two (Algiers and Oran) practicing bone marrow transplant. Adult hematologic malignancies account for 10% (about 4000 new cases/year) of the malignancy affecting in most cases young patients under 65years of age. In 2016, 270 transplants were performed in total (Algiers+Oran), including 149 allografts (related donor transplants: 99%) and 121 autografts. 98% of transplants are done in adults and only 2% in children with cord blood transplants. In summary for the two transplant centers, the predominant types of transplantation performed are allogeneic transplant in 55% and autologous transplant in 45%. The particularity of EHU1st November in Oran, is the use of non-cryopreserved stem cells. Stem cell was mobilized using G-CSF alone and the grafts were kept in a conventional blood bank refrigerator at +4°C until reinfusion on day 0. The outcome with non-cryopreserved stem cells are the same as those with cryopreserved stem cells and we conclude that autologous transplant with non cryopreserved hematopoietic stem cells (HSC) is a simple, effective and safe method and the cryopreservation is not necessary in our work conditions in developing countries. The projects are achieving the autograft in all University Hospitals with non cryopreserved HSC, achieving a center allograft in the east of the country and the development of bone marrow transplantation in children. Currently in Algeria, the number of transplantation is insufficient and the development of new transplant centers is essential. In the future, we hope to implement the National Society of Bone Marrow transplant and also the National recipient registry and Donor registry in Algeria. Copyright © 2017 King Faisal Specialist Hospital & Research

  20. Antioxidants and cryopreservation, the new normal? (United States)

    Cryopreservation protocols are established for many plant species. Cryopreservation provides a stable, long-term and low-cost backup that is safe from the diseases or environmental damage that challenge whole plants. However, many plants respond poorly to cryopreservation due to osmotic stress or la...

  1. Cryopreservation of eucalyptus genetic resources. (United States)

    Kaya, E; Alves, A; Rodrigues, L; Jenderek, M; Hernandez-Ellis, M; Ozudogru, A; Ellis, D


    The long-term preservation of forest genetic resources is a vital part of preserving our forest crops for future generations. Unfortunately, there are few genebanks dedicated to forest trees and very few methods for long-term preservation of forest genetic resources collections aside from field plantings of a limited number of seed-derived or elite clonal individuals. The use of cryopreservation for the long-term storage of elite germplasm is increasingly being used for the long-term preservation of clonal agronomic crops but for forest trees, such as Eucalyptus, the methodology for cryopreservation of diverse genetic resources collections has not been established. We report the successful cryopreservation of a germplasm collection of in vitro shoot cultures of thirteen Eucalyptus spp. lines consisting of two E. grandis x E. camaldulensis lines, seven E. urophylla x E. grandis lines, one E. grandis line, two E. grandis x E. urophylla lines, and one E. camaldulensis line. In a comparison of two cryopreservation methods, sucrose sensitivity limited the application of encapsulation-dehydration. However, with droplet-vitrification, all thirteen lines had good survival after cryopreservation in liquid nitrogen. A 30 min exposure to Plant Vitrification Solution 2 (PVS2) yielded post-liquid nitrogen survival between 38% and 85% depending on the line. One hundred shoot tips from all thirteen lines are currently in long-term storage as a germplasm collection.

  2. Autotransplantation of cryopreserved tooth in connection with orthodontic treatment. (United States)

    Schwartz, O; Rank, C P


    In orthodontic treatment of certain cases of tooth loss, aplasia, or ectopia, autotransplantation is sometimes a valid treatment alternative and often provides an improved result, compared to conventional orthodontic treatment, if an appropriate donor tooth is available and the anatomic circumstances permit it. However, in some cases autotransplantation is not immediately possible as a one-step procedure. In such cases cryopreservation provides a clinically useful technique when an extraoral storage period of months or years is needed to orthodontically prepare the recipient region. In the present case a mature upper first left premolar was stored for 18 months. During this period sufficient space was achieved in the contralateral recipient region between the upper left first and second premolars. The thawed graft was autotransplanted to this position. Endodontic treatment was initiated 4 weeks after transplantation. Four years after transplantation, the periodontal healing of the grafted tooth appeared clinically and radiographically normal without any signs of root resorption. The presented case demonstrates the capacity of a cryopreserved tooth to regenerate what seems to be a normal periodontium after transplantation. If these findings are confirmed in further clinical trials, the technique could be a valuable tool in future orthodontic treatment planning.

  3. Cryopreservation of mammalian semen. (United States)

    Curry, Mark R


    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.

  4. Subcutaneous phaeohyphomycosis due to Pyrenochaeta romeroi mimicking a synovial cyst

    Directory of Open Access Journals (Sweden)

    Aurelien Dinh


    Full Text Available Opportunistic subcutaneous fungal infections are increasing nowadays due to the growing number of medical conditions causing immunosuppression, especially organ transplant. The incidence rate of subcutaneous phaeohyphomycosis is very low. Most studies found are case reports. They showed a wide variation of clinical presentations. Pyrenochaeta romeroi, a fungus from the Dematiaceae group is a saprophyte found in soil and plants and a possible causative agent of phaeohyphomycosis. We present a rare case of subcutaneous phaeohyphomycosis caused by P. romeroi mimicking a synovial cyst in a diabetic patient.

  5. Effects of micro-encapsulation on morphology and endocrine function of cryopreserved neonatal porcine islet-like cell clusters. (United States)

    Murakami, M; Satou, H; Kimura, T; Kobayashi, T; Yamaguchi, A; Nakagawara, G; Iwata, H


    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.

  6. Successful cryopreservation of buffalo ovaries using in situ oocyte cryopreservation

    Directory of Open Access Journals (Sweden)

    Saber Mohammed Abd-Allah


    Full Text Available To improve the efficiency and efficacy of cryopreservation of ovaries, we developed a new method termed in situ oocyte (ISO cryopreservation. ISO cryopreservation is a multistep procedure that involves aspiration of follicular fluid and then perfusion of antral follicles and diffusion of whole buffalo ovaries with cryoprotectant agent (CPA, rapid cooling, storage, thawing and, finally, dilution and removal of the CPA with return to physiological environment. Our study compared ISO cryo ovaries with cryo-diffused ovaries. We systematically examined the effects of ISO cryo and diffuse cryo on ovaries by morphological examination and with viability tests. The percentages of morphologically normal and viable follicular oocytes from ISO cryo were significantly higher than those that resulted from the cryo-diffused method (p<0.01. The quality of follicular oocytes from ISO cryo ovaries appeared better than that achieved from cryo-diffused ovaries. In conclusion, this study shows that ISO cryo is highly efficient for cryopreservation of oocytes and ovarian tissue.

  7. Principles of cryopreservation. (United States)

    Pegg, David E


    Cryopreservation is the use of very low temperatures to preserve structurally intact living cells and tissues. Unprotected freezing is normally lethal and this chapter seeks to analyze some of the mechanisms involved and to show how cooling can be used to produce stable conditions that preserve life. The biological effects of cooling are dominated by the freezing of water, which results in the concentration of the solutes that are dissolved in the remaining liquid phase. Rival theories of freezing injury have envisaged either that ice crystals pierce or tease apart the cells, destroying them by direct mechanical action, or that damage is from secondary effects via changes in the composition of the liquid phase. Cryoprotectants, simply by increasing the total concentration of all solutes in the system, reduce the amount of ice formed at any given temperature; but to be biologically acceptable they must be able to penetrate into the cells and have low toxicity. Many compounds have such properties, including glycerol, dimethyl sulfoxide, ethanediol, and propanediol. In fact, both damaging mechanisms are important, their relative contributions depending on cell type, cooling rate, and warming rate. A consensus has developed that intracellular freezing is dangerous, whereas extracellular ice is harmless. If the water permeability of the cell membrane is known it is possible to predict the effect of cooling rate on cell survival and the optimum rate will be a trade-off between the risk of intracellular freezing and effects of the concentrated solutes. However, extracellular ice is not always innocuous: densely packed cells are more likely to be damaged by mechanical stresses within the channels where they are sequestered and with complex multicellular systems it is imperative not only to secure cell survival but also to avoid damage to the extracellular structure. Ice can be avoided by vitrification-the production of a glassy state that is defined by the viscosity

  8. Oocyte cryopreservation: where are we now? (United States)

    Argyle, Catrin E; Harper, Joyce C; Davies, Melanie C


    Since the first live birth from oocyte cryopreservation three decades ago, oocyte cryopreservation has become an important component of ART. Cryopreservation techniques have evolved, leading to higher success rates and the introduction of oocyte cryopreservation into IVF clinics worldwide. Concurrently, there has been an increase in patient demand, especially for so-called 'social egg freezing' that allows women to preserve their fertility in anticipation of age-related fertility decline. This review addresses a need to evaluate the current status of oocyte cryopreservation. It explores current techniques and success rates, clinical applications, the rise of elective oocyte cryopreservation, and future implications. A search was performed using Web of Science and PubMed databases for publications between January 1980 and December 2015. Keywords used included 'egg freezing', 'oocyte freezing', 'oocyte cryopreservation', 'oocyte vitrification', and 'fertility preservation'. The success rate of oocyte cryopreservation has risen, and the increasing use of vitrification offers has improved outcomes, with IVF pregnancy rates now similar to those achieved with fresh oocytes. There are conflicting opinions about the comparative success rates of open and closed vitrification. Patients are accessing and receiving oocyte cryopreservation for a wide range of indications, and there has been a marked increase in patient numbers and oocyte cryopreservation cycles. Oocyte cryopreservation for circumventing age-related infertility is becoming more widely accepted. Oocyte cryopreservation is an established component of ART, with vitrification now being the cryopreservation technique of choice. Increasing numbers of women undergo oocyte cryopreservation for both medical and social reasons. It is important to continue auditing outcomes and reporting long-term follow-up of children born from frozen-thawed oocytes. © The Author 2016. Published by Oxford University Press on behalf of

  9. Tectonic deep anterior lamellar keratoplasty in impending corneal perforation using cryopreserved cornea. (United States)

    Jang, Ji Hye; Chang, Sung Dong


    We report a case of tectonic corneal transplantation for impending corneal perforation to preserve anatomic integrity using cryopreserved donor tissue. An 82-year-old woman exhibiting impending corneal perforation suffered from moderate ocular pain in the left eye for one week. After abnormal tissues around the impending perforation area were carefully peeled away using a Crescent blade and Vannas scissors, the patient received tectonic deep anterior lamellar keratoplasty using a cryopreserved cornea stored in Optisol GS® solution at -70℃ for four weeks. At six months after surgery, the cornea remained transparent and restored the normal corneal thickness. There were no complications such as corneal haze or scars, graft rejection, recurrent corneal ulcer, and postoperative rise of intraocular pressure. Cryopreserved donor lamellar tissue is an effective substitute in emergency tectonic lamellar keratoplasty, such as impending corneal perforation and severe necrotic corneal keratitis. © 2011 The Korean Ophthalmological Society

  10. The effect of Setarud (IMODTM) on angiogenesis in transplanted human ovarian tissue to nude mice (United States)

    Hormozi, Maryam; Talebi, Saeed; Khorram Khorshid, Hamid Reza; Zarnani, Amir-Hassan; Kamali, Koorosh; Jeddi-Tehrani, Mahmood; Soltangoraee, Haleh; Akhondi, Mohammad Mehdi


    Background: One of the promising methods in fertility preservation among women with cancer is cryopreservation of ovarian cortex but there are many drawbacks such as apoptosis and considerable reduction of follicular density in the transplanted ovary. One solution to reduce ischemic damage is enhancing angiogenesis after transplantation of ovarian cortex tissue. Objective: The aim of this study was to investigate the effect of Setarud, on angiogenesis in transplanted human ovarian tissue. Materials and Methods: In this case control study, twenty four nude mice were implanted subcutaneously, with human ovarian tissues, from four women. The mice were randomly divided into two groups (n=12): the experimental group was treated with Setarud, while control group received only vehicle. Each group was divided into three subgroups (n=4) based on the graft recovery days post transplantation (PT). The transplanted fragments were removed on days 2, 7, and 30 PT and the expression of Angiopoietin-1, Angiopoietin-2, and Vascular endothelial growth factor at both gene and protein levels and vascular density were studied in the grafted ovarian tissues. Results: On the 2nd and 7th day PT, the level of Angiopoietin-1 gene expression in case group was significantly lower than that in control group, while the opposite results were obtained for Angiopoietin-2 and Vascular endothelial growth factor. These results were also confirmed at the protein level. The density of vessels in Setarud group elevated significantly on day 7 PT compared to pre-treatment state. Conclusion: Our results showed that administration of Setarud may stimulates angiogenesis in transplanted human ovarian tissues, although further researches are needed before a clear judgment is made. PMID:26644788

  11. Recent advances in the field of ovarian tissue cryopreservation and opportunities for research. (United States)

    Ladanyi, Camille; Mor, Amir; Christianson, Mindy S; Dhillon, Namisha; Segars, James H


    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.

  12. A Supramolecular Gel Approach to Minimize the Neural Cell Damage during Cryopreservation Process. (United States)

    Zeng, Jie; Yin, Yixia; Zhang, Li; Hu, Wanghui; Zhang, Chaocan; Chen, Wanyu


    The storage method for living cells is one of the major challenges in cell-based applications. Here, a novel supramolecular gel cryopreservation system (BDTC gel system) is introduced, which can observably increase the neural cell viability during cryopreservation process because this system can (1) confine the ice crystal growth in the porous of BDTC gel system, (2) decrease the amount of ice crystallization and cryopreservation system's freezing point, and (3) reduce the change rates of cell volumes and osmotic shock. In addition, thermoreversible BDTC supramolecular gel is easy to be removed after thawing so it does not hinder the adherence, growth, and proliferation of cells. The results of functionality assessments indicate that BDTC gel system can minimize the neural cell damage during cryopreservation process. This method will be potentially applied in cryopreservation of other cell types, tissues, or organs and will benefit cell therapy, tissue engineering, and organs transplantation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Cryopreservation of human limbal stem cells ex vivo expanded on amniotic membrane. (United States)

    Yeh, Hui-Jung; Yao, Chao-Ling; Chen, Hsin-I; Cheng, Huey-Chuan; Hwang, Shiaw-Min


    After cornea transplantation, the donor's limbal zone is currently discarded as medical waste. However, the limbal zone is rich in limbal stem cells and can be used in therapeutic applications of limbus loss. This study aimed to increase the availability of limbal stem cells and develop the optimal conditions of cryopreservation for ex vivo expanded limbal stem cells. Pieces of the limbus were cultured on amniotic membrane (AM) to outgrow limbal stem cells as cell sheets for 3 weeks. Different formulas of cryoprotectants were tested to preserve the expanded cell sheets in liquid nitrogen. Before and after cryopreservation, expanded cell sheets were assessed for cellular characteristics by viability, histologic examination, and expression of ABCG2, vimentin, and keratin 3. Expanded cell sheets usually exhibited 3-6 stratified layers after 3-week culture on AM and expressed specific markers of ABCG2 and vimentin for limbal stem cells. The effects of cryopreservation with different cryoprotectants were analyzed by histopathology, stem cell markers, and cell viability. The results showed that the optimal formula of cryoprotectants for expanded limbal cell sheets was 60% Dulbecco modified Eagle medium, 30% fetal bovine serum, and 10% dimethyl sulfoxide. After 8-week cryopreservation in liquid nitrogen, the characteristics of limbal stem cells were maintained, and the average viability of thawed cells was 53.8% +/- 5.8%. These results showed that limbal stem cells expanded on AM could be cryopreserved and provide a promising source without delay, if banking, for patients with limbal stem cell deficiency in the future.

  14. Subcutaneous encapsulated fat necrosis

    DEFF Research Database (Denmark)

    Aydin, Dogu; Berg, Jais O


    We have described subcutaneous encapsulated fat necrosis, which is benign, usually asymptomatic and underreported. Images have only been published on two earlier occasions, in which the necrotic nodules appear "pearly" than the cloudy yellow surface in present case. The presented image may help f...

  15. Ovarian tissue cryopreserved for fertility preservation from patients with Ewing or other sarcomas appear to have no tumour cell contamination

    DEFF Research Database (Denmark)

    Greve, Tine; Wielenga, Vera Timmermans; Grauslund, Morten


    The chemotherapy required to treat patients with sarcoma may as a side-effect induce infertility in girls and young women. If these patients have ovarian cortical tissue cryopreserved prior to chemotherapy, they may, if necessary, have the tissue transplanted and restore their fertility. The aim ...

  16. Parathyroid transplantation in thyroid surgery (United States)

    Gołkowski, Filip; Nawrot, Ireneusz


    Permanent hypoparathyroidism following thyroid surgery is rare. Its prevalence is reported to be below 1–2% if surgery is performed by experienced thyroid surgeons. Parathyroid identification and preservation in situ with good vascular supply is the mainstay of safe thyroid surgery. However, if the parathyroid glands are damaged, autotransplantation should be undertaken to preserve their function. Parathyroid transplantation can be considered in three distinct modes of application: (I) fresh parathyroid tissue autotransplantation during thyroidectomy in order to reduce the risk of permanent hypoparathyroidism; (II) cryopreserved parathyroid tissue autotransplantation in patients with permanent hypoparathyroidism; (III) parathyroid allotransplantation in patients with permanent hypoparathyroidism when cryopreserved parathyroid tissue is not available for grafting. Nowadays, allotransplantation of cultured parathyroid cells without immunosuppression should be taken into consideration in selected patients as an alternative to calcium and vitamin D3 supplementation in management of permanent hypoparathyroidism. This paper is aimed to provide a review of current status of various parathyroid transplantation techniques in thyroid surgery. PMID:29142845

  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


    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. Massive subcutaneous emphysema with pneumoscrotopenis ...

    African Journals Online (AJOL)

    Chest injury commonly leads to subcutaneous emphysema of the chest, neck and face. It is usually non-life threatening. Massive subcutaneous emphysema may occur and very rarely may spread to involve the scrotal sac and subcutaneous tissue planes of the penis to cause pneumoscrotopenis. This case report presents ...

  19. Tooth loss treatment in the anterior region: Autotransplantation of premolars and cryopreservation


    Hans Ulrik Paulsen; Jens Ove Andreasen; Ole Schwartz


    Avulsed and lost anterior teeth are common in young people. Using autotransplantation, it is possible to move problems in dental arches to regions where they are easier to solve orthodontically. Transplantation of premolars with three-quarter root formation or full root formation with wide open apical foramina provides the best prognosis for long-term survival. This article describes the use of autotransplantation and orthodontic treatment, together with cryopreservation, in connection with c...

  20. Subcutaneous bronchogenic cyst

    Directory of Open Access Journals (Sweden)

    Vivek Manchanda


    Full Text Available Bronchogenic cysts occur due to the anomalous development of the primitive tracheobronchial tree early in fetal life. They are usually present in middle mediastinum. Rarely, they have been found in other locations. We describe two patients with subcutaneous bronchogenic cysts located over manubrium sterni with special emphasis on the difficulties in pre-operative diagnosis. The two boys were managed by complete excision of the cysts. The children are well on follow-up.

  1. Cryopreservation of South African indigenous goat semen

    African Journals Online (AJOL)



    Dec 5, 2011 ... motility rates of South African indigenous goats. Reduction in the sperm cell motility after freeze/thawing is still a problem and requires further research on the diluents and techniques that give protection to sperm cells during cryopreservation. Key words: Cryopreservation, semen characteristics, indigenous ...

  2. Impact of cryopreservation on bull () semen proteome. (United States)

    Westfalewicz, B; Dietrich, M A; Ciereszko, A


    Cryopreservation of bull spermatozoa is a well-established technique, allowing artificial insemination of cattle on a commercial scale. However, the extent of proteome changes in seminal plasma and spermatozoa during cryopreservation are not yet fully known. The objective of this study was to compare the proteomes of fresh, equilibrated, and cryopreserved bull semen (spermatozoa and seminal plasma) to establish the changes in semen proteins during the cryopreservation process. Semen was collected from 6 mature Holstein Friesian bulls. After sample processing, comparative analysis and identification of proteins was performed using 2-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. Analysis of spermatozoa extracts revealed that 25 identified protein spots, representing 16 proteins, underwent significant ( cryopreservation. Eighteen protein spots decreased in abundance, 5 protein spots increased in abundance, and 2 protein spots showed different, specific patterns of abundance changes. Analysis of seminal fluid containing seminal plasma showed that 6 identified protein spots, representing 4 proteins, underwent significant ( cryopreservation. Two protein spots increased in abundance and 4 decreased in abundance. Semen extending and equilibration seems to be responsible for a significant portion of the proteome changes related to cryopreservation technology. Most sperm proteins affected by equilibration and cryopreservation are membrane bound, and loss of those proteins may reduce natural spermatozoa coating. Further research is needed to unravel the mechanisms of the particular protein changes described in this study and establish the relationship between those changes and sperm quality.

  3. Cryopreservation Maintains Functionality of Human iPSC Dopamine Neurons and Rescues Parkinsonian Phenotypes In Vivo

    Directory of Open Access Journals (Sweden)

    Dustin R. Wakeman


    Full Text Available A major challenge for clinical application of pluripotent stem cell therapy for Parkinson's disease (PD is large-scale manufacturing and cryopreservation of neurons that can be efficiently prepared with minimal manipulation. To address this obstacle, midbrain dopamine neurons were derived from human induced pluripotent stem cells (iPSC-mDA and cryopreserved in large production lots for biochemical and transplantation studies. Cryopreserved, post-mitotic iPSC-mDA neurons retained high viability with gene, protein, and electrophysiological signatures consistent with midbrain floor-plate lineage. To test therapeutic efficacy, cryopreserved iPSC-mDA neurons were transplanted without subculturing into the 6-OHDA-lesioned rat and MPTP-lesioned non-human-primate models of PD. Grafted neurons retained midbrain lineage with extensive fiber innervation in both rodents and monkeys. Behavioral assessment in 6-OHDA-lesioned rats demonstrated significant reversal in functional deficits up to 6 months post transplantation with reinnervation of the host striatum and no aberrant growth, supporting the translational development of pluripotent cell-based therapies in PD.

  4. Cryopreservation and revival of mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Kastrup, Jens


    ) 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...... is the lethality associated with the cooling and thawing processes. The major objective is to minimize damage to cells during low temperature freezing and storage and the use of a suitable cryoprotectant. The detrimental effects of cellular cryopreservation can be minimized by controlling the cooling rate, using...... better cryoprotective agents, maintaining appropriate storage temperatures, and controlling the cell thawing rate. As is described in this chapter, human MSCs can either be frozen in cryovials or in freezing bags together with cryopreserve solutions containing dimethyl sulfoxide (DMSO)....

  5. Cryopreservation for preservation of potato genetic resources (United States)

    Niino, Takao; Arizaga, Miriam Valle


    Cryopreservation is becoming a very important tool for the long-term storage of plant genetic resources and efficient cryopreservation protocols have been developed for a large number of plant species. Practical procedures, developed using in vitro tissue culture, can be a simple and reliable preservation option of potato genetic resources rather than maintaining by vegetative propagation in genebanks due their allogamous nature. Cryopreserved materials insure a long-term backup of field collections against loss of plant germplasm. Occurrence of genetic variation, in tissue culture cells during prolonged subcultures, can be avoided with suitable cryopreservation protocols that provide high regrowth, leading and facilitating a systematic and strategic cryo-banking of plant genetic resources. Cryopreservation protocols for potato reviewed here, can efficiently complement field and in vitro conservation, providing for preservation of genotypes difficult to preserve by other methods, wild types and other species decided as priority collections. PMID:25931979

  6. [52-year-old patient with subcutaneous space-occupying lesion in immunosuppression]. (United States)

    Kolligs, F T; Gerbes, A L; Dürr, E M; Schauer, R; Kessler, M; Jelinek, T; Löscher, T; Bilzer, M


    We report the case of a 52-years-old male patient, who was diagnosed with subcutaneous alveolar echinococcosis 6 months after liver transplantation for HCV-related cirrhosis. Nether the explanted nor the transplantated liver revealed an echinococcus focus. Therefore a rare primary extrahepatic manifestation was likely. Interestingly, the echinococcal larvae had developed protoscolices. The development of mature tapeworms in human is a rarity, which could be related to the immunosuppressive therapy after liver transplantation. The patient was curatively treated by surgical removal of the subcutaneous tumor and a postoperative therapy with albendazole. Furthermore, HCV reinfection (genotype 2b) was successfully treated with interferone alpha 2b and ribavirine for 6 months.

  7. Coconut (Cocos nucifera l.) pollen cryopreservation. (United States)

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


    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.

  8. Two-piece cryopreserved tracheal allotransplantation: an experimental study. (United States)

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


    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

  9. Long-term experiences in cryopreservation of mobilized peripheral blood stem cells using a closed-bag system: a technology with potential for broader application. (United States)

    Spoerl, Silvia; Peter, Robert; Wäscher, Dagmar; Verbeek, Mareike; Menzel, Helge; Peschel, Christian; Krackhardt, Angela M


    In several European countries, preparation of cellular products with open manufacturing systems as used for cryopreservation of peripheral blood stem cells (PBSCs) needs to be performed in a clean-room facility. However, this form of manufacturing is highly expensive and laborious. Thus, safe techniques providing improved efficacy regarding time and material, which are in accordance with legal requirements are highly desirable. We have developed, validated, and applied a simple method for cryopreservation of PBSCs within a functionally closed-bag system using the closed cryo freeze prep set. This process fulfills good manufacturing practice requirements and allows for the cryopreservation of PBSCs without a clean-room facility. In addition to cryopreservation of PBSCs, we have recently successfully modified our system for processing, portioning, and cryopreservation of allogeneic donor lymphocytes. Since 2010, cryopreservation of PBSCs using a closed-bag system has been performed in our facility on a routine basis and 210 patients and healthy donors have been included in this analysis. No significant reduction in viability of CD34+ cells and no process-related contamination were observed. Outcome of hematopoietic stem cell transplantation regarding time of engraftment and infectious complications is comparable to products manufactured in conventional clean-room facilities. Our data confirm that cryopreservation of PBSCs within a functionally closed-bag system is safe, effective, and economical. Furthermore, the system has the potential to be extended to other manufacturing processes of cellular products. © 2015 AABB.

  10. Cryopreservation of microencapsulated canine sperm. (United States)

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


    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.

  11. The influence of temperature treatment before cryopreservation on the viability and potency of cryopreserved and thawed CD34+and CD45+cord blood cells. (United States)

    Schwandt, Svenja; Liedtke, Stefanie; Kogler, Gesine


    Hematopoietic stem cell (HSC) viability and potency is crucial for qualified cord blood (CB) transplants. This study analyzes time and temperature condition before cryopreservation for the viability of CD34 + /CD45 + cells after cryopreservation. Cell viabilities were determined by antibody co-staining with 7-aminoactinomycin D detecting necrotic cells, and subsequent flow cytometric analysis. Additionally, Annexin V staining for determination of apoptotic cells and colony-forming unit (CFU) assays for testing functional potency of HSCs were performed. For all cell types assessed (CD45 + /CD34 + cells, lymphocytes and granulocytes), the highest viabilities were obtained for CB maintained at 4°C or room temperature (RT; 22 ± 4°C) and cryopreserved directly after collection. Starting material were CB units with an age of 24.7 ± 3.5 h after birth. Post-thaw CD34 + cell results were > 90% after temperature treatment of t = 24 h (48 h total age) and > 70% after t = 48 h (72 h total age) at 4°C (48 h, 91.4 ± 5.5%; 72 h, 75.0 ± 12.0%) and RT (48 h, 84.2 ± 9.7%; 72 h, 72.6 ± 0.6%). Viabilities for 30°C samples were cryopreservation should be minimized because increasing time reduces numbers of viable cells and CFUs before/after cryopreservation. CB units should be maintained at 4°C/RT to retain the highest possible potency of the cells after thawing. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  12. Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation (United States)

    Onofre, J.; Baert, Y.; Faes, K.; Goossens, E.


    BACKGROUND Germ cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex for preserving male fertility, this cannot help out prepubertal boys. Yet, these boys do have spermatogonial stem cells (SSCs) that able to produce sperm at the start of puberty, which allows them to safeguard their fertility through testicular tissue (TT) cryopreservation. SSC transplantation (SSCT), TT grafting and recent advances in in vitro spermatogenesis have opened new possibilities to restore fertility in humans. However, these techniques are still at a research stage and their efficiency depends on the amount of SSCs available for fertility restoration. Therefore, maintaining the number of SSCs is a critical step in human fertility preservation. Standardizing a successful cryopreservation method for TT and testicular cell suspensions (TCSs) is most important before any clinical application of fertility restoration could be successful. OBJECTIVE AND RATIONALE This review gives an overview of existing cryopreservation protocols used in different animal models and humans. Cell recovery, cell viability, tissue integrity and functional assays are taken into account. Additionally, biosafety and current perspectives in male fertility preservation are discussed. SEARCH METHODS An extensive PubMED and MEDline database search was conducted. Relevant studies linked to the topic were identified by the search terms: cryopreservation, male fertility preservation, (immature)testicular tissue, testicular cell suspension, spermatogonial stem cell, gonadotoxicity, radiotherapy and chemotherapy. OUTCOMES The feasibility of fertility restoration techniques using frozen-thawed TT and TCS has been proven in animal models. Efficient protocols for cryopreserving human TT exist and are currently applied in the clinic. For TCSs, the highest post-thaw viability reported after vitrification is 55.6 ± 23

  13. Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation. (United States)

    Onofre, J; Baert, Y; Faes, K; Goossens, E


    Germ cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex for preserving male fertility, this cannot help out prepubertal boys. Yet, these boys do have spermatogonial stem cells (SSCs) that able to produce sperm at the start of puberty, which allows them to safeguard their fertility through testicular tissue (TT) cryopreservation. SSC transplantation (SSCT), TT grafting and recent advances in in vitro spermatogenesis have opened new possibilities to restore fertility in humans. However, these techniques are still at a research stage and their efficiency depends on the amount of SSCs available for fertility restoration. Therefore, maintaining the number of SSCs is a critical step in human fertility preservation. Standardizing a successful cryopreservation method for TT and testicular cell suspensions (TCSs) is most important before any clinical application of fertility restoration could be successful. This review gives an overview of existing cryopreservation protocols used in different animal models and humans. Cell recovery, cell viability, tissue integrity and functional assays are taken into account. Additionally, biosafety and current perspectives in male fertility preservation are discussed. An extensive PubMED and MEDline database search was conducted. Relevant studies linked to the topic were identified by the search terms: cryopreservation, male fertility preservation, (immature)testicular tissue, testicular cell suspension, spermatogonial stem cell, gonadotoxicity, radiotherapy and chemotherapy. The feasibility of fertility restoration techniques using frozen-thawed TT and TCS has been proven in animal models. Efficient protocols for cryopreserving human TT exist and are currently applied in the clinic. For TCSs, the highest post-thaw viability reported after vitrification is 55.6 ± 23.8%. Yet, functional proof of fertility restoration in the

  14. Cryopreservation of epididymal stallion sperm. (United States)

    Olaciregui, M; Gil, L; Montón, A; Luño, V; Jerez, R A; Martí, J I


    Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion's breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen-thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose-egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Livebirth after uterus transplantation. (United States)

    Brännström, Mats; Johannesson, Liza; Bokström, Hans; Kvarnström, Niclas; Mölne, Johan; Dahm-Kähler, Pernilla; Enskog, Anders; Milenkovic, Milan; Ekberg, Jana; Diaz-Garcia, Cesar; Gäbel, Markus; Hanafy, Ash; Hagberg, Henrik; Olausson, Michael; Nilsson, Lars


    Uterus transplantation is the first available treatment for absolute uterine infertility, which is caused by absence of the uterus or the presence of a non-functional uterus. Eleven human uterus transplantation attempts have been done worldwide but no livebirth has yet been reported. In 2013, a 35-year-old woman with congenital absence of the uterus (Rokitansky syndrome) underwent transplantation of the uterus in Sahlgrenska University Hospital, Gothenburg, Sweden. The uterus was donated from a living, 61-year-old, two-parous woman. In-vitro fertilisation treatment of the recipient and her partner had been done before transplantation, from which 11 embryos were cryopreserved. The recipient and the donor had essentially uneventful postoperative recoveries. The recipient's first menstruation occurred 43 days after transplantation and she continued to menstruate at regular intervals of between 26 and 36 days (median 32 days). 1 year after transplantation, the recipient underwent her first single embryo transfer, which resulted in pregnancy. She was then given triple immunosuppression (tacrolimus, azathioprine, and corticosteroids), which was continued throughout pregnancy. She had three episodes of mild rejection, one of which occurred during pregnancy. These episodes were all reversed by corticosteroid treatment. Fetal growth parameters and blood flows of the uterine arteries and umbilical cord were normal throughout pregnancy. The patient was admitted with pre-eclampsia at 31 full weeks and 5 days, and 16 h later a caesarean section was done because of abnormal cardiotocography. A male baby with a normal birthweight for gestational age (1775 g) and with APGAR scores 9, 9, 10 was born. We describe the first livebirth after uterus transplantation. This report is a proof-of-concept for uterus transplantation as a treatment for uterine factor infertility. Furthermore, the results show the feasibility of live uterus donation, even from a postmenopausal donor. Jane and

  16. Subcutaneous adipose tissue classification

    Directory of Open Access Journals (Sweden)

    A. Sbarbati


    Full Text Available The developments in the technologies based on the use of autologous adipose tissue attracted attention to minor depots as possible sampling areas. Some of those depots have never been studied in detail. The present study was performed on subcutaneous adipose depots sampled in different areas with the aim of explaining their morphology, particularly as far as regards stem niches. The results demonstrated that three different types of white adipose tissue (WAT can be differentiated on the basis of structural and ultrastructural features: deposit WAT (dWAT, structural WAT (sWAT and fibrous WAT (fWAT. dWAT can be found essentially in large fatty depots in the abdominal area (periumbilical. In the dWAT, cells are tightly packed and linked by a weak net of isolated collagen fibers. Collagenic components are very poor, cells are large and few blood vessels are present. The deep portion appears more fibrous then the superficial one. The microcirculation is formed by thin walled capillaries with rare stem niches. Reinforcement pericyte elements are rarely evident. The sWAT is more stromal; it is located in some areas in the limbs and in the hips. The stroma is fairly well represented, with a good vascularity and adequate staminality. Cells are wrapped by a basket of collagen fibers. The fatty depots of the knees and of the trochanteric areas have quite loose meshes. The fWAT has a noteworthy fibrous component and can be found in areas where a severe mechanic stress occurs. Adipocytes have an individual thick fibrous shell. In conclusion, the present study demonstrates evident differences among subcutaneous WAT deposits, thus suggesting that in regenerative procedures based on autologous adipose tissues the sampling area should not be randomly chosen, but it should be oriented by evidence based evaluations. The structural peculiarities of the sWAT, and particularly of its microcirculation, suggest that it could represent a privileged source for

  17. Fetal bovine serum-free cryopreservation methods for clinical banking of human adipose-derived stem cells. (United States)

    Park, Seah; Lee, Dong Ryul; Nam, Ji Sun; Ahn, Chul Woo; Kim, Haekwon


    The use of fetal bovine serum (FBS) as a cryopreservation supplement is not suitable for the banking of mesenchymal stem cells (MSCs) due to the risk of transmission of disease as well as xenogeneic immune reactions in the transplanted host. Here, we investigated if human serum albumin (HSA), human serum (HS), or knockout serum replacement (KSR) can replace FBS for the cryopreservation of MSCs. In addition, we examined the characteristics of MSCs after multiple rounds of cryopreservation. Human adipose-derived stem cells (ASCs) cryopreserved with three FBS replacements, 9% HSA, 90% HS, or 90% KSR, in combination with 10% dimethyl sulfoxide (Me 2 SO) maintained stem cell properties including growth, immunophenotypes, gene expression patterns, and the potential to differentiate into adipogenic, osteogenic, and chondrogenic lineages, similar to ASCs frozen with FBS. Moreover, the immunophenotype, gene expression, and differentiation capabilities of ASCs were not altered by up to four freeze-thaw cycles. However, the performance of three or four freeze-thaw cycles significantly reduced the proliferation ability of ASCs, as indicated by the longer population doubling time and reduced colony-forming unit-fibroblast frequency. Together, our results suggest that HSA, HS, or KSR can replace FBS for the cryopreservation of ASCs, without altering their stemness, and should be processed with no more than two freeze-thaw cycles for clinical approaches. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Protectants used in the cryopreservation of microorganisms

    Czech Academy of Sciences Publication Activity Database

    Hubálek, Zdeněk


    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

  19. Cryopreservation of biopsied cleavage stage human embryos. (United States)

    Stachecki, James J; Cohen, Jacques; Munné, Santiago


    The aim was to develop a method to optimize cryopreservation of biopsied multi-celled human embryos. Human day 3 embryos that were donated to research, along with those found to be chromosomally abnormal after blastomere biopsy and fluorescence in-situ hyridization (FISH), were cryopreserved using a slow-freezing protocol in either standard embryo cryopreservation solution [embryo transfer freezing medium (ETFM), a conventional sodium-based medium] or CJ3 (a choline-based, sodium-free medium). After thawing, the number of intact cells was recorded and the previously biopsied embryos were re-analysed using FISH. Biopsied embryos had a lower proportion of intact blastomeres after cryopreservation as compared with intact embryos. However, a significantly (P < 0.05) higher proportion of blastomeres from intact and biopsied embryos cryopreserved in CJ3 (84.1 and 80.1% respectively) survived after thaw than those in ETFM (73.6 and 50.5% respectively). The proportion of aneuploid and mosaic embryos was not statistically different between the two groups. In addition, the frequency of lost cells by aneuploid and mosaic embryos was similar. This study describes a new method that improves the survival of cryopreserved biopsied embryos, and shows that it may also be beneficial for the storage of intact human multi-celled embryos.

  20. Production of functional sperm by subcutaneous auto-grafting of immature testes in rainbow trout. (United States)

    Hayashi, Makoto; Sakuma, Daika; Yoshizaki, Goro


    Sexually mature individuals are indispensable for breeding programs. Salmonids require a long period before reaching sexual maturity, so we aimed to shorten the period required to obtain functional sperm by grafting immature testicular fragments into mature recipients, which we predicted would allow the grafted testicular fragments to skip the long pre-pubertal period. First, we demonstrated successful subcutaneous auto-grafting of testicular fragments in rainbow trout. Unilateral testectomy was performed, and the isolated immature testicular fragment was auto-grafted into the subcutaneous space along the back of recipient fish. The grafted testicular fragments developed synchronously with the recipients' testis remaining in its body cavity, and both eventually produced functional sperm. Next, immature testicular fragments were auto-grafted into the subcutaneous space of sexually mature males. We achieved this, without immune rejection, by isolating and cryopreserving testes from immature fish, and rearing these unilaterally testectomized fish until sexual maturity. The cryopreserved testes were then auto-grafted into the original, now spermiating fish. The grated immature testicular fragments differentiated and produced functional sperm within 5 months after grafting. By combining this grafting method with a technique to avoid immune rejection, we expect to develop a practical method for producing sperm in a shorter period in salmonids. © 2017 Wiley Periodicals, Inc.

  1. Cryopreservation studies on the marine diatom Navicula subinflata Grun

    Digital Repository Service at National Institute of Oceanography (India)

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

    Very little work has been done on marine unicellular algae regarding cryopreservation. The present work was, therefore, undertaken to study the effect of different cryoprotectants and cryopreservation on the growth of marine diatom Navicula...

  2. Some quantitative studies on the transplantation of human tissues into nude mice

    International Nuclear Information System (INIS)

    Zietman, A.; Suit, H.D.; Sedlacek, R.


    Quantitative cell transplantation assays (TD/sub 50/) were performed for human tumors xenografted into athymic NCr(nμ/nμ) nude mice. Transplantation assays for FaDu when transplanted into brain and when transplanted into subcutaneous tissues are compared. Effects of immunization are discussed and results are given

  3. Historical perspectives and the future of adverse reactions associated with haemopoietic stem cells cryopreserved with dimethyl sulfoxide

    DEFF Research Database (Denmark)

    Cox, Michael A; Kastrup, Jens; Hrubiško, Mikulas


    A retrospective review of the published literature identified several hundred adverse reactions (e.g. nausea, chills, cardiac arrhythmias, neurological symptoms and respiratory arrest) associated with the transplantation of stem cells cryopreserved with dimethyl sulfoxide. The occurrences...... of these are generally accepted as commonplace, as the majority of reactions are transient, whilst a few patients may require clinical treatment. This exploratory study is a collation of the historical data and the expectations for the notification of serious adverse reactions. Outline information is presented...

  4. Extending the dormant bud cryopreservation method to new tree species (United States)

    In cryopreservation of germplasm, using dormant winter buds (DB) as source plant material is economically favorable over tissue culture options. Although the DB cryopreservation method has been known for many years, the approach is feasible only for cryopreserving a select number of temperate tree s...

  5. Current trends and progress in clinical applications of oocyte cryopreservation (United States)

    Cil, Aylin P.; Seli, Emre


    Purpose of review To delineate the current trends in the clinical application of oocyte cryopreservation. Recent findings Although the first live birth from oocyte cryopreservation was reported approximately three decades ago, significant improvement in the clinical application of oocyte cryopreservation took place only over the past decade. On the basis of the available evidence suggesting that success rates with donor oocyte vitrification are similar to that of IVF with fresh donor oocytes, the American Society of Reproductive Medicine has recently stated that oocyte cryopreservation should no longer be considered experimental for medical indications, outlying elective oocyte cryopreservation. Meanwhile, a few surveys on the attitudes toward oocyte cryopreservation revealed that elective use for the postponement of fertility is currently the most common indication for oocyte cryopreservation. Most recently, a randomized controlled trial revealed important evidence on the safety of nondonor oocyte cryopreservation, and confirmed that the clinical success of vitrification is comparable to that of IVF with fresh oocytes. Summary The evidence suggesting similar IVF success rates with both donor and nondonor cryopreserved oocytes compared with fresh oocytes will increase the utilization of elective oocyte cryopreservation. Appropriate counseling of women for oocyte cryopreservation requires the establishment of age-based clinical success rates with cryopreserved oocytes for various indications. PMID:23562954

  6. Stability of cryopreserved samples of mutant mice. (United States)

    Ramin, Michael; Bürger, Antje; Hörlein, Andreas; Kerkau, Dagmar; von Walcke-Wulffen, Vincent; Nicklas, Werner; Schenkel, Johannes


    Genetically modified animals are unique models with enormous scientific potential. Cryopreservation of pre-implantation embryos or of spermatozoa is a common approach to save those lines. The breeding of a line can be discontinued if a sufficient number of samples have been cryopreserved. To maintain the opportunity to recover a line, it is mandatory to assess the quality of the cryopreserved samples and to assure safe long-term storage conditions. Here, we investigated the revitalization rate of cryopreserved pre-implantation embryos stored in-house up to 158 months, of imported (and shipped) embryos, and of embryos received after in vitro fertilization. The storage period did not affect the revitalization rate, whereas the recovery of imported embryos was significantly reduced, possibly due to shipment conditions. The genotypes of genetically modified pups received following embryo-transfer were slightly smaller than expected by Mendelian laws. Intensive investigations of the hygienic state of the cryopreserved samples and the equipment used never showed microbiological contamination of a sample within a cryo-tube. However, environmental organisms were found frequently in the permanent freezers and dry shippers used. Since such contamination cannot be completely excluded and an embryo-transfer might not lead in all cases to a secure rederivation, foster mothers and revitalized pups should be housed in an intermediate facility and their health assessed before introducing them into the target facility.

  7. Kidney Transplant (United States)

    ... Menu Menu Search Home Prevention Kidney Disease Patients Organ Donation & Transplantation Professionals Events Advocacy Donate A to Z Health ... Tests for Transplant Care After Kidney Transplant Common Organ Donation and Transplantation Terms The National Kidney Foundation (NKF) is the ...

  8. Comparison of the Viability of Cryopreserved Fat Tissue in Accordance with the Thawing Temperature

    Directory of Open Access Journals (Sweden)

    So-Min Hwang


    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. Treatment history and outcome of 24 deliveries worldwide after autotransplantation of cryopreserved ovarian tissue, including two new Danish deliveries years after autotransplantation

    DEFF Research Database (Denmark)

    Macklon, Kirsten T; Jensen, Annette Klüver; Loft, Anne


    PURPOSE: To report another two successful pregnancies and deliveries resulting from autotransplanted cryopreserved ovarian tissue several years after the autotransplantation procedure took place. Further, to review the literature on the treatment history, number of live births and their outcome so......'s lymphoma. Both suffered from premature ovarian insufficiency after treatment. Because of a pregnancy wish they later had pieces of thawed cortical tissue transplanted to the remaining ovary and the anterior abdominal wall. PubMed was searched for reports of deliveries resulting from cryopreserved ovarian...

  10. Subcutaneous granuloma annulare: radiologic appearance

    International Nuclear Information System (INIS)

    Kransdorf, M.J.; Murphey, M.D.; Temple, H.T.


    Objective. Granuloma annulare is an uncommon benign inflammatory dermatosis characterized by the formation of dermal papules with a tendency to form rings. There are several clinically distinct forms. The subcutaneous form is the most frequently encountered by radiologists, with the lesion presenting as a superficial mass. There are only a few scattered reports of the imaging appearance of this entity in the literature. We report the radiologic appearance of five cases of subcutaneous granuloma annulare. Design and patients. The radiologic images of five patients (three male, two female) with subcutaneous granuloma annulare were retrospectively studied. Mean patient age was 6.4 years (range, 2-13 years). The lesions occurred in the lower leg (two), foot, forearm, and hand. MR images were available for all lesions, gadolinium-enhanced imaging in three cases, radiographs in four, and bone scintigraphy in one. Results. Radiographs showed unmineralized nodular masses localized to the subcutaneous adipose tissue. The size range, in greatest dimension on imaging studies, was 1-4 cm. MR images show a mass with relatively decreased signal intensity on all pulse sequences, with variable but generally relatively well defined margins. There was extensive diffuse enhancement following gadolinium administration. Conclusion. The radiologic appearance of subcutaneous granuloma annulare is characteristic, typically demonstrating a nodular soft-tissue mass involving the subcutaneous adipose tissue. MR images show a mass with relatively decreased signal intensity on all pulse sequences and variable but generally well defined margins. There is extensive diffuse enhancement following gadolinium administration. Radiographs show a soft-tissue mass or soft-tissue swelling without evidence of bone involvement or mineralization. This radiologic appearance in a young individual is highly suggestive of subcutaneous granuloma annulare. (orig.)

  11. Cryopreservation and revival of mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Kastrup, Jens


    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...... 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......) 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...

  12. Tooth loss treatment in the anterior region: Autotransplantation of premolars and cryopreservation

    Directory of Open Access Journals (Sweden)

    Hans Ulrik Paulsen


    Full Text Available Avulsed and lost anterior teeth are common in young people. Using autotransplantation, it is possible to move problems in dental arches to regions where they are easier to solve orthodontically. Transplantation of premolars with three-quarter root formation or full root formation with wide open apical foramina provides the best prognosis for long-term survival. This article describes the use of autotransplantation and orthodontic treatment, together with cryopreservation, in connection with complicated trauma in the anterior region of an 8-year-old female child.

  13. Tooth loss treatment in the anterior region: autotransplantation of premolars and cryopreservation. (United States)

    Paulsen, Hans Ulrik; Andreasen, Jens Ove; Schwartz, Ole


    Avulsed and lost anterior teeth are common in young people. Using autotransplantation, it is possible to move problems in dental arches to regions where they are more easy to solve orthodontically. Transplantation of premolars with three-quarter root formation or full root formation with wide-open apical foramina provides the best prognosis for long-term survival. This article describes the use of autotransplantation and orthodontic treatment, together with cryopreservation, in connection with complicated trauma in the anterior region of an 8-year-old girl.

  14. Karyotype of cryopreserved bone marrow cells

    Directory of Open Access Journals (Sweden)

    M.L.L.F. Chauffaille


    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.

  15. Strategies for commercialization of cryopreserved fish semen

    Directory of Open Access Journals (Sweden)

    Terrence R. Tiersch


    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

  16. Simplified cryopreservation of porcine cloned blastocysts

    DEFF Research Database (Denmark)

    Du, Yutao; Zhang, Yunhai; Li, Juan


    )â€"handmade cloning (HMC)â€"to establish a simplified and efficient cryopreservation system for porcine cloned embryos. In Experiment 1, zonae pellucidae of oocytes were partially digested with pronase, followed by centrifugation to polarize lipid particles. Ninety percent (173/192) oocytes were successfully......). Our results prove that porcine embryos produced from delipated oocytes by PA or HMC can be cryopreserved effectively by ultrarapid vitrification. Further experiments are required to assess the in vivo developmental competence of the cloned-vitrified embryos  ...

  17. Timing embryo biopsy for PGD - before or after cryopreservation? (United States)

    Shinar, S; Kornecki, N; Schwartz, T; Mey-Raz, N; Amir, H; Almog, B; Shavit, T; Hasson, J


    Pre-implantation genetic diagnosis (PGD) is required in order to screen and diagnose embryos of patients at risk of having a genetically affected offspring. A biopsy to diagnose the genetic profile of the embryo may be performed either before or after cryopreservation. The aim of this study was to determine which biopsy timing yields higher embryo survival rates. Retrospective cohort study of all PGD patients in a public IVF unit between 2010 and 2013. Inclusion criteria were patients with good-quality embryos available for cryopreservation by the slow freezing method. Embryos were divided into two groups: biopsy before and biopsy after cryopreservation. The primary outcome was embryo survival rates post thawing. Sixty-five patients met inclusion criteria. 145 embryos were biopsied before cryopreservation and 228 embryos were cryopreserved and biopsied after thawing. Embryo survival was significantly greater in the latter group (77% vs. 68%, p Cryopreservation preceding biopsy results in better embryo survival compared to biopsy before cryopreservation.

  18. 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.

  19. Cryopreservation of artificial gut microbiota produced with in vitro fermentation technology. (United States)

    Bircher, Lea; Schwab, Clarissa; Geirnaert, Annelies; Lacroix, Christophe


    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.

  20. 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

  1. Impact of Procedural Steps and Cryopreservation Agents in the Cryopreservation of Chlorophyte Microalgae (United States)

    Bui, Tony V. L.; Ross, Ian L.; Jakob, Gisela; Hankamer, Ben


    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 of each of

  2. [Cellular subcutaneous tissue. Anatomic observations]. (United States)

    Marquart-Elbaz, C; Varnaison, E; Sick, H; Grosshans, E; Cribier, B


    We showed in a companion paper that the definition of the French "subcutaneous cellular tissue" considerably varied from the 18th to the end of the 20th centuries and has not yet reached a consensus. To address the anatomic reality of this "subcutaneous cellular tissue", we investigated the anatomic structures underlying the fat tissue in normal human skin. Sixty specimens were excised from the surface to the deep structures (bone, muscle, cartilage) on different body sites of 3 cadavers from the Institut d'Anatomie Normale de Strasbourg. Samples were paraffin-embedded, stained and analysed with a binocular microscope taking x 1 photographs. Specimens were also excised and fixed after subcutaneous injection of Indian ink, after mechanic tissue splitting and after performing artificial skin folds. The aspects of the deep parts of the skin greatly varied according to their anatomic localisation. Below the adipose tissue, we often found a lamellar fibrous layer which extended from the interlobular septa and contained horizontally distributed fat cells. No specific tissue below the hypodermis was observed. Artificial skin folds concerned either exclusively the dermis, when they were superficial or included the hypodermis, but no specific structure was apparent in the center of the fold. India ink diffused to the adipose tissue, mainly along the septa, but did not localise in a specific subcutaneous compartment. This study shows that the histologic aspects of the deep part of the skin depend mainly on the anatomic localisation. Skin is composed of epidermis, dermis and hypodermis and thus the hypodermis can not be considered as being "subcutaneous". A difficult to individualise, fibrous lamellar structure in continuity with the interlobular septa is often found under the fat lobules. This structure is a cleavage line, as is always the case with loose connective tissues, but belongs to the hypodermis (i.e. fat tissue). No specific tissue nor any virtual space was

  3. [Hepatic cell transplantation. Technical and methodological aspects]. (United States)

    Pareja, Eugenia; Martínez, Amparo; Cortés, Miriam; Bonora, Ana; Moya, Angel; Sanjuán, Fernando; Gómez-Lechón, M José; Mir, José


    Hepatic cell transplantation consists of grafting already differentiated cells such as hepatocytes. Human hepatocytes are viable and functionally active. Liver cell transplantation is carried out by means of a 3-step method: isolation of hepatocytes from donor liver rejected for orthotopic transplantation, preparing a cell suspension for infusion and, finally, hepatocytes are implanted into the recipient. There are established protocols for the isolation of human hepatocytes from unused segments of donor livers, based on collagenase digestion of cannulated liver tissue at 37 degrees C. The hepatocytes can be used fresh or cryopreserved. Cryopreservation of isolated human hepatocytes would then be available for planned use. In cell transplant, the important aspects are: infusion route, number of cells, number of infusions and viability of the cells. The cells are infused into the patient through a catheter inserted via portal vein or splenic artery. Liver cell transplantation allows liver tissue to be used that would, otherwise, be discarded, enabling multiple patients to be treated with hepatocytes from a single tissue donor. Copyright 2009 AEC. Published by Elsevier Espana. All rights reserved.

  4. stausartikel: behandling af subcutane abscesser

    DEFF Research Database (Denmark)

    Hardgrib, Nina; Petersen, Klaus Kjær


    be administered, and the cavity should be closed without dead space. Antibiotics should not routinely be given, but always be considered in immunocompromised patients or patients with septicaemia. Routine examination of the microbiology has no influence on the treatment and is not recommended.......Simple subcutaneous abscesses are common, and we have examined the literature concerning the ideal treatment of subcutaneous abscesses. We recommend radical debridement with removal of all pus, the abscess wall and any necrosis. If primary suture is chosen, preoperative antibiotics should...

  5. Sea urchin (Paracentrotus lividus) cryopreserved embryos survival and growth: effects of cryopreservation parameters and reproductive seasonality. (United States)

    Paredes, P; Bellas, J


    The cryopreservation of embryos can be a powerful biotechnological tool to supply all year-round biological material for sea urchin aquaculture production. This study investigates different methodological and biological factors that may affect the result of the cryopreservation process of sea urchin (Paracentrotus lividus) embryos. Our data indicate that neither embryo density nor the use of different cryopreservation containers presented effect on the cryopreservation outcome. Contrary to other marine invertebrates, for sea urchin embryo cryopreservation ultrapure water cannot be used as CPA solvent, yielding zero survival. After studying the reproductive parameters along the reproductive season, we found a positive correlation between both male and female Condition Index (C.I.), and between the oocyte weight and C.I. Both the histology study of female gonads and the C.I. variation, suggest that the sea urchin natural spawning period in the Ría de Vigo occurs between June and July. We found no correlation between any of the reproductive parameters monitored and the cryopreservation outcome.

  6. Cells isolated from cryopreserved dental follicle display similar characteristics to cryopreserved dental follicle cells. (United States)

    Yang, Hefeng; Li, Jie; Sun, Jingjing; Guo, Weihua; Li, Hui; Chen, Jinlong; Hu, Yu; Tian, Weidong; Li, Song


    Dental follicle tissue is a promising resource of mesenchymal stem cells for cytotherapeutic approaches and tissue engineering applications. There are two procedures for banking of human dental follicle stem cells have been reported. Conventional method requires cell isolation, expansion and immediate cryopreservation. Whereas dental follicle stem cells can be isolated from cryopreserved dental follicle fragments. The aim of this study was to compare the characteristics of dental follicle cells isolated from cryopreserved fragments (DFCs-CF) with dental follicle cells recovered from cryopreserved cells (DFCs-CC). Dental follicle fragments obtained after mechanical disaggregation were divided into two parts, with one part maintained in culture, while another part underwent cryopreservation. Dental follicle fragments and dental follicle cells from fresh tissue were stored in liquid nitrogen for 3 months. After thawing, the isolation, morphology, proliferation, cell cycle, colony-forming-unit ability, stemness-related marker expression, apoptosis, and multi-lineage differentiation potential of DFCs-CF were tested compared with DFCs-CC. DFCs-CF expressed mesenchymal stem cells marker, proliferated well, showed similar levels of mRNA for stemness- and apoptosis-related genes and exhibited the capacity of multi-lineage differentiation similar to those of DFCs-CC. These results imply that cryopreservation of dental follicle fragments is an effective banking method for isolation of dental follicle cells. Copyright © 2017. Published by Elsevier Inc.

  7. Modeling the transport of cryoprotective agents in articular cartilage for cryopreservation (United States)

    Torqabeh, Alireza Abazari

    Loading vitrifiable concentrations of cryoprotective agents is an important step for cryopreservation of biological tissues by vitrification for research and transplantation purposes. This may be done by immersing the tissue in a cryoprotective agent (CPA) solution, and increasing the concentration, continuously or in multiple steps, and simultaneously decreasing the temperature to decrease the toxicity effects of the cryoprotective agent on the tissue cellular system. During cryoprotective agent loading, osmotic water movement from the tissue to the surrounding solution, and the resultant tissue shrinkage and stress-strain in the tissue matrix as well as on the cellular system can significantly alter the outcome of the cryopreservation protocol. In this thesis, a biomechanical model for articular cartilage is developed to account for the transport of the cryoprotective agent, the nonideal-nondilute properties of the vitrifiable solutions, the osmotic water movement and the resultant tissue shrinkage and stress-strain in the tissue matrix, and the osmotic volume change of the chondrocytes, during cryoprotective agent loading in the cartilage matrix. Four essential transport parameters needed for the model were specified, the values of which were obtained uniquely by fitting the model to experimental data from porcine articular cartilage. Then, it was shown that using real nonuniform initial distributions of water and fixed charges in cartilage, measured separately in this thesis using MRI, in the model can significantly affect the model predictions. The model predictions for dimethyl sulfoxide diffusion in porcine articular cartilage were verified by comparing to spatially and temporally resolved measurements of dimethyl sulfoxide concentration in porcine articular cartilage using a spectral MRI technique, developed for this purpose and novel to the field of cryobiology. It was demonstrated in this thesis that the developed mathematical model provides a novel tool

  8. 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.

  9. 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 ...

  10. Cryopreservation of medicinal plants: role of melatonin (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...

  11. Preliminary study on cryopreservation of Dendrobium Bobby ...

    African Journals Online (AJOL)

    Protocorm-like bodies of Dendrobium Bobby Messina were cryopreserved by vitrification method. In this study, protocorm-like bodies (PLBs) with the size range of 1-2 and 3-4 mm were selected from 4 weeks old culture, pretreated with half strength semi-solid Murashige and Skoog (MS) media supplemented with 0.5 M ...

  12. Cryopreservation and Cryotherapy of Citrus Cultivars (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...

  13. Fundamental concept of cryopreservation using Dendrobium sonia ...

    African Journals Online (AJOL)

    dehydration technique on cryopreservation protocorm-like bodies (PLBs) of Dendrobium sonia-17. The survival of the PLBs was assessed based on the effects of 4 dehydration periods (0, 1, 3 and 5 h) and 4 different concentrations of 24-h ...

  14. 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 ...

  15. Cryopreservation of embryonic axes of groundnut ( Arachis ...

    African Journals Online (AJOL)

    An efficient cryopreservation protocol was developed for groundnut embryonic axes using vitrification technique. Embryonic axes obtained from seeds of four groundnut genotypes were dehydrated in Plant Vitrification Solution (PVS2) solution for different durations (0, 1, 2, 3, 4 and 5 h) before plunged into liquid nitrogen ...

  16. The Second International Symposium on Plant Cryopreservation (United States)

    The Second International Symposium on Plant Cryopreservation was held in Fort Collins, Colorado, USA, from August 11-14, 2013, under the auspices of the International Society for Horticultural Science. The town of Fort Collins is home to the USDA-ARS, National Center for Genetic Resources Preservati...

  17. Cryopreservation: Vitrification and Controlled Rate Cooling. (United States)

    Hunt, Charles J


    Cryopreservation is the application of low temperatures to preserve the structural and functional integrity of cells and tissues. Conventional cooling protocols allow ice to form and solute concentrations to rise during the cryopreservation process. The damage caused by the rise in solute concentration can be mitigated by the use of compounds known as cryoprotectants. Such compounds protect cells from the consequences of slow cooling injury, allowing them to be cooled at cooling rates which avoid the lethal effects of intracellular ice. An alternative to conventional cooling is vitrification. Vitrification methods incorporate cryoprotectants at sufficiently high concentrations to prevent ice crystallization so that the system forms an amorphous glass thus avoiding the damaging effects caused by conventional slow cooling. However, vitrification too can impose damaging consequences on cells as the cryoprotectant concentrations required to vitrify cells at lower cooling rates are potentially, and often, harmful. While these concentrations can be lowered to nontoxic levels, if the cells are ultra-rapidly cooled, the resulting metastable system can lead to damage through devitrification and growth of ice during subsequent storage and rewarming if not appropriately handled.The commercial and clinical application of stem cells requires robust and reproducible cryopreservation protocols and appropriate long-term, low-temperature storage conditions to provide reliable master and working cell banks. Though current Good Manufacturing Practice (cGMP) compliant methods for the derivation and banking of clinical grade pluripotent stem cells exist and stem cell lines suitable for clinical applications are available, current cryopreservation protocols, whether for vitrification or conventional slow freezing, remain suboptimal. Apart from the resultant loss of valuable product that suboptimal cryopreservation engenders, there is a danger that such processes will impose a selective

  18. Slow Cooling Cryopreservation Optimized to Human Pluripotent Stem Cells. (United States)

    Miyazaki, Takamichi; Suemori, Hirofumi


    Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cells that form all three germ layers. Cryopreservation is one of the key processes for successful applications of hPSCs, because it allows semi-permanent preservation of cells and their easy transportation. Most animal cell lines, including mouse embryonic stem cells, are standardly cryopreserved by slow cooling; however, hPSCs have been difficult to preserve and their cell viability has been extremely low whenever cryopreservation has been attempted.Here, we investigate the reasons for failure of slow cooling in hPSC cryopreservation. Cryopreservation involves a series of steps and is not a straightforward process. Cells may die due to various reasons during cryopreservation. Indeed, hPSCs preserved by traditional methods often suffer necrosis during the freeze-thawing stages, and the colony state of hPSCs prior to cryopreservation is a major factor contributing to cell death.It has now become possible to cryopreserve hPSCs using conventional cryopreservation methods without any specific equipment. This review summarizes the advances in this area and discusses the optimization of slow cooling cryopreservation for hPSC storage.

  19. Cryopreservation of peach palm zygotic embryos. (United States)

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


    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.

  20. Mechanisms of Aeroallergen Immunotherapy: Subcutaneous Immunotherapy and Sublingual Immunotherapy. (United States)

    Ozdemir, Cevdet; Kucuksezer, Umut Can; Akdis, Mübeccel; Akdis, Cezmi A


    Allergen immunotherapy (AIT) is an effective way to treat allergic disorders, targeting the underlying mechanisms and altering the disease course by inducing a long-lasting clinical and immune tolerance to allergens. Although sublingual and subcutaneous routes are used in daily practice, many novel ways to decrease side effects and duration and increase efficacy have been pursued. Further studies are needed to develop biomarkers for the identification of AIT responder patients and also to use the developed knowledge in allergy prevention studies. Future directions in AIT include treatments for autoimmune diseases, chronic infections, organ transplantation, and breaking immune tolerance to cancer cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Hydrogel Encapsulation Facilitates Rapid-Cooling Cryopreservation of Stem Cell-Laden Core-Shell Microcapsules as Cell-Biomaterial Constructs. (United States)

    Zhao, Gang; Liu, Xiaoli; Zhu, Kaixuan; He, Xiaoming


    Core-shell structured stem cell microencapsulation in hydrogel has wide applications in tissue engineering, regenerative medicine, and cell-based therapies because it offers an ideal immunoisolative microenvironment for cell delivery and 3D culture. Long-term storage of such microcapsules as cell-biomaterial constructs by cryopreservation is an enabling technology for their wide distribution and ready availability for clinical transplantation. However, most of the existing studies focus on cryopreservation of single cells or cells in microcapsules without a core-shell structure (i.e., hydrogel beads). The goal of this study is to achieve cryopreservation of stem cells encapsulated in core-shell microcapsules as cell-biomaterial constructs or biocomposites. To this end, a capillary microfluidics-based core-shell alginate hydrogel encapsulation technology is developed to produce porcine adipose-derived stem cell-laden microcapsules for vitreous cryopreservation with very low concentration (2 mol L -1 ) of cell membrane penetrating cryoprotective agents (CPAs) by suppressing ice formation. This may provide a low-CPA and cost-effective approach for vitreous cryopreservation of "ready-to-use" stem cell-biomaterial constructs, facilitating their off-the-shelf availability and widespread applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Long-term duration of function of ovarian tissue transplants

    DEFF Research Database (Denmark)

    Andersen, Claus Yding; Silber, Sherman J; Berghold, Stinne Holm


    tissue transplanted, and two women underwent fresh ovarian tissue transplants. The function of ovarian cortical strips has continued for more than 7years in these three women, with the birth of eight healthy babies following a single graft per patient. In addition to these three cases, transplantation...... (repeatedly in some cases) of cryopreserved ovarian tissue has restored reproductive function to all other women in the study centres' programmes for some years. The sustained longevity of function of the transplanted tissue suggests that it may also be possible to postpone the normal time of menopause...... frozen ovarian tissue transplanted, and two women underwent fresh ovarian tissue transplants. Function of ovarian cortical strips has continued for more than 7years in these three women, with the birth of eight healthy babies following a single graft per patient. In addition to these three cases...

  3. Kidney Transplant (United States)

    ... type matches or is compatible to your own. Blood-type incompatible transplants are also possible but require additional medical treatment before and after transplant to reduce the risk of organ rejection. These are known as ABO incompatible kidney transplants. ...

  4. Kidney transplant (United States)

    ... place a healthy kidney into a person with kidney failure . ... Renal transplant; Transplant - kidney ... Becker Y, Witkowski P. Kidney and pancreas transplantation. In: Townsend CM ... Urology . 11th ed. Philadelphia, PA: Elsevier; 2016: ...

  5. Need for Comprehensive Counseling in Women Requesting Oocyte Cryopreservation. (United States)

    Bachmann, Gloria; MacArthur, Taleen A; Khanuja, Kavisha


    The aim of this study is to assess current counseling recommendations for women undergoing elective oocyte cryopreservation. PubMed and Clinical Key. A search of PubMed and Clinical Key was conducted to assess current counseling practices for elective oocyte cryopreservation. It is substantiated that uniform counseling guidelines are lacking for this group of assisted reproductive technology (ART) patients presenting only for cryopreserving their oocytes. However, although a woman may be a suitable candidate for pregnancy at the point that she undergoes oocyte cryopreservation, possibly many years later, at the time of oocyte thawing, this same woman may have multiple risk factors, which will increase her risk for pregnancy-related maternal and fetal morbidity and mortality. Given the increasing use of oocyte cryopreservation, data support that women be extensively counseled at the time they are requesting elective oocyte cryopreservation for future use in the same manner that they are counseled when requesting ART for pursuing an immediate pregnancy.

  6. Cryopreserved Dental Pulp Tissues of Exfoliated Deciduous Teeth Is a Feasible Stem Cell Resource for Regenerative Medicine (United States)

    Yamaza, Haruyoshi; Akiyama, Kentaro; Hoshino, Yoshihiro; Song, Guangtai; Kukita, Toshio; Nonaka, Kazuaki; Shi, Songtao; Yamaza, Takayoshi


    Human exfoliated deciduous teeth have been considered to be a promising source for regenerative therapy because they contain unique postnatal stem cells from human exfoliated deciduous teeth (SHED) with self-renewal capacity, multipotency and immunomodulatory function. However preservation technique of deciduous teeth has not been developed. This study aimed to evaluate that cryopreserved dental pulp tissues of human exfoliated deciduous teeth is a retrievable and practical SHED source for cell-based therapy. SHED isolated from the cryopreserved deciduous pulp tissues for over 2 years (25–30 months) (SHED-Cryo) owned similar stem cell properties including clonogenicity, self-renew, stem cell marker expression, multipotency, in vivo tissue regenerative capacity and in vitro immunomodulatory function to SHED isolated from the fresh tissues (SHED-Fresh). To examine the therapeutic efficacy of SHED-Cryo on immune diseases, SHED-Cryo were intravenously transplanted into systemic lupus erythematosus (SLE) model MRL/lpr mice. Systemic SHED-Cryo-transplantation improved SLE-like disorders including short lifespan, elevated autoantibody levels and nephritis-like renal dysfunction. SHED-Cryo amended increased interleukin 17-secreting helper T cells in MRL/lpr mice systemically and locally. SHED-Cryo-transplantation was also able to recover osteoporosis bone reduction in long bones of MRL/lpr mice. Furthermore, SHED-Cryo-mediated tissue engineering induced bone regeneration in critical calvarial bone-defect sites of immunocompromised mice. The therapeutic efficacy of SHED-Cryo transplantation on immune and skeletal disorders was similar to that of SHED-Fresh. These data suggest that cryopreservation of dental pulp tissues of deciduous teeth provide a suitable and desirable approach for stem cell-based immune therapy and tissue engineering in regenerative medicine. PMID:23251621

  7. Subcutaneous sarcoidosis associated with sarcoid tenosynovitis. (United States)

    Enzenauer, R J; Waterhouse, W J; West, S G


    Subcutaneous sarcoidosis and sarcoid tenosynovitis are unusual manifestations of systemic sarcoidosis. We report two Japanese women with disseminated sarcoidosis presenting with subcutaneous and tenosynovial involvement demonstrated by computed tomography and magnetic resonance imaging. Sarcoidosis must be considered in the differential diagnosis of unexplained subcutaneous nodulosis or tenosynovitis in patients with or without a previous diagnosis of sarcoidosis.

  8. Cryopreservation of human skeletal muscle impairs mitochondrial function

    DEFF Research Database (Denmark)

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


    Previous studies have investigated if cryopreservation is a viable approach for functional mitochondrial analysis. Different tissues have been studied, and conflicting results have been published. The aim of the present study was to investigate if mitochondria in human skeletal muscle maintain...... 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...... of oxidative phosphorylation was significantly (P cryopreserved human skeletal muscle samples. Cryopreservation impaired respiration with substrates linked to Complex I more than for Complex II (P

  9. Subcutaneous hemangiomas of the hand. (United States)

    Palmieri, T J


    Subcutaneous hemangiomas, the fourth most common tumor of the hand, consist primarily of proliferating blood vessels within the soft tissue. One hundred and sixty consecutive patients with the histologic diagnosis of subcutaneous hemangiomas were reviewed. Patients with diffuse hemangiomas, strawberry nevi, and arteriovenous fistulae were not included in the study. The median age was 32 years, the youngest being 2 years old and the oldest 68. Sixty-two percent of the patients were female. None had a history of trauma. The palm was the most common location. Progressive enlargement of the lesion and throbbing pain were the most common symptoms. As is characteristic of hemangiomas, they were readily compressible, poorly defined, bluish, subcutaneous masses that distended when the venous return was obstructed and contracted when the extremity was elevated. Roentgenographic evaluation rarely showed the typical calcification of phleboliths, but a soft tissue mass was almost always present. All of these hemangiomas were surgically excised, with the tributory vessels being identified and ligated as far distant from the tumor as possible in order to diminish the chances of recurrence.

  10. Subcutaneous implants of polypropylene filaments. (United States)

    Liebert, T C; Chartoff, R P; Cosgrove, S L; McCuskey, R S


    Extruded filaments of unmodified polypropylene (PP) with and without antioxidant were implanted subcutaneously in hamsters in order to determine their rate of degradation. Specimens were removed periodically during a 5 month test period and analyzed by infrared spectroscopy and dynamic mechanical testing. The analyses show that degradation beigns to occur after only a few days. Although the reaction sequence is not known, several factors suggest that the in vivo degradation process is similar to autoxidation which occurs in air or oxygen. The infrared data indicate that the hydroxyl content of the implants increases at a rate of 0.061 mg/g polypropylene per day during the initiation phase of the reaction. An induction time of 108 days was extablished. Carbonyl bonds appear after an implantation time of 50--90 days and increase therafter. Mechanical tests indicate a decrease in the dynamic loss tangent, tan delta, during the first month of implantation for unmodified polypropylene. No change in the infrared spectra or tan delta was observed, however, for implants containing an antioxidant. Thus, it is apparent that polypropylene filaments implanted subcutaneously in hamsters degrade by an oxidation process which is retarded effectively by using an antioxidant. While the findings reported are specific to subcutaneous polypropylene implants, they suggest that degradation of other systems may involve similar processes. This notion suggests directions for further research on increasing the in vivo stability of synthetic polymers. Long-term effects of polymer implantation upon tissue were not studied in this work.

  11. Study on cryopreservation of Porphyra yezoensis conchocelis (United States)

    Zhou, Wenjun; Li, Yun; Dai, Jixun


    Cryopreservation of Porphyra yezoensis conchocelis was conducted with cryoprotectants and a proposed pretreatment procedure and thawing methods explored. Six cryoprotectants combined by DMSO with ethylene glycol (EG), propylene glycol (PEG), sorbitol and sucrose were developed. The effect of prefreezing at -40°C or -20°C for different time durations was compared and the thawing methods were screened. It was shown that the cryoprotectant including 10% DMSO with 0.5 molL-1 sorbitol exhibited the optimal effect. The ideal pretreatment was that conchocelis segments were stayed for 20 min at -40°C before stored in liquid nitrogen, and 40°C water bath was proper for quick thawing. The highest recovery rate of cryopreserved P. yezoensis conchocelis reached 89.41%.

  12. 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.


    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

  13. Immunologic reaction and viability of cryopreserved homografts. (United States)

    Fischlein, T; Schütz, A; Haushofer, M; Frey, R; Uhlig, A; Detter, C; Reichart, B


    Homograft cell viability after cryopreservation was investigated and cytoimmunologic monitoring was performed during the early postoperative course to research possible immunologic reactions after allograft aortic valve replacement. After cryopreservation, morphologic observations were made, a nonradioactive cell proliferation assay was used, and prostaglandin I2 secretion of the remaining endothelial cells was determined. Cytoimmunologic monitoring was performed daily within the first 3 weeks postoperatively. An increase of the activation index greater than 1 was rated as an immunologic reaction. Maintained metabolic activity of graft endothelial cells after cryopreservation was confirmed by prostaglandin I2 release (9.24 +/- 3.48 ng/cm2 basic release and 20.1 +/- 5.76 ng/cm2 when stimulated with 25 mumol/L Na arachidonic acid). Cell proliferation was indicated after graft incubation with the nonradioactive viability kit (0.27 +/- 0.9 at 450 nm). Cytoimmunologic examinations (n = 861) after homograft implantation showed a more intense activation in patients with ABO-incompatible grafts (activation index 2.1 +/- 1.6, n = 16) than in those with ABO-compatible grafts (activation index 1.3 +/- 0.8, n = 17). In these groups, the duration of activation by cytoimmunologic monitoring was 2.8 +/- 1.5 days and 1.3 +/- 0.6 days, respectively (p < 0.041). No activation was observed in 8 patients after xenograft valve replacement (p < 0.01). Our data indicate that cryopreservation of homograft valves represents a cell- and tissue-protective preservation method. Postoperatively, all homograft valves caused immunologic reactions, which were reversible without immunosuppression treatment.

  14. Cryopreservation of South African indigenous goat semen ...

    African Journals Online (AJOL)

    Semen was thereafter cryopreserved in straws using liquid nitrogen. Data were analysed using Stata® V10 software. South African indigenous goats had total sperm cell motility rate of 83.1%, progressive sperm cell motility of 49.3% and non-progressive sperm cell motility of 33.9%. Moreover, acidic semen pH of 6.4 and ...

  15. Subcutaneous blood flow in psoriasis

    International Nuclear Information System (INIS)

    Klemp, P.


    The simultaneously recorded disappearance rates of 133 xe from subcutaneous adipose tissue in the crus were studied in 10 patients with psoriasis vulgaris using atraumatic labeling of the tissue in lesional skin (LS) areas and symmetrical, nonlesional skin (NLS) areas. Control experiments were performed bilaterally in 10 younger, healthy subjects. The subcutaneous washout rate constant was significantly higher in LS, 0.79 +/- 0.05 min-1 x 10(2) compared to the washout rate constant of NLS, 0.56 +/- 0.07 min-1. 10(2), or the washout rate constant in the normal subjects, 0.46 +/- 0.17 min-1 x 10(2). The mean washout rate constant in NLS was 25% higher than the mean washout rate constant in the normal subjects. The difference was, however, not statistically significant. Differences in the washout rate constants might be due to abnormal subcutaneous tissue-to-blood partition (lambda) in the LS--and therefore not reflecting the real differences in the subcutaneous blood flow (SBF). The lambda for 133 Xe was therefore measured--using a double isotope washout method ( 133 Xe and [ 131 I]antipyrine)--in symmetrical sites of the lateral crus in LS and NLS of 10 patients with psoriasis vulgaris and in 10 legs of normal subjects. In LS the lambda was 4.52 +/- 1.67 ml/g, which was not statistically different from that of NLS, 5.25 +/- 2.19 ml/g, nor from that of normal subcutaneous tissue, 4.98 +/- 1.04 ml/g. Calculations of the SBF using the obtained lambda values gave a significantly higher SBF in LS, 3.57 +/- 0.23 ml/100 g/min, compared to SBF in the NLS, 2.94 +/- 0.37 ml/100 g/min. There was no statistically significant difference between SBF in NLS and SBF in the normal subjects. The increased SBF in LS of psoriatics might be a secondary phenomenon to an increased heat loss in the lesional skin

  16. Cryopreservation of Arachis pintoi (leguminosae) somatic embryos. (United States)

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


    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.

  17. Cryopreservation of mutton snapper ( Lutjanus analis sperm

    Directory of Open Access Journals (Sweden)



    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.

  18. Prolonged Survival of Subcutaneous Allogeneic Islet Graft by Donor Chimerism without Immunosuppressive Treatment

    Directory of Open Access Journals (Sweden)

    Brend Ray-Sea Hsu


    Full Text Available The aim of this study was to investigate whether tolerance-induced protection of islets in the renal subcapsular space can also prevent subcutaneous allogeneic islets from being rejected. We used bone marrow stem cells from C57BL/6 (H2b mice to construct donor chimerism in conditioned diabetic BALB/c (H2d mice and investigated the effect of donor chimerism on engraftment and survival of subcutaneously transplanted allogeneic islets in streptozotocin-induced diabetic mice. We also studied the anti-inflammatory effect of mesenchymal stem cell on islet engraftment. Full but not low-grade or no donor chimerism was associated with successful engraftment of allogeneic islets and restoration of normoglycemia in the treated diabetic mice. The temporary hyperglycemia was 11 ± 1 versus 19 ± 5 days (p<0.05 for the mice with full donor chimerism with transplanted islets in the renal subcapsular space versus the subcutaneous space, respectively. Cotransplantation of mesenchymal stem cell did not enhance alloislet engraftment. Full multilineage donor chimerism was associated with a higher transient expansion of CD11b+ and Gr-1+ myeloid progenitor cells and effector memory CD4 and CD8 T cells. In conclusion, full donor chimerism protected both renal subcapsular and subcutaneous allogeneic islets in this rodent transplantation model.

  19. A case of subcutaneous zygomycosis

    Directory of Open Access Journals (Sweden)

    Usha Rani Anaparthy


    Full Text Available Zygomycosis is an acute or chronic infection caused by several fungal agents belonging to the phylum Zygomycota. These are saprophytic fungi and are found ubiquitously in the environment. These are emerging highly opportunistic pathogenic organisms. Basidiobolus ranarum (B. haptosporus, B. meristoporus is a fungus belonging to the order Entomophthorales under the family Zygomycota. Basidiobolomycosis is a predominantly subcutaneous infection involving the trunk and limbs in immunocompetent hosts. We hereby report a case of Basidiobolomycosis from the Department of Microbiology, Siddhartha Medical College, Vijayawada in a 6 month old child who presented to us with a painless swelling over her left knee following an insect bite.

  20. Permeability barriers to embryo cryopreservation of Pectinophora gossypiella (Lepidoptera: Gelechiidae) (United States)

    The aim of this study was to develop a method to cryopreserve the embryos of the pink bollworm moth, Pectinophora gossypiella (Saunders). Previously developed dipteran cryopreservation protocols were not directly adaptable to use with the embryos of this lepidopteran species. Physiochemical and ele...

  1. Evaluation of genetic stability in cryopreserved Solanum tuberosum ...

    African Journals Online (AJOL)

    In the present investigation, the genetic stability of potato (Solanum tuberosum L.) plantlets of the cultivars Agria and Marphona stored under cryopreservation and non-cryopreservation conditions was studied using amplified fragment length polymorphism (AFLP) technique. Also, flow cytometric studies were performed to

  2. Solea senegalensis sperm cryopreservation: New insights on sperm quality.

    Directory of Open Access Journals (Sweden)

    Marta F Riesco

    Full Text Available Cryopreservation of Senegalese sole sperm can represent an alternative to overcome some reproductive problems of this species. However, it is important to guarantee the safe use of cryopreserved sperm by selecting an appropriate protocol according to a high demand quality need to be ensured. It has been demonstrated that traditional assays such as motility and viability do not provide enough information to identify specific damage caused by cryopreservation process (freezing and thawing. Specific tests, including lipid peroxidation and DNA damage, should be performed. In the present study, motility and lipid peroxidation were performed as specific tests allowing us to discard cryopreservation conditions such as methanol as internal cryoprotectant and bovine serum albumin as external cryoprotectant. In addition, a caspase 3/7 detection by flow cytometry was performed to analyze apoptosis activity in the best selected conditions. Moreover, new highly sensitive tests based on transcript number detection have recently been described in fish sperm cryopreservation. For this reason, a transcript level detection assay was performed on certain oxidative and chaperone genes related to fertilization ability and embryo development (hsp70, hsp90BB, hsp90AA, gpx to select the best cryopreservation conditions. DMSO+ egg yolk proved to be the best cryoprotectant combination in terms of transcript level. This study describes an optimized cryopreservation protocol for Solea senegalensis sperm demonstrating for the first time that transcript degradation is the most sensitive predictor of cell status in this species after cryopreservation.

  3. Cryopreservation and plant regeneration of anther callus in Hevea ...

    African Journals Online (AJOL)

    Callus induced from anther of Hevea brasiliensis was successfully cryopreserved in liquid nitrogen (LN) by vitrification method and subsequently regenerated into plants. The effects of different preculture time, loading and dehydration duration on callus viability after cryopreservation were evaluated. The effective ...

  4. Medium-term cryopreservation of rabies virus samples

    Directory of Open Access Journals (Sweden)

    Tereza D'avila de Freitas Aguiar


    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.

  5. Cryopreservation of Cell/Scaffold Tissue-Engineered Constructs (United States)

    Costa, Pedro F.; Dias, Ana F.; Reis, Rui L.


    The aim of this work was to study the effect of cryopreservation over the functionality of tissue-engineered constructs, analyzing the survival and viability of cells seeded, cultured, and cryopreserved onto 3D scaffolds. Further, it also evaluated the effect of cryopreservation over the properties of the scaffold material itself since these are critical for the engineering of most tissues and in particular, tissues such as bone. For this purpose, porous scaffolds, namely fiber meshes based on a starch and poly(caprolactone) blend were seeded with goat bone marrow stem cells (GBMSCs) and cryopreserved for 7 days. Discs of the same material seeded with GBMSCs were also used as controls. After this period, these samples were analyzed and compared to samples collected before the cryopreservation process. The obtained results demonstrate that it is possible to maintain cell viability and scaffolds properties upon cryopreservation of tissue-engineered constructs based on starch scaffolds and goat bone marrow mesenchymal cells using standard cryopreservation methods. In addition, the outcomes of this study suggest that the greater porosity and interconnectivity of scaffolds favor the retention of cellular content and cellular viability during cryopreservation processes, when compared with nonporous discs. These findings indicate that it might be possible to prepare off-the-shelf engineered tissue substitutes and preserve them to be immediately available upon request for patients' needs. PMID:22676448

  6. Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia

    DEFF Research Database (Denmark)

    Rosendahl, Mikkel; Andersen, Morten Tolstrup; Ralfkiær, Elisabeth


    To systematically search for leukemic cells in cryopreserved ovarian cortex from Danish female patients with leukemia, who had ovarian cortex cryopreserved for fertility preservation before potentially sterilizing treatment.......To systematically search for leukemic cells in cryopreserved ovarian cortex from Danish female patients with leukemia, who had ovarian cortex cryopreserved for fertility preservation before potentially sterilizing treatment....

  7. RAPD and phytochemical analysis of Thymus moroderi plantlets after cryopreservation. (United States)

    Marco-Medina, Ana; Casas, José Luis


    Cryopreservation is at present the most reliable strategy to preserve plant germplasm. When aromatic plants are the object of conservation it is necessary to assess not only the genetic but also the phytochemical stability to ensure that plant material maintains its qualities after storage. In this work we present molecular and phytochemical stability data related to a previously described vitrification-based cryopreservation protocol for Thymus moroderi Pau ex Martínez. RAPD markers have been used to assess the genetic stability of T. moroderi explants and revealed 0.34 percent of variation in the cryopreserved material studied. Phytochemical data collected from GC-MS analysis of dichloromethane extracts from cryopreserved plantlets rendered a profile in which 1,8-cineole (14.5 percent), camphor (5.9 percent) and borneol (5.2 percent) were the major components. Both data confirmed the suitability of the cryopreservation protocol applied.

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

    Directory of Open Access Journals (Sweden)

    Ramírez Torrez, A.


    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. Cryopreservation of embryos and oocytes in human assisted reproduction. (United States)

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


    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.

  10. Cryopreservation of Embryos and Oocytes in Human Assisted Reproduction

    Directory of Open Access Journals (Sweden)

    János Konc


    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.

  11. Phenotypic and molecular characterization of plants regenerated from non-cryopreserved and cryopreserved wild Solanum lycopersicum mill. Seeds. (United States)

    Zevallos, B; Cejas, I; Engelmann, F; Carputo, D; Aversano, R; Scarano, M T; Yanes, E; Martinez-Montero, M; Lorenzo, J C


    Before cryopreservation is routinely used, its effect on the trueness-to-type of the regenerated plant material needs to be evaluated. In this work, we studied the effect of seed cryopreservation on the phenotypic and molecular characteristics of wild Solanum lycopersicum Mill. plants. Thirty-five morphological traits of plants regenerated from cryopreserved seeds were compared to those measured on plants regenerated from non-cryopreserved seeds. No statistically significant differences were observed between cryopreserved and non-cryopreserved samples, either in the first or in the second generation post-liquid nitrogen exposure. However, at the molecular level, the genetic analyses performed on the second generation plants germinated from control and cryopreserved seeds using 14 nuclear Simple Sequences Repeats (SSR) markers uncovered some changes in microsatellite length between control and cryopreserved samples. These results confirm at the botanical phenotype level the effectiveness of seed cryostorage for conservation and regeneration of true-to-type S. lycopersicum plants. Further experiments are required to clarify potential phenotypic effects of the changes observed in the DNA.

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

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


    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.

  13. Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation. (United States)

    Yango, Pamela; Altman, Eran; Smith, James F; Klatsky, Peter C; Tran, Nam D


    To determine whether optimal human spermatogonial stem cell (SSC) cryopreservation is best achieved with testicular tissue or single cell suspension cryopreservation. This study compares the effectiveness between these two approaches by using testicular SSEA-4+ cells, a known population containing SSCs. In vitro human testicular tissues. Academic research unit. Adult testicular tissues (n=4) collected from subjects with normal spermatogenesis and normal fetal testicular tissues (n=3). Testicular tissue versus single cell suspension cryopreservation. Cell viability, total cell recovery per milligram of tissue, as well as viable and SSEA-4+ cell recovery. Single cell suspension cryopreservation yielded higher recovery of SSEA-4+ cells enriched in adult SSCs, whereas fetal SSEA-4+ cell recovery was similar between testicular tissue and single cell suspension cryopreservation. Adult and fetal human SSEA-4+ populations exhibited differential sensitivity to cryopreservation based on whether they were cryopreserved in situ as testicular tissues or as single cells. Thus, optimal preservation of human SSCs depends on the patient's age, type of samples cryopreserved, and end points of therapeutic applications. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  14. Cryopreservation of coffee zygotic embryos: dehydration and osmotic rehydration

    Directory of Open Access Journals (Sweden)

    Maísa de Siqueira Pinto

    Full Text Available ABSTRACT Conservation of plant genetic resources is important to prevent genetic erosion. Seed banks are the most common method of ex situ conservation; however, coffee seeds can not be stored by conventional methods. Cryopreservation is a viable alternative for long-term conservation of species that produce intermediate or recalcitrant seeds, as coffee. The aim of this work was to cryopreserve Coffea arabica L. cv Catuaí Vermelho IAC 144 zygotic embryos, and analyse the effects of dehydration prior cryopreservation and osmotic rehydration after thawing, in embryos germination and seedlings formation after cryopreservation. Prior to cryopreservation, different dehydration times (0, 15, 30, 60 and 120 min were tested. Dehydrated embryos were cryopreserved in liquid nitrogen for 1 hour, and after thawing were rehydrated by osmotic solutions. Dehydrated and non-cryopreserved embryos were also analysed. The test with 2,3,5 triphenyl tetrazolium chloride (TTC was used to evaluate the embryos viability. Non-dehydrated embryos did not survive after freezing. Embryos that were dehydrated until 20% of the moisture content did not germinate when osmotic rehydration was not performed. In contrast, cryopreserved embryos with the same moisture content presented 98% germination when they were rehydrated slowly in osmotic solution. According to tetrazolium tests, embryos presented maximum viability (75% after dehydration for 60 minutes (23% moisture content. Therefore, coffee zygotic embryos (Coffea arabica L. cv. Catuaí Vermelho can be successfully cryopreserved using physical dehydration in silica gel for 60 minutes (23% moisture content, followed by osmotic rehydration after thawing. This method allowed a germination of 98% of cryopreserved zygotic embryos.

  15. Impact of age and diagnosis on viability during centrifugation and cryopreservation of peripheral blood stem cell products. (United States)

    Civriz Bozdag, S; Bay, M; Ayyıldız, E; Topcuoglu, P; Ilhan, O


    The viability of the hematopoietic stem cells infused to the patient is important for transplant outcome. We evaluated 31 peripheral blood stem cell product collected from 15 patients. We aimed to check the viabilities of the cells from patients with different age and diagnosis, in different stages of the cryopreservation procedure. We showed a markedly decreased viability rate after centrifugation and addition of DMSO. Percentages of viabilities were similar between young and old patients in each step. Type of hematological malignancy did not make a significant influence on the viability. High speed centrifugation has a negative impact on the viability. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Sperm cleanup and centrifugation processing for cryopreservation. (United States)

    Sieme, Harald; Oldenhof, Harriëtte


    Fertility rates with artificial insemination are highest with good-quality sperm samples. Therefore, nonviable sperm, cellular debris, and seminal plasma are preferably removed from semen samples prior to use or for preservation. Such compounds are sources where reactive oxygen species are generated during storage or upon cryopreservation, impairing sperm function. In this chapter we describe methods to remove seminal plasma and cellular debris from sperm samples, and for selecting morphologically normal motile sperm. The methods that are described here include: ordinary centrifugation, sperm swim-up, glass wool and Sephadex filtration/adherence, and single-layer as well as discontinuous two-layer iodixanol density gradient centrifugation.

  17. The choice of cryopreservation method affects immune compatibility of human cardiovascular matrices. (United States)

    Schneider, Maria; Stamm, Christof; Brockbank, Kelvin G M; Stock, Ulrich A; Seifert, Martina


    Conventional frozen cryopreservation (CFC) is currently the gold standard for cardiovascular allograft preservation. However, inflammation and structural deterioration limit transplant durability. Ice-free cryopreservation (IFC) already demonstrated matrix structure preservation combined with attenuated immune responses. In this study, we aim to explore the mechanisms of this diminished immunogenicity in vitro. First, we characterized factors released by human aortic tissue after CFC and IFC. Secondly, we analyzed co-cultures with human peripheral blood mononuclear cells, purified monocytes, T cells and monocyte-derived macrophages to examine functional immune effects triggered by the tissue or released cues. IFC tissue exhibited significantly lower metabolic activity and release of pro-inflammatory cytokines than CFC tissue, but surprisingly, more active transforming growth factor β. Due to reduced cytokine release by IFC tissue, less monocyte and T cell migration was detected in a chemotaxis system. Moreover, only cues from CFC tissue but not from IFC tissue amplified αCD3 triggered T cell proliferation. In a specifically designed macrophage-tissue assay, we could show that macrophages did not upregulate M1 polarization markers (CD80 or HLA-DR) on either tissue type. In conclusion, IFC selectively modulates tissue characteristics and thereby attenuates immune cell attraction and activation. Therefore, IFC treatment creates improved opportunities for cardiovascular graft preservation.

  18. Effects of 3, 4-Dihydroxyflavone on Cryopreserved Testicular Tissue of Neonatal Mouse

    Directory of Open Access Journals (Sweden)

    Afshin Pirnia


    Full Text Available Background and Aim: Infertility is a side effect of cancer treatment because of chemotherapy or radiotherapy. Cryopreservation of testicular tissue or spermatogonial stem cells before cancer treatment and their transplantation may preserve the natural fertility. Cryopreservation is a damaging process due to free radicals and toxic effect of frozen solution. The purpose of this paper is to study the antioxidant and antiapoptotic effects of 3',4'-dihydroxyflavone on the structure of frozen-thawed testicular tissue of neonatal mouse. Materials and Methods: Testes of 6-day-old NMRI mice (N = 20 were isolated. Testicles were randomly divided into four groups: two groups as control groups for quick and slow freezing-melting process, and two others as treatment groups that underwent quick and slow freezing-melting with addition of 3',4'-dihydroxyflavone (10µM in frozen solution. For the assessment of structural changes and apoptosis in the frozen-thawed testes, the hematoxylin-eosin staining and tunnel pod kits were used respectively. In order to analyze the data, we used Kruskal-Wallis Test and Mann-Whitney Test. The statistical analysis was entirely done by SPSS software. Results: Histopathological changes and apoptosis rate were significantly decreased after thawing in both treatment groups in comparison with control groups. Conclusion: Adding 3',4'-dihydroxyflavone (10µM to freezing-melting environment and the use of quick freezing-melting method can reduce the histopathological and apoptotic changes.

  19. Hair transplant (United States)

    ... this procedure: Scarring Unnatural-looking tufts of new hair growth It is possible that the transplanted hair will ... Most hair transplants result in excellent hair growth within several ... may be needed to create best results. The replaced hairs are ...

  20. Transplant rejection (United States)

    ... Shortness of breath and less ability to exercise (heart transplant) Yellow skin color and easy bleeding (liver transplant) ... accountability. A.D.A.M. is among the first to achieve this important distinction for online health ...

  1. Intestine Transplant (United States)

    ... to know FAQ Living donation What is living donation? Organs Types Being a living donor First steps Being ... Nursing Care of the Renal Transplant Recipient." UNOS Donation and Transplantation Nursing Curriculum . 1996 This Web site is intended ...

  2. Islet Transplantation (United States)

    ... be successful. However, transplanting islet cells has several advantages over transplanting a pancreas. First, unlike the pancreas ... Email: Sign Up Thank you for signing up ' + ' '); $('.survey-form').show(); }, success: function (data) { $('#survey-errors').remove(); $('. ...

  3. Improved tissue cryopreservation using inductive heating of magnetic nanoparticles. (United States)

    Manuchehrabadi, Navid; Gao, Zhe; Zhang, Jinjin; Ring, Hattie L; Shao, Qi; Liu, Feng; McDermott, Michael; Fok, Alex; Rabin, Yoed; Brockbank, Kelvin G M; Garwood, Michael; Haynes, Christy L; Bischof, John C


    Vitrification, a kinetic process of liquid solidification into glass, poses many potential benefits for tissue cryopreservation including indefinite storage, banking, and facilitation of tissue matching for transplantation. To date, however, successful rewarming of tissues vitrified in VS55, a cryoprotectant solution, can only be achieved by convective warming of small volumes on the order of 1 ml. Successful rewarming requires both uniform and fast rates to reduce thermal mechanical stress and cracks, and to prevent rewarming phase crystallization. We present a scalable nanowarming technology for 1- to 80-ml samples using radiofrequency-excited mesoporous silica-coated iron oxide nanoparticles in VS55. Advanced imaging including sweep imaging with Fourier transform and microcomputed tomography was used to verify loading and unloading of VS55 and nanoparticles and successful vitrification of porcine arteries. Nanowarming was then used to demonstrate uniform and rapid rewarming at >130°C/min in both physical (1 to 80 ml) and biological systems including human dermal fibroblast cells, porcine arteries and porcine aortic heart valve leaflet tissues (1 to 50 ml). Nanowarming yielded viability that matched control and/or exceeded gold standard convective warming in 1- to 50-ml systems, and improved viability compared to slow-warmed (crystallized) samples. Last, biomechanical testing displayed no significant biomechanical property changes in blood vessel length or elastic modulus after nanowarming compared to untreated fresh control porcine arteries. In aggregate, these results demonstrate new physical and biological evidence that nanowarming can improve the outcome of vitrified cryogenic storage of tissues in larger sample volumes. Copyright © 2017, American Association for the Advancement of Science.

  4. Addition of ascorbate during cryopreservation stimulates subsequent embryo development. (United States)

    Lane, Michelle; Maybach, Jeffery M; Gardner, David K


    Embryo development following cryopreservation is reduced compared with fresh embryos. One of the traumas that cryopreservation imparts on embryos is an increase in oxidative stress. Therefore, this study investigated the effects of the addition of the antioxidant ascorbate to the cryopreservation solutions on subsequent embryo development. Mouse embryos at the 2-cell and blastocyst stages were either slow-frozen or vitrified in solutions containing either no ascorbate or 0.1 or 0.5 mmol/l ascorbate. The effects on the levels of hydrogen peroxide and subsequent embryo development and physiology were assessed. Addition of ascorbate to the cryopreservation solutions reduced the levels of hydrogen peroxide in embryos. Furthermore, addition of 0.1 mmol/l ascorbate significantly enhanced inner cell mass development in blastocysts. Embryos cryopreserved with ascorbate had significantly lower levels of lactate dehydrogenase leakage, and increased rates of metabolism compared with those cryopreserved in the absence of ascorbate. The benefits of ascorbate were significantly greater in embryos that were slow-frozen compared with those that were vitrified. These data indicate that the addition of 0.1 mmol/l ascorbate to the cryopreservation solutions for the mammalian embryo would be of significant value.

  5. Cryopreservation and Revival of Human Mesenchymal Stromal Cells. (United States)

    Haack-Sørensen, Mandana; Ekblond, Annette; Kastrup, Jens


    Cell-based therapy is a promising and innovative new treatment for different degenerative and autoimmune diseases, and mesenchymal stromal cells (MSCs) from the bone marrow have demonstrated great therapeutic potential due to their immunosuppressive and regenerative capacities.The establishment of methods for large-scale expansion of clinical-grade MSCs in vitro has paved the way for their therapeutic use in clinical trials. However, the clinical application of MSCs also requires cryopreservation and banking of the cell products. To preserve autologous or allogeneic MSCs for future clinical applications, a reliable and effective cryopreservation method is required.Developing a successful cryopreservation protocol for clinical stem cell products, cryopreservation media, cryoprotectant agents (CPAs), the freezing container, the freezing temperature, and the cooling and warming rate are all aspects which should be considered.A major challenge is the selection of a suitable cryoprotectant which is able to penetrate the cells and yet has low toxicity.This chapter focuses on recent technological developments relevant for the cryopreservation of MSCs using the most commonly used cryopreservation medium containing DMSO and animal serum or human-derived products for research use and the animal protein-free cryopreservation media CryoStor (BioLife Solutions) for clinical use.

  6. Cryopreservation of Byrsonima intermedia embryos followed by room temperature thawing

    Directory of Open Access Journals (Sweden)

    Luciano Coutinho Silva


    Full Text Available Byrsonima intermedia is a shrub from the Brazilian Cerrado with medicinal properties. The storage of biological material at ultra-low temperatures (-196°C is termed cryopreservation and represents a promising technique for preserving plant diversity. Thawing is a crucial step that follows cryopreservation. The aim of this work was to cryopreserve B. intermedia zygotic embryos and subsequently thaw them at room temperature in a solution rich in sucrose. The embryos were decontaminated and desiccated in a laminar airflow hood for 0-4 hours prior to plunging into liquid nitrogen. The embryo moisture content (% MC during dehydration was assessed. Cryopreserved embryos were thawed in a solution rich in sucrose at room temperature, inoculated in a germination medium and maintained in a growth chamber. After 30 days, the embryo germination was evaluated. No significant differences were observed between the different embryo dehydration times, where they were dehydrated for at least one hour. Embryos with a MC between 34.3 and 20.3% were germinated after cryopreservation. In the absence of dehydration, all embryos died following cryopreservation. We conclude that B. intermedia zygotic embryos can be successfully cryopreserved and thawed at room temperature after at least one hour of dehydration in a laminar airflow bench.

  7. Successful Oocyte Cryopreservation in Reproductive-Aged Cancer Survivors. (United States)

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


    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.

  8. Recent Progress in Cryopreservation of Bovine Oocytes

    Directory of Open Access Journals (Sweden)

    In-Sul Hwang


    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.

  9. Cryopreservation of Human Pluripotent Stem Cells in Defined Medium (United States)

    Liu, Weiwei; Chen, Guokai


    This protocol describes a cryopreservation procedure using an enzyme-free dissociation method to harvest cells and preserve cells in albumin-free chemically defined E8 medium for human pluripotent stem cells (hPSCs). The dissociation by EDTA/PBS produces small cell aggregates that allow high survival efficiency in passaging and cryopreservation. The preservation in E8 medium eliminates serum or other animal products, and is suitable for the increasing demand for high quality hPSCs in translational research. In combination with the special feature of EDTA/PBS dissociation, this protocol allows efficient cryopreservation in more time-saving manner. PMID:25366897

  10. Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization. (United States)

    Nejlund, Sarah; Smith, Julie; Kraan, Jaco; Stender, Henrik; Van, Mai N; Langkjer, Sven T; Nielsen, Mikkel T; Sölétormos, György; Hillig, Thore


    A blood sample containing circulating tumor cells (CTCs) may serve as a surrogate for metastasis in invasive cancer. Cryopreservation will provide new opportunities in management of clinical samples in the laboratory and allow collection of samples over time for future analysis of existing and upcoming cancer biomarkers. Blood samples from healthy volunteers were spiked with high (∼500) and low (∼50) number of tumor cells from culture. The samples were stored at -80C with cryopreservative dimethyl sulfoxide mixed with Roswell Park Memorial Institute 1640 medium. Flow cytometry tested if cryopreservation affected specific biomarkers regularly used to detect CTCs, i.e. cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM) and white blood cell specific lymphocyte common antigen (CD45). After various time intervals (up to 6 months), samples were thawed and tumor cell recovery (enumeration) was examined. Clinical samples may differ from cell line studies, so the cryopreservation protocol was tested on 17 patients with invasive breast cancer and tumor cell recovery was examined. Two blood samples were drawn from each patient. Biomarkers, CK, CD45, and EpCAM, were not affected by the freezing and thawing procedures. Cryopreserved samples (n = 2) spiked with a high number of tumor cells (∼500) had a ∼90% recovery compared with the spiked fresh samples. In samples spiked with lower numbers of tumor cells (median = 43 in n = 5 samples), the recovery was 63% after cryopreservation (median 27 tumor cells), p = 0.03. With an even lower number of spiked tumor cells (median = 3 in n = 8 samples), the recovery rate of tumor cells after cryopreservation did not seem to be affected (median = 8), p = 0.09. Time of cryopreservation did not affect recovery. When testing the effect of cryopreservation on enumeration in clinical samples, no difference was observed in the number of CTCs between the fresh and the cryopreserved samples based

  11. Use of cryopreserved peripheral mononuclear blood cells in biomonitoring

    DEFF Research Database (Denmark)

    Risom, Lotte; Knudsen, Lisbeth E.


    This study was performed to investigate the effect of storing blood samples by freezing on selected biomarkers and possible implications for biomonitoring. Comparative measurements were performed in order to investigate the use of cryopreserved vs. freshly separated peripheral mononuclear blood....../T-lymphocytes and monocytes was measured in phytohemaglutinin (PHA)-stimulated cultures at different time intervals. The results showed a higher DNA repair activity in cryopreserved samples compared with fresh samples. We also found differences in mutant frequencies with higher values in fresh samples. A significant...... correlation of frequencies was seen when comparing fresh with cryopreserved samples. Furthermore we recommend fresh human plasma used in UDS incubation media....

  12. Cryopreservation of human skeletal muscle impairs mitochondrial function

    DEFF Research Database (Denmark)

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


    Previous studies have investigated if cryopreservation is a viable approach for functional mitochondrial analysis. Different tissues have been studied, and conflicting results have been published. The aim of the present study was to investigate if mitochondria in human skeletal muscle maintain...... 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...... of oxidative phosphorylation was significantly (P skeletal muscle samples. Cryopreservation impaired respiration with substrates linked to Complex I more than for Complex II (P

  13. Cryopreservation of ovarian tissue for fertility preservation in a large cohort of young girls

    DEFF Research Database (Denmark)

    Jensen, A K; Rechnitzer, C; Macklon, K T


    risk of POI should be offered ovarian tissue cryopreservation (OTC). This includes girls who are planned to receive either high doses of alkylating agents, conditioning regimen before stem cell transplantation (SCT), total body irradiation (TBI) or high radiation doses to the craniospinal, abdominal...... tumours and malignant haematological diseases. Among the 32 prepubertal girls, 12 received high dose chemotherapy and either TBI prior to SCT or irradiation to the pelvis, abdomen or the spinal axis, 13 received high dose alkylating agents but no irradiation prior to SCT, six received alkylating agents...... received high dose alkylating agents and five had received conventional chemotherapy. LIMITATIONS REASONS FOR CAUTION: All information was retrieved retrospectively from patient records, and thus some information was not available. WIDER IMPLICATIONS OF THE FINDINGS: OTC should be recommended to all young...

  14. Assessment of the permeability properties of cryopreservation outer bags used in NHSBT. (United States)

    Hogg, P; Rooney, P; Lomas, R; Kearney, J N


    This study was carried out to investigate leakage/transport across the bag material of six outer cryopreservation bags in common use within NHS Blood and Transplant. In order to do this two different leak testing procedures; coloured dye and hydrogen tracer gas, were used. The data obtained show that a coloured dye cannot permeate through the materials both at room temperature and following storage at liquid nitrogen temperature (- 196 °C). In addition, when filled with the smallest elemental molecule, hydrogen, in the form of a tracer gas, all of the bags only allowed trace amounts of hydrogen to escape, either through the seal or the bag material. The data indicated that each of the bag materials tested would be capable of preventing bacterial or viral cross-contamination as long as the material remained intact.

  15. Facilitated subcutaneous immunoglobulin administration (fSCIg)

    DEFF Research Database (Denmark)

    Blau, Igor-Wolfgang; Conlon, Niall; Petermann, Robert


    and diverse medical needs that treatments for SID management should strive to meet. In this special report, we study the opportunities provided by facilitated subcutaneous immunoglobulin administration (fSCIg) to treat patients for whom the conventional routes (intravenous and subcutaneous) are sub...

  16. "Extended subcutaneous route" technique: a quick subcutaneous tunnelling technique for PICC insertion. (United States)

    Elli, Stefano; Abbruzzese, Chiara; Cannizzo, Luigi; Vimercati, Simona; Vanini, Stefania; Lucchini, Alberto


    To describe a quick tunnelling technique for peripherally inserted central catheter (PICC) insertion called the "extended subcutaneous route" technique. The "extended subcutaneous route" technique is described step by step. In 18 consecutive PICCs, inserted with extended route technique in ASST Monza, no complications during insertion were registered. In 969 catheter days observed, we identified only one accidental dislodgement. No other mid-term complications were observed. Extended subcutaneous route technique allows the creation of a subcutaneous tunnel <5 cm, without skin incision and additional manipulation. Extended subcutaneous route technique may be feasible and useful, particularly for patients with high risk of bleeding or infection.

  17. Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos by vitrification. (United States)

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


    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.

  18. Cryopreservation of Mammalian Oocyte for Conservation of Animal Genetics

    Directory of Open Access Journals (Sweden)

    Jennifer R. Prentice


    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.

  19. Chapter 1 Historical Background on Gamete and Embryo Cryopreservation. (United States)

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


    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.

  20. Cryopreservation Strategies for Farm Animal Genetic Resources in Europe

    NARCIS (Netherlands)

    Hiemstra, S.J.


    European countries have developed national strategies and action plans implementing the Global Plan of Action for animal genetic resources. National action plans include development and implementation of cryopreservation strategies for animal genetic resources. Although some cross-border

  1. Fertility preservation and refreezing of transplanted ovarian tissue-a potential new way of managing patients with low risk of malignant cell recurrence

    DEFF Research Database (Denmark)

    Kristensen, Stine Gry; Giorgione, Veronica; Humaidan, Peter


    OBJECTIVE: To report the first successful refreezing of ovarian tissue recovered more than 3 years after transplantation in a woman previously treated for early-stage ovarian cancer. DESIGN: Evaluation of cryopreserved and grafted ovarian tissue. SETTING: University hospital. PATIENT(S): A 23-year......-old woman diagnosed with stage 1C ovarian mucinous cystadenocarcinoma. INTERVENTION(S): The patient underwent ovarian tissue cryopreservation for fertility preservation and subsequent heterotopic transplantation for fertility restoration 9 years after freezing. After a successful IVF twin pregnancy, grafted...

  2. Ultrastructural changes associated with cryopreservation of banana ( Musa spp.) highly proliferating meristems. (United States)

    Helliot, B; Swennen, R; Poumay, Y; Frison, E; Lepoivre, P; Panis, B


    Cryopreservation has been shown to improve the frequency of virus elimination - specifically cucumber mosaic virus and banana streak virus - from banana ( Musa spp.) plants. To understand the mode of action of cryopreservation for the eradication of viral particles, we examined the ultrastructure of meristem tips at each step of the cryopreservation process. Excised meristematic clumps produced from infected banana plants belonging to cv. Williams (AAA, Cavendish subgroup) were cryopreserved through vitrification using the PVS-2 solution. We demonstrated that the cryopreservation method used only allowed survival of small areas of cells in the meristematic dome and at the base of the primordia. Cellular and subcellular changes occurring during the cryopreservation process are discussed.

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

    Directory of Open Access Journals (Sweden)

    Morse Michael A


    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.

  4. Application of antioxidants and centrifugation for cryopreservation of boar spermatozoa. (United States)

    Zhang, Wei; Yi, Kangle; Chen, Chao; Hou, Xiaofeng; Zhou, Xu


    Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred due to the high susceptibility of boar spermatozoa to damage during cryopreservation and the complicated process required for deep freezing. In recent years, the application of antioxidants during the cryopreservation of boar semen has been the subject of considerable research aimed at improving the quality of post-thaw semen. Centrifugation is necessary before using cryopreservation protocols for freezing boar spermatozoa. Studies of the effect of different centrifugation regimens on boar sperm recovery, yield and cryosurvival have made significant contributions. Therefore this review elucidates results of recent applications of various antioxidants and centrifugation regimens used in efforts to improve cryopreservation of boar spermatozoa. This review is intended to enhance understanding of the roles of these antioxidants and centrifugation regimens with respect to mechanisms that increase resistance to cryodamage of boar spermatozoa. In addition, the discussion addresses the need for developing an objective evaluation of effectiveness and estimating the prospect of application of new techniques for the cryopreservation of boar semen and its use in artificial insemination. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Oocyte cryopreservation for donor egg banking. (United States)

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


    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

  6. Bentall Procedure Using Cryopreserved Valved Aortic Homografts (United States)

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


    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

  7. Methods for cryopreservation of guinea fowl sperm.

    Directory of Open Access Journals (Sweden)

    Éva Váradi

    Full Text Available Conservation of indigenous poultry species is an important part of the new Hungarian agricultural strategy. Semen cryopreservation is the most practical method for the long term storage of poultry genetic material. The objective was to compare four protocols for cryopreservation of guinea fowl sperm (slow and fast programmable, freezing in nitrogen vapor, and pellet and three cryoprotectants (10% ethylene glycol, 6% dimethyl-formamide and 6% dimethyl-acetamide. The efficiency of the methods was examined by in vitro tests (subjective motility scoring, sperm concentration, morphological and live/dead sperm analysis with eosin-aniline staining. Thereafter, the two most promising methods were tested by artificial insemination of frozen-thawed semen (3 times a week for 3 weeks using 300 million spermatozoa/hen, followed by candling of incubated eggs, assessment of fertilization, embryonic death, and hatching rate. The survival rate of live, intact spermatozoa was greatest (p≤0.05 in pellet method and the slow programmable protocol (with 10% ethylene glycol (28.6 and 23.5%. The two best protocols (based on in vitro assessment of post-thaw semen quality were subsequently tested in vivo with artificial insemination. The pellet method yielded a 64% fertility rate compared to slow protocol with only 30% fertility. Regardless, both freezing protocols significantly increased embryonic deaths compared to the control group (16,7; 9,1 and 8,3%, respectively. During the 3-week in vivo trial, fertility increased and early embryonic death decreased over time. According to the results the guinea fowl sperm could tolerate the fast freezing in pellet better than the slower freezing rates and resulted acceptable fertility rate.

  8. Massive Preperitoneal Hematoma after a Subcutaneous Injection

    Directory of Open Access Journals (Sweden)

    Hideki Katagiri


    Full Text Available Preperitoneal hematomas are rare and can develop after surgery or trauma. A 74-year-old woman, receiving systemic anticoagulation, developed a massive preperitoneal hematoma after a subcutaneous injection of teriparatide using a 32-gauge, 4 mm needle. In this patient, there were two factors, the subcutaneous injection of teriparatide and systemic anticoagulation, associated with development of the hematoma. These two factors are especially significant, because they are widely used clinically. Although extremely rare, physicians must consider this potentially life-threatening complication after subcutaneous injections, especially in patients receiving anticoagulation.

  9. Initial Results of Peripheral-Blood Stem-Cell Mobilization, Collection, Cryopreservation, and Engraftment After Autologous Transplantation Confirm That the Capacity-Building Approach Offers Good Chances of Success in Critical Contexts: A Kurdish-Italian Cooperative Project at the Hiwa Cancer Hospital, Sulaymaniyah

    Directory of Open Access Journals (Sweden)

    Ignazio Majolino


    Full Text Available Introduction: At Hiwa Cancer Hospital (Sulaymaniyah, Iraqi Kurdistan after the center was started by a cooperative project in June 2016, autologous transplantation was developed. Patients and Methods: To develop the project, the capacity-building approach was adopted, with on-site training and coaching of personnel, educational meetings, lectures, on-the-job training, and the implementation of quality management planning. Results: Here, we report initial results of peripheral-blood stem-cell mobilization and collection of the first 27 patients (age 12 to 61 years; 19 males and 8 females; multiple myeloma, n = 10; plasma cell leukemia, n = 1; Hodgkin lymphoma, n = 12; non-Hodgkin lymphoma, n = 3; and acute myeloid leukemia, n = 1. Only three (11.5% of 26 patients experienced a failure of mobilization. A median of 6.1 × 106/kg CD34-positive cells per patient were collected (range, 2.4 to 20.8, with two apheretic runs. Twenty-four patients underwent autologous transplantation. All but one transplantation engrafted fully and steadily, with 0.5 and 1.0 × 109/L polymorphonucleates on day 10.5 (range, 8 to 12 and day 11 (range, 9 to 15, respectively, and with 20 and 50 × 109/L platelets on day 13 (range, 10 to 17 and day 17 (range, 2 to 44, respectively. More than 95% of patients are projected to survive 1 year after autograft. Conclusion: These data are the result of an Italian effort to establish in Iraqi Kurdistan a leading center for hemopoietic stem-cell transplantation. The capacity building approach was used, with on-site training and coaching as instruments for the development of provider ability and problem solving. With future limitations for immigration, this method will be helpful, especially in the field of high-technology medicine.

  10. Initial Results of Peripheral-Blood Stem-Cell Mobilization, Collection, Cryopreservation, and Engraftment After Autologous Transplantation Confirm That the Capacity-Building Approach Offers Good Chances of Success in Critical Contexts: A Kurdish-Italian Cooperative Project at the Hiwa Cancer Hospital, Sulaymaniyah. (United States)

    Majolino, Ignazio; Mohammed, Dereen; Hassan, Dastan; Ipsevich, Francesco; Abdullah, Chra; Mohammed, Rebar; Palmas, Angelo; Possenti, Marco; Noori, Diana; Ali, Dlir; Karem, Harem; Salih, Salah; Vacca, Michele; Del Fante, Claudia; Ostuni, Angelo; Frigato, Andrea; Massei, Maria Speranza; Manna, Annunziata; Vasta, Stefania; Gabriel, Marcela; Verna, Marta; Rovelli, Attilio; Conter, Valentino; Ali, Kosar; Othman, Dosti


    Introduction At Hiwa Cancer Hospital (Sulaymaniyah, Iraqi Kurdistan) after the center was started by a cooperative project in June 2016, autologous transplantation was developed. Patients and Methods To develop the project, the capacity-building approach was adopted, with on-site training and coaching of personnel, educational meetings, lectures, on-the-job training, and the implementation of quality management planning. Results Here, we report initial results of peripheral-blood stem-cell mobilization and collection of the first 27 patients (age 12 to 61 years; 19 males and 8 females; multiple myeloma, n = 10; plasma cell leukemia, n = 1; Hodgkin lymphoma, n = 12; non-Hodgkin lymphoma, n = 3; and acute myeloid leukemia, n = 1). Only three (11.5%) of 26 patients experienced a failure of mobilization. A median of 6.1 × 10 6 /kg CD34-positive cells per patient were collected (range, 2.4 to 20.8), with two apheretic runs. Twenty-four patients underwent autologous transplantation. All but one transplantation engrafted fully and steadily, with 0.5 and 1.0 × 10 9 /L polymorphonucleates on day 10.5 (range, 8 to 12) and day 11 (range, 9 to 15), respectively, and with 20 and 50 × 10 9 /L platelets on day 13 (range, 10 to 17) and day 17 (range, 2 to 44), respectively. More than 95% of patients are projected to survive 1 year after autograft. Conclusion These data are the result of an Italian effort to establish in Iraqi Kurdistan a leading center for hemopoietic stem-cell transplantation. The capacity building approach was used, with on-site training and coaching as instruments for the development of provider ability and problem solving. With future limitations for immigration, this method will be helpful, especially in the field of high-technology medicine.

  11. Cryopreservation of zebrafish (Danio rerio) primordial germ cells by vitrification of yolk-intact and yolk-depleted embryos using various cryoprotectant solutions. (United States)

    Higaki, Shogo; Kawakami, Yutaka; Eto, Yoshiki; Yamaha, Etsuro; Nagano, Masashi; Katagiri, Seiji; Takada, Tatsuyuki; Takahashi, Yoshiyuki


    The aim of this study was to examine the effects of partial removal of yolk and cryoprotectant mixtures on the viability of cryopreserved primordial germ cells (PGCs) and elucidated the differentiation ability of cryopreserved PGCs in zebrafish. First, dechorionated yolk-intact and yolk-depleted (partially yolk removed) embryos, PGCs of which were labeled with green fluorescence protein (GFP), were vitrified after serial exposures to pretreatment solution (PS) and vitrification solution (VS) that contained ethylene glycol (EG), dimethyl sulfoxide (Me 2 SO) or propylene glycol at 3 and 5 M, respectively. Although partial removal of yolk improved the viability of cryopreserved PGCs, numbers of PGCs with pseudopodial movement were limited (0 to 2.6 cells/embryo). Next, yolk-depleted embryos were cryopreserved using mixtures of two types of cryoprotectants. The maximum survival rate of PGCs (81%; 9.6 cells/embryo) was obtained from the yolk-depleted embryos vitrified using PS containing 2 M EG + 1 M Me 2 SO and VS containing 3 M EG + 2 M Me 2 SO and 56% (5.3 cells/embryo) of PGCs showed pseudopodial movement. Finally, PGCs recovered from yolk-depleted embryos (wild-type) that were vitrified under the optimum condition were transplanted individually into 236 sterilized recipient blastulae (recessive light-colored). Seven recipients matured and generated progeny with characteristics inherited from the PGC donor. In conclusion, the authors confirmed the beneficial effects of partial removal of yolk on the viability of cryopreserved PGCs and that the viability of the PGCs was improved by using PS and VS that contained two types of cryoprotectants, especially PS containing 2 M EG + 1 M Me 2 SO and VS containing 3 M EG + 2 M Me 2 SO, and that recovered PGCs retained ability to differentiate into functional gametes. Copyright © 2013. Published by Elsevier Inc.

  12. Subcutaneous growth of human acoustic schwannomas in athymic nude mice

    DEFF Research Database (Denmark)

    Charabi, S.; Rygaard, J.; Klinken, Leif


    Neuropathology, acoustic schwannomas, subcutaneous implantation, nude mouse, growth, pcna, ki-67......Neuropathology, acoustic schwannomas, subcutaneous implantation, nude mouse, growth, pcna, ki-67...

  13. Carinal transplantation.


    Ueda, H; Shirakusa, T


    BACKGROUND: Current techniques of management of carinal lesions are not always satisfactory. Carinal transplantation, if feasible, would be valuable in certain circumstances. METHODS AND RESULTS: Carinal transplantation experiments were performed in dogs. In early cross transplant experiments there were problems in controlling ventilation and in obtaining satisfactory anastomoses, and the animals failed to live for even a few days. In seven subsequent experiments the carinal graft was removed...

  14. Tracheal transplantation for carinal reconstruction in dogs. (United States)

    Kawahara, K; Inutsuka, K; Hiratsuka, M; Makihata, S; Okabayashi, K; Shiraishi, T; Shirakusa, T


    Experimental carinal allotransplantation has been performed with tracheocarinal Y-shaped allografts in dogs. In this study we tried canine carinal reconstruction with cylindrical allografts. Carinal reconstruction was performed with allotransplantation of cylindrical trachea in dogs, and graft healing was evaluated by bronchoscopic observation, mucosal blood flow measurement, and histologic examination. A section of the recipient carina containing five tracheal rings and two main stem bronchi was removed, and a donor trachea seven rings long was inserted between the recipient trachea and the left main stem bronchus; then side-to-end anastomosis was performed between the graft midportion and recipient right main stem bronchus (new carina). The grafts were wrapped with pedicled omentum. Fresh grafts were transplanted into one group of dogs (n=8 ), and grafts cryopreserved for 1 week were transplanted into another group (n=7). No anastomotic leakage occurred in any dog. Excellent healing of grafts and graft anastomoses was observed by fiberoptic bronchoscopy in six dogs (75%) in the fresh graft group and in four dogs (57%) in the cryopreserved graft group. The mucosal blood flow in the new carina decreased remarkably and, although it recovered, mucosal blood flow remained under the preoperative level on day 28 after the operation. Cylindrical tracheal allotransplantation is useful for carinal reconstruction, and the method of side-to-end anastomosis between the donor trachea and recipient bronchus is a feasible and accessible procedure in dogs.

  15. First Production of Larvae Using Cryopreserved Sperm: Effects of Preservation Temperature and Cryopreservation on European Eel Sperm Fertilization Capacity. (United States)

    Asturiano, J F; Sørensen, S R; Pérez, L; Lauesen, P; Tomkiewicz, J


    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 have been described, but until recently fertilization trials were not feasible. This study evaluated the effect of cold storage of diluted sperm prior to fertilizations and tested whether a previously defined protocol for European eel sperm cryopreservation can be successfully applied in fertilization trials to produce viable offspring. In our experiment, the sperm motility was evaluated after the extraction and the best samples were selected and pooled. Until stripping of eggs and fertilization, diluted sperm samples were maintained at either 4 or 20°C, or cryopreserved, following existing protocols. 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. © 2016 Blackwell Verlag GmbH.

  16. Generation of live piglets for the first time using sperm retrieved from immature testicular tissue cryopreserved and grafted into nude mice.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Kaneko

    Full Text Available Cryopreservation of immature testicular tissues is essential for increasing the possibilities of offspring generation by testicular xenografting for agricultural or medical purposes. However, successful production of offspring from the sperm involved has never been reported previously. In the present study, therefore, using intracytoplasmic sperm injection (ICSI, we examined whether xenogeneic sperm obtained from immature pig testicular tissue after cryopreservation would have the capacity to produce live piglets. Testicular fragments from 9- to 11-day-old piglets were vitrified after 10- or 20-min immersion in vitrification solution containing ethylene glycol (EG, polyvinyl pyrrolidone (PVP and trehalose as cryoprotectants, and then stored in liquid nitrogen for more than 140 days. Thirty nude mice were assigned to each immersion-time group. Testicular fragments were transplanted under the back skin of castrated mice immediately after warming and removal of the cryoprotectants. Blood and testicular grafts were then recovered from the recipient mice on days 60, 120, 180 and 230-350 (day 0 =  grafting. Histological assessment of the testicular grafts and analyses of inhibin and testosterone production revealed no significant differences between the two immersion-time groups, indicating equal growth activity of the cryopreserved tissues. A single sperm obtained from a mouse in each group on day 230-350 was injected into an in vitro-matured porcine oocyte, and then the ICSI oocytes were transferred to the oviducts of estrus-synchronized recipient gilts. One out of 4 gilts that had received oocytes fertilized using sperm from the 10-min immersion group delivered 2 live piglets, and one of another 4 gilts from the 20-min group delivered 4 live piglets. Thus, we have successfully generated porcine offspring utilizing sperm from immature testicular tissues after cryopreservation and transplantation into nude mice. The present model using pigs will

  17. Liver Transplant (United States)

    ... Nutrition Clinical Trials Primary Biliary Cholangitis Definition & Facts Symptoms & Causes Diagnosis Treatment Eating, Diet, & Nutrition Clinical Trials Wilson Disease Liver Transplant View or Print All Sections Definition & ...

  18. Injection site reactions after subcutaneous oligonucleotide therapy

    NARCIS (Netherlands)

    van Meer, L. (Leonie); M. Moerland (Matthijs); Gallagher, J. (Jolie); M.B.A. van Doorn (Martijn); E.P. Prens (Errol); A.F. Cohen; Rissmann, R. (Robert); J. Burggraaf (Jacobus)


    textabstractOligonucleotides (ONs) are short fragments of nucleic acids, currently being investigated as therapeutic agents. When administered subcutaneously (sc), ONs cause a specific local reaction originating around the injection site, such as erythema, itching, discomfort and pain, including

  19. Subcutaneous nodule: A case of Dirofilaria (United States)

    D’Souza, Reshmina; Jakribettu, Ramakrishna Pai; Sudharsana, Sunil H; Aithala, Sathyamoorthy P


    Human subcutaneous dirofilariasis is a rare helminthic infection. It is caused by filarial worms of the Genus Dirofilaria, which is the natural parasites of dogs, cats, foxes, and wild mammals. Zoonotic filariasis caused by Dirofilaria species is rarely seen in the region of Dakshina Kannada. Most of them are seen in patients who are along the border of the district which shares its boundary with the state of Kerala, where dirofilariasis is constantly reported to occur. Here, we report a case of subcutaneous dirofilariasis, presenting as a subcutaneous nodule on the left cheek of a 28-year-old male from Cochin (Ernakulam), Kerala, South India. Two live worms were recovered from the subcutaneous nodular swelling. PMID:23776842

  20. Removal of subcutaneous lipomas: Interest of liposuction. (United States)

    Gaucher, Sonia; Maladry, David; Silitra, Ana Maria; Documet, David; Philippe, Henri-Jean


    Subcutaneous lipomas are very common tumors. We report our experience of treatment by liposuction in carefully selected patients, initially referred for conventional surgical excision. © 2017 Wiley Periodicals, Inc.

  1. Interferon Beta-1a Subcutaneous Injection (United States)

    Interferon beta-1a subcutaneous injection is used to reduce episodes of symptoms and slow the development of disability in ... problems with vision, speech, and bladder control). Interferon beta-1a is in a class of medications called ...

  2. Primary Kaposi sarcoma of the subcutaneous tissue

    Directory of Open Access Journals (Sweden)

    Dezube Bruce J


    Full Text Available Abstract Background Involvement of the subcutis by Kaposi sarcoma (KS occurs primarily when cutaneous KS lesions evolve into deep penetrating nodular tumors. Primary KS of the subcutaneous tissue is an exceptional manifestation of this low-grade vascular neoplasm. Case presentation We present a unique case of acquired immune deficiency syndrome (AIDS-associated KS manifesting primarily in the subcutaneous tissue of the anterior thigh in a 43-year-old male, which occurred without overlying visible skin changes or concomitant KS disease elsewhere. Radiological imaging and tissue biopsy confirmed the diagnosis of KS. Conclusion This is the first documented case of primary subcutaneous KS occurring in the setting of AIDS. The differential diagnosis of an isolated subcutaneous lesion in an human immunodeficiency virus (HIV-infected individual is broad, and requires both imaging and a histopathological diagnosis to guide appropriate therapy.

  3. Recurrent, giant subcutaneous leiomyosarcoma of the thigh

    Directory of Open Access Journals (Sweden)

    Gao Chuanping, MD


    Full Text Available We present a case of recurrent, massive subcutaneous leiomyosarcoma involving the left thigh in a 29-year-old male from Madagascar. The patient had earlier undergone local resection of subcutaneous leiomyosarcoma a half year before. After surgical intervention, local recurrence developed at this site and was rapidly growing. The patient was surgically treated with a 2-cm-wide margin local excision in our hospital. The patient has remained recurrence free at 1-year follow-up.

  4. Cytological diagnosis of microfilariae in subcutaneous nodule

    Directory of Open Access Journals (Sweden)

    Narayanan K Panicker


    Full Text Available Microfilariaemia presenting with subcutaneous nodule is rare. We report a case of a 22-year-old female patient who presented with a subcutaneous nodule on right forearm. Fine-needle aspiration cytology from the nodule showed many sheathed motile microfilaria on wet mount preparation during day time. Wuchereria loa loa is known to be associated with soft tissue swellings as reported earlier. Nocturnal motility and cytomorphological features differentiate Wuchereria bancrofti from Wuchereria loa loa.

  5. Intrauterine insemination or intracervical insemination with cryopreserved donor sperm in the natural cycle: a cohort study

    NARCIS (Netherlands)

    Kop, P. A. L.; van Wely, M.; Mol, B. W.; de Melker, A. A.; Janssens, P. M. W.; Arends, B.; Curfs, M. H. J. M.; Kortman, M.; Nap, A.; Rijnders, E.; Roovers, J. P. W. R.; Ruis, H.; Simons, A. H. M.; Repping, S.; van der Veen, F.; Mochtar, M. H.


    Does intrauterine insemination in the natural cycle lead to better pregnancy rates than intracervical insemination (ICI) in the natural cycle in women undergoing artificial insemination with cryopreserved donor sperm. In a large cohort of women undergoing artificial insemination with cryopreserved

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

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

  8. Safety of ovarian tissue transplantation in patients with borderline ovarian tumors. (United States)

    Masciangelo, Rossella; Bosisio, Chiara; Donnez, Jacques; Amorim, Christiani A; Dolmans, Marie-Madeleine


    Is transplantation of cryopreserved ovarian tissue from patients with borderline ovarian tumors (BOTs) a safe procedure? BOT cells were found in frozen-thawed and xenografted ovarian tissue in 1 of 11 BOT patients. The risk of reintroducing malignant cells upon ovarian tissue transplantation has been subject of debate for many years. Reimplantation of cryopreserved ovarian tissue from leukemia patients is unsafe, while results from studies of cryopreserved ovarian tissue from other forms of cancer, such as Hodgkin's lymphoma, are reassuring. Prospective experimental study conducted in an academic research unit using ovarian tissue from 11 patients undergoing cryopreservation for BOTs. Histology, immunohistochemistry (IHC) for mucin 1 (MUC1) and cytokeratin 7 (CK7) and molecular analysis by reverse transcription quantitative polymerase chain reaction (RT-qPCR) for CK7 and MUC1 were performed on frozen-thawed ovarian tissue from 11 patients. Long-term (5 months) xenografting of ovarian tissue in immunodeficient mice was performed. The xenografts were analyzed by histology, IHC and RT-qPCR, furthermore IHC for CD10, a marker of endometriosis, was performed on a selected sample. Analysis by histology, IHC and RT-qPCR indicated 10 of the ovarian tissue samples were negative. Analysis of the xenograft samples indicated nine were negative for malignant cells but in two xenografts glandular lesions were detected by histology. In these two xenografts, CK7 and MUC1 markers were demonstrated by IHC and CK7 expression also by RT-qPCR. A BOT was confirmed in the xenograft in which the original ovarian tissue was positive, while in the other case IHC demonstrated expression of endometriosis marker CD10. Cryopreserved ovarian fragments cannot be tested before transplantation, therefore the preimplantation analysis cannot guarantee that all cryopreserved fragments will be free of BOT cells. BOT cells can be found in cryopreserved ovarian tissue from BOT patients, therefore

  9. Frontal subcutaneous blood flow, and epi- and subcutaneous temperatures during scalp cooling in normal man

    DEFF Research Database (Denmark)

    Bülow, J; Friberg, L; Gaardsting, O


    Cooling of the scalp has been found to prevent hair loss following cytostatic treatment, but in order to obtain the hair preserving effect the subcutaneous temperature has to be reduced below 22 degrees C. In order to establish the relationship between epicutaneous and subcutaneous temperatures...... epicutaneous and subcutaneous temperatures could be demonstrated with the regression equation: s = 0.9 c + 4.9 (r = 0.99). In eight of the 10 subjects the subcutaneous temperature could be reduced below 22 degrees C with the applied technique. It is concluded that the hair preserving effect of scalp cooling...

  10. Cryopreservation Causes Genetic and Epigenetic Changes in Zebrafish Genital Ridges.

    Directory of Open Access Journals (Sweden)

    Marta F Riesco

    Full Text Available Cryopreservation is an important tool routinely employed in Assisted Reproduction Technologies (ARTs and germplasm banking. For several years, the assessment of global DNA fragmentation seemed to be enough to ensure the integrity of genetic material. However, cryopreservation can produce molecular alterations in key genes and transcripts undetectable by traditional assays, such modifications could interfere with normal embryo development. We used zebrafish as a model to study the effect of cryopreservation on key transcripts and genes. We employed an optimized cryopreservation protocol for genital ridges (GRs containing primordial germ cells (PGCs considered one of the best cell sources for gene banking. Our results indicated that cryopreservation produced a decrease in most of the zebrafish studied transcripts (cxcr4b, pou5f1, vasa and sox2 and upregulation of heat shock proteins (hsp70, hsp90. The observed downregulation could not always be explained by promoter hypermethylation (only the vasa promoter underwent clear hypermethylation. To corroborate this, we used human spermatozoa (transcriptionally inactive cells obtaining a reduction in some transcripts (eIF2S1, and LHCGR. Our results also demonstrated that this effect was caused by freezing/thawing rather than exposure to cryoprotectants (CPAs. Finally, we employed real-time PCR (qPCR technology to quantify the number of lesions produced by cryopreservation in the studied zebrafish genes, observing very different vulnerability to damage among them. All these data suggest that molecular alterations caused by cryopreservation should be studied in detail in order to ensure the total safety of the technique.

  11. Evaluation of two human dental pulp stem cell cryopreservation methods. (United States)

    Munévar, Juan C; Gutiérrez, Nicole; Jiménez, Nury T; Lafaurie, Gloria I


    Dental pulp is a promising source of mesenchymal stem cells for use in cell therapy and regenerative medicine. Methods for storing stem cells with minimum compromise of cell viability, differentiation capacity and function should be developed for clinical and research applications. The aim of this study was to evaluate whether human dental pulp stem cells (hDPSCs) isolated and cryopreserved for 1, 7 and 30 days maintain viability and expression of specific stem cell markers. Human dental pulp stem cells were isolated from 23 healthy patients aged 18 to 31 years. Dental pulp was enzymatically dissociated, and CD105+ cells were separated using the Miltenyi™ system. The hDPSCs were cryopreserved using the Kamath and Papaccio methods. Post-cryopreservation viability was measured by flow cytometry (7AAD) and by the expression of the phenotype markers CD105+/ CD73+, CD34-/CD45-. The Papaccio method showed greater cell viability for cells that had been frozen for 30 days (59.5%) than the Kamath method (56.2%), while the Kamath method provided better results for 1 day (65.5%) and 7 days (56%). Post-cryopreservation expression of the markers CD105+/CD34- was greater after 1 and 7 days with the Kamath method and CD105+/CD45- were expressed after all 3 cryopreservation times. There was greater expression of CD73+ in the hDPSCs after 1 and 7 days with the Kamath method, and after 30 days with the Papaccio method. These results suggest that hDPSCs express mesenchymal stem cell markers after cryopreservation. However, cryopreservation time may affect marker expression, probably by altering the spatialconfiguration of cell membrane proteins or by compromising cells at a certain level of differentiation.


    Ahmad, Hajira F.; Sambanis, Athanassios


    Cell encapsulation in hydrogels is widely used in tissue engineering applications, including encapsulation of islets or other insulin-secreting cells in pancreatic substitutes. Use of adhesive, bio-functionalized hydrogels is receiving increasing attention, as cell-matrix interactions in 3-D can be important for various cell processes. With pancreatic substitutes, studies have indicated benefits of 3-D adhesion on the viability and/or function of insulin-secreting cells. As long-term storage of microencapsulated cells is critical for their clinical translation, cryopreservation of cells in hydrogels is actively being investigated. Previous studies have examined the cryopreservation response of cells encapsulated in non-adhesive hydrogels using conventional freezing and/or vitrification (ice-free cryopreservation), however, none have systematically compared the two cryopreservation methods with cells encapsulated within an adhesive 3-D environment. The latter would be significant, as evidence suggests adhesion influences cellular response to cryopreservation. Thus, the objective of this study was to determine the response to conventional freezing and vitrification of insulin-secreting cells encapsulated in an adhesive biomimetic hydrogel. Recombinant insulin-secreting C2C12 myoblasts were encapsulated in oxidized RGD-alginate and cultured 1 or 4 days post-encapsulation, cryopreserved, and assessed up to 3 days post-warming for metabolic activity and insulin secretion, and one day post-warming for cell morphology. Besides certain transient differences of the vitrified group relative to the Fresh control, both conventional freezing and vitrification maintained metabolism, secretion and morphology of the recombinant C2C12 cells. Thus, due to a simpler procedure and slightly superior results, conventional freezing is recommended over vitrification for the cryopreservation of C2C12 cells in oxidized RGD-modified alginate. PMID:23499987

  13. Corneal Transplantation

    DEFF Research Database (Denmark)

    Hjortdal, Jesper Østergaard

    Corneal transplantation has been performed for more than 100 years. Until 15 years ago the state-of-the art type of transplantation was penetrating keratoplasty, but since the start of this millennium, newly designed surgical techniques have developed considerably. Today, the vast majority of ker...

  14. Heart transplant (United States)

    ... hospital for 7 to 21 days after a heart transplant. The first 24 to 48 hours will likely be in ... follow your self-care instructions. Biopsies of the heart muscle are ... after transplant, and then less often after that. This helps ...

  15. Lung transplant (United States)

    ... transplant surgery include: You are placed on the heart-lung machine. One or both of your lungs are removed. For people who are having a double lung transplant, most or all of the steps from the first side are completed before the second side is ...

  16. Carinal transplantation. (United States)

    Ueda, H; Shirakusa, T


    BACKGROUND: Current techniques of management of carinal lesions are not always satisfactory. Carinal transplantation, if feasible, would be valuable in certain circumstances. METHODS AND RESULTS: Carinal transplantation experiments were performed in dogs. In early cross transplant experiments there were problems in controlling ventilation and in obtaining satisfactory anastomoses, and the animals failed to live for even a few days. In seven subsequent experiments the carinal graft was removed from one dog and transplanted into a second dog. Two dogs lived for over four months with immunosuppression. CONCLUSION: The results suggest that carinal transplantation can succeed if (1) the calibre of the graft is matched with that of the recipient; (2) there is an abundant blood supply to the graft; (3) appropriate immunosuppression is provided; (4) ventilation is adequate during surgery. Images PMID:1465758

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

  18. New technique for more rapid cryopreservation of dormant vegetative tree buds (United States)

    Cryopreservation of dormant buds of temperate trees in liquid nitrogen can provide a safe backup of field germplasm collections. However the process requires several months of preparation before buds can be cryopreserved. Cryopreservation at the natural moisture content (MC) would greatly accelerate...

  19. Dehydration improves cryopreservation of coconut (Cocos nucifera L.). (United States)

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


    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

  20. Gamma-Irradiated Sterile Cornea for Use in Corneal Transplants in a Rabbit Model. (United States)

    Yoshida, Junko; Heflin, Thomas; Zambrano, Andrea; Pan, Qing; Meng, Huan; Wang, Jiangxia; Stark, Walter J; Daoud, Yassine J


    Gamma irradiated corneas in which the donor keratocytes and endothelial cells are eliminated are effective as corneal lamellar and glaucoma patch grafts. In addition, gamma irradiation causes collagen cross inking, which stiffens collagen fibrils. This study evaluated gamma irradiated corneas for use in corneal transplantations in a rabbit model comparing graft clarity, corneal neovascularization, and edema. Penetrating keratoplasty was performed on rabbits using four types of corneal grafts: Fresh cornea with endothelium, gamma irradiated cornea, cryopreserved cornea, and fresh cornea without endothelium. Slit lamp examination was performed at postoperative week (POW) one, two, and four. Corneal clarity, edema, and vascularization were graded. Confocal microscopy and histopathological evaluation were performed. A P cornea with endothelium compared to the other three groups (P cornea scored better than the cryopreserved and fresh cornea without endothelium groups in clarity (0.9 vs. 1.5 and 2.6, respectively), and edema (0.6 vs. 0.8 and 2.0, respectively). The gamma irradiated corneas, cryopreserved corneas and the fresh corneas without endothelium, developed haze and edema after POW 2. Gamma irradiated cornea remained statistically significantly clearer than cryopreserved and fresh cornea without endothelium during the observation period (P cornea. Gamma irradiated corneas remained clearer and thinner than the cryopreserved cornea and fresh cornea without endothelium. However, this outcome is transient. Gamma irradiated corneas are useful for lamellar and patch grafts, but cannot be used for penetrating keratoplasty.

  1. Clinical aspects of the 360 º conjunctival graft and amniotic membrane cryopreserved transplantation in corneal ulcers in dogsAspectos clínicos do enxerto conjuntival 360º e do implante da membrana amniótica criopreservada no tratamento de úlceras de córnea em cães

    Directory of Open Access Journals (Sweden)

    Gabriel Thadeu Nogueira Martins Ferreira


    Full Text Available To evaluate the clinical application of implant of the canine cryopreserved amniotic membrane (DMEM plus DMSO 1:1 and 360° conjunctival flap in the treatment of progressive corneal ulceration. 10 dogs of the different breeds, males and females, aging four months to four years old with deep corneal ulceration and different clinical progression were divided in two groups: G1=360° conjunctival graft (n=5 and G2=implant of amniotic membrane, sutured at the edge of the ulcer with epithelial side facing up, associated with the third eyelid flap (n=5. The comparative analysis between groups was: complications, blepharospasm, ocular secretion, corneal vascularization, epithelial defect and corneal opacification in six moments (first emergency care, surgery and 3, 7, 15 and 30 days of postoperative. Without epithelial defect was evaluated quality of the scar. It was used score scale for subjective to qualify of the ocular signs. In G1, it was observed the non-adherence of the conjunctival graft to the ulcer (n=2, dehiscence of the suture (n=2, anterior synechia (n=2 and intense chemosis (n=1. In G2, it was not observed these complications. It was not significant difference between the groups to others ocular parameters, but it was different among the start and end moments of the same groups (ocular secretion, corneal vascularization, epithelial defect. The corneal opacity was more intense in G1. According to the clinical results, the cryopreserved amniotic membrane implant proved to be as effective in the corneal ulceration in comparison to the 360° conjunctival flap, because probably, the membrane promoted a trophic support for epithelialization, anti-inflamatory effect associated with important to the end result phenotype. Avaliar a aplicação clínica do implante da membrana amniótica canina criopreservada em DMEM (Dulbecco’s Modified Eagle’s Médium e DMSO4 (dimetilsulfóxido na proporção 1:1 e do enxerto conjuntival 360º no tratamento de

  2. Social oocyte cryopreservation: a portrayal of Brazilian women. (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


    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.

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

    Directory of Open Access Journals (Sweden)

    Ghasem Saki


    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.

  4. Cryo-mesh: a simple alternative cryopreservation protocol. (United States)

    Funnekotter, B; Bunn, E; Mancera, R L

    The continued development of new cryopreservation protocols has improved post-cryogenic success rates for a wide variety of plant species. Methods like the cryo-plate have proven beneficial in simplifying the cryopreservation procedure. This study assessed the practicality of a stainless steel mesh strip (cryo-mesh) for cryopreserving shoot tips from Anigozanthos viridis. Shoot tips of A. viridis (Kangaroo Paw) were precultured on 0.4 M sucrose medium for 48 h. Precultured shoot tips were coated in a 2% alginate solution and placed onto the cryo-mesh (a 25 x 7 mm, 0.4 mm aperture, 0.224 mm diameter wire stainless steel mesh strip). The alginate was set for 20 min in a loading solution containing 100 mM CaCl2, anchoring the shoot tips to the cryo-mesh. The cryo-mesh was then transferred to PVS2 on ice for 20, 30 or 40 min prior to plunging the cryo-mesh into liquid nitrogen. The cryo-mesh protocol was compared to the droplet-vitrification protocol. A maximum of 83% post-cryogenic regeneration was achieved with the cryo-mesh when exposed to PVS2 for 30 min. No significant difference in post-cryogenic regeneration was observed between the cryo-mesh and droplet-vitrification protocols. Anigozanthos viridis shoot tips were successfully cryopreserved utilising the new cryo-mesh. The cryo-mesh thus provides a simple and successful alternative for cryopreservation.

  5. Role of Membrane Lipid Fatty Acids in Sperm Cryopreservation

    Directory of Open Access Journals (Sweden)

    Rajes Mandal


    Full Text Available Lipid is an important constituent of cell membrane. Membrane lipid composition of spermatozoa has been correlated to different function. Many researchers have related membrane lipid with survival success after cryopreservation or cold shock. Sperm maturation and acrosome reactions are natural phenomenon, but cryopreservation or cold shock is not. Therefore, sperm cells are not programmed for such change and undergo stress. So the change in membrane lipid composition due to cold shock or cryopreservation may be looked upon as response of spermatozoa to a certain stressed condition. A significant body of research worked on the relationship between membrane lipid and fatty acid composition and ability of cell to tolerate adverse change in temperature. However, as the approach of different research groups was different, it is very difficult to compare the changes. Studies have been done with different species, ejaculated/seminal or epididymal sperm. Lipid analyses have been done with whole cell membrane isolated by different methods. Fatty acids estimated were from whole cell, plasma membrane, head membrane, or phospholipids. The cryopreservation condition, media composition, and diluents/cryoprotectants were also different. At this onset a comprehensive review is needed to cover changes of sperm membrane lipid composition of different species under different cryopreservation conditions.

  6. Serum-Free Cryopreservation of Human Amniotic Epithelial Cells

    Directory of Open Access Journals (Sweden)

    H. Niknejad


    Full Text Available Introduction & Objective: One of the important issues in long term storage of cells is removal of animal serum from cell culture environments. The aim of this study was to evaluate amni-otic fluid (AF, which is full of growth factors, as substitute for fetal bovine serum (FBS in the cryopreservation protocol. Materials & Methods: In this experimental study human amniotic epithelial cells were isolated from placentas which were seronegative for microbial infections. The cells were preserved in 24 different patterns for 12 months in -196 ?C (liquid nitrogen and viability of cells were determined before and after cryopreservation by trypan blue and MTT assay. Moreover, Oct-4 expression was studied to determine pluripotency before and after cryopreservation with immunocytochemistry. Results were compared between groups with ANOVA (Tukey Post-Test. P.value under 0.01 and 0.05 was considered statistically significant. Results: The presence of DMEM, FBS or AF is necessary for amniotic cell cryopreservation. Trypan-blue, MTT and immunocytochemistry showed that there isn’t significant difference between using AF and FBS in viability and pluripotency of cells. Moreover, results showed that DMSO is a better cryoprotectant compared to glycerol. Conclusion : Results showed that amniotic fluid can be a proper substitute for FBS in amniotic epithelial cells cryopreservation. (Sci J Hamadan Univ Med Sci 2013; 20 (1:15-24

  7. Frontal subcutaneous blood flow, and epi- and subcutaneous temperatures during scalp cooling in normal man

    DEFF Research Database (Denmark)

    Bülow, J; Friberg, L; Gaardsting, O


    Cooling of the scalp has been found to prevent hair loss following cytostatic treatment, but in order to obtain the hair preserving effect the subcutaneous temperature has to be reduced below 22 degrees C. In order to establish the relationship between epicutaneous and subcutaneous temperatures d...

  8. Cryopreservation of canine ovarian and testicular fibroblasts. (United States)

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


    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.

  9. Cryopreservation of spin-dried mammalian cells.

    Directory of Open Access Journals (Sweden)

    Nilay Chakraborty

    Full Text Available This study reports an alternative approach to achieve vitrification where cells are pre-desiccated prior to cooling to cryogenic temperatures for storage. Chinese Hamster Ovary (CHO cells suspended in a trehalose solution were rapidly and uniformly desiccated to a low moisture content (<0.12 g of water per g of dry weight using a spin-drying technique. Trehalose was also introduced into the cells using a high-capacity trehalose transporter (TRET1. Fourier Transform Infrared Spectroscopy (FTIR was used to examine the uniformity of water concentration distribution in the spin-dried samples. 62% of the cells were shown to survive spin-drying in the presence of trehalose following immediate rehydration. The spin-dried samples were stored in liquid nitrogen (LN(2 at a vitrified state. It was shown that following re-warming to room temperature and re-hydration with a fully complemented cell culture medium, 51% of the spin-dried and vitrified cells survived and demonstrated normal growth characteristics. Spin-drying is a novel strategy that can be used to improve cryopreservation outcome by promoting rapid vitrification.

  10. Cryopreservation of Tetraclinis articulata (vahl.) Masters. (United States)

    Serrano-Martinez, Francisco; Casas, José Luis


    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.

  11. Meniscal transplantation: still experimental surgery? A review. (United States)

    Moens, K; Dhollander, A; Moens, P; Verdonk, K; Verdonk, R; Almqvist, K F; Victor, J


    The objective of this review is to give a state of affairs of meniscal transplantation, with the accent on preservation and surgical techniques. All articles were selected by performing a search on the literature by using relevant keywords. The most relevant articles were selected with close attention to the publication date. When a meniscal tear is diagnosed, suture can be an option in the vascular zone, whereas the more frequently affected avascular zone heals poorly. A meniscectomy however is not without consequences, wherefore meniscal transplantation can be seen as a therapeutic option for pain reduction and improvement of function when the meniscus is lost. The meniscal scaffold, allograft and autograft can be currently withheld as possible grafts, where the meniscal scaffolds hold great promise as an alternative to the allograft. Various fixation techniques are therefore developed, where viable, deep frozen as well as cryopreservated allografts seem to give the most promising short term results. The transplantation can be performed using an open as well as an arthroscopic technique, using soft tissue fixation, bone plugs or blocks. De primacy of one technique can't be proven. In general meniscal transplantation can be considered as an acceptable procedure. Since the outcomes of different studies are difficult to compare, an attempt should be made to limit new studies to the comparison of one aspect. We can conclude that larger, more comparative randomised controlled long-term studies are necessary to resolve which techniques can give the best long-term results.

  12. Subcutaneous tissue flaps for hallux covering. (United States)

    Vaienti, Luca; Urzola, Victor; Scotti, Andrea; Masetto, L


    With the understanding of the extensive vascular supply of the subcutaneous tissue, of its efficacy in the protection of the anatomical structures and of its capability of promoting the adequate functioning of very stressed regions of the human body, the use of subcutaneous adipose flaps has become a valid and sometimes the only reasonable therapeutic weapon in the treatment of small and medium-sized tissue loss. Such a defect represents a common complication of great toe injuries and surgery. Here subcutaneous flap reconstruction is proposed for the treatment of dorsal and medial soft tissue losses of the hallux complicated with infection. Two case are reported. To the best of the authors' knowledge, this application has not been reported in this anatomical site so far. The technique might be worth knowing both for orthopedic and plastic surgeons, as it may represent a safe, less invasive solution for most tegumentary problems of the dorso-medial side of the first ray.

  13. [Subcutaneous phaeohyphomycosis caused by Phaeoacremonium parasiticum]. (United States)

    Alayeto Ortega, Jose; Alier Fabregó, Albert; Puig Verdie, Lluis; Sorli Redo, Maria Luisa; Horcajada Gallego, Juan Pablo; Portillo Bordonabe, M Eugenia


    From the available literature, it is demonstrated that dematiaceous fungal infections mostly affect immunosuppressed patients. These infections can occur in different forms, from subcutaneous infection to disseminated forms that may compromise the life of the patient. In many cases the infection is related to the inoculation of the microorganism by diverse traumatic mechanisms, which determines the course of the infection to be slower in some cases. We describe two cases of phaeohyphomycosis caused by Phaeoacremonium parasiticum: A cancer patient with subcutaneous lesions affecting the left hand and forearm, and a patient who presented with subcutaneous abscesses in the left leg. These cases confirm the presence of this type of fungus in Spain. In the second case a combination of amphotericin B lipid complex and posaconazole, together with several surgical resections, were necessary in order to overcome the infection. Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

  14. Hypertrophic Obesity and Subcutaneous Adipose Tissue Dysfunction

    Directory of Open Access Journals (Sweden)

    Anna Meiliana


    Full Text Available BACKGROUND: Over the past 50 years, scientists have recognized that not all adipose tissue is alike, and that health risk is associated with the location as well as the amount of body fat. Different depots are sufficiently distinct with respect to fatty-acid storage and release as to probably play unique roles in human physiology. Whether fat redistribution causes metabolic disease or whether it is a marker of underlying processes that are primarily responsible is an open question. CONTENT: The limited expandability of the subcutaneous adipose tissue leads to inappropriate adipose cell expansion (hypertrophic obesity with local inflammation and a dysregulated and insulin-resistant adipose tissue. The inability to store excess fat in the subcutaneous adipose tissue is a likely key mechanism for promoting ectopic fat accumulation in tissues and areas where fat can be stored, including the intra-abdominal and visceral areas, in the liver, epi/pericardial area, around vessels, in the myocardium, and in the skeletal muscles. Many studies have implicated ectopic fat accumulation and the associated lipotoxicity as the major determinant of the metabolic complications of obesity driving systemic insulin resistance, inflammation, hepatic glucose production, and dyslipidemia. SUMMARY: In summary, hypertrophic obesity is due to an impaired ability to recruit and differentiate available adipose precursor cells in the subcutaneous adipose tissue. Thus, the subcutaneous adipose tissue may be particular in its limited ability in certain individuals to undergo adipogenesis during weight increase. Inability to promote subcutaneous adipogenesis under periods of affluence would favor lipid overlow and ectopic fat accumulation with negative metabolic consequences. KEYWORDS: obesity, adipogenesis, subcutaneous adipose tissue, visceral adipose tissue, adipocyte dysfunction.

  15. Organ Transplantation (United States)

    ... here for a deeper conversation on this topic led by Hastings Director of Research Josephine Johnston. Sorry, ... accept people without insurance. Transplant teams rarely consider anyone over 75 years of age. Some centers exclude ...

  16. Hair Transplants (United States)

    ... for Every Season How to Choose the Best Skin Care Products In This Section Dermatologic Surgery What is dermatologic ... for Every Season How to Choose the Best Skin Care Products Hair Transplants Before (left) and after (right) - top ...

  17. Cornea Transplant (United States)

    ... Swelling of the cornea Signs and symptoms of cornea rejection In some cases, your body's immune system ... the risks of the procedure. Finding a donor cornea Most corneas used in cornea transplants come from ...

  18. Liver Transplant (United States)

    ... Legacy Society Make Gifts of Stock Donate Your Car Personal Fundraising Partnership & Support Share Your Story Spread the Word Give While You Shop Contact Us Donate Now Liver Transplant Back In ...

  19. Application of new phenolic antioxidants for cryopreservation of sturgeon sperm. (United States)

    Osipova, V P; Berberova, N T; Gazzaeva, R A; Kudryavtsev, K V


    Heterocyclic derivatives of butylated hydroxytoluene (BHT) were studied as cryoprotectants of the basic media for cryopreservation of the Russian sturgeon sperm. Rates of lipid peroxidation of sturgeon sperm before and after cryopreservation were reduced in the presence of the studied compounds, exceeding the effects of BHT and water-soluble analogue of vitamin E, trolox. The most efficient antioxidant has the effective concentration of 0.1 mM. Novel antioxidant agents as cryomedium supplements not only reduced the level of lipid peroxidation, but also enhanced the translational motility of the sperm of the Russian sturgeon after defrosting. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Differentiation-Promoting Medium Additives for Hepatocyte Cultivation and Cryopreservation. (United States)

    Gouliarmou, Varvara; Pelkonen, Olavi; Coecke, Sandra


    Isolated primary hepatocytes are considered as the reference system for in vitro hepatic methods. Following the isolation of primary hepatocytes from liver tissue, an unfavorable process named dedifferentiation is initiated leading to the attenuation of the hepatocellular phenotype both at the morphological and functional level. Freshly isolated hepatocytes can be used immediately or can be cryopreserved for future purposes. Currently, a number of antidedifferentiation strategies exist to extend the life span of isolated hepatocytes. The addition of differentiation-promoting compounds to the hepatocyte culture medium is the oldest and simplest antidedifferentiation approach applied. In the present chapter, the most commonly used medium additives for cultivation and cryopreservation of primary hepatocytes are reviewed.

  1. Use of cryopreserved transiently transfected cells in high-throughput pregnane X receptor transactivation assay. (United States)

    Zhu, Zhengrong; Puglisi, Jaime; Connors, David; Stewart, Jeremy; Herbst, John; Marino, Anthony; Sinz, Michael; O'Connell, Jonathan; Banks, Martyn; Dickinson, Kenneth; Cacace, Angela


    Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.

  2. Changes in rat spermatozoa function after cooling, cryopreservation and centrifugation processes. (United States)

    Kim, Suhee; Agca, Cansu; Agca, Yuksel


    Rat sperm cryopreservation is an effective method of archiving valuable strains for biomedical research and handling of rat spermatozoa is very important for successful cryopreservation. The aim of this study was to evaluate changes in rat sperm function during cryopreservation and centrifugation. Epididymal rat spermatozoa were subjected to cooling and freezing-thawing processes and then motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were compared before and after minimum centrifugation force (200×g). Cryopreservation decreased sperm motility, PMI, and MMP (PCentrifugation decreased motility and PMI of frozen-thawed spermatozoa (PCentrifugation decreased basal ROS of all spermatozoa (PCentrifugation affected function of cryopreserved spermatozoa. These data suggest that centrifugation makes rat spermatozoa susceptible to external ROS source, in particular during cooling process. Thus, protection from ROS damage and minimizing centrifugation should be considered during cryopreservation and post-thaw use of cryopreserved epididymal rat spermatozoa. Published by Elsevier Inc.

  3. 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.


    have been described, but until recently fertilization trials were not feasible. This study evaluated the effect of cold storage of diluted sperm prior to fertilizations and tested whether a previously defined protocol for European eel sperm cryopreservation can be successfully applied in fertilization....... 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...

  4. Subcutaneous peritoneal access device for type I diabetic patients nonresponsive to subcutaneous insulin. (United States)

    Schade, D S; Eaton, R P; Warhol, R M; Gregory, J A; Doberneck, R C


    Three type I diabetic patients nonresponsive to subcutaneous insulin were implanted with a subcutaneous peritoneal access device. In these patients, multiple subcutaneous injections had been unable to prevent recurrent hospital admissions for diabetic ketoacidosis. The patients were responsive to intravenous insulin but had limited accessible peripheral veins. Complications of thrombosis and/or septicemia from permanent central venous catheters prevented the long-term use of this route. The peritoneal access device was implanted subcutaneously adjacent to the umbilicus with its insulin delivery catheter terminating in the peritoneal space. Transcutaneous injection of insulin into the subcutaneous access port resulted in the same quantity of insulin entering the peritoneal space. Using a mixture of regular and protamine zinc insulin in a ration of 1:1 resulted in acute increases in plasma free insulin concentration with meals and a declining background level postprandially. All peritoneal access devices have been functioning well for at least 2 mo and in one of the implanted diabetic subjects, it has been in continuous use for 5 mo with no evidence of peritonitis or resistance to peritoneal insulin. These results suggest that a subcutaneous peritoneal access device may provide an alterative insulin delivery route for patients who are nonresponsive to subcutaneous insulin injections.

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


    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.

  6. Cryopreservation of Sambar deer semen in Thailand. (United States)

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


    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.

  7. Effects of cryopreservation at various temperatures on the survival of kelp grouper (Epinephelus moara) embryos from fertilization with cryopreserved sperm. (United States)

    Tian, Yongsheng; Chen, Zhangfan; Tang, Jiang; Duan, Huimin; Zhai, Jieming; Li, Bo; Ma, Wenhui; Liu, Jiangchun; Hou, Yunxia; Sun, Zhengxiang


    Fish embryo cryopreservation is highly important for the long-term preservation of genomic and genetic information; however, few successful cases of fish embryo cryopreservation have been reported over the past 60 years. This is the first study to use Epinephelus moara embryos from fertilization with cryopreserved sperm as experimental material. Embryos that developed to the 16-22 somite stage and tail-bud stage were treated with the vitrification solution PMG3T according to a five-step equilibration method and cryopreserved at various temperatures and storage duration. Only 19.9 ± 9.2% of 16-22 somite stage embryos and 1.3 ± 1.1% of tail-bud stage embryos survived when cooled at 4 °C for 60 min. In total, 8.0 ± 3.0% of 16-22 somite stage embryos survived when cooled at -25.7 °C for 30 min, 22.4 ± 4.7% of tail-bud stage embryos survived after 45 min of cooling at -25.7 °C, and none survived after 60 min. Only 2.0 ± 2.7% of embryos survived when cryopreserved at -140 °C for 20 min. However, 9.7% of tail-bud stage embryos survived after cryopreservation in liquid nitrogen (-196 °C) for 2 h. Most surviving embryos developed normally. Embryonic volume decreased and spherical segments appeared when embryos were treated with higher concentrations of vitrification solution. Additionally, the volume recovered gradually after rinsing with sucrose and seawater. This is the first estimate of the survival of E. moara embryos and larvae after cryopreservation. These findings provide a foundation for further explorations of fish embryo cryopreservation techniques. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Kidney-Pancreas Transplant (United States)

    ... Menu Menu Search Home Prevention Kidney Disease Patients Organ Donation & Transplantation Professionals Events Advocacy Donate A to Z Health ... Tests for Transplant Care After Kidney Transplant Common Organ Donation and Transplantation Terms The National Kidney Foundation (NKF) is the ...

  9. Subcutaneous fibrosis after whole neck irradiation

    International Nuclear Information System (INIS)

    Hirota, Saeko; Tsujino, Kayoko; Oshitani, Takashi; Hishikawa, Yoshio; Takada, Yoshiki; Kono, Michio; Abe, Mitsuyuki


    Purpose: To identify the risk factors for moderate to severe subcutaneous fibrosis after whole neck irradiation. Methods and Materials: We analyzed 233 cases of patients who had undergone whole neck irradiation with 4-MV X-ray or 8-10-MeV electrons, or both, and had been followed with regard to their skin condition for at least 1 year. The prescribed dose to the whole neck ranged from 19.2 to 72.4 Gy (median 50). The skin-absorbed dose was specified as that at a depth of 4.1 mm (d4.1-mm depth ), and a biologically equivalent dose (BED) of d4.1-mm depth was also estimated (BED 1.8 4.1-mm depth ). Results: Univariate analysis revealed that previous neck dissection, concurrent chemotherapy, corticosteroid administration as a part of chemotherapy, fractionation, and BED 1.8 4.1-mm depth were significant prognostic variables. Multivariate analysis showed that BED 1.8 4.1-mm depth and previous neck dissection were the only prognostic variables for moderate to severe subcutaneous fibrosis. Conclusion: A high dose to a 4.1-mm depth of the skin and a history of neck dissection were identified as the predominant risk factors for moderate to severe subcutaneous fibrosis after whole neck irradiation. A subcutaneous dose should be considered in radiotherapy treatment planning involving the whole neck, especially in cases in which patients have undergone previous neck dissection

  10. An entirely subcutaneous implantable cardioverter-defibrillator

    NARCIS (Netherlands)

    G.H. Bardy (Gust); W.M. Smith (Warren); A.M. Hood (Margaret); I.G. Crozier (Ian); I.C. Melton (Iain Craig); L.J.L.M. Jordaens (Luc); D.A.M.J. Theuns (Dominic); E. Park (Robert); D.J. Wright (David Justin); D.T. Connelly (Derek); S.P. Fynn (Simon Patrick); F.D. Murgatroyd (Francis); J. Sperzel (Johannes); J. Neuzner (Jörg); S.G. Spitzer (Stefan); A.V. Ardashev (Andrey); A. Oduro (Amo); L. Boersma (Lucas); A.H. Maass (Alexander); I.C. van Gelder (Isabelle); A.A.M. Wilde (Arthur); P.F.H.M. van Pascal; R.E. Knops (Reinoud); C.S. Barr (Craig); P. Lupo (Pierpaolo); R. Cappato (Riccardo); A.A. Grace (Andrew)


    textabstractBACKGROUND: Implantable cardioverter-defibrillators (ICDs) prevent sudden death from cardiac causes in selected patients but require the use of transvenous lead systems. To eliminate the need for venous access, we designed and tested an entirely subcutaneous ICD system. METHODS: First,

  11. Transmissible Venereal Tumor with Subcutaneous and Bone ...

    African Journals Online (AJOL)

    A five year old entire mixed breed dog was admitted to the University of Nairobi's small animal clinic with a 5-months history of subcutaneous masses. Physical examination revealed firm and mobile masses in the subcuticular tissues, on the mandible and the transverse processes of the lumbar vertebrae. Visual inspection ...

  12. Case Report Pneumomediastinum and Subcutaneous Emphysema ...

    African Journals Online (AJOL)

    oxygen may enhance faster absorption of air from extra-pulmonary tissues while needle aspiration and/ or surgical decompression may be useful if mediastinal structures are compressed [2,9,]. Conclusion. Extra-pulmonary extravasations of air manifested as subcutaneous emphysema and pneumomediastinum.

  13. Subcutaneous endoscopically assisted ligation using miniport for ...

    African Journals Online (AJOL)

    Background This report describes the first miniport method using subcutaneous endoscopically assisted ligation (SEAL) for the treatment of girls with inguinal hernia. To validate its safety and efficacy, the authors evaluated their early experiences. Methods Between April 2014 and December 2014, 19 SEALs using miniport ...

  14. Case Report: Pneumomediastinum and subcutaneous cervical ...

    African Journals Online (AJOL)

    The occurrence of pneumomediastinum and subcutaneous cervical emphysema as complications of childhood pneumonia is very unusual. They results most often from respiratory manoeuvres that produce high intrathoracic pressure. Although they are largely benign, pneumomediastinum can cause compression of major ...

  15. Anthropometrical Profile, Skinfold Tickness and Subcutaneous Fat ...

    African Journals Online (AJOL)

    Background: The threatening health problems resulting from excess subcutaneous fat depositions have been reported by the world Health Organization. Also noteworthy is that childhood obesity is a pointer to adult obesity. This necessitated a study on the anthropometrical profiles of adolescents of Southeast Nigeria using ...

  16. [Investigation of follicular development and oocyte maturation after cryopreservation and xenograft of newborn mouse ovaries]. (United States)

    Qin, Bo-Lin; Chen, Xue-Jin; Shi, Zhen-Dan; Li, Wan-Li; Tian, Yun-Bo


    In order to explore the feasibility of cryopreserving primordial follicles in attaining their developmental competence following freezing and thawing, ovaries from newborn mice were cryopreserved and the thawed ovaries were xenografted into kidney capsules of adult female mice. Ovaries were isolated from newborn B6C2F(1) female mice, infiltrated by Leibovitz 15 (L-15) medium containing 10% (V/V) fetal bovine serum (FBS) and 1.5 mol/L dimethylsulfoxide (DMSO), and then packed into 0.25 ml plastic straws. The ovaries contained in straws were frozen under nitrogen vapour at -40 degrees C in Cryocell 1200 programmable freezer, and stored in liquid nitrogen for periods ranging from 1 week to 6 months. Upon thawing, the straws were dipped into room temperature water for 10~20 s, after which the ovaries were collected and washed in L-15 buffer containing 10% (V/V) FBS without DMSO to remove cryoprotectant. The thawed ovaries were transplanted into kidney capsules of 8~12-week old adult B6C2F(1) female recipient mice by two protocols, with either 1 or 2 ovaries in each capsule. Upon withdrawal after at least 14 d of transplantation, only 45.00% (72/160) of the ovaries were recovered from 40 recipients transplanted with 2 ovaries in each capsule, compared to 82.50% (33/40) in 20 recipients with only 1 ovary in each capsule. The grafted ovaries exhibited similar follicular developmental progression to that of natural ovaries. There were antral follicles present in the transplanted ovaries on day 14, whose number increased more substantially on day 19 after transplantation. Following stimulation of the recipient mice with 10 IU PMSG on day 19 after xenografting, follicles further developed to preovulatory stage with appearance of cumulus oocytes and enlarged antrum. Oocytes from these fully grown antral follicles were collected and matured in vitro in modified essential medium-alpha (MEMalpha). After 16~17 h of culture, 40.90% of the oocytes exhibited germinal vesicle

  17. Conditioned Medium from Placental Mesenchymal Stem Cells Reduces Oxidative Stress during the Cryopreservation of Ex Vivo Expanded Umbilical Cord Blood Cells. (United States)

    Kadekar, Darshana; Rangole, Sonal; Kale, Vaijayanti; Limaye, Lalita


    The limited cell dose in umbilical cord blood (UCB) necessitates ex vivo expansion of UCB. Further, the effective cryopreservation of these expanded cells is important in widening their use in the clinics. During cryopreservation, cells experience oxidative stress due to the generation of reactive oxygen species (ROS). Conditioned medium from mesenchymal stem cells (MSCs-CM) has been shown to alleviate the oxidative stress during wound healing, Alzheimer's disease and ischemic disease. This premise prompted us to investigate the influence of MSCs-CM during cryopreservation of expanded UCB cells. CM-was collected from cord/placental MSCs(C-MSCs-CM, P-MSC-CM). UCB CD34+cells were expanded as suspension cultures in serum free medium containing cytokines for 10 days. Cells were frozen with/without C-MSCs-CM and or P-MSCs-CM in the conventional freezing medium containing 20%FCS +10%DMSO using a programmable freezer and stored in liquid nitrogen. Upon revival, cells frozen with MSCs-CM were found to be superior to cells frozen in conventional medium in terms of viability, CD34+content and clonogenecity. Priming of revived cells for 48 hrs with MSCs-CM further improved their transplantation ability, as compared to those cultured without MSCs-CM. P-MSCs-CM radically reduced the oxidative stress in cryopreserved cells, resulting in better post thaw functionality of CD34+ cells than with C-MSCs-CM. The observed cryoprotective effect of MSCs-CM was primarily due to anti-oxidative and anti-apoptotic properties of the MSCs-CM and not because of the exosomes secreted by them. Our data suggest that MSCs-CM can serve as a valuable additive to the freezing or the priming medium for expanded UCB cells, which would increase their clinical applicability.

  18. Conditioned Medium from Placental Mesenchymal Stem Cells Reduces Oxidative Stress during the Cryopreservation of Ex Vivo Expanded Umbilical Cord Blood Cells.

    Directory of Open Access Journals (Sweden)

    Darshana Kadekar

    Full Text Available The limited cell dose in umbilical cord blood (UCB necessitates ex vivo expansion of UCB. Further, the effective cryopreservation of these expanded cells is important in widening their use in the clinics. During cryopreservation, cells experience oxidative stress due to the generation of reactive oxygen species (ROS. Conditioned medium from mesenchymal stem cells (MSCs-CM has been shown to alleviate the oxidative stress during wound healing, Alzheimer's disease and ischemic disease. This premise prompted us to investigate the influence of MSCs-CM during cryopreservation of expanded UCB cells.CM-was collected from cord/placental MSCs(C-MSCs-CM, P-MSC-CM. UCB CD34+cells were expanded as suspension cultures in serum free medium containing cytokines for 10 days. Cells were frozen with/without C-MSCs-CM and or P-MSCs-CM in the conventional freezing medium containing 20%FCS +10%DMSO using a programmable freezer and stored in liquid nitrogen. Upon revival, cells frozen with MSCs-CM were found to be superior to cells frozen in conventional medium in terms of viability, CD34+content and clonogenecity. Priming of revived cells for 48 hrs with MSCs-CM further improved their transplantation ability, as compared to those cultured without MSCs-CM. P-MSCs-CM radically reduced the oxidative stress in cryopreserved cells, resulting in better post thaw functionality of CD34+ cells than with C-MSCs-CM. The observed cryoprotective effect of MSCs-CM was primarily due to anti-oxidative and anti-apoptotic properties of the MSCs-CM and not because of the exosomes secreted by them.Our data suggest that MSCs-CM can serve as a valuable additive to the freezing or the priming medium for expanded UCB cells, which would increase their clinical applicability.

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

    DEFF Research Database (Denmark)

    Herrmann, Janne Rothmar; Kroløkke, Charlotte


    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...

  20. Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization

    DEFF Research Database (Denmark)

    Nejlund, Sarah; Smith, Julie; Kraan, Jaco


    BACKGROUND: A blood sample containing circulating tumor cells (CTCs) may serve as a surrogate for metastasis in invasive cancer. Cryopreservation will provide new opportunities in management of clinical samples in the laboratory and allow collection of samples over time for future analysis of exi...

  1. Collection, analysis and cryopreservation of semen from Malayan ...

    African Journals Online (AJOL)

    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 ...

  2. Cryopreservation of dammar (Agathis damara Salisb. seeds in liquid nitrogen

    Directory of Open Access Journals (Sweden)



    Full Text Available Dammar (Agathis damara seeds categorized as an intermediate seeds since their viability tend to decrease when subjected to storage more than 2 weeks conventionally. Storage period of seeds could be prolonged when the seeds cryopreserved in liquid nitrogen since the metabolism of the cells could be minimized without loss of viability. The objective of the study was to identify suitable vitritification method for dammar storage seeds. Seed water content was decreased gradually from initial water content (28.48% as a control using desiccators and vacuum method. Vitrification solution (PVS2 containing glycerol 30%, ethylene glycol 15% and dimethylsulfoxide (DMSO 15% in 0.4M sucrose solution was used as a cryoprotectant of peeled or unpeeled dammar seeds during freezing process. The samples were soaked in PVS2 for 1 hour followed by exposure to liquid nitrogen in cryotube for 1 hour. The samples were then thawed in water bath at 28°C for 1 hour prior to germination in IPB-78 germinator with UDK and UKdDp germination methods. Results showed that the highest viability of cryopreserved dammar seeds (22.32% moisture content was 100% obtained from those germinated with UKdDp method. A negative effect of cryoprotectant was occurred in both peeled and unpeeled seeds cryopreserved for 1 hour. However, it was effective for seeds cryopreserved for 4 weeks which indicate the possibility to preserve for a longer period in the future.

  3. Sperm cryopreservation before cancer treatment: a 15-year monocentric experience. (United States)

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


    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.

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

    International Nuclear Information System (INIS)

    Martinez, Jose Gregorio; Pardo Carrasco, Sandra


    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.

  5. Application of functional genomics and proteomics to plant cryopreservation (United States)

    Plant cryobiology has primarily emerged from the classical fields of cryobiology and plant stress physiology. Cryopreservation tools are now available to geneticists for germplasm preservation and the field itself is advancing significantly through the use of molecular techniques. Long-term preser...

  6. Cryopreservation of achenes of caju-de-árvore-docerrado ...

    African Journals Online (AJOL)

    Cryopreservation of achenes of caju-de-árvore-docerrado (Anacardium othonianum Rizz). Lílian Abadia da Silva, Juliana de Fátima Sales, Fabiano Guimarães Silva, Pedro Henrique Castro Magalhães Ferreira ...

  7. Cryopreservation of specialized chicken lines using cultured primordial germ cells. (United States)

    Nandi, S; Whyte, J; Taylor, L; Sherman, A; Nair, V; Kaiser, P; McGrew, M J


    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells ( PGCS: ). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- ( MHC-: ) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. © The Author 2016. Published by Oxford University Press on behalf of Poultry Science Association.

  8. Cryopreservation of Filamentous Micromycetes and Yeasts Using Perlite

    Czech Academy of Sciences Publication Activity Database

    Homolka, Ladislav; Lisá, Ludmila; Kubátová, A.; Váňová, M.; Janderová, B.; Nerud, František


    Roč. 51, č. 2 (2007), s. 153-157 ISSN 0015-5632 R&D Projects: GA MŠk(CZ) 0021620828 Institutional research plan: CEZ:AV0Z50200510 Source of funding: V - iné verejné zdroje Keywords : cryopreservation * perlite * basidiomycetes Subject RIV: EE - Microbiology, Virology Impact factor: 0.989, year: 2007

  9. 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, ...

  10. Use of plumules cryopreservation to save coconut germplasm in ...

    African Journals Online (AJOL)

    Plumules excised from zygotic embryos through the largest representative diversity of four of the five different areas of coconut cash and food crops were used in a cryopreservation process using encapsulation-dehydration technique. Five accessions of coconut trees were used [Panama Tall (PNT/GPA), Brazilian Green ...

  11. Midterm Results of Aortic Valve Replacement with Cryopreserved Homografts

    Directory of Open Access Journals (Sweden)

    Emre Özker


    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.

  12. Seasonal and cryopreservation impacts on semen quality in boars (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...

  13. Cryopreservation of intact testicular tissue from boys with cryptorchidism

    DEFF Research Database (Denmark)

    Kvist, K; Thorup, Jørgen Mogens; Byskov, A G


    Boys with cryptorchidism often face fertility problems in adult life despite having orchiopexy performed at a very young age. During this operation, a biopsy of the testis is normally taken in order to evaluate their infertility potential and the presence of malignant cells. This study evaluated ...... the morphology and functional capacity of cryopreserved testes biopsies and their possible use in fertility preservation....

  14. Cryopreservation of embryonic axes of maize (Zea mays L.) by ...

    African Journals Online (AJOL)



    Dec 21, 2010 ... Cryopreservation of embryonic axes of maize (Zea mays L.) by vitrification protocol. I. S. Usman* and M. M. Abdulmalik. Department of Plant Science, Ahmadu Bello University,PMB 1044, Zaria, Kaduna State, Nigeria. Accepted 27 April, 2010. A storage protocol at cryogenic temperature was established for ...

  15. Studies on the motility and cryopreservation of rainbow trout

    African Journals Online (AJOL)

    Studies on the motility and cryopreservation of rainbow trout (Salmo gairdnerl) spermatozoa. G. van der Horst, H.M. Dott and G.C. Foster. ARC, Institute of Animal Physiology, Animal Research Station, Cambridge, United Kingdom. The very short duration of vigorous movement (1'12 to 7 min) in fresh water and physiological ...

  16. Cellular response of Murine Osteoblasts to Cryopreservation: the ...

    African Journals Online (AJOL)



    Nov 2, 2006 ... engineered bone. The effects of cell-scaffold interactions and cell-cell interactions on osteoblast viability and attachment to hydroxyapatite (HA) scaffolds following cryopreservation ... servation methods for bio-artificial tissue products can be ... tissue-engineered bone constructs involve osteoblasts attached ...

  17. Cardiac mitochondrial oxidative capacity is partly preserved after cryopreservation with dimethyl sulfoxide. (United States)

    Meyer, A; Charles, A L; Singh, F; Zoll, J; Talha, S; Enache, I; Chaarloux, A; Inser-Horobeti, M E; Geny, B


    Cardiac muscle cryopreservation is a challenge for both diagnostic procedure requiring viable tissues and therapeutic advance in regenerative medicine. Mitochondria are targets of both direct and indirect damages, secondary to congelation per se and/or to cryoprotectant's toxic effects, which participate to diminution of viability and/or functioning of cells after freezing. At the cardiac muscle level, only one study had investigated mitochondrial respiration after cryopreservation. To determine the effect of cryopreservation on mitochondrial respiration of cardiac muscle. We recorded mitochondrial respiration through complexes I, II, III and IV along with mitochondrial coupling in fresh and cryopreserved rat left ventricles samples and assessed difference of the means, correlation and agreement between the measures in all samples. Mitochondrial respiration was partly maintained up to 70% in cryopreserved samples whatever the substrate. A significant correlation was observed between fresh and cryopreserved samples (r = 0.71, p cryopreservation (- 1.44 ± 0.15; p cryopreservation. Further, the fluctuations around the mean difference were wide (-14.06, +5.08 µmol/min/g), increasing with respiration rates (p cryopreservation using DMSO partly protect cardiac mitochondrial respiration and coupling. These data support the interest to further refine cryopreservation methods.

  18. Abdominal wall phlebitis due to Prevotella bivia following renal transplantation in a patient with an occluded inferior vena cava

    NARCIS (Netherlands)

    Janssen, S.; van Donselaar-van der Pant, K. A. M. I.; van der Weerd, N. C.; Develter, W.; Bemelman, F. J.; Grobusch, M. P.; Idu, M. M.; ten Berge, I. J. M.


    Pre-existing occlusion of the inferior vena cava may complicate renal transplantation. Suppurative abdominal wall phlebitis following renal transplantation was diagnosed in a patient with pre-existing thrombosis of the inferior vena cava of unknown cause. The phlebitis developed in the subcutaneous

  19. Cryopreservation of preimplantation embryos of cattle, sheep, and goats. (United States)

    Youngs, Curtis R


    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.

  20. Cryopreservation of boar sperm induces differential microRNAs expression. (United States)

    Zhang, Yan; Dai, Dinghui; Chang, Yu; Li, Yuan; Zhang, Ming; Zhou, Guangbin; Peng, Zhanghua; Zeng, Changjun


    Lower conception rates and litter sizes limit the wide use of artificial insemination with frozen-thawed boar sperm, due to a lack of understanding of the mechanisms that cause cryodamage and cryoinjury to sperm during cryopreservation. CryoMiRs, a family of freeze-related microRNAs (miRNAs), are associated with freeze tolerance, and regulate metabolism in mammalian hibernators and insects. Thus, we speculate that miRNAs maybe involved in the regulation of the freeze-thaw process and may affect boar sperm function. In this study, we studied the differential expression of 46 miRNAs that have roles in spermatogenesis, sperm maturation, and sperm quality in response to cryopreservation (with or without 3% glycerol). The results indicated that, in response to cryopreservation with 3% glycerol, 14 miRNAs were significantly up-regulated, but only two miRNAs (miR-22 and miR-450b-5p) were significantly down-regulated, relative to fresh sperm. Preservation with 3% glycerol caused up-regulation of 17 miRNAs, but only caused down-regulation of one miRNA (miR-24), relative to sperm cryopreserved without glycerol. Functional annotations of these differentially expressed miRNAs indicated that these miRNAs and their targets are mainly associated with metabolic and cellular processes. Therefore, our findings show that cryopreservation results in changes in miRNA expression, and suggest that the anti-freeze mechanisms of boar sperm need to be studied further. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats (United States)

    Youngs, Curtis R.


    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. PMID:21847080

  2. Cryopreserved semilunar heart valve allografts: leaflet surface damage in scanning electron microscopy. (United States)

    Burkert, J; Krs, O; Vojácek, J; Mokrácek, A; Slízová, D; Hlubocký, J; Kobylka, P; Spatenka, J


    Allograft heart valves (AHV), biological valves of human origin, offer potential advantages over conventional xenografts in terms of superior hemodynamics and, perhaps, better durability. The most important factors for long-term AHV clinical performance are the processing and cryopreservation methods. The aim of this study was to evaluate the impact of current processing protocol on valve tissue morphology, mainly to address the effect of successive processing steps on the leaflet surface structure. For the detection of fine changes in endothelial covering and underlying layers, our own modification of the scanning electron microscopy (SEM) technique was utilized. The study was based on an investigation of 20 AHV (40 specimens). Fourteen valves came from heart-beating donors (multiorgan harvesting) when the heart could not be transplanted for any reason (donor criteria, availability of recipient and/or logistics). Six were obtained at the time of routine postmortems--non heart-beating donors (NHBD). All specimens were initially fixed in Baker's solution. Tissue samples were dissected, dried with hexamethyldisilazane (HMDS), gold-coated, studied and photographed by SEM (Tesla BS 301). In order to define the integrity of the endothelium, subendothelial layers and the quality of the surface under SEM, a special six-level score system was introduced: 1-intact endothelium, 2-confluent endothelium with structural inhomogeneity, 3-disruption of intercellular contacts, 4-separation of endothelial cells, 5-complete loss of endothelium, 6-damage of subendothelial layers). AHV samples were divided into 4 groups for comparison. One aortic AHV "fresh" control sample obtained from a heart-beating donor was evaluated without any processing and was compared with (i) tissue from AHV obtained from NHBD with warm ischemia of 12 and 48 hours, (ii) samples stored at +4 degrees C in saline for 24 h, (iii) antibiotic-treated tissue for 24 h at 37 degrees C and finally with (iv


    NARCIS (Netherlands)


    OBJECTIVE- To compare the subcutaneous glucose sensor measurements with two reference methods. Previous studies provide conflicting findings about the real glucose concentrations in subcutaneous tissue. Some suggest substantially lower concentration, whereas others measure proportionally higher

  4. Cadaveric transplantation.

    Directory of Open Access Journals (Sweden)

    Gokal R


    Full Text Available Transplantation is already the optimum treatment for terminal renal failure. Donor organ shortage means that there are large number of patients on dialysis awaiting this treatment. This has in some countries led to unacceptable unscrupulous practices of live non-related graft donation. The outcome of graft and patient after transplantation has improved significantly based on a better understanding of immunopathology, immunosuppression and tissue typing. The future is promising and xenografting may well solve the organ shortage but undoubtedly will raise other issues.

  5. Pancreas transplantation

    International Nuclear Information System (INIS)

    Snider, J.F.; Hunter, D.W.; Castaneda-Zuniga, W.R.; Letourneau, J.G.


    Pancreas transplantation can be complicated by vascular thrombosis, stenosis, or anastomotic leak, complications that predispose to transplant pancreatectomy. The relative roles of noninvasive radiologic studies in such vascular complications have been correlated with angiographic or pathologic data. The results of 54 scintigraphic studies, 25 CT studies, 16 sonograms, and 23 color Doppler examinations have been correlated with those of 40 angiograms and 28 pathologic studies in a population of 185 recipients. CT (sensitivity, 100%; specificity, 75%; accuracy, 92%) and US (sensitivity, 88%; specificity, 80%; accuracy, 85%) were most helpful in noninvasive screening for vascular complications, while angiography remains nearly definite in the radiographic diagnosis of these problems

  6. Gene expression profiles of cryopreserved CD34{sup +} human umbilical cord blood cells are related to their bone marrow reconstitution abilities in mouse xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Sudo, Kazuhiro [Cell Engineering Division, RIKEN BioResource Center, Tsukuba (Japan); Yasuda, Jun, E-mail: [Omics Science Center, RIKEN, Yokohama (Japan); Department of Cell Biology, The JFCR-Cancer Institute (Japan); Nakamura, Yukio, E-mail: [Cell Engineering Division, RIKEN BioResource Center, Tsukuba (Japan)


    Human umbilical cord blood (UCB) cells are an alternative source of hematopoietic stem cells for treatment of leukemia and other diseases. It is very difficult to assess the quality of UCB cells in the clinical situation. Here, we sought to assess the quality of UCB cells by transplantation to immunodeficient mice. Cryopreserved CD34{sup +} UCB cells from twelve different human donors were transplanted into sublethally irradiated NOD/shi-scid Jic mice. In parallel, the gene expression profiles of the UCB cells were determined from oligonucleotide microarrays. UCB cells from three donors failed to establish an engraftment in the host mice, while the other nine succeeded to various extents. Gene expression profiling indicated that 71 genes, including HOXB4, C/EBP-{beta}, and ETS2, were specifically overexpressed and 23 genes were suppressed more than 2-fold in the successful UCB cells compared to those that failed. Functional annotation revealed that cell growth and cell cycle regulators were more abundant in the successful UCB cells. Our results suggest that hematopoietic ability may vary among cryopreserved UCB cells and that this ability can be distinguished by profiling expression of certain sets of genes.

  7. Subcutaneous emphysema in cavitary pulmonary tuberculosis without pneumothorax or pneumomediastinum

    Directory of Open Access Journals (Sweden)

    Ramakant Dixit


    Full Text Available Extra-alveolar air in the form of subcutaneous tissue emphysema is observed in a variety of clinical settings. Spontaneous subcutaneous emphysema in the absence of pneumothorax or pneumomediastinum is very rare. We report a case of spontaneous subcutaneous emphysema secondary to cavitary pulmonary tuberculosis in the absence of pneumothorax or pneumomediastinum.

  8. Patient selection for subcutaneous versus sublingual immunotherapy. (United States)

    Larenas-Linnemann, Désirée


    With the Food and Drug Administration's approval of sublingual allergen-specific immunotherapy (SL-AIT) tablets for grass and ragweed pollen, SL-AIT is progressively gathering importance not only in Europe, but also in the United States and other parts of the world. We reviewed issues related to the selection of patients for the sublingual or the subcutaneous route for allergic patients, based on what has been published since January 2014 on subcutaneous-versus-SL-AIT efficacy, safety and other issues. (Figure is included in full-text article.) As patient's adherence seems one of the major problems in real-life AIT, investigators have sought how to enhance AIT simplicity by changing the route to home-administrated SL-AIT, and by shortening the subcutaneous-allergen-specific immunotherapy (SC-AIT) build-up or maintenance phase. The latter was safe with several hypoallergenic extracts. As for SL-AIT, double blind placebo-controlled large trials in patients with allergic rhinitis and asthma have shown the efficacy and safety of ragweed pollen and house dust mite SLIT tablets and highly concentrated liquid formulations, primarily in adults. A large trial with SLIT in 3-year-old children was effective. With the improvement of SL-AIT efficacy, the selection of SC-versus-SL-AIT will probably increasingly be based not on efficacy, but on practical aspects, without losing sight of which SL-AIT products have proven efficacy.

  9. Nanotechnology-based Cryopreservation of Cell-Scaffold Constructs: A New Breakthrough to Clinical Application. (United States)

    Chen, G; Lv, Y

    The developments of "off-the-shelf" cell-scaffold constructs received an increasing interest in tissue engineering and regenerative medicine. Although the direct cryopreservation of a single-cell suspension in the tube is a relative mature technology, the cryopreservation of cell-scaffold constructs remains a challenge. Nanotechnology shows tremendous potential for cryopreservation in regulating of freezing and thawing processes. For example, nanoparticles have been reported to modify the cryoprotective agent (CPA), adjust the process of cooling and warming cycles. In this review, we provide an overview of cryopreservation of cell-scaffold constructs firstly. The review further focuses on the effects of nanotechnology on cryopreservation of cell-scaffold constructs, including the nanostructure of scaffold, nanoparticles in cooling and warming process in cryopreservation. The perspectives on future challenges in this filed are also pointed out.

  10. Application of cryopreservation to genetic analyses of a photosynthetic picoeukaryote community. (United States)

    Kawachi, Masanobu; Kataoka, Takafumi; Sato, Mayumi; Noël, Mary-Hélène; Kuwata, Akira; Demura, Mikihide; Yamaguchi, Haruyo


    Cryopreservation is useful for long-term maintenance of living strains in microbial culture collections. We applied this technique to environmental specimens from two monitoring sites at Sendai Bay, Japan and compared the microbial diversity of photosynthetic picoeukaryotes in samples before and after cryopreservation. Flow cytometry (FCM) showed no considerable differences between specimens. We used 2500 cells sorted with FCM for next-generation sequencing of 18S rRNA gene amplicons and after removing low-quality sequences obtained 10,088-37,454 reads. Cluster analysis and comparative correlation analysis of observed high-level operational taxonomic units indicated similarity between specimens before and after cryopreservation. The effects of cryopreservation on cells were assessed with representative culture strains, including fragile cryptophyte cells. We confirmed the usefulness of cryopreservation for genetic studies on environmental specimens, and found that small changes in FCM cytograms after cryopreservation may affect biodiversity estimation. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Eyebrow transplantation. (United States)

    Goldman, G D


    Reconstruction of the eyebrow has historically been accomplished with temporal scalp pedicle flap formation or free composite scalp grafts. These two techniques may be associated with substantial morbidity and a false, overly dense eyebrow appearance. Hair transplantation of the eyebrows has been described with excellent results, but is relatively underreported in the literature. To determine whether modern techniques of micrograft hair transplantation can suitably re-create an aesthetic eyebrow in a case of iatrogenic eyebrow alopecia. A 33-year-old woman with iatrogenic eyebrow alopecia underwent four sessions of eyebrow micrograft hair transplantation to re-create both eyebrows. Suitable aesthetic eyebrows were re-created in a symmetric fashion with proper hair orientation. The process was time consuming and tedious, but highly effective. Eyebrow transplantation is a suitable alternative to pedicle flap formation and composite scalp grafting. It is a straightforward procedure that can be performed in the office under local anesthesia with minimal attendant morbidity. The result may be superior to that seen with more involved eyebrow replacement procedures.

  12. Intestine transplantation

    Directory of Open Access Journals (Sweden)

    Tadeja Pintar


    Conclusion: Intestine transplantation is reserved for patients with irreversible intestinal failure due to short gut syndrome requiring total paranteral nutrition with no possibility of discontinuation and loss of venous access for patient maintenance. In these patients complications of underlying disease and long-term total parenteral nutrition are present.

  13. Heart Transplantation (United States)

    A heart transplant removes a damaged or diseased heart and replaces it with a healthy one. The healthy heart comes from a donor who has died. It is the last resort for people with heart failure when all other treatments have failed. The ...

  14. Successful delivery derived from cryopreserved rare human spermatozoa with novel cryopiece. (United States)

    Sun, J; Chen, W; Zhou, L; Hu, J; Li, Z; Zhang, Z; Wu, Y


    Herein, we report the clinical outcomes following intracytoplasmic sperm injection (ICSI) with cryopiece cryopreserved rare human spermatozoa from severe male factor infertility patients. We established a novel cryopiece system on the basis of previous studies. In this study, 126 spermatozoa from four patients with non-obstructiveazoospermia (NOA) or severe oligozoospermia were stored in cryopiece and then thawed on the day of the oocyte retrieval, 88 (83%) spermatozoa were recovered with a 47.5% (38 of 80) motile rate. Routine ICSI were performed in enrolled 30 MII oocytes from their spouse with their own spermatozoa, respectively. Twenty-two (73%) fertilization and 19 (86%) zygote cleavage were observed. Finally, a total of 11 embryos were achieved and each female patient received a transplantation of two fresh embryos. Four healthy babies were born at term. In conclusion, our novel cryopiece can be applied in assisted reproduction through ICSI with an effective motile spermatozoa recovery rate, high fertilization rate, and successful pregnancy result. © 2017 American Society of Andrology and European Academy of Andrology.

  15. Measurement of Thermal Conductivities of Two Cryoprotective Agent Solutions for Vitreous Cryopreservation of Organs at the Temperature Range of 77 K-300 K Using a Thermal Sensor Made of Microscale Enamel Copper Wire. (United States)

    Li, Yufang; Zhao, Gang; Hossain, S M Chapal; Panhwar, Fazil; Sun, Wenyu; Kong, Fei; Zang, Chuanbao; Jiang, Zhendong


    Biobanking of organs by cryopreservation is an enabling technology for organ transplantation. Compared with the conventional slow freezing method, vitreous cryopreservation has been regarded to be a more promising approach for long-term storage of organs. The major challenges to vitrification are devitrification and recrystallization during the warming process, and high concentrations of cryoprotective agents (CPAs) induced metabolic and osmotic injuries. For a theoretical model based optimization of vitrification, thermal properties of CPA solutions are indispensable. In this study, the thermal conductivities of M22 and vitrification solution containing ethylene glycol and dimethyl sulfoxide (two commonly used vitrification solutions) were measured using a self-made microscaled hot probe with enameled copper wire at the temperature range of 77 K-300 K. The data obtained by this study will further enrich knowledge of the thermal properties for CPA solutions at low temperatures, as is of primary importance for optimization of vitrification.

  16. Update on cryopreservation of adipose tissue and adipose-derived stem cells. (United States)

    Shu, Zhiquan; Gao, Dayong; Pu, Lee L Q


    This article first discusses some fundamentals of cryobiology and challenges for cell and tissue cryopreservation. Then, the results of cryopreservation of adipose cells and tissues, including adipose-derived stem cells, in the last decade are reviewed. In addition, from the viewpoint of cryobiology, some desired future work in fat cryopreservation is proposed that would benefit the optimization, standardization, and better application of such techniques. Copyright © 2015 Elsevier Inc. All rights reserved.

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


    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


    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...

  18. Improvement of cryopreservation protocols: systematic parametric analysis; Optimierung von Kryokonservierungsprotokollen: Systematische Parameteranalyse

    Energy Technology Data Exchange (ETDEWEB)

    Bernemann, I.; Hofmann, N.; Glasmacher, B.; Szentivanyi, A.; Kuberka, M. [Leibniz Univ. Hannover (Germany). Inst. fuer Mehrphasenprozesse


    Effective long-term storage of cells in suspension by cryopreservation needs optimization of freezing and thawing parameters to guarantee high product quality after recultivation. Different cell types need special conditions to minimize cell stress and to enhance cell survival after cryopreservation. Therefore, adaptation of cooling rates and concentration of cryoprotectant are important to deliver satisfactorily results after longterm preservation. To improve the cryopreservation of cells, it is expedient to develop a systematic factor optimization system. (orig.)

  19. Subcutaneous versus subcutaneous and intraperitoneal local anaesthetic in the management of post appendicectomy pain

    International Nuclear Information System (INIS)

    Qureshi, K.Z.; Gondal, Z.I.; Raza, A.


    To compare the efficacy of subcutaneous only and combined subcutaneous and peritoneal infiltration of 0.5% bupivacaine during appendicectomy for the management of early post operative pain. Study Design: Randomized controlled study. Place and Duration of Study: Department of Surgery, CMH Kohat from 13th December 2007 to 20th December 2008. Patients and Methods: Sixty patients of a cute appendicitis, divided into two groups of 30 each, were included in the study. Group A was given 0.5% bupivacaine subcutaneously, whereas group B was given the anaesthetic subcutaneously as well as intraperitoneally during appendectomy. Results: In group A, 24 (80%) were VAS (visual analoguescoring) 3 (uncomfortable) and 6 (20%) were VAS 2 (mild pain) whereas in study group B, 11 (36.6%) were VAS 3, 19 (63.3%) were VAS 2 and 19 (63.3%) were VAS 2 during 1st 12 hrs postoperatively (p=0.001). In 12-24 hrs post operatively, 15 (50%) patients were VAS 3 in group A and same number was VAS 2 and in group B, only 3 (10%) were in VAS 3 and 27 (90%) were VAS 2 (p=0.001). Conclusion: A combination of subcutaneous and peritoneal infiltration with bupivacaine is superior in relieving post appendectomy pain so patients require less dosage of analgesics in early post operative period along with early mobilization. (author)

  20. Efficacy of postal communication with patients who have cryopreserved pre-embryos. (United States)

    Brzyski, R G


    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.

  1. The Relationship of a Combination of Human Adipose Tissue-Derived Stem Cells and Frozen Fat with the Survival Rate of Transplanted Fat. (United States)

    Ha, Ki-Young; Park, Hojin; Park, Seung-Ha; Lee, Byung-Il; Ji, Yi-Hwa; Kim, Tae-Yeon; Yoon, Eul-Sik


    The survival rate of grafted fat is difficult to predict, and repeated procedures are frequently required. In this study, the effects of the freezing period of harvested adipose tissue and the addition of human adipose tissue-derived stem cells (ASCs) on the process of fat absorption were studied. Adipose tissue was obtained from patients who underwent a lipoaspirated fat graft. The fat tissue was cryopreserved at -20℃ in a domestic refrigerator. A total of 40 nude mice were used. The mice in the experimental group received three different subcutaneous injections in the back: an injection of fresh fat and ASCs, an injection of fat that had been frozen for one month and ASCs, and an injection of fat that had been frozen for two months and ASCs. The control mice received fat grafts without ASCs. The mice were sacrificed at four or eight weeks after the procedure, and the grafted fat tissues were harvested. The extracted fat was evaluated using photographic analysis, volume measurements, and histological examination. In the control group, the fat resorption rates four weeks after transplantation in the grafts of fresh fat, fat that had been frozen for one month, and fat that had been frozen for two months were 21.14%, 22.46%, and 42.56%, respectively. In the experimental group, the corresponding resorption rates were 6.68%, 13.0%, and 33.9%, respectively. ASCs can increase the fat graft survival rate. The use of ASCs in fat grafting can reduce the need for repeated fat grafts and provide good long term results.

  2. Segregated water observed in a putative fish embryo cryopreservative. (United States)

    Kirichek, O; Soper, A K; Dzyuba, B; Holt, W V


    Development of new cryopreservation strategies has major potential in medicine and agriculture and is critical to the conservation of endangered species that currently cannot be preserved. A critical property of any potential cryopreservative solution is its ability to prevent cell-damaging ice formation during cooling and subsequent heating. This study focuses on the freezing behaviour of promising model cryoprotective solutions. We perform neutron scattering analysis, combined with computer modelling, of the water structure after quench cooling these solutions. It is found that water in this solution forms nano-clusters encapsulated by the surrounding matrix of cryoprotectant solute molecules. We posit that these small volumes inhibit ice formation, because water does not have space for the structural relaxation required to crystallize on the timescale of the cooling process.

  3. Cryopreservation of somatic embryos of paradise tree (Melia azedarach L.)


    Scocchi, A.; Vila, S.; Mroginski, L.; Engelmann, Florent


    In paradise tree (Melia azedarach L.), immature zygotic embryos sampled from immature fruits are the starting material for the production of somatic embryos. These somatic embryos are employed for freezing experiments. Immature fruits could be stored at 25 degrees C for up to 80 days without impairing the embryogenic potential of zygotic embryos, which represents a four-fold increase in immature fruit storage duration, compared with previous studies. Among the three cryopreservation technique...

  4. Cryopreservation of zebrafish (Danio rerio) oocytes by vitrification. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Robert F. Casper


    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.

  6. [Cryopreservation study on seeds and embryos in Dalbergia odorifera]. (United States)

    Zeng, Lin; He, Ming-Jun; Chen, Kui; Wei, Jian-He


    The mature seeds and excised embryos of Dalbergia odorifera were used as materials to study the effect of moisture content on their survival, as well as the effect of rapid freezing and vitrification freezing method on seeds and in vitro embryos cryopreservation. The results showed that the germination rate and vigor decreased from 82.67%, 85% to 18.35%, 25% respectively, when the seed moisture content decreased from 15.04% to 8.14%; and the germination rate decreased from 82.67% to 37.50%, 25.37% respectively by vitrification freezing method and rapid freezing method, when the seed moisture content decreased from 15.04% to 9.37%. Among all the moisture content gradient, 12.35% moisture reached the maximal germination rate, which were 63.58% and 50.45% respectively by vitrification freezing and rapid freezing; and when the embryo moisture content was 26.32%, the germination rate decreased from 95.67% to 58.31% and 33.82% respectively by vitrification freezing and rapid freezing. And when the moisture content was in the range of 14.17% -21.34%, the germination rate was a bit of decrease. The experiment results showed that the optimum conditions of seed cryopreservation were: moisture content 12.35%, vitrification freezing; and the optimum conditions of in vitro embryo cryopreservation were: moisture 15.04%, vitrification freezing. In conclusion, the effects of moisture content on germination rate after cryopreservation in D. odorifera seeds and embryo were significant, and vitrification freezing method is much better than rapid freezing method.


    Pablos, María Teresa Prieto; Saragusty, Joseph; Santiago-Moreno, Julián; Stagegaard, Julia; Göritz, Frank; Hildebrandt, Thomas Bernd; Hermes, Robert


    Genetic diversity is a primary component of adaptive evolution, and its loss or reduction can decrease the long-term survival probability of populations. Utilization of cryopreserved semen may be considered a perfect tool to improve genetic diversity, reduce inbreeding, and avoid animal translocation for breeding. The present study aimed at finding a reliable epididymal sperm freezing protocol for the critically endangered onager (Equus hemionus onager). Six testicles from three animals were processed postmortem. The effects of two transportation temperatures (22°C and 4°C; testicles submerged in saline), two cryopreservation techniques (conventional liquid nitrogen vapor freezing in straws and directional freezing in 8-ml HollowTubes(TM)), and two postthaw incubation temperatures (22°C and 37°C; evaluated after 0.5, 1, 2, and 3 hr) were tested in a 2×2×2 experimental design. Sperm samples were evaluated for motility, viability, acrosome integrity, and sperm morphology. The resulting optimal freezing protocol includes transportation of testicles at 4°C, cryopreservation by directional freezing, and, if needed, postthaw incubation at 22°C. With this combination of transportation temperature and cryopreservation technique, the authors obtained the following postthaw values normalized to prefreezing values: 60.3±8.8% motility, 60.7±13.3% viability, 75.3±9.5% acrosome integrity, and 94.7±2.9% normal morphology (excluding defects due to the epididymal origin of the sperm). After incubation at 22°C, motility values for the above combination were 40±5.7%, 30.3±5.2%, 28.3±4.4%, and 16.7±4.4% for 0.5, 1, 2, and 3 hr, respectively. In conclusion, with this protocol, good quality semen can be stored for future use in artificial inseminations when and where needed.

  8. Serum-Free Cryopreservation of Human Amniotic Epithelial Cells


    H. Niknejad; H. Peirovi; B. Jambar Noushin


    Introduction & Objective: One of the important issues in long term storage of cells is removal of animal serum from cell culture environments. The aim of this study was to evaluate amni-otic fluid (AF), which is full of growth factors, as substitute for fetal bovine serum (FBS) in the cryopreservation protocol. Materials & Methods: In this experimental study human amniotic epithelial cells were isolated from placentas which were seronegative for microbial infections. The cells were preserved ...

  9. [Current status and future perspectives of hepatocyte transplantation]. (United States)

    Pareja, Eugenia; Cortés, Miriam; Gómez-Lechón, M José; Maupoey, Javier; San Juan, Fernando; López, Rafael; Mir, Jose


    The imbalance between the number of potential beneficiaries and available organs, originates the search for new therapeutic alternatives, such as Hepatocyte transplantation (HT).Even though this is a treatment option for these patients, the lack of unanimity of criteria regarding indications and technique, different cryopreservation protocols, as well as the different methodology to assess the response to this therapy, highlights the need of a Consensus Conference to standardize criteria and consider future strategies to improve the technique and optimize the results.Our aim is to review and update the current state of hepatocyte transplantation, emphasizing the future research attempting to solve the problems and improve the results of this treatment. Copyright © 2013 AEC. Published by Elsevier Espana. All rights reserved.

  10. Transplant Ethics. (United States)

    Altınörs, Nur; Haberal, Mehmet


    The aim of this study was to review and discuss the great variety of ethical issues related to organ donation, organ procurement, transplant activities, and new ethical problems created as a result of technologic and scientific developments. An extensive literature survey was made, and expert opinions were obtained. The gap between demand and supply of organs for transplant has yielded to organ trafficking, organ tourism, and commercialism. This problem seems to be the most important issue, and naturally there are ethical dilemmas related to it. A wide number of ideas have been expressed on the subject, and different solutions have been proposed. The struggle against organ trafficking and commercialism should include legislation, efforts to increase deceased-donor donations, and international cooperation. China's policy to procure organs from prisoners sentenced to death is unethical, and the international community should exert more pressure on the Chinese government to cease this practice. Each particular ethical dilemma should be taken separately and managed.

  11. Cryopreservation of somatic embryos of paradise tree (Melia azedarach L.). (United States)

    Scocchi, Adriana; Vila, Silvia; Mroginski, Luis; Engelmann, Florent


    In paradise tree (Melia azedarach L.), immature zygotic embryos sampled from immature fruits are the starting material for the production of somatic embryos. These somatic embryos are employed for freezing experiments. Immature fruits could be stored at 25 degrees C for up to 80 days without impairing the embryogenic potential of zygotic embryos, which represents a four-fold increase in immature fruit storage duration, compared with previous studies. Among the three cryopreservation techniques tested for freezing paradise tree somatic embryos, namely desiccation, encapsulation-dehydration and pregrowth-dehydration, only encapsulation-dehydration and pregrowth-dehydration led to successful results. The optimal protocol was the following: i) somatic embryos (encapsulated or not) pretreated in liquid Murashige & Skoog medium with daily increasing sucrose concentration (0.5 M/0.75 M/1.0 M); ii) dehydrated with silica gel to 21 - 26% moisture content (fresh weight basis), for encapsulation-dehydration, or to 19% moisture content, for pregrowth-dehydration; iii) frozen at 1 degree C/min from 20 degrees C to -30 degrees C with a programmable freezing apparatus; iv) rapid immersion in liquid nitrogen. The highest recovery achieved was 36% with encapsulation-dehydration and 30% with pregrowth-dehydration. Regrowth of frozen embryos was direct in most cases, as secondary embryogenesis originating from the root pole was observed on only around 10% of cryopreserved somatic embryos. Plants recovered from cryopreserved embryos presented the same phenotypic traits as non-frozen control plants.

  12. Highly efficient vitrification method for cryopreservation of human oocytes. (United States)

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


    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.

  13. Cryopreservation: a cold look at technology for fertility preservation. (United States)

    Gosden, Roger


    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.

  14. Current status and future direction of cryopreservation of camelid embryos. (United States)

    Herrid, M; Vajta, G; Skidmore, J A


    Over the past 3 decades, and similar to the horse industry, fresh embryo transfer has been widely practiced on large commercial scales in different camelid species, especially the dromedary camel and alpaca. However, the inability to cryopreserve embryos significantly reduces its broader application, and as such limits the capacity to utilize elite genetic resources internationally. In addition, cryopreservation of the semen of camelids is also difficult, suggesting an extreme sensitivity of the germplasm to cooling and freezing. As a result, genetic resources of camelids must continue to be maintained as living collections of animals. Due to concerns over disease outbreaks such as that of the highly pathogenic Middle East Respiratory Syndrome in the Middle East and Asia, there is an urgent need to establish an effective gene banking system for camelid species, especially the camel. The current review compares and summarizes recent progress in the field of camelid embryo cryopreservation, identifying four possible reasons for the slow development of an effective protocol and describing eight future directions to improve the current protocols. At the same time, the results of a recent dromedary camel embryo transfer study which produced a high morphologic integrity and survival rate of Open Pulled Straw-vitrified embryos are also discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Inhibiting ice recrystallization and optimization of cell viability after cryopreservation. (United States)

    Chaytor, Jennifer L; Tokarew, Jacqueline M; Wu, Luke K; Leclère, Mathieu; Tam, Roger Y; Capicciotti, Chantelle J; Guolla, Louise; von Moos, Elisabeth; Findlay, C Scott; Allan, David S; Ben, Robert N


    The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220 mM solutions of disaccharides; however, the best cell viability was obtained when a 200 mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.

  16. Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos by vitrification. (United States)

    Adu-Gyamfi, Raphael; Wetten, Andy


    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.

  17. Switching between intravenous and subcutaneous trastuzumab

    DEFF Research Database (Denmark)

    Gligorov, Joseph; Curigliano, Giuseppe; Müller, Volkmar


    AIM: To assess the safety and tolerability of switching between subcutaneous (SC) and intravenous (IV) trastuzumab in the PrefHer study (NCT01401166). PATIENTS AND METHODS: Patients with HER2-positive early breast cancer completed (neo)adjuvant chemotherapy and were randomised to receive four....... Rates of clinically important events, including grade ≥3 AEs, serious AEs, AEs leading to study drug discontinuation and cardiac AEs, were low and similar between treatment arms (safety signals for trastuzumab were observed. CONCLUSIONS: PrefHer revealed...... that switching from IV to SC trastuzumab (hand-held syringe or SID) or vice versa did not impact the known safety profile of trastuzumab....

  18. Subcutaneous myiasis caused by Dermatobia hominis. (United States)

    Logar, J; Beović, B; Triller, C; Rakovec, S


    A case of subcutaneous myiasis caused by the larvae of the Dermatobia hominis fly is described, involving the ankle region of a 25-y-old man who had returned from Peru. After removal of 4 larvae from the affected sites, the lesions healed in 2 weeks without further treatment. Because of the increasing number of people travelling to tropical America, physicians in Slovenia will have to consider Dermatobia myiasis in the differential diagnosis of furuncular lesions in patients with a relevant travel history.

  19. Stem Cell Transplant (United States)

    ... Graft-versus-host disease: A potential risk when stem cells come from donors If you receive a transplant ... medications and blood products into your body. Collecting stem cells for transplant If a transplant using your own ...

  20. 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.


    The usefulness of five extenders for cryopreservation of muskellunge semen was studied in fertlization trials and computer-assisted semen analyses (CASA) of postthaw sperm motility. The effect of pre-freezing storage time before cryopreservation on success of cryopreservation was...

  2. Cryopreserved cultured epithelial allografts for pediatric deep partial dermal burns: Early wound closure and suppression of scarring

    Directory of Open Access Journals (Sweden)

    Hiroko Yanaga


    Conclusion: Cryopreserved allo-CEG contains growth factors that promote wound healing and factors that suppress scarring. Three effects, namely (1 early wound closure, (2 scarring suppression, and (3 pain relief were seen with grafts of cryopreserved allo-CEG in cases of childhood DDB. These observations show that cryopreserved allo-CEG is clinically useful and effective for the treatment of childhood DDB.

  3. Facial transplantation: a review of ethics, progress, and future targets

    Directory of Open Access Journals (Sweden)

    Edwards JA


    Full Text Available James A Edwards1, David W Mathes21Department of Plastic and Reconstructive Surgery, Skagit Valley Hospital, Mount Vernon, WA, USA; 2Division of Plastic Surgery, University of Washington Medical Center, Seattle, WA, USAAbstract: The surgical history of transplantation in the modern era begins in 1956 with the successful transplantation of a kidney between identical twins. Since then the field of transplantation has seen remarkable advancements in both surgical techniques and our understanding and ability to manipulate the immune response. Composite tissue allotransplantation involves the transplantation of any combination of vascularized skin, subcutaneous tissue, blood vessels, nerves, muscle, and bone. Orthotopic hand transplantation is considered the first clinical example of CTA and has seen success at many different centers worldwide. Facial allotransplantation is a recent development in the field of CTA and the first successful case was performed as recently as November 2005. Since then there have been a number of successful facial transplants. The purpose of this paper is to examine some of the issues surrounding facial transplantation including the complex ethical issues, the surgical and clinical issues, cost and administrative issues, and future directions for this new, exciting, and controversial field.Keywords: composite tissue allograft, facial transplantation

  4. Kinetics of subcutaneous NPH insulin in diabetics

    Energy Technology Data Exchange (ETDEWEB)

    Kolendorf, K.; Bojsen, J.


    Kinetics of subcutaneous NPH insulin were studied in newly diagnosed insulin-dependent diabetics. Using a biotelemetric technique with small Geiger-Mueller detectors applied to the skin surface, the disappearance of 125I-NPH insulin fron subcutis was monoexponential with a mean half-life of 6.6 +/- 3.3 (SD) hr. A model is presented to compare the disappearance rates of subcutaneous 125I-NPH insulin and the calculated plasma appearance insulin curve derived from the actual plasma insulin concentration measurements, assuming a one-compartment model and first-order kinetics. Areas under the absorption- and appearance-time curves calculated from external measurements and from plasma insulin concentrations were identical. There was a strong correlation between plasma concentrations and absorbed amounts of labeled insulin (r . 0.8782, P less than 0.001), as there was between the blood glucose-lowering effects and percent absorbed insulin per hour (r . 0.7659, P less than 0.001). Our results indicate that the disappearance rate of iodine-labeled insulin was a relevant biological expression of insulin absorption from subcutis and a reliable noninvasive method of quantitative determination of insulin concentration in blood.

  5. Recovery of fertility from adult ovarian tissue transplanted into week-old Japanese quail chicks. (United States)

    Liu, Jianan; Cheng, Kimberly M; Silversides, Frederick G


    Fertility of cryopreserved ovarian tissue from immature chickens and Japanese quail has been recovered by transplantation. This is of special importance for non-mammalian vertebrates in which cryopreservation and in vitro maturation of oocytes are challenging because their oogenesis is characterised by vitellogenesis. This study tested whether fertility of adult quail ovarian tissue could be recovered by transplantation. Ovaries were isolated from mature Japanese quail hens, trimmed, cut into 3- to 4-mm2 pieces and transplanted into ovariectomised, week-old chicks. Recipients were administered an immunosuppressant for two weeks. Ten of 12 recipients survived until sexual maturity and seven laid eggs, but all stopped laying by 17 weeks of age. The age at first egg of recipients laying eggs (75.7±4.2 days) was greater than that of untreated hens (51.8±1.7 days) and egg production of recipients during the laying period (21.7±5.7) was less than that of untreated hens (60.8±3.5). Recipients were paired with males from the WB line for test mating. Only two hens laid eggs during the test period but both produced 100% donor-derived offspring. This research demonstrated that the reproductive potential of ovarian tissue from adult quail hens can be restored by transplantation.

  6. Cryopreservation of periodontal ligament cells with magnetic field for tooth banking. (United States)

    Kaku, M; Kamada, H; Kawata, T; Koseki, H; Abedini, S; Kojima, S; Motokawa, M; Fujita, T; Ohtani, J; Tsuka, N; Matsuda, Y; Sunagawa, H; Hernandes, R A M; Ohwada, N; Tanne, K


    The purpose of this study was to establish a long-term tooth cryopreservation method that can be used for tooth autotransplantation. Human periodontal ligament (PDL) cells were frozen in 10% dimethyl sulfoxide (Me(2)SO) using a programmed freezer with a magnetic field. Cells were cryopreserved for 7 days at -150 degrees C. Immediately after thawing, the number of surviving cells was counted and the cells were cultured; cultured cells were examined after 48 h. Results indicated that a 0.01 mT of a magnetic field, a 15-min hold-time, and a plunging temperature of -30 degrees C led to the greatest survival rate of PDL cells. Based on these findings, whole teeth were cryopreserved under the same conditions for 1 year. The organ culture revealed that the PDL cells of cryopreserved tooth with a magnetic field could proliferate as much as a fresh tooth, although the cells did not appear in the cryopreserved tooth without a magnetic field. Histological examination and the transmission electron microscopic image of cryopreserved tooth with a magnetic field did not show any destruction of cryopreserved cells. In contrast, severe cell damage was seen in cells frozen without a magnetic field. These results indicated that a magnetic field programmed freezer is available for tooth cryopreservation. (c) 2010 Elsevier Inc. All rights reserved.

  7. Effect of storage time on the viability of cryopreserved bovine spermatozoa (United States)

    Long term cryopreserved semen viability can impact the National Animal Germplasm Program’s (NAGP) sampling strategy and ability to reconstitute livestock populations. Therefore, the purpose of this project was to determine if prolonged storage of cryopreserved sperm impacts cell viability. Cryoprese...


    Computer-assisted motility analysis (CASA) was used to evaluate the effect of cryopreservation and theophylline treatment on sperm motility of lake sturgeon (Acipenser fulvescens).Motility was recorded at 0 and 5 min postactivation.The effect of cryopreservation on sperm acrosin-...

  9. Enhanced fertility prediction of cryopreserved boar spermatozoa using novel sperm function assessment (United States)

    Cryopreserved semen is seldom used for commercial porcine artificial insemination (AI) despite many advantages that cryopreservation provides. Compared to fresh semen, the fertility of frozen-thawed boar sperm is more variable but usually less. Predicting the fertility of individual ejaculates for s...

  10. 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

  11. Effects of Seed Cryopreservation and Priming on Germination in Several Cultivars of Apium graveolens. (United States)

    Gonzalez-Benito, M E; Iriondo, J M; Pita, J M; Pérez-García, F


    Seed germination of seven celery cultivars was studied after storage in liquid nitrogen for 1 or 30 d. Cryopreservation was also carried out on pelleted and primed seeds. None of the treatments applied reduced germination percentages. T(50) (time for germination to reach 50%) significantly decreased in Florida, Utah and Istar cultivars when priming, alone or in combination with cryopreservation, was used.

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

    Directory of Open Access Journals (Sweden)



    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.;

  13. Effect of pre-storage on Lilium martagon L. seed longevity following cryopreservation. (United States)

    Urbaniec-Kiepura, M; Bach, A


    The aim of the study was to compare different strategies for the cryopreservation of Lilium martagon L. seeds. The starting material was seeds of the martagon lily (without or with a special pretreatment involving sucrose pre-culture dehydratation and desiccation) subjected to cryopreservation after preliminary storage under conventional conditions (-5 degree C, 5 degree C, or 15 degree C). The effects of storage, pretreatment and cryopreservation on the seeds were assessed in terms of seed germination and phenotypic changes in the seedlings derived from those seeds. Subjecting the seeds to cold hardening or pretreatment did not affect significantly their germination capacity or the average germination time after cryopreservation. Combination of these strategies, however, significantly affected the capacity of the seeds to germinate. The highest germinability (100%) after cryopreservation was observed in the seeds stored at 15 degree C and subjected to sucrose pre-culture dehydration and air flow desiccation (seed moisture content [MC] 13.1%), and in those seeds stored at the same temperature but subjected subsequently to 5 h desiccation, not pretreated, with MC of 7.6 degree. Cryopreservation did not affect the germination capacity of the seeds but shortened the average germination time from 40.7 to 34.4 days. The obtained results demonstrate that a simple cryopreservation method without any special pretreatment of seeds can be used successfully for preserving L. martagon seeds. Neither the low temperature nor the sucrose concentration used affected the germination capacity or the average germination time of L. martagon seeds following cryopreservation.

  14. Bioprocessing of Cryopreservation for Large-Scale Banking of Human Pluripotent Stem Cells (United States)

    Ma, Teng


    Abstract Human pluripotent stem cell (hPSC)-derived cell therapy requires production of therapeutic cells in large quantity, which starts from thawing the cryopreserved cells from a working cell bank or a master cell bank. An optimal cryopreservation and thaw process determines the efficiency of hPSC expansion and plays a significant role in the subsequent lineage-specific differentiation. However, cryopreservation in hPSC bioprocessing has been a challenge due to the unique growth requirements of hPSC, the sensitivity to cryoinjury, and the unscalable cryopreservation procedures commonly used in the laboratory. Tremendous progress has been made to identify the regulatory pathways regulating hPSC responses during cryopreservation and the development of small molecule interventions that effectively improves the efficiency of cryopreservation. The adaption of these methods in current good manufacturing practices (cGMP)-compliant cryopreservation processes not only improves cell survival, but also their therapeutic potency. This review summarizes the advances in these areas and discusses the technical requirements in the development of cGMP-compliant hPSC cryopreservation process. PMID:23515461

  15. Cryopreservation affects ROS-induced oxidative stress and antioxidant response in Arabidopsis seedlings (United States)

    Plant recovery status after cryopreservation by vitrification had a negative relationship to the oxidative stress induced by reactive oxygen species (ROS). Arabidopsis thaliana seedlings germinated for 48-h or 72-h with different cryopreservation survival tolerances were examined at five steps of a ...

  16. Developing a Clinical-Grade Cryopreservation Protocol for Human Testicular Tissue and Cells (United States)

    Pacchiarotti, Jason; Ramos, Thomas; Howerton, Kyle; Greilach, Scott; Zaragoza, Karina; Olmstead, Marnie; Izadyar, Fariborz


    Recent work in preservation of female fertility as well as new information on the nature of spermatogonial stem cells has prompted an investigation into the possibility of an effective clinical-grade procedure for the cryopreservation of testicular cells and/or tissue. Clinical-grade reagents, validated equipment, and protocols consistent with cGTP/cGMP standards were used in developing a procedure suitable for the safe and effective cryopreservation of human testicular cells and tissues. These procedures were designed to be compliant with the relevant FDA regulations. The procedure proved to effectively cryopreserve both testicular cells and tissue. The cryopreservation of testicular tissue was comparable in most aspects we measured to the cryopreservation of isolated cells, except that the viability of the cells from cryopreserved testicular tissue was found to be significantly higher. On the other hand, cryopreservation of cells is preferred for cell analysis, quality control, and sterility testing. This study demonstrates that testicular tissue and cells from sexual reassignment patients can be successfully cryopreserved with a clinical-grade procedure and important cell populations are not only preserved but also enriched by the process. Further studies will determine whether these findings from hormone-treated patients can be generalized to other patients. PMID:23509810

  17. Impact of current cryopreservation procedures on mechanical and functional properties of human aortic homografts

    NARCIS (Netherlands)

    Langerak, S. E.; Groenink, M.; van der Wall, E. E.; Wassenaar, C.; VanBavel, E.; van Baal, M. C.; Spaan, J. A.


    We evaluated the impact f standard cryopreservation on mechanical and functional properties of human aortic homografts. From 14 human heart-valve donors, the thoracic descending aorta was obtained. Effects of cryopreservation on mechanical (elastic properties and breaking stress) and smooth muscle

  18. Cryopreservation of precision-cut tissue slices for application in drug metabolism research

    NARCIS (Netherlands)

    de Graaf, I.A.M.; Koster, H

    Cryopreservation of tissue slices greatly facilitates their use in drug metabolism research, leading to efficient use of human organ material and a decrease of laboratory animal use. In the present review, various mechanisms of cryopreservation such as equilibrium slow freezing, rapid freezing and

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

    NARCIS (Netherlands)

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


    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

  20. Recurrent subcutaneous emphysema of the face: a challenging clinical problem. (United States)

    Hojjati, Hossein; Davani, Sam Zeraatian Nejad; Johari, Hamed Ghoddusi


    In the neck or face, there are different causes for subcutaneous emphysema such as injury to the sinuses, the hypopharynx, the laryngotracheal complex, the pulmonary parenchyma, the esophagus or the presence of gas-forming organisms. However, factitious subcutaneous emphysema, a rare cause, must be considered in the differential diagnosis. In this clinical report, we discuss a 20-year-old girl who was under follow-up because of recurrent subcutaneous emphysema of the face and periorbital area. After 2 years of work-ups, including a period of close observation in the intensive care unit, self air injection by syringe was found as the cause of recurrent subcutaneous emphysema of the face, and the patient was labeled as having factitious recurrent subcutaneous emphysema. Therefore, when a patient presents with unexplained recurrent subcutaneous emphysema, one should suspect self-infliction and examine for puncture marks.

  1. Transplante uterino


    Carvalho, Margarida Teixeira Farias Meira de, 1992-


    Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2016 A infertilidade de causa uterina afecta cerca de 3-5% da população geral, sendo a adopção e a gestação de substituição as únicas opções para estes casais atingirem a parentalidade, dado que é um problema ainda sem tratamento. Nas últimas décadas, foram realizados diversos estudos animais com o objectivo de demonstrar a viabilidade, segurança e eficácia do transplante uterino como ...


    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.

  3. Evolving trends in cryopreservation and parameters influencing semen extender preparation - a prospective review. (United States)

    Sridharan, T B; Vickram, A S


    Cryopreservation is a technique by which, semen can be preserved to subzero temperature, usually at -196° C. The freezing of semen desires vitrification mediators that diminish wreck to the cells (spermatozoan) during the freeze and thaw process. Using cryopreservation, the quality of the semen has been increased in the latest years, by which the achievement rate for the insemination techniques has increased in an agreed way. The area need to be focused is to enhance the quality of the semen extender preparation before cryopreservation. Many researchers are working in the area of cryopreservation of human semen with different semen extenders. Several parameters influence the properties of semen extender essential for better post thaw results. This review is mainly focused on a range of parameters which influence the best semen extender for cryopreservation that includes glycerol and its importance, buffer and novel usage of antimicrobial peptides as antimicrobial agents.

  4. Cryopreservation of banana’s cv Grand Naine in vitro rhizomes

    Directory of Open Access Journals (Sweden)



    Full Text Available ABSTRACT The preservation of banana genetic material is usually performed through seedlings. However, most banana cultivars do not produce seed and are propagated vegetatively. Therefore, cryopreservation is a feasible technique that allows the preservation of banana genotypes indefinitely. For the success of cryopreservation protocols, the selection of cryoprotectants and pre-freezing techniques are important factor. Therefore, the objective of this study was to verify the effects of different cryoprotectants with and without 1% phloroglucinol and pre-cooling periods on the development of a protocol for cryopreservation of in vitro rhizomes ofMusa accuminata(AAA cv Grand Naine banana. The addition of 1% phloroglucinol to the cryoprotective solutions, such as PVS2 enhanced recovery of cryopreserved banana rhizomes. In addition, pre-cooling of explants in ice for 3 hours in PVS2 + 1% of phloroglucinol allowed efficient cryopreservation of banana rhizomes, followed by successful recovery and regeneration of in vitro shoots of banana cv Grand Naine.

  5. Character, distribution and biological implications of ice crystallization in cryopreserved rabbit ovarian tissue revealed by cryo-scanning electron microscopy. (United States)

    Gosden, Roger G; Yin, Hang; Bodine, Richard J; Morris, G John


    Ovarian tissue banking is an emerging strategy for fertility preservation which has led to several viable pregnancies after transplantation. However, the standard method of slow cooling was never rigorously optimized for human tissue nor has the extent and location of ice crystals in tissue been investigated. To address this, we used cryo-scanning electron microscopy (cryo-SEM) to study ice formation in cryopreserved ovarian tissue. Rabbit ovarian tissue slices were equilibrated in 1,2-propanediol-sucrose solution and cooled at either 0.3 degrees C/min or 3.0 degrees C/min after nucleating ice at -7 degrees C, or snap-frozen by plunging in liquid nitrogen. Frozen tissues were fractured, etched and coated with gold or prepared by freeze substitution and sectioning for cryo-SEM. The size, location and orientation of extracellular ice crystals were revealed as pits and channels that had grown radially between freeze-concentrated cellular materials. They represented 60% of the total volume in slowly cooled samples that were nucleated at -7 degrees C and the crystals, often >30 microm in length, displaced cells without piercing them. Samples cooled more rapidly were much less dehydrated, accounting for the presence of small ice crystals inside cells and possibly in organelles. Cryo-SEM revealed the internal structure of ovarian tissue in the frozen state was dominated by elongated ice crystals between islands of freeze-concentrated cellular matrix. Despite the grossly distorted anatomy, the greater degree of dehydration and absence of intracellular ice confirmed the superiority of a very slow rate of cooling for optimal cell viability. These ultrastructural methods will be useful for validating and improving new protocols for tissue cryopreservation.

  6. Subcutaneous Immunoglobulins: A Promising Alternative for Immunomodulation? (United States)

    Sanchez-Ramon, Silvia; Corbi, Angel L; Fidalgo, Agueda Garcia; Dominguez-Soto, Angeles


    Cumulative recent evidence from clinical trials, observational studies and case reports has shown that subcutaneous administration of immunoglobulin (SCIg) exerts similar immunomodulatory capacity than intravenous immunoglobulin (IVIg) in autoimmune neurological diseases. Besides the beneficial clinical effects, the profile of safety and autonomy for the patient is higher for SCIg, while it is cost-saving in terms of the health resources used. However, there are still very few approved indications for SCIg and a certain resistance to choose SCIg for other autoimmune conditions even despite patients' interests. Here we present an updated review of the known immunomodulatory mechanisms of action of Ig and the current hypothesis supporting the clinical and immunological advantages of SCIg over IVIg that derive from their specific pharmacokinetic features. Copyright© Bentham Science Publishers; For any queries, please email at

  7. [ANSYS simulation of subcutaneous pustule electrical characteristics]. (United States)

    Liu, Baohua; Wang, Xuan; Zhu, Honglian; Wang, Guoyong


    With the growing number of clinical surgery, post-operative surgical wound infection has become a very difficult clinical problem. In the treatments of it, non-invasive test of wound infection and healing status has a significance in clinical medicine practice. In this paper, beginning with the electrical properties of skin tissue structure and on the basis of the electromagnetism and the human anatomy, using the finite element analysis software, we applied safe voltage on the 3D skin model, performed the subcutaneous pustule simulation study and gained the relational curve between depth and radius of the pustule model. The simulation results suggested that the method we put forward could be feasible, and it could provide basis for non-invasive detection of wound healing and wound infection status.

  8. Eczematous dermatitis due to subcutaneous teriparatide injection. (United States)

    Chu, Howard; Kim, Dae Suk


    Teriparatide, or recombinant human parathyroid hormone, is approved for the treatment of osteoporosis. Possible cutaneous adverse events of teriparatide are urticaria, injection site pain, swelling, bruising, and pruritus. However, there have been no reports of widespread eczematous reactions caused by teriparatide. A 47-year-old male was recently diagnosed with osteogenesis imperfecta and prior to teeth extractions, he was subcutaneously injected with teriparatide. The patient developed multiple pruritic erythematous papules and plaques on his abdomen, around the site of the injection. A skin biopsy was done, which showed mild spongiosis and superficial perivenular lymphocytic infiltration with a few eosinophils. Drug-related eczematous changes were most likely suspected and in addition to the discontinuation of the injection, topical steroid was prescribed, in which dramatic improvements were observed. We report the eczematous hypersensitivity reaction caused by teriparatide, which is an adverse reaction that has not been reported before.

  9. Cryopreservation of mouse embryos by ethylene glycol-based vitrification. (United States)

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


    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

  10. Cryopreservation of sperm bundles (spermatozeugmata) from endangered livebearing goodeids. (United States)

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


    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. Published by Elsevier Inc.

  11. Cryopreservation of captive roe deer (Capreolus capreolus) semen. (United States)

    Prieto-Pablos, M T; Sánchez-Calabuig, M J; Hildebrandt, T B; Göritz, F; Ortmann, S; Eder, S; Santiago-Moreno, J; Hermes, R; Saragusty, J


    To address the need to preserve current genetic diversity before it is lost forever; further studies to adapt assisted reproductive technologies to various endangered species are needed, among other things. Roe deer (Capreolus capreolus), an over abundant wild deer, can serve as model species to develop or improve sperm cryopreservation of threatened or endangered deer species. The aim of this study was to compare the ability of three diluents (Berliner Cryomedium [BC]; Tris, citric acid, glucose [TCG]; TES, Tris, glucose) to support chilling, cryopreservation (with 5% glycerol; G) and postthaw incubation (at 22 °C and 37 °C) of roe deer spermatozoa collected by electroejaculation. Berliner Cryomedium was the diluent that better preserved roe deer spermatozoa during refrigeration, able to maintain motility for at least 14 days, longer than the other extenders. BC + G was the extender of choice for cryopreservation, showing higher viability compared with TCG + G (66.7 ± 3.4 vs. 54.5 ± 6.5; P < 0.05) and higher level of acrosome integrity compared with TES, Tris, glucose + G (79.4 ± 3.4 vs. 67.9 ± 5.0; P < 0.05). Maintaining the samples at 22 °C after thawing presented higher values in various parameters compared with 37 °C. The knowledge gained through this study can potentially act as a preliminary step toward development of new protocols to help increase the reproductive success of biologically similar, yet endangered, wild species. Copyright © 2016 Elsevier Inc. All rights reserved.


    Directory of Open Access Journals (Sweden)

    Jessica Neri Nascimento


    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.

  13. Regulatory considerations for global transfer of cryopreserved fish gametes (United States)

    Jenkins, Jill A.; Tiersch, Terrence R.; Green, Christopher C.


    Federal and state resource managers, scientists, lawmakers, business and development investors, and the general public all struggle with issues surrounding the conservation of our biological heritage, especially in the face of increased population growth and consequent anthropogenic disturbances. Conservation interests include recovering exploited aquatic populations, decreasing the loss of genetic diversity, and reintroducing locally depleted species. However, research on husbandry and other techniques critical to implementing conservation strategies is often not started until few individuals remain. A program in the cryopreservation of gametes and embryos from aquatic species would address several of these conservation concerns by allowing the establishment of gene banks

  14. Use of cryopreserved peripheral mononuclear blood cells in biomonitoring

    DEFF Research Database (Denmark)

    Risom, Lotte; Knudsen, Lisbeth E.


    This study was performed to investigate the effect of storing blood samples by freezing on selected biomarkers and possible implications for biomonitoring. Comparative measurements were performed in order to investigate the use of cryopreserved vs. freshly separated peripheral mononuclear blood...... cells (PMBC) obtained from donor blood. Measurements of DNA-repair, mutant frequency, and subcell content were included. Samples for large biomonitoring studies are usually taken from study groups within a short time period of days/weeks and storing of study material for later analysis can be necessary...

  15. Human hepatocyte transplantation for liver disease: current status and future perspectives. (United States)

    Iansante, V; Mitry, R R; Filippi, C; Fitzpatrick, E; Dhawan, A


    Liver transplantation is the accepted treatment for patients with acute liver failure and liver-based metabolic disorders. However, donor organ shortage and lifelong need for immunosuppression are the main limitations to liver transplantation. In addition, loss of the native liver as a target organ for future gene therapy for metabolic disorders limits the futuristic treatment options, resulting in the need for alternative therapeutic strategies. A potential alternative to liver transplantation is allogeneic hepatocyte transplantation. Over the last two decades, hepatocyte transplantation has made the transition from bench to bedside. Standardized techniques have been established for isolation, culture, and cryopreservation of human hepatocytes. Clinical hepatocyte transplantation safety and short-term efficacy have been proven; however, some major hurdles-mainly concerning shortage of donor organs, low cell engraftment, and lack of a long-lasting effect-need to be overcome to widen its clinical applications. Current research is aimed at addressing these problems, with the ultimate goal of increasing hepatocyte transplantation efficacy in clinical applications.Pediatric Research advance online publication, 6 December 2017; doi:10.1038/pr.2017.284.

  16. Role of Liposuction Combined with Subcutaneous Mastectomy in ...

    African Journals Online (AJOL)

    were ruled out. Subcutaneous mastectomy using infraareolar skin incision combined with liposuction was performed on. 26 patients. ... Subcutaneous mastectomy is one of the common procedures performed with an infraareolar ... Mean age of the patients was 24 (±6) years; most of them were between 20 and 30 years old.

  17. Absorption Kinetics of Subcutaneously Administered Ceftazidime in Hypoperfused Guinea Pigs

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Ebihara, MS


    Conclusions: The application of MWCs or WCs enhanced subcutaneous CAZ absorption by increasing blood flow. MWCs and WCs are considered to be safe and routine methods to induce defecation after surgery on the digestive system; thus, the combination of these methods and subcutaneous CAZ administration is a potential method for treating pneumonia in patients with SMID.

  18. The comparison of the intestinal adaptation effects of subcutaneous ...

    African Journals Online (AJOL)

    Aim: Insulin has been reported to have positive effects on intestinal adaptation after short bowel syndrome when applicated oral or subcutaneously. The purpose of this study is to compare the intestinal adaptation effects of subcutaneous and oral routes of insulin in rats with short bowel syndrome. Materials and Methods: ...

  19. Cost-minimization of mabthera intravenous versus subcutaneous administration

    NARCIS (Netherlands)

    Bax, P.; Postma, M.J.


    Objectives: To identify and compare all costs related to preparing and administrating MabThera for the intravenous and subcutaneous formulations in Dutch hematological patients. The a priori notion is that the costs of subcutaneous MabThera injections are lower compared to intravenous infusion due

  20. IVF recovery of mutant mouse lines using sperm cryopreserved with mtg in cryovials. (United States)

    Li, Ming-Wen; Vallelunga, Jadine M; Kinchen, Kristy L; Rink, Karina L; Zarrabi, Jasmin; Shamamian, Armen O; Lloyd, K C Kent


    Modification of cryoprotective medium (CPM) R18S3 (18% raffinose and 3% skim milk) by addition of monothioglycerol (MTG) or L-glutamine (Glu) has been shown to improve in vitro fertilization (IVF) using mouse sperm cryopreserved in cryostraws. However, whether these CPMs can be applied effectively to sperm cryopreserved in cryovials is unknown. The study was to determine the comparative effectiveness of using R18S3, R18S3+Glu (100mM and 87 mM), or R18S3+MTG (477 µM) to cryopreserve various sample volumes of mouse sperm in cryovials and cryostraws. This study compared the effects of different CPMs on motility of fresh and frozen-thawed C57BL/6J sperm and on IVF rate of C57BL/6J sperm cryopreserved in different CPMs and containers with different volumes, and then used technologies developed to cryopreserve and recover sperm of knockout mouse lines on inbred C57BL/6 backgrounds. Glutamine at 100 mM inhibited, but MTG at 477 µM protected, fresh sperm motility significantly (P 0.05). Sperm from 63 knockout mouse lines on C57BL/6 backgrounds cryopreserved using R18S3+MTG in cryovials were all recovered successfully to genotypically-confirmed offspring. Mouse sperm on C56BL/6 backgrounds can be successfully cryopreserved in cryovials using R18S3+MTG.

  1. Cryopreservation Method for the Effective Collection of Dental Pulp Stem Cells. (United States)

    Takebe, Yusuke; Tatehara, Seiko; Fukushima, Tatsuhiro; Tokuyama-Toda, Reiko; Yasuhara, Rika; Mishima, Kenji; Satomura, Kazuhito


    Dental pulp stem cells (DPSCs) are an attractive cell source for use in cell-based therapy, regenerative medicine, and tissue engineering because DPSCs have a high cell proliferation ability and multidifferentiation capacity. However, several problems are associated with the collection and preservation of DPSCs for use in future cell-based therapy. In particular, the isolation of DPSCs for cryopreservation is time consuming and expensive. In this study, we developed a novel cryopreservation method (NCM) for dental pulp tissues to isolate suitable DPSCs after thawing cryopreserved tissue. Using the NCM, dental pulp tissues were cultured on adhesion culture dishes for 5 days and then cryopreserved. After thawing, the cryopreserved dental pulp tissue fragments exhibited cell migration. We evaluated each property of DPSCs isolated using the NCM (DPSCs-NCM) and the explant method alone without cryopreservation (DPSCs-C). DPSCs-NCM had the same proliferation capacity as DPSCs-C. Flow cytometry (FACS) analysis indicated that both DPSCs-NCM and DPSCs-C were positive for mesenchymal stem cell markers at the same level but negative for hematopoietic cell markers. Moreover, both DPSCs-NCM and DPSCs-C could differentiate into osteogenic, chondrogenic, and adipogenic cells during culture in each induction medium. These results suggest that DPSCs-NCM may be mesenchymal stem cells. Therefore, our novel method might facilitate the less expensive cryopreservation of DPSCs, thereby providing suitable DPSCs for use in patients in future cell-based therapies.

  2. Cord Blood Banking for Potential Future Transplantation. (United States)

    Shearer, William T; Lubin, Bertram H; Cairo, Mitchell S; Notarangelo, Luigi D


    This policy statement is intended to provide information to guide pediatricians, obstetricians, and other medical specialists and health care providers in responding to parents' questions about cord blood donation and banking as well as the types (public versus private) and quality of cord blood banks. Cord blood is an excellent source of stem cells for hematopoietic stem cell transplantation in children with some fatal diseases. Cord blood transplantation offers another method of definitive therapy for infants, children, and adults with certain hematologic malignancies, hemoglobinopathies, severe forms of T-lymphocyte and other immunodeficiencies, and metabolic diseases. The development of universal screening for severe immunodeficiency assay in a growing number of states is likely to increase the number of cord blood transplants. Both public and private cord blood banks worldwide hold hundreds of thousands of cord blood units designated for the treatment of fatal or debilitating illnesses. The procurement, characterization, and cryopreservation of cord blood is free for families who choose public banking. However, the family cost for private banking is significant and not covered by insurance, and the unit may never be used. Quality-assessment reviews by several national and international accrediting bodies show private cord blood banks to be underused for treatment, less regulated for quality control, and more expensive for the family than public cord blood banks. There is an unquestionable need to study the use of cord blood banking to make new and important alternative means of reconstituting the hematopoietic blood system in patients with malignancies and blood disorders and possibly regenerating tissue systems in the future. Recommendations regarding appropriate ethical and operational standards (including informed consent policies, financial disclosures, and conflict-of-interest policies) are provided for physicians, institutions, and organizations that

  3. Methodology for reliable and reproducible cryopreservation of human cervical tissue. (United States)

    Fox, James M; Wiggins, Rebecca C; Moore, John W J; Brewer, Christine; Andrew, Alison C; Martin, Fabiola


    In order to conduct laboratory studies on donated cervical tissue at suitable times an effective and reliable cryopreservation protocol for cervical tissue is required. An active freezing approach was devised utilising 10% dimethyl sulfoxide in foetal bovine serum as a cryoprotective agent with a cooling rate of 1 °C/min to -50 °C then 10 °C/min to -120 °C; a related thawing protocol was also optimised which would allow for the bio-banking of cervical tissue. Viability of freshly harvested cervical tissue was compared to frozen-thawed samples utilising colorimetric MTT assay. In parallel, fresh and freeze-thawed samples were cultured and tested on days 1, 7 and 14 to determine whether bio-banking had detrimental effects on tissue viability over time. Repeat testing revealed that tissue viability between fresh and freeze-thawed samples was comparable at all four time points (days 0, 1, 7 and 14) with no apparent reductions of viability, thus demonstrating this method of cryopreserving cervical tissue is reliable and reproducible, without detrimental effects on live tissue culture. We believe this methodology creates the opportunity for bio-banking donated cervical tissues, which aids improved experimental design and reduces time pressures and wastage. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Cryopreservation of seeds of Brazilian edelweiss (Sinningia leucotricha

    Directory of Open Access Journals (Sweden)

    Vanessa Stegani


    Full Text Available The objective was to evaluate the use of cryogenic solutions in cryopreservation Sinningia leucotricha seeds by the vitrification method in liquid nitrogen. The treatments were: T1 - control: without cryoprotective solution; T2 - PVS1; T3 - modified PVS1; T4 - PVS2; T5 - modified PVS2; T6 - PVS3; T7 - PVS3 modified; T8 - PVS2 + 1% phloroglucinol. After 15 days of immersion of seeds in LN, the cryotubes were removed and rapidly reheated to a temperature of 40 °C water bath for 1.5 minutes. Then, the seeds were washed with wash solution for 20 min. Later they were submitted to the germination test which was conducted on blotting paper moistened with distilled water packaged in crystal polystyrene boxes kept in a growth chamber at 25 ± 2 °C and 16 hours photoperiod. Was evaluated the germination percentage the germination speed index (GSI, and at the end of the experiment will determine the length of shoot (LS and root (LR, and dry mass of seedlings (DMS. We used a completely randomized design with eight treatments and five replications, consisting of 100 seeds. The direct submission of the queen of the abyss seeds in liquid nitrogen provided the highest germination values, GSI, LS, LR and DMS. The queen of the abyss seeds can be cryopreserved in liquid nitrogen directly without the need to cryoprotectant solutions.


    Directory of Open Access Journals (Sweden)

    E. N. Ponomareva


    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.

  6. Experimental autotransplantation and cryopreservation of the thyroid gland. (United States)

    Yüce, İmdat; Okuducu, Hacı; Çağlı, Sedat; Vural, Alperen; Gündoğdu, Ramazan; Abdülrezzak, Ümmühan; Arlı, Turan; Aydın, Mesut; Güney, Ercihan


    The purpose of this study was to investigate the functionality of autotransplanted thyroid tissues immediately or after cryopreservation in rabbits. The study was completed with 12 rabbits randomized in 2 groups. Preoperative scintigraphies were performed for all subjects. The rabbits underwent total thyroidectomy. The first group underwent immediate thyroid autotransplantation. Thyroid tissues of the second group were cryopreserved and autoimplanted at the eighth postoperative week. The free triiodothyronine (fT3) and thyroxine (fT4) levels were monitored for 8 weeks. Postoperative scintigraphies were performed at the eigth week after autoimplantation. The subjects in the first group reached euthyroid levels at the eighth week while none of the second group reached that level, but all showed continuous increase. Although implanted thyroid tissues of 5 of the 6 rabbits in the first group were demonstrated during the first scintigraphy, the number was only 1 in the second group. Thyroid autografts were found to be functional and thought to have a potential preventing postoperative hypothyroidism. © 2014 Wiley Periodicals, Inc.

  7. Implementing Best Practices and Validation of Cryopreservation Techniques for Microorganisms

    Directory of Open Access Journals (Sweden)

    David Smith


    Full Text Available Authentic, well preserved living organisms are basic elements for research in the life sciences and biotechnology. They are grown and utilized in laboratories around the world and are key to many research programmes, industrial processes and training courses. They are vouchers for publications and must be available for confirmation of results, further study or reinvestigation when new technologies become available. These biological resources must be maintained without change in biological resource collections. In order to achieve best practice in the maintenance and provision of biological materials for industry, research and education the appropriate standards must be followed. Cryopreservation is often the best preservation method available to achieve these aims, allowing long term, stable storage of important microorganisms. To promulgate best practice the Organisation for Economic Development and Co-operation (OECD published the best practice guidelines for BRCs. The OECD best practice consolidated the efforts of the UK National Culture Collections, the European Common Access to Biological Resources and Information (CABRI project consortium and the World Federation for Culture Collections. The paper discusses quality management options and reviews cryopreservation of fungi, describing how the reproducibility and quality of the technique is maintained in order to retain the full potential of fungi.

  8. Implementing best practices and validation of cryopreservation techniques for microorganisms. (United States)

    Smith, David; Ryan, Matthew


    Authentic, well preserved living organisms are basic elements for research in the life sciences and biotechnology. They are grown and utilized in laboratories around the world and are key to many research programmes, industrial processes and training courses. They are vouchers for publications and must be available for confirmation of results, further study or reinvestigation when new technologies become available. These biological resources must be maintained without change in biological resource collections. In order to achieve best practice in the maintenance and provision of biological materials for industry, research and education the appropriate standards must be followed. Cryopreservation is often the best preservation method available to achieve these aims, allowing long term, stable storage of important microorganisms. To promulgate best practice the Organisation for Economic Development and Co-operation (OECD published the best practice guidelines for BRCs. The OECD best practice consolidated the efforts of the UK National Culture Collections, the European Common Access to Biological Resources and Information (CABRI) project consortium and the World Federation for Culture Collections. The paper discusses quality management options and reviews cryopreservation of fungi, describing how the reproducibility and quality of the technique is maintained in order to retain the full potential of fungi.

  9. Blastocyst cryopreservation using solid surface vitrification: A preliminary study

    Directory of Open Access Journals (Sweden)

    Mohan S Kamath


    Full Text Available Objective: The objective was to evaluate the effectiveness of a blastocyst cryopreservation program using solid surface vitrification. Setting: This study took place in a university teaching hospital. Study Design: Retrospective observational study. Materials and Methods: Women undergoing frozen embryo transfer cycles over a 4-year period between 2006 and 2010 were studied. The cryopreservation policy followed was a vitrification protocol performed at the blastocyst stage, using a solid surface (nonimmersion method. The post-thaw survival rate, implantation rate, clinical pregnancy rate, live birth rate, and neonatal outcome were recorded. Results: Eighty-one women underwent 86 frozen embryo transfer cycles. Of the 240 blastocysts warmed, 204 survived giving a cryosurvival rate of 85% (204/240. The clinical pregnancy, implantation, miscarriage, ongoing pregnancy, and live birth rates per transfer were 47%, 29%, 12%, 16%, and 23% respectively. Of the 20 live births, there were 16 singletons and 4 twins. Eleven boys and 13 girls were delivered with no major or minor abnormality detected. Conclusion(s: The blastocyst vitrification protocol using the solid surface method is effective with results comparable to fresh blastocyst transfers. While retaining the rapid cooling effect, the nonimmersion technique eliminates the risk of contamination and disease transmission. Larger studies with long-term follow-up data would further confirm the efficacy and safety of this method of vitrification.

  10. The reality, use and potential for cryopreservation of coral reefs. (United States)

    Hagedorn, Mary; Spindler, Rebecca


    Throughout the world coral reefs are being degraded at unprecedented rates. Locally, reefs are damaged by pollution, nutrient overload and sedimentation from out-dated land-use, fishing and mining practices. Globally, increased greenhouse gases are warming and acidifying oceans, making corals more susceptible to stress, bleaching and newly emerging diseases. The coupling of climate change impacts and local anthropogenic stressors has caused a widespread and well-recognized reef crisis. Although in situ conservation practices, such as the establishment and enforcement of marine protected areas, reduce these stressors and may help slow the loss of genetic diversity on reefs, the global effects of climate change will continue to cause population declines. Gamete cryopreservation has already acted as an effective insurance policy to maintain the genetic diversity of many wildlife species, but has only just begun to be explored for coral. Already we have had a great deal of success with cryopreserving sperm and larval cells from a variety of coral species. Building on this success, we have now begun to establish genetic banks using frozen samples, to help offset these threats to the Great Barrier Reef and other areas.

  11. Organ transplantation in Tunisia. (United States)

    El Matri, Aziz; Ben Abdallah, Taieb


    Kidney transplants were first performed in Tunisia in 1986, and transplants soon extended to other organs including the heart, liver, and pancreas. Live-related donor and deceased-donor kidney transplants were both began in the summer of 1986. An organ procurement and transplant law was passed in March 1991, and the National Centre for Advancement of Organ Transplantation was created in 1995. The number of transplantation units has increased to 7 throughout the country, and the yearly transplant number has progressively increased to 139 in 2010, including 20% from deceased kidney donors. Despite these gains, the need continues to grow. Heart transplants began in January 1993, and Tunisia and Jordan are currently the only Arab countries where it is practiced. However, only 16 patients have received a heart transplant as of 2004, and the number of recipients has decreased in the past 10 years. Liver transplants are rare in other Arab countries, but began in Tunisia in January 1998. Over 10 years, 38 patients benefited from this procedure. After a few years of stagnation, the number of liver transplants is increasing. While all types of transplantation are needed, kidney transplantation is a priority in Tunisia. The target is to perform 400 transplants annually, which would require a long-term strategy to provide full financial coverage using the National Health Insurance Funds in both the public and private sectors.

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

    Directory of Open Access Journals (Sweden)

    Corsini Joe


    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.

  13. Decoding mechanisms of loss of fertilization ability of cryopreserved mouse sperm (United States)

    Gray, Jeffrey Earl

    Cryopreservation of mouse sperm is an important technology for management of biomedical research resources. Dramatic progress has been made recently in the development of protocols that combat mouse-strain specific reduction of IVF after cryopreservation. Equal emphasis, however, has not been placed on investigating the biological mechanisms underlying these improvements to IVF. This dissertation broadly investigates the basic question of how mouse-strain specific reduction of IVF occurs after cryopreservation, and how recently developed protocols prevent this process. My research investigated the effects of antioxidants, the cholesterol-acceptor CD, reduced calcium media, and TYH capacitation media on sperm function and oxidative stress after cryopreservation in a variety of mouse strains. I found that reduced IVF was associated with loss of capacitation-dependent sperm function in three strains, B6/J, B6/N, and 129X1, and CD improved sperm function and IVF in all three strains. These findings suggest that cryopreservation inhibits cholesterol efflux resulting in reduced IVF of many mouse strains. I also found that cryopreservation induces uniquely high production of mitochondrial H2O2 by B6/J sperm. H2O2 present in other cellular compartments of B6/J sperm was not elevated compared to other strains. High levels of mitochondrial H2O2 were associated with lipid peroxidation of the sperm head and inability to acrosome react. Antioxidants reduced mitochondrial H2O2 production, decreased sperm head lipid peroxidation, and improved acrosome reaction. The cryopreservation-induced increase in mitochondrial H2O2 production of B6/J and B6129XF1 sperm was associated with elevation of intracellular calcium after cryopreservation and dependent on mitochondrial metabolic substrates. Reducing intracellular calcium levels or removing mitochondrial metabolic substrates decreased mitochondrial H2O2 production and increased IVF rates of cryopreserved B6/J sperm. Many of the strains

  14. Cryopreservation with dimethyl sulfoxide prevents accurate analysis of skinned skeletal muscle fibers mitochondrial respiration. (United States)

    Meyer, Alain; Charles, Anne-Laure; Zoll, Joffrey; Guillot, Max; Lejay, Anne; Singh, François; Schlagowski, Anna-Isabel; Isner-Horobeti, Marie-Eve; Pistea, Cristina; Charloux, Anne; Geny, Bernard


    Impact of cryopreservation protocols on skeletal muscle mitochondrial respiration remains controversial. We showed that oxygen consumption with main mitochondrial substrates in rat skeletal muscles was higher in fresh samples than in cryopreserved samples and that this difference was not fixed but grow significantly with respiration rates with wide fluctuations around the mean difference. Very close results were observed whatever the muscle type and the substrate used. Importantly, the deleterious effects of ischemia-reperfusion observed on fresh samples vanished when cryopreserved samples were studied. These data demonstrate that this technic should probably be performed only extemporaneously. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    María Fernanda Lazo-Javalera


    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.

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


    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.

  17. [Comparative studies on different cryopreservation protocols of human amniotic fluid-derived mesenchymal stem cells]. (United States)

    Wang, Yiru; Bai, Jing; Chen, Jie; Liu, Lifeng; Wang, Yu


    To isolate and culture human amniotic fluid-derived mesenchymal stem cells (HAFMSCs), to investigate a better cryopreservation protocol of HAFMSCs and to observe the bio-characteristics and the multi-potential of HAFMSCs after cryopreservation for the further fundamental researches and clinical applications. HAFMSCs were isolated from the amniotic fluid of pregnant women during the second trimester by the improved two-step method. HAFMSCs were cryopreserved with different cryopreservation protocols (containing different contents of FBS and DMSO at cryoprotectant) in liquid nitrogen for 12 weeks. The bio-characteristics of the HAFMSCs after cryopreservation were analyzed. The growth characteristics were observed by MTT method and the growth curves were drawn. The surface antigens of HAFMSCs were detected using flow cytometry, including CD29, CD34, CD44, CD45, CD73, and CD90. The adipogenic and osteogenic differentiation abilities of HAFMSCs were observed. The mRNA levels of Oct-4 and Nanog of the HAFMSCs were compared between before and after cryopreservation. At 12 weeks after cryopreservation, different protocols had different effects on the cell viability; the better formula of cryoprotectant was 50% DMEM, 40% FBS, and 10% DMSO. After cryopreservation, the cells proliferated rapidly and the growth curves showed "S" shape, which was the same as the cells before cryopreservation. Phenotype showed that HAFMSCs were positive for the surface markers CD29, CD44, CD73, and CD90, and negative for CD34 and CD45. After 21 days of adipogenic differentiation, the lipid droplets were observed by oil red O staining. After 21 days of osteogenic differentiation, the calcium mineralization were verified by von Kossa staining. There was no significant difference (P > 0.05) in the mRNA levels of Oct-4 and Nanog between before and after cryopreservation. HAFMSCs have rapid proliferation and multi-potential in vitro. The cells have high viabilities and no changes of the bio

  18. Three cases of systemic amyloidosis successfully diagnosed by subcutaneous fat tissue biopsy of the hip

    Directory of Open Access Journals (Sweden)

    Arahata M


    Full Text Available Masahisa Arahata,1 Shigeru Shimadoi,1 Satosi Yamatani,1 Shin-ichi Hayashi,2 Shigeharu Miwa,2 Hidesaku Asakura,3 Shinji Nakao4 1Department of Internal Medicine, Nanto Municipal Hospital, Nanto, 2Department of Diagnostic Pathology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 3Department of Internal Medicine (III, 4Department of Cellular Transplantation Biology, Division of Cancer Medicine, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan Abstract: Fine-needle aspiration biopsy of the abdominal fat pad is considered to be a minimally invasive procedure for diagnosing systemic amyloidosis. However, this procedure is sometimes difficult and can be dangerous for elderly patients whose abdominal fat layer is thin because of malnutrition. In such cases, alternative diagnostic methods are required. We report three elderly patients with heart failure complicated by malnutrition. In all cases, electrocardiogram showed low voltage in the limb leads and a pseudoinfarct pattern in the chest leads, and echocardiography showed left ventricular wall thickening with granular sparkling appearance. These patients were suspected of having amyloid cardiomyopathy but could not undergo myocardial biopsies because of their poor conditions. After failed attempts at biopsy of the abdominal fat pad or the other organs, subcutaneous fat tissue biopsy over the hip led to the diagnosis of systemic amyloidosis with cardiomyopathy. The resultant diagnosis guided us to choose the appropriate treatment for the patients. This article illustrates that subcutaneous fat tissue biopsy of the hip could be a useful procedure for diagnosing systemic amyloidosis in elderly patients, particularly when a fat tissue biopsy of the abdomen is associated with a high risk of complications because of malnutrition. Keywords: systemic amyloidosis, amyloid cardiomyopathy, fine-needle aspiration biopsy, subcutaneous fat tissue, hip

  19. Sublingual or subcutaneous immunotherapy for allergic rhinitis? (United States)

    Durham, Stephen R; Penagos, Martin


    Allergen immunotherapy is effective in patients with allergic rhinitis (AR) and, unlike antiallergic drugs, has been shown to modify the underlying cause of the disease, with proved long-term benefits. Subcutaneous immunotherapy (SCIT) has been the gold standard, whereas sublingual immunotherapy (SLIT) has emerged as an effective and safe alternative. Previous Cochrane systematic reviews and meta-analyses have confirmed that both SLIT and SCIT are effective in patients with seasonal AR, whereas evidence for their efficacy in patients with perennial disease has been less convincing. Recent large, adequately powered trials have demonstrated reductions in both symptoms and use of rescue medication in patients with seasonal and those with perennial AR. Here we appraise evidence for SCIT versus SLIT based on indirect evidence from Cochrane reviews and recent well-powered double-blind, randomized controlled trials versus placebo and the limited direct evidence available from randomized blind head-to-head comparisons. At present, based on an overall balance of efficacy and side effects, the patient is in equipoise. Pending definitive comparative trials, choice might be determined largely by the local availability of SCIT and SLIT products of proved value and personal (patient) preference. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  20. Recovery and characterisation of hybrid firs (Abies alba x A. cephalonica, Abies albax A. numidica) embryogenic tissues after cryopreservation. (United States)

    Salaj, Terezia; Matusikov, Ildiko; Panis, Bart; Swennen, Rony; Salaj, Jan


    Embryogenic tissues of hybrid firs were cryopreserved using a slow freezing protocol. The procedure involved preculture of tissues for 24, 48 or 72 h in media with different sorbitol concentrations (0.4 or 0.8 M) and addition of 5% (v/v) DMSO as cryoprotectant. The cell lines tested withstood cryopreservation, even though tissue regrowth after thawing was dependent on treatment and cell line. For cell line AN72, regrowth was 100% for all experimental conditions tested. With cell line AC78, regrowth was 100% except after shorter pretreatment durations, which produced 83% and 86% regrowth for 0.4 M and 0.8 M sorbitol pretreatment, respectively. Cell lines AC1 and AC4 were more sensitive to cryopreservation with 37.5 to 100% regrowth, respectively. Growth parameters evaluated 3 months after cryopreservation showed cell line and treatments effects. In most cases, cryopreservation had no negative effect on growth of tissues. Statistically significant differences in fresh mass accumulation were found for four samples out of 24 investigated, although growth increase of these tissues still reached 79.4-84.6%, compared with non-cryopreserved ones (100% increase). Maturation capacity and genetic fidelity were studied in tissues whose growth was not negatively influenced by cryopreservation. Maturation capacity of embryogenic tissues cryopreserved using the optimal protocol was comparable to that of non-frozen controls. RAPD analysis of 88 genomic regions per cell line did not reveal any changes in genetic fidelity of cryopreserved tissues compared to non-cryopreserved controls.

  1. Diabetic ketoacidosis associated with tacrolimus in solid organ transplant recipients

    International Nuclear Information System (INIS)

    Maqsood, M.Q.; Rabbani, M.; Habib, M.; Saleem, T.


    Diabetic ketoacidosis in patients receiving tacrolimus in the post-transplant setting is rare. We describe two such cases in solid-organ transplant recipients. The first patient, a 17-year-old male, presented with severe diabetic ketoacidosis and was managed with intravenous fluids and insulin infusion. He was a known case of Laurence-Moon-Bardet-Biedl syndrome and had received a renal transplant 2 years ago and was receiving tacrolimus since then. Although diabetic ketoacidosis resolved in 24 hours, large doses of subcutaneous insulin (unto 130 units per day) were needed to keep serum glucose within the normal range. Substitution of tacrolimus with cyclosporine obviated the need for insulin or oral hypoglycaemics. The second patient, a 55-year-old woman, presented with a history of polyuria for 3 days. She had received a hepatic transplant 2 years ago and tacrolimus was being used since then. Mild diabetic ketoacidosis was managed with fluid resuscitation and subcutaneous insulin. Her insulin requirement after an uneventful recovery has been 54 - 70 units per day. Clinicians should be cognizant of the possibility of hyperglycaemic crisis presenting as sudden onset of diabetic ketoacidosis in patients receiving tacrolimus. Use of an alternative calcineurin inhibitor may provide a safer solution to minimize future morbidity in such patients. (author)

  2. Hematopoietic stem cell transplantation induces immunologic tolerance in renal transplant patients via modulation of inflammatory and repair processes

    Directory of Open Access Journals (Sweden)

    Wu Duojiao


    Full Text Available Abstract Background Inducing donor-specific tolerance in renal transplant patients could potentially prevent allograft rejection and calcineurin inhibitor nephrotoxicity. Combined kidney and hematopoietic stem cell transplant from an HLA-matched donor is an exploratory and promising therapy to induce immune tolerance. Investigtion of molecular mechanisms involved in the disease is needed to understand the potential process of cell therapy and develop strategies to prevent this immunologic rejection. Methods We enrolled nine patients in a clinical study in which cryopreserved donor hematopoietic stem cells were infused on days 2, 4, and 6 after kidney transplantation. One month post-transplant, 4 plasma samples were collected from combined transplants (C + Tx, and 8 plasma samples from patients with kidney transplantation alone (Tx. High abundance proteins in plasma were depleted and the two-dimensional liquid chromatography-tandem mass spectrometry coupled with iTRAQ labeling was utilized to identify the protein profiling between the two groups. Clusters of up- and down-regulated protein profiles were submitted to MetaCore for the construction of transcriptional factors and regulation networks. Results and Discussion Among the 179 identified proteins, 65 proteins were found in C + Tx with at least a 2-fold change as compared with Tx. A subset of proteins related to the complement and coagulation cascade, including complement C3a,complement C5a, precrusors to fibrinogen alpha and beta chains,was significantly downregulated in C + Tx. Meanwhile, Apolipoprotein-A1(ApoA1, ApoC1, ApoA2, ApoE, and ApoB were significantly lower in Tx compared to C + Tx. Gene ontology analysis showed that the dominant processes of differentially expressed proteins were associated with the inflammatory response and positive regulation of plasma lipoprotein particle remodeling. Conclusions Thus, our study provides new insight into the molecular events in

  3. Transplante de intestino delgado Small intestine transplantation

    Directory of Open Access Journals (Sweden)

    Flávio Henrique Ferreira Galvão


    Full Text Available RACIONAL: Avanços da biotecnologia e o desenvolvimento de novas drogas imunossupressoras melhoraram os resultados do transplante de intestino delgado. Esse transplante é atualmente indicado para casos especiais da falência intestinal. OBJETIVO: A presente revisão realça os recentes desenvolvimentos na área do transplante de intestino delgado. MATERIAL E MÉTODO: Mais de 600 publicações de transplante de intestino delgado foram revisadas. O desenvolvimento da pesquisa, novas estratégias de imunossupressão, monitorização do enxerto e do receptor, e avanços na técnica cirúrgica são discutidos. RESULTADOS: Realizaram-se cerca de 700 transplante de intestino delgado em 55 centros: 44% intestino-fígado, 41% enxerto intestinal isolado e 15% transplante multivisceral. Rejeição e infecção são as principais limitações desse transplante. Sobrevida de 5 anos na experiência internacional é de 46% para o transplante de intestino isolado, 43% para o intestino-fígado e de cerca de 30% para o transplante multivisceral. Sobrevidas prolongadas são mais freqüentes nos centros com maior experiência. Em série de 165 transplantes intestinais na Universidade de Pittsburgh, PA, EUA, foi relatada sobrevida do paciente maior do que 75% no primeiro ano, 54% em 5 anos e 42% em 10 anos. Mais de 90% desses pacientes assumem dieta oral irrestrita. CONCLUSÃO: O transplante de intestino delgado evoluiu de estratégia experimental para uma alternativa viável no tratamento da falência intestinal permanente. Promover o refinamento da terapia imunossupressora, do manejo e prevenção de infecções, da técnica cirúrgica e da indicação e seleção adequada dos pacientes é crucial para melhorar a sobrevida desse transplante.BACKGROUND: Significant progress has been made in clinical small bowel transplantation over the last decade mainly due advances in biotechnology and new immunosuppressive regiments. This transplantation has now been indicated

  4. Subcutaneous blood flow during insulin-induced hypoglycaemia

    DEFF Research Database (Denmark)

    Hilsted, J; Madsbad, S; Sestoft, L


    Subcutaneous blood flow was measured preceding insulin-induced hypoglycaemia, at the onset of hypoglycaemic symptoms and 2 h later in juvenile diabetics with and without autonomic neuropathy and in normal males. In all groups subcutaneous blood flow decreased at the onset of hypoglycaemic symptoms...... compared with pre-hypoglycaemic flow. Two hours after onset of hypoglycaemic symptoms, subcutaneous blood flow was still significantly decreased compared with pre-hypoglycaemic flow. In normal subjects local nerve blockade had no effect on blood flow changes during hypoglycaemia, whereas local alpha...

  5. Subcutaneous Zygomycosis : Current Indian Scenario With A Review

    Directory of Open Access Journals (Sweden)

    Thappa Devinder Mohan


    Full Text Available Zygomycosis broadly consists of mucormycosis and subcutaneous zygomycosis. Subcutaneous zygomycosis can further be subdivided into conidiobolomycosis and basidiobolomycosis. We have done retrospective analysis of data of cases of subcutaneous zygomycosis (basidiobolomycosis, diagnosis based on clinical features, histopathology and/or culture. The cases were treated in Dermatology and STD department of Jawaharlal Institute of postgraduate Medical Education and Research (JIPMER, Pondicherry between 1990 to 2002. Case reported in India has been summarized and relevant literature on this entity has been reviewed.

  6. Subcutaneous versus intravenous immunoglobulin in multifocal motor neuropathy

    DEFF Research Database (Denmark)

    Harbo, T; Andersen, Henning; Hess, A


    Background and purpose: For treatment of multifocal motor neuropathy (MMN), we hypothesized that (i) infusion of equivalent dosages of subcutaneous immunoglobulin (SCIG) is as effective as intravenous immunoglobulin (IVIG) and that (ii) subcutaneous infusion at home is associated with a better...... at the injection sites for a few weeks. All other adverse effects during SCIG were mild and transient. No differences between treatments of health-related quality of life occurred. Conclusion: In MMN, short-term subcutaneous infusion of immunoglobulin is feasible, safe and as effective as intravenous infusion...

  7. An evaluation of the effects of long term cryopreservation, cause of death, and time between death and donation on heart valve leaflet viability

    International Nuclear Information System (INIS)

    Strachan, K.


    The protocol for cryopreservation of allograft heart valves at the Donor Tissue Bank of Victoria was based on validation studies on the viability of the heart valve leaflets at the time of processing. The heart block is removed within 24 hours of death and the aor-tic and pulmonary valves trimmed immediately following retrieval. Following this processing, the valves are incubated in antibiotics at 30 degree C for 6 to 8 hours before being frozen in 10% DMSO at a controlled rate. A sample of tricuspid valve leaflet is placed in Krebs solution at the time of trimfning and is used for viability studies. Leaflet viability studies have been perfon-ned on all heart valves retrieved from 1993 to the present day at the Donor Tissue Bank of Victoria. Viability involves a qualitative assessment of the cellular outgrowth by leaflet fibroblasts, this assessment ranging from '-' for no outgrowth to '++++' for maximum outgrowth. Surgeons do not request valves with any particular viability and will use them whether they are viable or not. This evaluation was to determine the effects of long-term cryopreservation, cause of death, and also time lapse of heart removal following death on the viability of the retrieved leaflets. The aim of investigating the effects of long-term cryopreservation was to determine whether there was any correlation between initial viability and viability following storage for several months to several years. It was also decided to investigate whether there was any correlation between time length between death and heart retrieval and the viability. It was also thought that the cause of death may have had an effect on the viability, for example, did death by carbon monoxide poisoning have an effect on the viability of heart valve cells. Heart valves, which had been cryopreserved but could not be transplanted for various reasons were used to study the effects of cryopreservation in this study. These were thawed according to protocol and a sample of the valve

  8. Intrauterine insemination or intracervical insemination with cryopreserved donor sperm in the natural cycle : A cohort study

    NARCIS (Netherlands)

    Kop, P. A L; Van Wely, M.; Mol, B. W.; De Melker, A. A.; Janssens, P. M W; Arends, B.; Curfs, M. H J M; Kortman, M.; Nap, A.; Rijnders, E.; Roovers, J. P W R; Ruis, H.; Simons, A. H M; Repping, S.; Van Der Veen, F.; Mochtar, M. H.


    studyquestion: Does intrauterine insemination in the natural cycle lead to better pregnancy rates than intracervical insemination (ICI) in the natural cycle in women undergoing artificial insemination with cryopreserved donor sperm. summaryanswer: In a large cohort of women undergoing artificial

  9. 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.


    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

  10. Vitrification of mosses: a useful method for the cryopreservation of Splachnum ampullaceum Hedw. (United States)

    Mallon, R; Rodriguez-Oubina, J; Luz Gonzalez, M


    The source of germplasm as well as the technique used for storage of mosses can enhance survival after cryopreservation. Samples of gametophores, protonemata and protonemal brood cells from in vitro cultures of Splachnum ampullaceum were cryopreserved following exposure to a plant vitrification solution (PVS2) for two different times (5 and 10 min) at 0 degree C. Half of the samples were pretreated with a loading solution containing 2 M glycerol and 0.4 M sucrose before exposure to PVS2. After one week storage in liquid nitrogen, S. ampullaceum samples were regenerated on Gamborg's B5 mineral medium with B5 vitamins. Exposure to a loading solution was a prerequisite for high survival in all samples. Four weeks after cryopreservation, 92.3 percent brood cells, 60.0 percent gametophores and 46.0 percent protonemata pretreated with a loading solution had regenerated, displaying normal growth and development, thus demonstrating that vitrification is a useful method for moss cryopreservation.

  11. Artificial cryopreserved embryo transfer cycle success depends on blastocyst developmental rate and progesterone timing

    DEFF Research Database (Denmark)

    Ozgur, Kemal; Bulut, Hasan; Berkkanoglu, Murat


    This retrospective cohort analysis compared the developmental competence of cryopreserved day-4 and 5 blastocysts, and investigated the effect of progesterone administration duration on the success of artificial frozen embryo transfers. Between October 2015 and March 2016, 868 intracytoplasmic sp...

  12. Genetic and epigenetic stability of cryopreserved and cold-stored hops (Humulus lupulus L.). (United States)

    Peredo, Elena L; Arroyo-García, Rosa; Reed, Barbara M; Revilla, M Angeles


    Conventional cold storage and cryopreservation methods for hops (Humulus lupulus L.) are available but, to our knowledge, the genetic and epigenetic stability of the recovered plants have not been tested. This study analyzed 51 accessions of hop using the molecular techniques, Random Amplified DNA Polymorphism (RAPD) and Amplified Fragment Length Polymorphism (AFLP), revealing no genetic variation among greenhouse-grown controls and cold stored or cryopreserved plants. Epigenetic stability was evaluated using Methylation Sensitive Amplified Polymorphism (MSAP). Over 36% of the loci were polymorphic when the cold and cryo-treated plants were compared to greenhouse plants. The main changes were demethylation events and they were common to the cryopreserved and cold stored plants indicating the possible effect of the in vitro establishment process, an essential step in both protocols. Protocol-specific methylation patterns were also detected indicating that both methods produced epigenetic changes in plants following cold storage and cryopreservation.

  13. Addition of Tempol in semen cryopreservation medium improves the post-thaw sperm function. (United States)

    Bateni, Zahra; Azadi, Leila; Tavalaee, Marziyeh; Kiani-Esfahani, Abbas; Fazilati, Mohammad; Nasr-Esfahani, Mohammad Hossein


    Despite extensive research carried out for optimization and commercialization of sperm cryopreservation media, percentage of motility and viability remain low following cryopreservation. These adverse effects have been partially ascribed to reactive oxygen species (ROS) production during cryopreservation. Therefore, we proposed that addition of a cell permeable antioxidant like Tempol, with superoxide dismutase (SOD) mimetic action, may overcome these effects in an optimized commercially available cryo-protective medium. Therefore, semen samples were cryopreserved in the presence or absence of Tempol. A concentration of 5 μM Tempol was defined as optimal since it significantly improved motility and viability post thawing and reduced DNA fragmented sperm. In addition, percentage of ROS positive sperm was reduced. These effects of Tempol can be attributed to cell permeability characteristic and ability to reduce superoxide production both at intra- and extra-cellular levels. Tempol may hold the potential for clinical applications.

  14. Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant

    Directory of Open Access Journals (Sweden)

    Hak Jun Kim


    Full Text Available Antifreeze proteins (AFPs are biological antifreezes with unique properties, including thermal hysteresis(TH,ice recrystallization inhibition(IRI,and interaction with membranes and/or membrane proteins. These properties have been utilized in the preservation of biological samples at low temperatures. Here, we review the structure and function of marine-derived AFPs, including moderately active fish AFPs and hyperactive polar AFPs. We also survey previous and current reports of cryopreservation using AFPs. Cryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs. Cryopreserved biological samples mainly originate from mammals. Most cryopreservation trials using marine-derived AFPs have demonstrated that addition of AFPs can improve post-thaw viability regardless of freezing method (slow-freezing or vitrification, storage temperature, and types of biological sample type.

  15. 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.


    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

  16. [Promoting Living Kidney Transplantation]. (United States)

    Lin, Chiu-Chu


    Kidney transplantation is the best approach for treating patients with end stage renal disease, offering patients the best chance of returning to normal health. While the techniques used in kidney transplantation surgery are mature and highly successful, there is a severe shortage of donor organs. Statistics show a serious imbalance between organ donations and patients on the waiting list for organ transplantation. Moreover, evidence from empirical studies has shown a better transplantation outcome for patients who receive living donor transplantation than for those who receive organs from cadavers. Although using relatives as donors offers an effective way to reduce the problem of organ shortage, this strategy faces many challenges and many other factors affect the promotion of living donor transplantation. This article elaborates how cultural and psychological factors, kidney transplantation awareness, and ethics and laws impact upon living kidney donations and then proposes coping strategies for promoting living kidney transplantation.


    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. BONE MARROW TRANSPLANTATION. AUTOLOGOUS TRANSPLANTS: Oct 1986 - Dec 2007. Multiple Myeloma 90. NHL 39. Hodgkins lymphoma 19. AML 36. APML 9. ALL 2. Amyloidosis 2. Granulocytic Sarcoma 1.

  18. Life After Transplant (United States)

    ... however you can Daughter's dying wish became mother's motivation Be The Match Blog Stories Anna, transplant recipient ... Jobs Job application FAQs E-Verify Career events Employee benefits About us Bea, transplant recipient Be The ...

  19. Choosing a Transplant Center (United States)

    ... however you can Daughter's dying wish became mother's motivation Be The Match Blog Stories Anna, transplant recipient ... Jobs Job application FAQs E-Verify Career events Employee benefits About us Bea, transplant recipient Be The ...

  20. Treatment Before Transplant (United States)

    ... however you can Daughter's dying wish became mother's motivation Be The Match Blog Stories Anna, transplant recipient ... Jobs Job application FAQs E-Verify Career events Employee benefits About us Bea, transplant recipient Be The ...

  1. Cord Blood and Transplants (United States)

    ... however you can Daughter's dying wish became mother's motivation Be The Match Blog Stories Anna, transplant recipient ... Jobs Job application FAQs E-Verify Career events Employee benefits About us Bea, transplant recipient Be The ...

  2. Microsurgical tunica albuginea transplantation in an animal model

    Directory of Open Access Journals (Sweden)

    Salvatore Sansalone


    Full Text Available Several andrological diseases require surgical repair or reconstruction of tunica albuginea, which envelops the corpora cavernosa penis. Despite intense research efforts involving a variety of biological materials, such as skin, muscle aponeurosis, human dura mater, tunica vaginalis, and pericardium, engineered tunica albuginea suitable for graft use is yet to be obtained. The study investigates microsurgical tunica albuginea allotransplantation in an animal model with the purpose of creation of an organ-specific tissue bank to store penile tissue, from cadaveric donors and male-to-female trans-sexual surgery, for allogeneic transplantation. Materials were tunica albuginea tissue explanted from 15 donor rats, cryopreserved at −80°C, gamma-irradiated, and implanted in 15 recipient rats, of which three rats were used as controls. Penile grafts were explanted at different time intervals; after macroscopic evaluation of the organ, the grafts were processed to morphological, histochemical, and immunohistochemical examinations by light microscopy. Detection of pro-inflammatory cytokines was also performed. Examination of the tunica albuginea allografts collected 1, 3, or 6 months after surgery and of control tunica albuginea fragments showed that tunica albuginea implants achieved biointegration with adjacent tissue at all-time points. The integration of cryopreserved rat tunica albuginea allografts, documented by our study, encourages the exploration of tunica albuginea allotransplantation in humans. In conclusion, the effectiveness and reliability of the tunica albuginea conditioning protocol described here suggest the feasibility of setting up a tunica albuginea bank as a further tissue bank.

  3. Microsurgical tunica albuginea transplantation in an animal model (United States)

    Sansalone, Salvatore; Loreto, Carla; Leonardi, Rosario; Vespasiani, Giuseppe; Musumeci, Giuseppe; Lombardo, Claudia; Castorina, Sergio; Cardile, Venera; Caltabiano, Rosario


    Several andrological diseases require surgical repair or reconstruction of tunica albuginea, which envelops the corpora cavernosa penis. Despite intense research efforts involving a variety of biological materials, such as skin, muscle aponeurosis, human dura mater, tunica vaginalis, and pericardium, engineered tunica albuginea suitable for graft use is yet to be obtained. The study investigates microsurgical tunica albuginea allotransplantation in an animal model with the purpose of creation of an organ-specific tissue bank to store penile tissue, from cadaveric donors and male-to-female trans-sexual surgery, for allogeneic transplantation. Materials were tunica albuginea tissue explanted from 15 donor rats, cryopreserved at −80°C, gamma-irradiated, and implanted in 15 recipient rats, of which three rats were used as controls. Penile grafts were explanted at different time intervals; after macroscopic evaluation of the organ, the grafts were processed to morphological, histochemical, and immunohistochemical examinations by light microscopy. Detection of pro-inflammatory cytokines was also performed. Examination of the tunica albuginea allografts collected 1, 3, or 6 months after surgery and of control tunica albuginea fragments showed that tunica albuginea implants achieved biointegration with adjacent tissue at all-time points. The integration of cryopreserved rat tunica albuginea allografts, documented by our study, encourages the exploration of tunica albuginea allotransplantation in humans. In conclusion, the effectiveness and reliability of the tunica albuginea conditioning protocol described here suggest the feasibility of setting up a tunica albuginea bank as a further tissue bank. PMID:28139472

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

    Directory of Open Access Journals (Sweden)

    Indra Kusuma


    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.

  6. L-carnitine Supplemented Extender Improves Cryopreserved-thawed Cat Epididymal Sperm Motility


    S. Manee-in; S. Parmornsupornvichit; S. Kraiprayoon; T. Tharasanit; P. Chanapiwat; K. Kaeoket


    Cryopreservation of epididymal sperm is an effective technique to preserve genetic materials of domestic cats and wild felids when they unexpectedly die. However, this technique inevitably causes detrimental changes of cryopreserved-thawed spermatozoa, for example, by physical damage and excessive oxidative stress. L-carnitine is an antioxidant that has been used to improve sperm motility in humans and domestic animals. This study aimed to investigate the effects of L-carnitine on cat epididy...

  7. 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.

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




    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. Blood and Bone MarrowTransplant? (United States)

    ... Topics / Blood and Bone Marrow Transplant Blood and Bone Marrow Transplant Also known as Hematopoietic Stem Cell Transplant , Hematopoietic Cell Transplant , Autologous Transplant , Allogeneic Transplant A blood or bone marrow ...

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

    Directory of Open Access Journals (Sweden)

    Safrina Rahmah


    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.

  11. Organ Transplants in Kazakhstan. (United States)

    Baigenzhin, Abay; Doskaliyev, Zhaksylyk; Tuganbekova, Saltanat; Zharikov, Serik; Altynova, Sholpan; Gaipov, Abduzhappar


    The Republic of Kazakhstan is one of the fastest developing countries in the world and has a health care system that is unique in Central Asia. Its organ transplant services are also developing rapidly. We aimed to analyze and briefly report on the current status of organ transplant in the Republic of Kazakhstan. We analyzed organ transplant activities in that country for the period 2012 to 2014. All data were collected from the official database of the National Transplant Coordinating Center of the Republic of Kazakhstan. At the end of 2014, the number of transplant centers had increased to 10, three of which could perform multiorgan transplants; during the same period, the number of deceased-donor organ-donating hospitals increased up to 37. By 2013, the transplant activity rate for all centers had reached 9.22 per million population. During the previous 3 years (2012-2014), there was a 3-fold increase in the number of living donors and an 18-fold increase in the number of kidney transplants. Between 2012 and 2014, the number of living-donor liver transplants increased from 17 to 25, and the number of deceased-donor transplants increased from 3 to 7. During the last 3 years (2012-2014), the number of heart transplants increased to 7 cases. During the last 3 years (2012-2014), Kazakhstan achieved a significant improvement in the organization of its transplant services, and a noticeable upward trend in the system continues.

  12. Subcutaneous blood flow during insulin-induced hypoglycaemia

    DEFF Research Database (Denmark)

    Hilsted, J; Madsbad, S; Sestoft, L


    Subcutaneous blood flow was measured preceding insulin-induced hypoglycaemia, at the onset of hypoglycaemic symptoms and 2 h later in juvenile diabetics with and without autonomic neuropathy and in normal males. In all groups subcutaneous blood flow decreased at the onset of hypoglycaemic symptoms...... compared with pre-hypoglycaemic flow. Two hours after onset of hypoglycaemic symptoms, subcutaneous blood flow was still significantly decreased compared with pre-hypoglycaemic flow. In normal subjects local nerve blockade had no effect on blood flow changes during hypoglycaemia, whereas local alpha......-receptor blockade abolished the vasoconstrictor response. We suggest that circulating catecholamines stimulating vascular alpha-receptors are probably responsible for flow reduction in the subcutaneous tissue during hypoglycaemia....

  13. Chordoma with postoperative subcutaneous implantation and meningeal dissemination: MRI

    International Nuclear Information System (INIS)

    Kinoshita, T.; Okudera, T.; Shimosegawa, E.; Hatazawa, J.; Yoshida, Y.; Yasui, N.; Ogawa, T.


    Chordomas are histologically benign tumours which are locally invasive. We present an unusual case of recurrent chordoma with subcutaneous implantation and widespread meningeal dissemination after surgery. Contrast-enhanced MRI was useful for determining the extent of the tumour. (orig.)

  14. Cryopreservation of Lilium martagon l. meristems by droplet-vitrification and evaluation of their physiological stability. (United States)

    Urbaniec-Kiepura, M; Bach, A

    The aim of the present study was to compare different strategies for cryopreservation of martagon lily meristems and to evaluate the physiological status of the regenerants. The bulblets were stored at 5 degree C or 20 degree C and pretreated with 3% or 6% sucrose prior to droplet-vitrification. The meristems were then assessed for their survival and regeneration. Their photochemical activity was investigated using a Photosynthesis Yield Analyzer MINI PAM 2000 Portable Chlorophyll Fluorometer and their photosynthesis oxygen production was evaluated with a Plant Vital 5030 device. The plant material stored at 5 degree C on medium containing 3% sucrose exhibited lower survival (40.8%) and regeneration (75%) of meristems following cryopreservation compared with material stored at 20 degree C on medium containing 3% sucrose, for which survival was 65% and regeneration 87%. Treatment of lily meristems for 30 min with PVS2 yielded high survival and regeneration. The implemented cryopreservation protocol did not induce any physiological changes in regenerants. Chlorophyll fluorescence (Fv/Fm) was 0.822 for cryopreserved samples (+LN) and 0.824 for non-cryopreserved ones (-LN). Photosynthetic oxygen production (KphA) was 1.531 (+LN) and 1.410 (-LN). Droplet-vitrification seems to be an effective method for cryopreservation of martagon lily meristems with the aim of its ex situ protection.

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

    Directory of Open Access Journals (Sweden)

    Jana Žáková


    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.

  16. Cryopreservation of non-human primate sperm: priorities for future research. (United States)

    Morrell, J M; Hodges, J K


    Wild populations of many non-human primate species have declined alarmingly due to habitat destruction, hunting and genetic isolation. Captive breeding programmes to aid species survival could be enhanced by the use of assisted reproductive techniques, such as artificial insemination (AI), if a source of viable sperm was readily accessible. Cryobanks of primate sperm could provide such a supply if techniques for freezing sperm could be developed. Although sporadic attempts to cryopreserve primate sperm have been reported for some of the more frequently encountered zoo-maintained species, there is limited information available on techniques for sperm collection and storage. It is vital that adequate reporting of all cryopreservation attempts be made to avoid repetition of inappropriate methodologies and wastage of valuable genetic material from rare or endangered animals. An integrated approach to the cryobanking of non-human primate sperm is considered to be essential for species conservation. In this review, the factors affecting the success of sperm cryopreservation are outlined, existing information is compiled from previous reported attempts at cryopreservation, and suggestions are made for cryopreserving sperm in further non-human primate species. Moreover, recommendations are given for additional studies to augment existing data. It is intended that this information should serve as a guide for developing cryopreservation protocols in the future, particularly for endangered species.

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

    Directory of Open Access Journals (Sweden)

    N. Prapaiwan


    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.

  18. An overview of sperm cryopreservation services for adolescent cancer patients in the United Kingdom. (United States)

    Wilford, Howard; Hunt, Jane


    For young men, a consequence of surviving childhood malignancy can be iatrogenic infertility. Current health policies focus on the elimination of "post code lotteries" in cancer services. The extent to which sperm cryopreservation services for young men at risk of infertility from cancer treatment are provided and standardised within the United Kingdom and Ireland, was therefore the subject of this research. This paper draws on data from a three-stage study to describe sperm cryopreservation services for adolescent males, identifying current provision of sperm cryopreservation at the majority of United Kingdom Children's Cancer Study Group (UKCCSG) centres. In particular, the ways in which services are managed and written information provided to patients and their families was focused upon. Nurses from 18 of 22 UKCCSG centres responded to a questionnaire, six nurses from the replying centres participated in further, focused interviews. Results suggested that, during a 1 year period, approximately 118 adolescent males within the United Kingdom and Ireland could potentially have benefited from cryopreservation services. Of the responding centres, 15 offered a cryopreservation service. However, the majority (n=14) lacked consistency and co-ordination in their service provision. The provision of written information to this patient group was limited and analysis revealed all was of a poor quality. Findings from the study led the researchers to conclude that all young men at risk of iatrogenic sterility from cancer treatment could benefit from the production and systematic application of national guidelines and that standardising sperm cryopreservation services would fit with current health policy.

  19. Effect of sugars on characteristics of Boer goat semen after cryopreservation. (United States)

    Naing, S W; Wahid, H; Mohd Azam, K; Rosnina, Y; Zuki, A B; Kazhal, S; Bukar, M M; Thein, M; Kyaw, T; San, M M


    In order to improve Boer goat semen quality during cryopreservation process, the influence of sugar supplementation on semen characteristics of sperm were investigated. Three experiments were carried out to investigate the effect of (a) addition of two monosaccharides (fructose and glucose) and two disaccharides sugars (trehalose and sucrose) (b) sugar combination (fructose and trehalose, sucrose and trehalose, glucose and trehalose), and control (glucose without trehalose) (c) different concentrations of trehalose on cryopreservation using Tris based extender. The total motility, forward motility, viability, normal spermatozoa, acrosome integrity and membrane integrity were assessed subjectively. Differences were not detected among monosaccharides, but glucose increased (Pdiffer (P>0.05) among disaccharide sugar supplementation. Combination of glucose and trehalose significantly improved the characteristics of Boer spermatozoa after cryopreservation (P<0.05). Supplementation of trehalose (198.24mM) into the glucose extender significantly increased total motility, forward motility, live spermatozoa, acrosome integrity and membrane integrity following cryopreservation (P<0.05). In conclusion, glucose had the better ability to support Boer sperm motility and movement patterns. Combination of monosaccharide (glucose) and disaccharide (trehalose) improved semen quality following cryopreservation. Trehalose supplementation at the concentration of 198.24mM to the glucose extender conferred the greater improvement of semen quality for Boer semen cryopreservation. Copyright © 2010 Elsevier B.V. All rights reserved.

  20. Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing. (United States)

    Kim, Ki-Jung; Lee, Yong-An; Kim, Bang-Jin; Kim, Yong-Hee; Kim, Byung-Gak; Kang, Hyun-Gu; Jung, Sang-Eun; Choi, Sun-Ho; Schmidt, Jonathan A; Ryu, Buom-Yong


    Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200 mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Sensitivity of human embryonic stem cells to different conditions during cryopreservation. (United States)

    Xu, Yanqing; Zhang, Liang; Xu, Jiandong; Wei, Yuping; Xu, Xia


    Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study, sensitivity of human embryonic stem cells (hESCs) to different cooling rates, ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS, and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs, but no increase for Me2SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type, cooling rate and ice seeding. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Full-term potential of goat in vitro produced embryos after different cryopreservation methods. (United States)

    Ferreira-Silva, José Carlos; Moura, Marcelo Tigre; Silva, Túlio Diego; Oliveira, Luis Rennan Sampaio; Chiamenti, Adauto; Figueirêdo Freitas, Vicente José; Oliveira, Marcos Antonio Lemos


    Cryopreservation of preimplantation embryos represents a major challenge due to their shape and relatively large cells. Embryo source and cryopreservation method are key factors to cryotolerance efficiency and few reports have investigated more promising protocols for goat embryos. The study was aimed to compare different cryopreservation methods for goat in vitro produced (IVP) embryos. Goat blastocysts were subjected to conventional freezing (CF), Dimethyl sulfoxide vitrification (DMSO-V) and Dimethylformamide vitrification (DMF-V). Cryopreserved blastocysts were assessed for re-expansion, cell viability and in vivo development rates. Blastocyst re-expansion after cryopreservation was similar between groups, but cell viability was lower for DMF-V (32%) than CF (68%) and DMSO-V (60%). Pregnancy and delivery rates were similar for CF (60% and 50%) and DMSO-V (50% and 45%) and higher then DMF-V (20% and 15%), respectively. Finally, kidding rates were also indistinguishable for CF (40%) and DMSO-V (35%), but higher then DMF-V (12.5%). In conclusion, conventional freezing and vitrification using DMSO have similar efficiencies for cryopreservation of goat IVP embryos and cryoprotectant for vitrification affects its outcome. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. The effects of cryopreservation on the expression of canine regulatory T-cell markers. (United States)

    Tarpataki, Noemi; Wawrzyniak, Marcin; Akdis, Cezmi A; Rückert, Beate; Meli, Marina L; Fischer, Nina M; Favrot, Claude; Rostaher, Ana


    Regulatory T (Treg) cells have been described as key regulators in various immunological processes and are of growing interest in veterinary allergy. Cryopreservation of immune cells is performed routinely in human basic science research and in clinical studies. As such, it allows batch testing of collected samples at a single time point, resulting in a significant reduction in sample variability. Data which describe the effects of cryopreservation on Treg cell frequency and functionality in the canine species are important to inform future research. The purpose of this study was to establish a robust freeze/thaw procedure and flow cytometric staining protocol for canine Treg cells, and to compare the frequencies of different canine Treg cell phenotypes before and after cryopreservation. Nine privately owned dogs. Peripheral blood mononuclear cells were isolated and Treg cells stained and analysed by flow cytometry, before and after three months of cryopreservation. The recovery percentages and the corresponding correlations (fresh versus cryopreserved) for CD4 + CD25 + , CD4 + FOXP3 + and CD4 + CD25 + FOXP3 + cell populations were calculated. A high recovery rate of 97.2 (r = 0.94, P cryopreservation does not substantially affect the expression of surface and intracellular markers of canine Treg cells; however, additional studies will be necessary to assess whether functionality of the cells is also maintained. © 2017 ESVD and ACVD.

  4. Natural zwitterionic l-Carnitine as efficient cryoprotectant for solvent-free cell cryopreservation. (United States)

    Zhai, Hongwen; Yang, Jing; Zhang, Jiamin; Pan, Chao; Cai, Nana; Zhu, Yingnan; Zhang, Lei


    Organic solvents, such as dimethyl sulfoxide (DMSO) and glycerol, have been commonly used as cryoprotectants (CPAs) in cell cryopreservation. However, their cytotoxicity and need of complex freezing protocols have impeded their applications especially in clinical cell therapy and regenerative medicine. Trehalose has been explored as a natural CPA to cryopreserve cells, but its poor cell permeability frequently results in low cryopreservation efficacy. In this work, we presented that a natural zwitterionic molecule-l-carnitine-could serve as a promising CPA for solvent-free cryopreservation. We demonstrated that l-carnitine possessed strong ability to depress water freezing point, and with ultrarapid freezing protocol, we studied the post-thaw survival efficiency of four cell lines (GLC-82 cells, MCF-7 cells, NIH-3T3 cells and Sheep Red Blood Cells) using l-carnitine without addition of any organic solvents. At the optimum l-carnitine concentration, all four cell lines could achieve above 80% survival efficiency, compared with the significantly lower efficiency using organic CPAs and trehalose. After cryopreservation, the recovered cell behaviors including cell attachment and proliferation were found to be similar to the normal cells, indicating that the cell functionalities were not affected. Moreover, l-carnitine showed no observable cytotoxicity, which was superior to the organic CPAs. This work offered an attractive alternative to traditional CPAs and held great promise to revolutionize current cryopreservation technologies, to benefit the patients in various cell-based clinical applications. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART

    Directory of Open Access Journals (Sweden)

    Marlea Di Santo


    Full Text Available Cryopreservation of human spermatozoa—introduced in the 1960's—has been recognized as an efficient procedure for management of male fertility before therapy for malignant diseases, vasectomy or surgical infertility treatments, to store donor and partner spermatozoa before assisted reproduction treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility. Despite the usefulness of it, cryopreservation may lead to deleterious changes of sperm structure and function: while the effects of cryopreservation on cells are well documented, to date there is no agreement in the literature on whether or not cryopreservation affects sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-thawing procedure. Therefore, sperm cryopreservation is an important component of fertility management and much of its successful application seems to affect the reproductive outcome of assisted reproduction technologies (ART: appropriate use of cryoprotectants before and sperm selection technologies after cryopreservation seem to have the greatest impact on preventing DNA fragmentation, thus improving sperm cryosurvival rates.

  6. A cryopreservation method for Pasteurella multocida from wetland samples (United States)

    Moore, Melody K.; Shadduck, D.J.; Goldberg, Diana R.; Samuel, M.D.


    A cryopreservation method and improved isolation techniques for detection of Pasteurella multocida from wetland samples were developed. Wetland water samples were collected in the field, diluted in dimethyl sulfoxide (DMSO, final concentration 10%), and frozen at -180 C in a liquid nitrogen vapor shipper. Frozen samples were transported to the laboratory where they were subsequently thawed and processed in Pasteurella multocida selective broth (PMSB) to isolate P. multocida. This method allowed for consistent isolation of 2 to 18 organisms/ml from water seeded with known concentrations of P. multocida. The method compared favorably with the standard mouse inoculation method and allowed for preservation of the samples until they could be processed in the laboratory.

  7. Fertility in cancer patients after cryopreservation of one ovary

    DEFF Research Database (Denmark)

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


    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......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...... 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....

  8. Expansion of Human Tregs from Cryopreserved Umbilical Cord Blood for GMP-Compliant Autologous Adoptive Cell Transfer Therapy

    Directory of Open Access Journals (Sweden)

    Howard R. Seay


    Full Text Available Umbilical cord blood is a traditional and convenient source of cells for hematopoietic stem cell transplantation. Thymic regulatory T cells (Tregs are also present in cord blood, and there is growing interest in the use of autologous Tregs to provide a low-risk, fully human leukocyte antigen (HLA-matched cell product for treating autoimmune diseases, such as type 1 diabetes. Here, we describe a good manufacturing practice (GMP-compatible Treg expansion protocol using fluorescence-activated cell sorting, resulting in a mean 2,092-fold expansion of Tregs over a 16-day culture for a median yield of 1.26 × 109 Tregs from single-donor cryopreserved units. The resulting Tregs passed prior clinical trial release criteria for Treg purity and sterility, including additional rigorous assessments of FOXP3 and Helios expression and epigenetic analysis of the FOXP3 Treg-specific demethylated region (TSDR. Compared with expanded adult peripheral blood Tregs, expanded cord blood Tregs remained more naive, as assessed by continued expression of CD45RA, produced reduced IFN-γ following activation, and effectively inhibited responder T cell proliferation. Immunosequencing of the T cell receptor revealed a remarkably diverse receptor repertoire within cord blood Tregs that was maintained following in vitro expansion. These data support the feasibility of generating GMP-compliant Tregs from cord blood for adoptive cell transfer therapies and highlight potential advantages in terms of safety, phenotypic stability, autoantigen specificity, and tissue distribution.

  9. Artificial insemination and cryopreservation of semen from nondomestic birds (United States)

    Gee, G.F.; Bakst, M.R.; Wishart, G.J.


    Studies of Al and cryopreservation of semen from nondomestic birds began because of the increased emphasis on conservation of avian species threatened with extinction. Over the years, aviculturists have developed techniques for Al and cryopreservation of semen obtained from a variety of birds ranging from passerines to Andean condors. Generally, for each new species, we develop a practical semen collection technique and then evaluate the semen. A commercial semen extender (Beltsville Poultry Semen Extender) is modified and used to dilute the semen and provide support for the sperm during the freezing process (the pH and osmolality of the extender is adjusted to reflect the pH and osmolality of the semen being frozen). We find that the freezing schedule developed by Sexton (1977), which utilizes dimethylsulfoxide (DMS0) as cryoprotectant, works well for many species. We cool the sample sequentially in an ethanol bath, in liquid nitrogen vapor, and lastly in liquid nitrogen. Although we have experimented with a variety of freezing protocols, we prefer a 15-min equilibration period in DMSO at 5 C. We begin the freezing process by cooling at -1 C/min from 5 to -20 C in the ethanol bath. The samples are transferred into a vapor tank at a location just above liquid nitrogen and frozen at -50 C/min to -80 C. To complete the freezing process, the samples are plunged into the liquid nitrogen in the bottom of the vapor tank. The samples remain in liquid nitrogen until they are thawed just before insemination. If necessary, the freezing equipment can be transported in a van to remote locations.

  10. Sperm cryopreservation in brown bear (Ursus arctos): preliminary aspects. (United States)

    Anel, L; Alvarez, M; Martínez-Pastor, F; Gomes, S; Nicolás, M; Mata, M; Martínez, A F; Borragán, S; Anel, E; de Paz, P


    The development of sperm cryopreservation procedures in brown bear is the basis for establishing a specific genetic resource bank aimed at the preservation of a Cantabric brown bear population, which is seriously threatened. Several issues complicate the development of these cryopreservation procedures: lack of previous specific studies, a high incidence of urospermia and spermagglutination observed in bear ejaculates. Moreover, the availability of individuals for research from these threatened populations is problematic. In the case of the Cantabric brown bear, we have used males from other populations, but of the same species, as surrogates, to carry out a direct extrapolation of the results. Urospermia-- Moreover, 70% of the ejaculates are urine contaminated and spermagglutination have a detrimental effect on post-thawing cell quality recovery in this species. Considering the high value of these samples (autochthonous population with few individuals), a pre-selection of the ejaculates is not a viable alternative. Preventive methods reducing the mentioned detrimental effects need to be developed. On the basis of previous data, we can suppose that bear spermatozoa resist freezing injuries well. Nevertheless, because of the scarcity of this information, it is necessary to conduct further research on bear semen freezing under field conditions. Epidydimal spermatozoa can be important for genetic resource banking of threatened populations and thus specific cryobiological protocols need to be assayed. To date, 168 brown bear ejaculates have been frozen by the ITRA-ULE group at the University of León (Spain) in the development of methodologies for the preservation of brown bear sperm.

  11. Methods of cryopreservation of Tambaqui semen, Colossoma macropomum. (United States)

    Varela Junior, A S; Goularte, K L; Alves, J P; Pereira, F A; Silva, E F; Cardoso, T F; Jardim, R D; Streit, D P; Corcini, C D


    This study compared three different techniques for sperm cryopreservation of Tambaqui (Colossoma macropomum). Semen was diluted in Beltsville Thawing Solution with the addition of dimethyl sulfoxide (DMSO) at various concentrations (5%, 10%, 15% and 20%). Cryopreservation was performed using three methods: Box Conditioner Method with straws at a 5 cm distance from liquid nitrogen vapor (N2L); Dry Shipper Method placing the straws inside the machine; Vitrification Method placing the straws directly into N2L, amounting to 12 treatments (four DMSO concentrations×three freezing methods). The samples were evaluated for analysis of sperm quality in vivo and in vitro. Use of the Vitrification Method at different concentrations of DMSO provided the least values in the different evaluations. Fertilization, hatching rates and plasma membrane integrity using the Box Conditioner Method with 5% and 10% DMSO did not differ (P>0.05) but use of the concentration of 5% DMSO resulted in greater values than the other treatments (P<0.05) as well as for sperm motility and latency time (P<0.05), although sperm viability was superior using the Dry Shipper Method with 20% of the cryoprotectant. Mitochondrial functionality was impaired by use of the Vitrification Method with all DMSO concentration tested showing the most desirable values when the Box Conditioner Method was used with 5%, 10%, 15% DMSO and the Dry Shipper Method was used with 10% and 15% DMSO. Considering the variables evaluated, the use of the Box Conditioner Method is associated with enhanced Tambaqui semen quality with freeze concentrations of 5% and 10% DMSO. Copyright © 2015. Published by Elsevier B.V.

  12. Gender differences in subcutaneous and perirenal fat distribution


    Eisner, BH; Zargooshi, J; Berger, AD; Cooperberg, MR; Doyle, SM; Sheth, S; Stoller, ML


    Purpose: Body mass index (BMI) has been shown to influence the outcome of various surgical procedures. The purpose of this study is to assess the relationship between BMI, gender, and the distribution of subcutaneous and perirenal fat. Methods: A retrospective review was performed for 123 patients who underwent radical or partial nephrectomy. Preoperative CT scans were reviewed by two independent observers. Subcutaneous fat was measured at three locations and perirenal fat was measured at six...

  13. Subcutaneous autologous serum therapy in chronic spontaneous urticaria

    Directory of Open Access Journals (Sweden)

    Kiran Vasant Godse


    Full Text Available Background: There is a felt need for trying newer therapeutic modalities in patients with chronic spontaneous urticaria, especially in the subset of patients classified as non-responders to antihistamines. Autologous serum therapy is an upcoming modality of treatment, and we decided to study its efficacy by subcutaneous route. Aims: To evaluate the effectiveness of subcutaneous autologous serum therapy (AST in CSU. Methods: This was a single blind, placebo-controlled parallel group, randomized, controlled study. Twenty-four patients with CSU (11M: 13 F were given subcutaneous AST and seventeen patients (7 M: 10F patients were given subcutaneous injection normal saline (placebo, along with levocetirizine in an on-demand basis in both groups. Results: Urticaria activity score (UAS came down from 35.74 to 7 at the end of 9 weeks and the patients' requirement of antihistamines also reduced remarkably from 5.8 to 1.7 per week in the serum group. Sub-cutaneous saline group did not show statistically significant fall in UAS. Saline group showed UAS 32.8 at zero week to 22.1 at the end of 9 weeks. DLQI showed significant fall in serum group, from 14.26 to 4 at the end of 9 weeks. Conclusion: Subcutaneous autoserum therapy is effective in treatment of CSU.

  14. Gender differences in subcutaneous and perirenal fat distribution. (United States)

    Eisner, Brian H; Zargooshi, Javaad; Berger, Aaron D; Cooperberg, Matthew R; Doyle, Sean M; Sheth, Sonali; Stoller, Marshall L


    Body mass index (BMI) has been shown to influence the outcome of various surgical procedures. The purpose of this study is to assess the relationship between BMI, gender, and the distribution of subcutaneous and perirenal fat. A retrospective review was performed for 123 patients who underwent radical or partial nephrectomy. Preoperative CT scans were reviewed by two independent observers. Subcutaneous fat was measured at three locations and perirenal fat was measured at six locations. Statistical analysis was performed using the Student's t test and the Pearson's correlation coefficient. Mean anterior subcutaneous fat was significantly greater in females than in males (2.54 vs. 1.78 cm, p fat (2.78 vs. 2.21 cm, p = 0.03). With regard to perirenal fat, men were greater than women for all perirenal locations around the left kidney. For the right kidney, men were greater than women for four out of six perirenal positions. In both men and women, BMI was strongly correlated with subcutaneous fat. However, BMI was weakly correlated with perirenal fat. Women exceed men in subcutaneous fat, while men exceed women in perirenal fat. Obese patients are very likely to have large amounts of subcutaneous fat, but will not necessarily have proportionally increased fat around their kidneys when compared to the patients with lower BMI. These differences may have important implications for surgical approaches to the kidney.

  15. Pediatric Liver Transplantation. (United States)

    Rawal, Nidhi; Yazigi, Nada


    Excellent outcomes over the last 3 decades have made liver transplantation the treatment of choice for many advanced liver disorders. This success also opened liver transplantation to new indications such as liver tumors and metabolic disorders. The emergence of such new indications for liver transplantation is bringing a new stream of patients along with disease-specific challenges. The cumulative number of liver transplant recipients is peaking, requiring novel systems of health care delivery that meet the needs of this special patient population. This article reviews updates and new development in pediatric liver transplantation. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Long-Term Results of Surgical Treatment for Herniated Discs Using the Technique of Autologous Chondrocyte Transplantation

    Directory of Open Access Journals (Sweden)

    E.G. Pedachenko


    Full Text Available Objective: to evaluate the effectiveness of autologous chondrocyte (AC transplantation in patients after lumbar microdiscectomy in the long-term period. Materials and methods. Transplantation of cryopreserved AC has been carried out in 6 patients 3 months after lumbar microdiscectomy. Evaluation of clinical status was performed using VAS and NASS scales, before and 3 months after microdiscectomy, 1 and 2 years after AC transplantation. In the same period, there were also evaluated magnetic resonance characteristics of hydration of intervertebral disc. Results. According to magnetic resonance imaging, recovery of nucleus pulposus hydrophilicity was found in 4 of 6 patients (66.7 %, who underwent AC transplantation. Pain severity decreased significantly in the early period after microdiscectomy, and 3 months after (by the time of AC transplantation it has reduced by almost 6 times. One and 2 years after this procedure, pain continued to decline steadily. Functional status and quality of life after microdiscectomy significantly improved during first 3 months, and after transplantation they have not changed. Conclusions. Autologous chondrocyte transplantation after lumbar microdiscectomy is a safe method of surgical treatment for degenerative diseases of the spine, it helps to recover the biomechanical properties of the operated intervertebral disc.

  17. Transplantation of Adipose Tissue and Adipose-Derived Stem Cells as a Tool to Study Metabolic Physiology and for Treatment of Disease


    Tran, Thien T.; Kahn, C. Ronald


    Humans and other mammals have three main fat depots - visceral white fat, subcutaneous white fat, and brown fat - each possessing unique cell-autonomous properties. In contrast to visceral fat which can induce detrimental metabolic effects, subcutaneous white fat and brown fat have potential beneficial metabolic effects, including improved glucose homeostasis and increased energy consumption, which might be transferred by transplantation of these fat tissues. In addition, fat contains adipose...

  18. Stage selection and restricted oviposition period improves cryopreservation of dipteran embryos. (United States)

    Rajamohan, Arun; Rinehart, Joseph P; Leopold, Roger A


    Embryos of two dipteran species (Musca domestica and Lucilia sericata) were assessed for an effective sampling time that would result in the highest post-cryopreservation hatch rate, with a primary goal to define species-specific egg collection periods and the effects of manual stage selection on post cryopreservation yield. The effects of the time taken to collect eggs on, (a) the proportion of embryos reaching a specific developmental stage between 17 and 20 h of development, and (b) the post-cryopreservation hatch rate were assessed. Permeabilization treatment applied at any stage of embryonic development did not significantly reduce embryo viability. Eggs collected over longer durations significantly reduced the number of embryos available in a specific developmental stage amenable to cryopreservation. Hatch percentage after cryopreservation of the embryos of M. domestica collected over a 60 min period was 10.7 ± 8.7% compared to 31 ± 5% for the eggs collected for just 15 min. Similarly, percent hatch in L. sericata resulted in 17.0 ± 3.9 and cryopreservation after manual selection of specific embryonic developmental stages from the dechorionated samples. Post-cryopreservation hatching rate for stage-selected M. domestica embryos was 86.5 ± 5.5% compared to 33.3 ± 4.5% for embryos staged only by an overall visual confirmation. In the case of L. sericata, the hatching percentage was 79.0 ± 11.1 for stage-selected embryos compared to 17.0 ± 3.9% without individual selection. Published by Elsevier Inc.

  19. Cryopreservation of bull semen is associated with carbonylation of sperm proteins. (United States)

    Mostek, Agnieszka; Dietrich, Mariola Aleksandra; Słowińska, Mariola; Ciereszko, Andrzej


    Artificial insemination with cryopreserved semen enables affordable, large-scale dissemination of gametes with superior genetics. However, cryopreservation can cause functional and structural damage to spermatozoa that is associated with reactive oxygen species (ROS) production, impairment of sperm motility and decreased fertilizing potential, but little attention has been paid to protein changes. The goal of this study was to investigate the oxidative modifications (measured as carbonylation level changes) of bull spermatozoa proteins triggered by the cryopreservation process. Flow cytometry and computer-assisted sperm analysis were used to evaluate changes in viability, ROS level and motility of spermatozoa. Western blotting, in conjunction with two-dimensional electrophoresis (2D-oxyblot) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight spectrometry, was employed to identify and quantify the specifically carbonylated spermatozoa proteins. Cryopreservation decreased motility and viability but increased the number of ROS-positive cells. We identified 11 proteins (ropporin-1, outer dense fiber protein 2, glutathione S-transferase, triosephosphate isomerase, capping protein beta 3 isoform, actin-related protein M1, actin-related protein T2, NADH dehydrogenase, isocitrate dehydrogenase, cilia- and flagella-associated protein 161, phosphatidylethanolamine-binding protein 4) showing differences in protein carbonylation in response to cryopreservation. The identified proteins are associated with cytoskeleton and flagella organization, detoxification and energy metabolism. Moreover, almost all of the identified carbonylated proteins are involved in capacitation. Our results indicate for the first time that cryopreservation induces oxidation of selected sperm proteins via carbonylation. We suggest that carbonylation of sperm proteins could be a direct result of oxidative stress and potentially lead to disturbances of capacitation

  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


    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. MRI of pancreatic transplants

    International Nuclear Information System (INIS)

    Studniarek, M.; Deschamps, P.Y.; Finas, B.; Pinet, A.


    The value of MRI in assessing pancreatic transplants was studied in 37 patients. Sixty-seven MRI examinations were performed in patients with an uncomplicated transplant, 14 in patients with poorly functioning transplant, and 10 in patients with a non-functioning graft. On the basis of 54 follow-up studies, it was shown that the volume of the graft decreased systematically during the 8 months after transplantation. On T1-weighted images the normal transplant was poorly delineated, with an almost homogeneous isointense or slightly hyperintense structure when compared with either renal transplant cortex or muscle. On T2-weighted images the organ was isointense or slightly hypointense compared with fat and hyperintense compared with muscle. T2-weighted delayed echo time image (TR = 2000 ms, TE = 150-200 ms) showed transplanted pancreas as well-delineated, hypointense and with a lobulated structure. This structure was characteristic of normal whole pancreatic grafts. Patent transplant vessels were seen as tubular structures of low signal intensity on T2-weighted short echo time images (TR = 2000 ms, TE = 50 ms). In the 10 patients with a non-functioning pancreatic transplant there were: 4 cases of focal intraparenchymal abnormalities, 6 cases in which the lobular structure was absent, and 4 cases of absence of patent graft main vessels (3 thromboses). There was no configuration of signal intensity of pancreatic parenchyma on MRI which could be considered typical for normal or non-functioning transplant. (orig.)

  2. Beneficial effect of directional freezing on in vitro viability of cryopreserved sheep whole ovaries and ovarian cortical slices. (United States)

    Maffei, S; Pennarossa, G; Brevini, T A L; Arav, A; Gandolfi, F


    Does directional freezing improve the structural and functional integrity of ovarian fragments compared with conventional slow freezing and to whole ovary cryopreservation? Compared with slow freezing, the use of directional freezing significantly improves all structural and functional parameters of ovarian fragments assessed in vitro and, overall, whole ovaries were better preserved than ovarian fragments. Directional freezing has been developed to provide an alternative way to cryopreserve large biological samples and it is known to improve the structural and functional integrity of whole ovaries. Conventional slow freezing of ovarian fragments is the procedure more widely used in clinical settings but it causes substantial structural damage that limits the functional period after transfer back into the patient. We performed a 2 × 2 factorial design experiment on a total of 40 sheep ovaries, divided into four groups (n = 10 ovaries per group): (i) directional freezing of whole ovary (DFwo); (ii) directional freezing of ovarian fragments (DFof); (iii) conventional freezing of whole ovary (CFwo); (iv) conventional freezing of ovarian fragments (CFof). An additional eight ovaries were used as fresh controls. Ewe ovaries were randomly assigned to one of the experimental groups and frozen accordingly. Upon thawing, ovarian tissue was examined morphologically and cultured in vitro for 7 days. Samples were analyzed for cell proliferation and apoptosis, for DNA damage and repair activity, and for the presence of a panel of heat shock proteins (HSPs) by immunohistochemistry. Most studied parameters were significantly improved (P sheep ovaries, which are smaller than human ovaries and therefore may withstand the procedures better. Improved integrity of ovarian morphology may translate to improved outcomes after transplantation. Alternatively, the particularly good preservation of whole ovaries suggests they could provide a source of ovarian follicles for in vitro culture

  3. Morphology and function of cryopreserved whole ovine ovaries after heterotopic autotransplantation

    Directory of Open Access Journals (Sweden)

    Sharma Rakesh K


    Full Text Available Abstract Background The objective of this study was to perform complex characterization of cryopreserved and then autotransplanted ovaries including determination of the ability to respond to in vivo follicle stimulating hormone (FSH-treatment, fertilizability of retrieved oocytes, and morphology, vascularization, cellular proliferation and apoptosis in sheep. Methods Mature crossbred ewes were divided into two groups; an intact (control group (n = 4, and autotransplanted group (n = 4 in which oophorectomy was performed laparoscopically and ovaries with intact vascular pedicles frozen, thawed and transplanted back into the same animal at a different site. Approximately five months after autotransplantation, estrus was synchronized, ewes were treated with FSH, and ovaries were collected. For all ovaries, number of visible follicles was determined, and collected cumulus oocyte complexes (COC were matured and fertilized in vitro. Remaining ovarian tissues were fixed for evaluation of morphology, expression of factor VIII (marker of endothelial cells, vascular endothelial growth factor (VEGF; expressed by pericytes and smooth muscle cells, and smooth muscle cell actin (SMCA; marker of pericytes and smooth muscle cells, and cellular proliferation and apoptosis. Two fully functional ovaries were collected from each control ewe (total 8 ovaries. Results Out of eight autotransplanted ovaries, a total of two ovaries with developing follicles were found. Control ewes had 10.6 +/- 2.7 follicles/ovary, oocytes were in vitro fertilized and developed to the blastocyst stage. One autotransplanted ewe had 4 visible follicles from which 3 COC were collected, but none of them was fertilized. The morphology of autotransplanted and control ovaries was similar. In control and autotransplanted ovaries, primordial, primary, secondary, antral and preovulatory follicles were found along with fully functional vascularization which was manifested by expression of factor

  4. Scheduled transplantation of human umbilical cord blood to severe combined immunodeficient mice

    International Nuclear Information System (INIS)

    Wu Jianqiu; Yang Yunfang; Jin Zhijun; Cai Jianming; Yang Rujun; Xiang Yingsong


    Objective: To explore a new method for developing the efficiency of human umbilical cord blood (UCB) cells engraftment, and further understand the growth characteristic of hematopoietic stem cells (HSC) in vivo. Methods: Sublethally irradiated severe combined immunodeficient (SCID) mice were transplanted i.v. with UCB cells which had been cryo-preserved at -80 degree C. The human cells in recipient mice were detected by flow cytometry and CFU-GM assay. Results: In contrast to the single transplantation, scheduled engraftment of similar numbers of UCB cells resulted in a proportionally obvious increase in the percentages of CD45 + , CD34 + cells produced in SCID mouse bone marrow (BM). When the donor cells were reduced to 20 percent, an identical reconstitution of both hematopoietic and part of immunologic functions was achieved. Conclusion: Scheduled engraftment improves the repopulating ability of HSC, which would provide a novel way for clinical cord blood engraftment in adult objects

  5. A Chemically Defined Medium for Rabbit Embryo Cryopreservation (United States)

    Bruyère, Pierre; Baudot, Anne; Joly, Thierry; Commin, Loris; Pillet, Elodie; Guérin, Pierre; Louis, Gérard; Josson-Schramme, Anne; Buff, Samuel


    This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in rabbit embryo cryopreservation solutions. This evaluation was performed using two approaches: a thermodynamic approach using differential scanning calorimetry and a biological approach using rabbit embryo slow-freezing. During the experiment, foetal calf serum (FCS) was used as a reference. Because FCS varies widely by supplier, three different FCS were selected for the thermodynamic approach. The rabbit embryo slow-freezing solutions were made from Dulbecco's phosphate buffer saline containing 1.5 M Dimethyl Sulfoxide and 18% (v.v−1) of CRYO3 or 18% (v.v−1) of FCS. These solutions were evaluated using four characteristics: the end of melting temperature, the enthalpy of crystallisation (thermodynamic approach) and the embryo survival rates after culture and embryo transfer (biological approach). In the thermodynamic approach, the solutions containing one of the three different FCS had similar mean thermodynamic characteristics but had different variabilities in the overall data with aberrant values. The solution containing CRYO3 had similar thermodynamic properties when compared to those containing FCS. Moreover, no aberrant value was measured in the solution containing CRYO3. This solution appears to be more stable than the solutions containing a FCS. In the biological approach, the in vitro embryo survival rates obtained with the solution containing CRYO3 (73.7% and 81.3%) and with the solution containing a FCS (77.6% and 71.9%) were similar (p = 0.7). Nevertheless, during the in vivo evaluation, the implantation rate (21.8%) and the live-foetuses rate (18.8%) of the CRYO3 group were significantly higher than the implantation rate (7.1%, p = 0.0002) and the live-foetuses rate (5.3%, p = 0.0002) of the FCS group. The pregnancy rate was also higher in the CRYO3 group compared to the FCS group (81.3% and 43.8%, respectively, p = 0

  6. High-Yield Purification, Preservation, and Serial Transplantation of Human Satellite Cells

    Directory of Open Access Journals (Sweden)

    Steven M. Garcia


    Full Text Available Summary: Investigation of human muscle regeneration requires robust methods to purify and transplant muscle stem and progenitor cells that collectively constitute the human satellite cell (HuSC pool. Existing approaches have yet to make HuSCs widely accessible for researchers, and as a result human muscle stem cell research has advanced slowly. Here, we describe a robust and predictable HuSC purification process that is effective for each human skeletal muscle tested and the development of storage protocols and transplantation models in dystrophin-deficient and wild-type recipients. Enzymatic digestion, magnetic column depletion, and 6-marker flow-cytometric purification enable separation of 104 highly enriched HuSCs per gram of muscle. Cryostorage of HuSCs preserves viability, phenotype, and transplantation potential. Development of enhanced and species-specific transplantation protocols enabled serial HuSC xenotransplantation and recovery. These protocols and models provide an accessible system for basic and translational investigation and clinical development of HuSCs. : Garcia and colleagues report methods for efficient purification of satellite cells from human skeletal muscle. They use their approaches to demonstrate stem cell functions of endogenous satellite cells and to make human satellite cells accessible for sharing among researchers. Keywords: human satellite cell purification, serial transplantation, satellite cell cryopreservation

  7. Cryopreservation of in vitro grown shoot tips and apical meristems of the forage legume Arachis pintoi. (United States)

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


    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.

  8. Cryopreservation of Acropora digitifera sperm with use of sucrose and methanol based solution. (United States)

    Ohki, Shun; Morita, Masaya; Kitanobo, Seiya; Kowalska, Agata A; Kowalski, Radosław Kajetan


    Coral biodiversity has recently been considered an important topic in environmental studies. Biodiversity could be preserved with successful cryopreservation of endangered species gametes or embryos. Herein, we developed cryopreservation protocols for Acropora digitifera sperm with use of sucrose and methanol based extender. We studied cryopreservation of A. digitifera sperm with floating frames, allowing the placement of 250 μl French straws 4 cm above the liquid nitrogen surface, resulting in a 40 °C/min freezing rate. This method enabled the successful cryopreservation of sperm in 0.9 M sucrose supplemented with 20% methanol. In this protocol, we used a 1:3 (sperm:extender) dilution ratio. The fertilization ratios of freezing:thawed sperm were similar to the control and reached 63%. This method might be a valuable option in the formation of A. digitifera gene banking. Further studies are needed to explore possibilities of using this method in cryopreservation of other coral's sperm. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Cryopreservation of Populus trichocarpa and Salix dormant buds with recovery by grafting or direct rooting. (United States)

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


    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.

  10. Cryopreservation of chicken primordial germ cells by vitrification and slow freezing: A comparative study. (United States)

    Tonus, C; Connan, D; Waroux, O; Vandenhove, B; Wayet, J; Gillet, L; Desmecht, D; Antoine, N; Ectors, F J; Grobet, L


    In the present study, we compare a classical slow freezing (SLF) method and an aseptic vitrification (Vitrif) technique to cryopreserve a stable primordial germ cell (PGCs) line issued from the Ardennaise chicken breed. Viability immediately after warming was close to 80% and did not differ between the two cryopreservation methods. Proliferation tended to be slower for both cryopreservation methods compared with controls, but the difference was significant only for Vitrif. No difference was found between the two methods after flow cytometry analysis of stage-specific embryonic antigen-1 expression and reverse transcription-polymerase chain reaction on several factors related to PGC phenotype. After 1 week in culture, all cryopreserved cells reached controls' main morphologic and expanding (viability/proliferation) features. However, SLF generated more unwanted cells clusters than Vitrif. After injection of the PGCs into recipient embryos, vitrified PGCs reported a clear, yet not significant, tendency to colonize the gonad at a higher rate than slow frozen PGCs. SLF in cryovials remains simple, inexpensive, and less technically demanding than Vitrif. Nevertheless, the intrinsic advantages of our aseptic Vitrif method and the present study suggest that this should be considered as safer than classical SLF for cryopreserving chicken PGCs. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Arulvilee Rajasegar


    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

  12. Effectiveness of glucose-methanol extender for cryopreservation of Huso huso spermatozoa. (United States)

    Aramli, Mohammad Sadegh; Golshahi, Karim; Nazari, Rajab Mohammad; Aramli, Salim; Banan, Ashkan


    The present approach was designed to evaluate the methanol-glucose extender effects on sperm cryopreservation in beluga sturgeon, Huso huso. Sperm quality was examined by measuring post-thaw sperm motility and fertilizing rate at hatching stage. We first tested the effect of glucose concentration (0, 0.10, 0.15, 0.20 and 0.30M) in a methanol extender on post-thaw sperm motility. The optimal cryopreservation conditions were found to be 0.2M glucose in the extender. Then, motility and fertilization rates of sperm cryopreserved with 0.2M glucose and 10% methanol (GM) were compared to Tris-sucrose-KCl in 10% methanol extender (TSKM). Additionally, sperm motility and fertilizing ability in relation to 15 and 30min equilibration in GM extender before and after cryopreservation were measured. Higher post-thaw sperm motility duration and percentage as well as fertilization rate were obtained with the GM extender when compared to TSKM extender. Equilibration of sperm in extender did not affect the motility quality of either fresh-diluted or frozen/thawed sperm, while fertilization rate showed a significant decline alone after 30min of post-thaw storage. Our results indicated that the use of a simple extender consisting of 0.2M glucose in 10% methanol can be an alternative cryopreservation method to those previously described for sturgeons. Copyright © 2015 Elsevier B.V. All rights reserved.

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


    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.

  14. Effect of subcutaneous treatment with human umbilical cord blood-derived multipotent stem cells on peripheral neuropathic pain in rats. (United States)

    Lee, Min Ju; Yoon, Tae Gyoon; Kang, Moonkyu; Kim, Hyun Jeong; Kang, Kyung Sun


    In this study, we aim to determine the in vivo effect of human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs) on neuropathic pain, using three, principal peripheral neuropathic pain models. Four weeks after hUCB-MSC transplantation, we observed significant antinociceptive effect in hUCB-MSC-transplanted rats compared to that in the vehicle-treated control. Spinal cord cells positive for c-fos, CGRP, p-ERK, p-p 38, MMP-9 and MMP 2 were significantly decreased in only CCI model of hUCB-MSCs-grafted rats, while spinal cord cells positive for CGRP, p-ERK and MMP-2 significantly decreased in SNL model of hUCB-MSCs-grafted rats and spinal cord cells positive for CGRP and MMP-2 significantly decreased in SNI model of hUCB-MSCs-grafted rats, compared to the control 4 weeks or 8weeks after transplantation (ppain signaling during pain signal processing within the nervous system, especially for CCI model. Thus, subcutaneous administration of hUCB-MSCs might be beneficial for improving those patients suffering from neuropathic pain by decreasing neuropathic pain activation factors, while increasing neuropathic pain inhibition factor.

  15. Effects of cryopreservation on excretory function, cellular adhesion molecules and vessel lumen formation in human umbilical vein endothelial cells. (United States)

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


    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.

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

    NARCIS (Netherlands)

    Long, J.A.; Purdy, P.H.; Zuidberg, C.A.; Hiemstra, S.J.; Velleman, S.G.; Woelders, H.


    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

  17. Effects of chilled storage and cryopreservation on sperm characteristics, antioxidant enzyme activities, and lipid peroxidation in Pacific cod Gadus microcephalus (United States)

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


    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.

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

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


    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. Isolation and Characterization of Human Dental Pulp Stem Cells from Cryopreserved Pulp Tissues Obtained from Teeth with Irreversible Pulpitis. (United States)

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


    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.

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

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


    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

  2. Skin temperature and subcutaneous adipose blood flow in man

    DEFF Research Database (Denmark)

    Astrup, A; Bülow, J; Madsen, J


    The abdominal subcutaneous adipose tissue blood flow (ATBF) was measured bilaterally by the 133Xe washout method. At one side of the skin (epicutaneous) temperature was varied with a temperature blanket, the other side served as control. There was a significant (P less than 0.001) positive...... correlation between skin temperature and ATBF. In the range from 25 to 37 degrees CATBF increased 9% of the control flow on average per centigrade increase in skin temperature. ATBF at the control side was uninfluenced by the contralateral variations in skin temperature. Although no better correlation could...... be demonstrated between ATBF and subcutaneous temperature than between ATBF and skin temperature, arguments are presented in favour of the hypothesis that ATBF is influenced by the subcutaneous temperature rather than via reflexes from the skin. Infiltration of the 133Xe depots with 20 microgram histamine...

  3. Successful treatment of systemic lupus erythematosus with subcutaneous immunoglobulin. (United States)

    Brasileiro, A; Fonseca Oliveira, J; Pinheiro, S; Paiva-Lopes, M J


    The therapeutic efficacy of high-dose intravenous immunoglobulin in systemic lupus erythematosus (SLE) patients is well established. However, side effects might limit its use and lead to the consideration of therapeutic alternatives, such as the subcutaneous formulation of immunoglobulin, which has been used in some patients with other autoimmune diseases. We report a case of SLE refractory to classical therapies. High-dose intravenous immunoglobulin was effective, but gave rise to significant side effects. The patient was successfully treated with subcutaneous human immunoglobulin, achieving and maintaining clinical and laboratory remission. A lower immunoglobulin dose was needed and no side effects were observed, compared to the intravenous administration. Subcutaneous immunoglobulin could be a better-tolerated and cost-saving therapeutic option for select SLE patients. © The Author(s) 2016.

  4. Subcutaneous steroid injection as treatment for chalazion: prospective case series. (United States)

    Ho, S Y; Lai, J S M


    To study the efficacy of subcutaneous steroid injection in the treatment of chalazion. Prospective consecutive case series. University teaching hospital, Hong Kong. Patients with chalazion presenting to the out-patient clinic of the Department of Ophthalmology at the Prince of Wales Hospital from January to June 1998. Size of the chalazion after steroid injection treatment. Forty-eight consecutive patients with chalazion were treated with injection of triamcinolone into the subcutaneous tissue around the lesion. In 43 (89.6%) patients, the lesion subsided completely. Twenty-six (54.2%) patients had lesions that subsided with one injection. The size and duration of the chalazion at presentation did not significantly affect the outcome of the treatment. Two patients developed depigmentation of the skin at the site of injection. No other major complications were encountered. Subcutaneous injection of the steroid triamcinolone acetonide appears to be a simple and effective treatment for chalazion. Further comparative clinical trials are indicated.

  5. Subcutaneous Emphysema, Pneumomediastinum and Pneumothorax in a Patient with Dermatomyositis

    Directory of Open Access Journals (Sweden)

    Mehdi Bakhsaee


    Full Text Available Introduction:Spontaneous pneumomediastinum, pneumothorax, and subcutaneous emphysema are rare, but serious complications of inflammatory myopathies and occur more commonly in DM than PM. complications of dermatomyositis (DM and polymyositis (PM, both of which can be fatal. Case Report:A 20-year-old woman was admitted with neck pain, dyspnea, cough, and fever. She had been diagnosed with dermatomyositis 21 months prior. A thorax computed tomography (CT scan revealed ground glass opacities in her lungs, pneumomediastinum, pneumothorax, and subcutaneous emphysema. Despite intensive immunosuppressive therapy, clinical deterioration and radiological progression were observed, ultimately the patient died. Conclusion:During the care for a patient with dermatomyositis, the otorhinolaryngologist should be cautious of rapidly progressive and fatal neck subcutaneous emphysema. For a patient with dermatomyositis and with normal bronchoscopy and esophagoscopy, the main treatment is control of dermatomyositis with medical therapy. Therefore, a tracheostomy and/or mechanical ventilation may not be necessary.

  6. [Measles and subcutaneous emphysema. Presentation of 3 cases]. (United States)

    Montesano-Delfín, J R; Mascareñas-Ponce, A


    This is a three case study report of children with measles which later progressed to bronchopneumonia and subcutaneous emphysema. All three children were from farming families, and none had been previously vaccinate against measles. For a period of six months, 183 cases of measles were treated at our hospital of which only three worsened to subcutaneous emphysema, demonstrating an incidence rate of 1.6%; they also showed to have bronchopneumonia, with severe coughing episodes; which made us recall the possible physiopathology principle of the pressure gradient theory behind this complication proposed by Bloch in 1968. The factors related to our patients suggested a more severe and aggresive type of measles with a greater probability of having complications. The prognostic value of the severity of this type of measles in the presence of subcutaneous emphysema is limited and its management should be primarly focused on treating the added bronchial problem.

  7. Successful hematopoietic reconstitution with transplantation of erythrocyte-depleted allogeneic human umbilical cord blood cells in a child with leukemia. (United States)

    Pahwa, R N; Fleischer, A; Than, S; Good, R A


    Cord blood, a potent source of hematopoietic stem cells, has been shown to successfully reconstitute hematopoiesis following allogeneic transplantation in a variety of disorders. A major drawback of cord blood has been the risk of transfusion reactions in ABO blood group incompatibility and drastic reduction in the stem cell pool if the cord blood is manipulated to remove red cells prior to cryopreservation or after thawing. This report describes an erythrocyte depletion method employing 3% gelatin-induced erythrocyte sedimentation for the selective removal of red cells from cord blood. The red cell-depleted fraction was shown to be enriched in progenitor cells and in cells secreting hematopoietic cytokines interleukin 3, granulocyte/macrophage colony-stimulating factor, and interleukin 6; a major source for cytokines was from cord T cells. This preparative technique was employed to separate out red cells from cord blood of an infant delivered by cesarean section who had an 8-year-old sibling with leukemia. Histocompatibility testing of cord cells revealed complete matching with the patient. A cord cell transplant of cryopreserved and thawed cells consisting of 4 x 10(7) nucleated cells per kg was administered to the patient following myeloablative chemotherapy. The patient's quick hematologic recovery and 9-month disease-free period to date suggest that 3% gelatin separation of erythrocytes is a simple method that can be successfully used for transplanting cord cells for malignant/nonmalignant diseases.

  8. Thoracic organ transplantation. (United States)

    Pierson, Richard N; Barr, Mark L; McCullough, Keith P; Egan, Thomas; Garrity, Edward; Jessup, Mariell; Murray, Susan


    This article presents an overview of factors associated with thoracic transplantation outcomes over the past decade and provides valuable information regarding the heart, lung, and heart-lung waiting lists and thoracic organ transplant recipients. Waiting list and post-transplant information is used to assess the importance of patient demographics, risk factors, and primary cardiopulmonary disease on outcomes. The time that the typical listed patient has been waiting for a heart, lung, or heart-lung transplant has markedly increased over the past decade, while the number of transplants performed has declined slightly and survival after transplant has plateaued. Waiting list mortality, however, appears to be declining for each organ and for most diseases and high-severity subgroups, perhaps in response to recent changes in organ allocation algorithms. Based on perceived inequity in organ access and in response to a mandate from Health Resources and Services Administration, the lung transplant community is developing a lung allocation system designed to minimize deaths on the waiting list while maximizing the benefit of transplant by incorporating post-transplant survival and quality of life into the algorithm. Areas where improved data collection could inform evolving organ allocation and candidate selection policies are emphasized.

  9. Subcutaneous Implants of Buprenorphine-Cholesterol-Triglyceride Powder in Mice


    L. DeTolla; R. Sanchez; E. Khan; B. Tyler; M. Guarnieri


    Subcutaneous drug implants are convenient systems for the long-term delivery of drugs in animals. Lipid carriers are logical tools because they generally allow for higher doses and low toxicity. The present study used an US Food and Drug Administration Target Animal Safety test system to evaluate the safety of a subcutaneous implant of a cholesterol-triglyceride-buprenorphine powder in 120 BALB/c mice. Mice were evaluated in 4- and 12-day trials with 1- and 5-fold doses of the intended 3 mg/k...

  10. Anisotropic bioelectrical impedance determination of subcutaneous fat thickness (United States)

    Hernández-Becerra, P. A. I.; Delgadillo-Holtfort, I.; Balleza-Ordaz, M.; Huerta-Franco, M. R.; Vargas-Luna, M.


    Preliminary results have shown that bioelectrical impedance measurements performed on different parts of the human body strongly depend upon the subcutaneous fat of the considered region. In this work, a method for the determination of subcutaneous fat thickness is explored. Within this method the measurement of the bioelectrical impedance response of the fat-muscle system, both along the direction defined by the muscle fibers and along the corresponding perpendicular direction, are performed. Measurements have been carried out on human female and male subjects of ages around 25 years old at the region of the biceps. Correlation has been performed with skinfold caliper measurements.

  11. Cryopreservation of Autologous Blood (Red Blood Cells, Platelets and Plasma) (United States)

    Ebine, Kunio

    Prevention of post-transfusion hepatitis is still a problem in cardiovascular surgery. We initiated the cryopreservation of autologous blood for the transfusion in elective cardiovascular surgery since 1981. This study includes 152 surgical cases in which autologous frozen, allogeneic frozen, and/or allogeneic non-frozen blood were used. In the 152 surgical cases, there were 69 cases in which autologous blood only (Group I) was used; 12 cases with autologous and allogeneic frozen blood (Group II); 46 cases with autologous and allgeneic frozen plus allogeneic non-frozen blood (Group III); and 25 cases with allogeneic frozen plus allogeneic non-frozen blood (Group IV). No hepatitis developed in Groups I (0%) and II (0%), but there was positive hepatitis in Groups III (4.3%) and IV (8.0%) . In 357 cases of those who underwent surgery with allogeneic non-frozen whole blood during the same period, the incidence rate of hepatitis was 13.7% (49/357). Patients awaiting elective surgery can store their own blood in the frozen state. Patients who undergo surgery with the cryoautotransfusion will not produce any infections or immunologic reactions as opposed to those who undergo surgery with the allogeneic non-frozen blood.

  12. Different extenders in the cryopreservation of bovine epididymal spermatozoa. (United States)

    Papa, Patrícia M; Papa, Frederico O; Oliveira, Letícia A; Guasti, Priscilla N; Castilho, Caliê; Giometti, Ines Cristina


    The objective of this study was to evaluate the effects of two different egg yolk extenders incubated with or without Sperm Talp on the motility and plasma membrane integrity of cryopreserved bovine epididymal spermatozoa after freezing. Twenty-five testicles with epididymides from mature bulls were collected at the abattoir. Epididymal sperm recovery was performed by retrograde flushing using a skim milk-extender (Botu-Semen™). After recovery, sperm were incubated either without or with Sperm Talp and then submitted to centrifugation. For the freezing process, half of the testes were processed with Tris egg yolk extender, and half were processed with Botu-Bov™ egg yolk extender. Samples incubated in Sperm Talp exhibited better results than epididymal spermatozoa that were incubated without Sperm Talp (psperm from the bovine epididymides if the sperm were previously incubated with Sperm Talp. The extenders examined in this work did not differ in their effect on plasma membrane integrity after freezing. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Cryopreservation of 'Nabali' olive (Olea europea l.) somatic embryos by encapsulation-dehydration and encapsulation-vitrification. (United States)

    Shibli, R A; Al-Juboory, K H


    Olive (Olea europea L.) somatic embryos were successfully cryopreserved using encapsulation-dehydration and encapsulation-vitrification. In the encapsulation-dehydration procedure, a maximum of 48% embryo survival was obtained when bead moisture content was decreased to 21.1% after 4 h dehydration. Preculture of embryos for 4 d in medium containing 0.75 to 1.25 M sucrose produced higher (40 to 34 %, respectively) regrowth after cryopreservation using encapsulation-dehydration procedure. Dehydration of beads for 3 h in PVS2 ensured higher survival (64%) of encapsulated-vitrified and cryopreserved (EVN) somatic embryos. Thermal treatment of embryogenic callus for 1 d at 30 degree C was very effective to increase survival of encapsulated-dehydrated and cryopreserved (EDN) (58%) and EVN (68%) embryos. Plantlets produced from control and cryopreserved embryos were phenotypically similar.

  14. Use of Viable Cryopreserved Placental Membrane as an Adjunct to Facial Keloid Resection

    Directory of Open Access Journals (Sweden)

    Rishi J. Gupta, DDS, MD, MBA


    Full Text Available Summary:. Keloids are the physical manifestation of an exaggerated inflammatory response resulting in excess collagen deposition. The resulting fibroproliferative mass can be distressing for patients due to appearance, pruritus, and/or pain. Despite extensive research into the pathophysiology of keloid formation and the development of numerous treatments, keloids remain a challenge to treat. Even when the initial treatment is successful, a risk of recurrence remains. Basic science research into viable cryopreserved placental membranes and viable cryopreserved umbilical tissue has demonstrated their anti-inflammatory and anti-fibrotic effects, which may decrease keloid recurrence after excision. In this article, we present the first-reported case of viable cryopreserved placental membrane, with living mesenchymal stem cells, to treat a painful preauricular keloid in conjunction with surgical resection.

  15. Cryopreservation by encapsulation-dehydration of plumules of coconut (Cocos nucifera L.). (United States)

    N'Nan, Oulo; Hocher, Valérie; Verdeil, Jean-Luc; Konan, Jean-Louis; Ballo, Koffi; Mondeil, Fanja; Malaurie, Bernard


    This study describes the use of an encapsulation-dehydration cryopreservation technique on coconut plumules (apical dome with three or four leaf primordia) excised from embryos. In order to establish a reliable cryopreservation process for plumules, several different key factors were tested: pretreatment duration, sugar concentration, dehydration period and freezing. In parallel, histological studies were performed to describe the structural changes of tissues and plumule cells subjected to dehydration and freezing. A good survival level of around 60% was obtained. However, after 8 months culture regrowth, this level decreased to a maximum of 20 % which was achieved using sucrose treatment. In this paper we report for the first time the regeneration of leafy shoots from coconut plumules after cryopreservation.

  16. Cryopreservation of animal oocytes and embryos: Current progress and future prospects. (United States)

    Mandawala, A A; Harvey, S C; Roy, T K; Fowler, K E


    Cryopreservation describes techniques that permit freezing and subsequent warming of biological samples without loss of viability. The application of cryopreservation in assisted reproductive technology encompasses the freezing of gametes, embryos, and primordial germ cells. Whilst some protocols still rely on slow-freezing techniques, most now use vitrification, or ultra-rapid freezing, for both oocytes and embryos due to an associated decreased risk of damage caused by the lack of ice crystal formation, unlike in slow-freezing techniques. Vitrification has demonstrated its use in many applications, not only following IVF procedures in human embryology clinics but also following in vitro production of embryos in agriculturally important, or endangered animal species, before embryo transfer. Here, we review the various cryopreservation and vitrification technologies that are used in both humans and other animals and discuss the most recent innovations in vitrification with a particular emphasis on their applicability to animal embryology. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Children born after cryopreservation of embryos or oocytes: a systematic review of outcome data

    DEFF Research Database (Denmark)

    Wennerholm, U-B; Söderström-Anttila, V; Bergh, C


    BACKGROUND: An estimated 3.5 million children have been born to date using assisted reproduction technologies. We reviewed the data in order to evaluate current knowledge of medical outcome for IVF/ICSI children born after cryopreservation, slow freezing and vitrification of early cleavage stage...... of blastocysts and for vitrification of early cleavage stage embryos, blastocysts and oocytes, limited neonatal data was reported. We found no long-term child follow-up data for any cryopreservation technique. CONCLUSION: Data concerning infant outcome after slow freezing of embryos was reassuring. Properly...... controlled follow-up studies of neonatal outcome are needed after slow freezing of blastocysts and after vitrification of early cleavage stage embryos, blastocysts and oocytes. In addition, child long-term follow-up studies for all cryopreservation techniques are essential....

  18. [Semen cryopreservation in adolescent with cancer: at which age can it be proposed?]. (United States)

    Laverdure, Noémie; d'Estaing, Sandrine Giscard; Marec-Berard, Perrine


    The increased incidence of tumors in children and adolescents (Lacour, 2010), as well as therapeutic advances in recent decades, have led to an increase of fertility disorders in young adult cancer survivors. In pubescent boys, the use of cryopreserved sperm and assisted reproductive technology (ART) is a validated preventive option. Currently, there is no consensus on the age at which the semen cryoconservation has to be proposed. There is a wide disparity of care among centers in France. Based on the observation of Nathan,11 years old, in whom semen cryopreservation was performed at his request, we analyze local practices and discuss the indications for cryopreservation of sperm in young teenagers with cancer treated by potentially sterilizing treatment.

  19. Kidney transplantation after previous liver transplantation: analysis of the organ procurement transplant network database. (United States)

    Gonwa, Thomas A; McBride, Maureen A; Mai, Martin L; Wadei, Hani M


    Patients after liver transplant have a high incidence of chronic kidney disease and end-stage renal disease (ESRD). We investigated kidney transplantation after liver transplantation using the Organ Procurement Transplant Network database. The Organ Procurement Transplant Network database was queried for patients who received kidney transplantation after previous liver transplantation. These patients were compared with patients who received primary kidney transplantation alone during the same time period. Between 1997 and 2008, 157,086 primary kidney transplants were performed. Of these, 680 deceased donor kidney transplants and 410 living donor kidney transplants were performed in previous recipients of liver transplants. The number of kidney after liver transplants performed each year has increased from 37 per year to 124 per year in 2008. The time from liver transplant to kidney transplant increased from 8.2 to 9.0 years for living donor transplants and from 5.4 to 9.6 years for deceased donor. The 1, 3, and 5 year actuarial graft survival in both living donor kidney after liver transplant and deceased donor kidney after liver transplant are less than the kidney transplant alone patients. However, the death-censored graft survivals are equal. The patient survival is also less but is similar to what would be expected in liver transplant recipients who did not have ESRD. In 2008, kidney after liver transplantation represented 0.9% of the total kidney alone transplants performed in the United States. Kidney transplantation is an appropriate therapy for selected patients who develop ESRD after liver transplantation.

  20. 86 successful births and 9 ongoing pregnancie s worldwide in women transplanted with frozen-thawed ovarian tissue

    DEFF Research Database (Denmark)

    Jensen, Annette Klüver; Macklon, Kirsten Tryde; Fedder, Jens


    . The mean gestational age was 39 weeks and the mean birth weight was 3168 g of the singleton pregnancies, which is within internationally recognized normal standards. Furthermore, half the singletons resulted from natural conception and all twins resulted from in vitro fertilization treatment. All seven...... Danish women became pregnant within 1-3 years after transplantation. They gave birth to nine healthy children. CONCLUSION: The data is reassuring and further suggests that cryopreservation of ovarian tissue is becoming an established fertility preservation method. The seven Danish women reported...