Interaction of estradiol and high density lipoproteins on proliferation of the human breast cancer cell line MCF-7 adapted to grow in serum free conditions

International Nuclear Information System (INIS)

Jozan, S.; Faye, J.C.; Tournier, J.F.; Tauber, J.P.; David, J.F.; Bayard, F.

1985-01-01

The responsiveness of the human mammary carcinoma cell line MCF-7 to estradiol and tamoxifen treatment has been studied in different culture conditions. Cells from exponentially growing cultures were compared with cells in their initial cycles after replating from confluent cultures (''confluent-log'' cells). It has been observed that estradiol stimulation of tritiated thymidine incorporation decreases with cell density and that ''confluent-log'' cells are estrogen unresponsive for a period of four cell cycles in serum-free medium conditions. On the other hand, growth of cells replated from exponentially growing, as well as from confluent cultures, can be inhibited by tamoxifen or a combined treatment with tamoxifen and the progestin levonorgestrel. This growth inhibitory effect can be rescued by estradiol when cells are replated from exponentially growing cultures. The growth inhibitory effect cannot be rescued by estradiol alone (10(-10) to 10(-8) M) when cells are replated from confluent cultures. In this condition, the addition of steroid depleted serum is necessary to reverse the state of estradiol unresponsiveness. Serum can be replaced by high density lipoproteins but not by low density lipoproteins or lipoprotein deficient serum. The present data show that estradiol and HDL interact in the control of MCF-7 cell proliferation

Active Stat3 is required for survival of human squamous cell carcinoma cells in serum-free conditions

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DiGiovanni John

2006-04-01

Full Text Available Abstract Background Squamous cell carcinoma (SCC of the skin is the most aggressive form of non-melanoma skin cancer (NMSC, and is the single most commonly diagnosed cancer in the U.S., with over one million new cases reported each year. Recent studies have revealed an oncogenic role of activated signal transducer and activator of transcription 3 (Stat3 in many human tumors, especially in those of epithelial origin, including skin SCC. Stat3 is a mediator of numerous growth factor and cytokine signaling pathways, all of which activate it through phosphorylation of tyrosine 705. Results To further address the role of Stat3 in skin SCC tumorigenesis, we have analyzed a panel of human skin-derived cell lines ranging from normal human epidermal keratinocytes (NHEK, to non-tumorigenic transformed skin cells (HaCaT, to highly tumorigenic cells (SRB1-m7 and SRB12-p9 and observed a positive correlation between Stat3 phosphorylation and SCC malignancy. We next determined the role of Stat3 activity in cell proliferation and viability under serum-free culture conditions. This was accomplished by suppressing Stat3 activity in the SRB12-p9 cells through stable expression of a dominant negative acting form of Stat3β, which contains a tyrosine 705 to phenylalanine mutation (S3DN. The S3DN cells behaved similar to parental SRB12-p9 cells when cultured in optimal growth conditions, in the presence of 10% fetal calf serum. However, unlike the SRB12-p9 cells, S3DN cells underwent apoptotic cell death when cultured in serum-free medium (SFM. This was evidenced by multiple criteria, including accumulation of sub-G1 particles, induced PARP cleavage, and acquisition of the characteristic morphological changes associated with apoptosis. Conclusion This study provides direct evidence for a role for Stat3 in maintaining cell survival in the conditions of exogenous growth factor deprivation produced by culture in SFM. We also propose that delivery of the S3DN gene or

Defined culture medium for stem cell differentiation: applicability of serum-free conditions in the mouse embryonic stem cell test.

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Riebeling, Christian; Schlechter, Katharina; Buesen, Roland; Spielmann, Horst; Luch, Andreas; Seiler, Andrea

2011-06-01

The embryonic stem cell test (EST) is a validated method to assess the developmental toxicity potency of chemicals. It was developed to reduce animal use and allow faster testing for hazard assessment. The cells used in this method are maintained and differentiated in media containing foetal calf serum. This animal product is of considerable variation in quality, and individual batches require extensive testing for their applicability in the EST. Moreover, its production involves a large number of foetuses and possible animal suffering. We demonstrate the serum-free medium and feeder cell-free maintenance of the mouse embryonic stem cell line D3 and investigate the use of specific growth factors for induction of cardiac differentiation. Using a combination of bone morphogenetic protein-2, bone morphogenetic protein-4, activin A and ascorbic acid, embryoid bodies efficiently differentiated into contracting myocardium. Additionally, examining levels of intracellular marker proteins by flow cytometry not only confirmed differentiation into cardiomyocytes, but demonstrated significant differentiation into neuronal cells in the same time frame. Thus, this approach might allow for simultaneous detection of developmental effects on both early mesodermal and neuroectodermal differentiation. The serum-free conditions for maintenance and differentiation of D3 cells described here enhance the transferability and standardisation and hence the performance of the EST. Copyright © 2011 Elsevier Ltd. All rights reserved.

The B-domain of factor VIII reduces cell membrane attachement to host cells in serum free conditions

DEFF Research Database (Denmark)

Kolind, Mille Petersen; Nørby, Peder Lisby; Flintegaard, Thomas Veje

2010-01-01

engineered extensively throughout the years to increase the low production yields that initially were obtained from mammalian cell cultures. The scope of this work was to investigate the interaction of rFVIII with the cell membrane surface of the producing cells in serum free medium. We wondered whether...... binding of rFVIII to the cell membrane could be a factor diminishing the production yield. We studied the contribution of the rFVIII B-domain to membrane attachment by transfecting several constructs containing increasing lengths of the B-domain into cells under serum free conditions. We found that 90......% of rFVIII is attached to the cell membrane of the producing cell when the rFVIII variant contains a short B-domain (21 aa). By increasing the length of the B-domain the membrane attached fraction can be reduced to 50% of the total expressed rFVIII. Further, our studies show that the N...

Growth in serum-free medium improves isolation of Chlamydia pneumoniae.

OpenAIRE

Maass, M; Essig, A; Marre, R; Henkel, W

1993-01-01

Infectivity titers were determined for eight Chlamydia pneumoniae strains simultaneously grown in serum-free and serum-supplemented cell culture media. Use of serum-free medium resulted in a 10- to 50-fold increase in the susceptibility of HL cells to chlamydial infection. Comparative primary isolation of a wild-type strain also produced higher inclusion counts in a serum-free environment. Serum-free cultivation is recommended to increase the efficiency of C. pneumoniae isolation from clinica...

Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.

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Kaneko, Ai; Sankai, Yoshiyuki

2014-01-01

The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10(4) cells/mL (8.9×10(3) cells/cm2) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).

Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.

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Ai Kaneko

Full Text Available The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10(4 cells/mL (8.9×10(3 cells/cm2 without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm, greater cell viability (≥30% for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks.

Compound serum and hemin free medium for cultivation of ...

African Journals Online (AJOL)

Serum free cultivation of Leishmania is cost-effective and improves large scale production of well defined parasite material. Moreover, the production of recombinant pharmaceutical proteins requires cultivation of the host in a culture medium free of animal materials, so several culture media for Leishmania tarentolae ...

In vitro maintenance of spermatogenesis in Xenopus laevis testis explants cultured in serum-free media

International Nuclear Information System (INIS)

Risley, M.S.; Miller, A.; Bumcrot, D.A.

1987-01-01

Spermatogenesis has been maintained for extended periods in Xenopus laevis testis explants cultured in serum-free media supplemented with bovine serum albumin, insulin, transferrin, follicle-stimulating hormone, dihydrotestosterone, testosterone, retinol, ascorbate, and tocopherol. The organization of the testis fragments was maintained for 28 days, and all stages of development were present throughout the culture period. 3 H-Thymidine-labeled secondary (Type B) spermatogonia developed in 28 days into spermatids at the acrosomal vesicle stage whereas labeled zygotene spermatocytes became mature spermatids in 28 days. Spermatogonial proliferation also continued in vitro for 28 days. Germ cell differentiation was not dependent upon exogenous testosterone, ascorbate, or tocopherol since 3 H-labeled spermatogonia became mature spermatids in testes cultured 35 days in media lacking these supplements. Autoradiography demonstrated that 55% of the luminal sperm present in explants cultured 10 days had differentiated in vitro. Sperm from testes cultured 10-35 days were similar to sperm from freshly dissected testes with regard to motility and fecundity, and eggs fertilized with sperm from explant cultures developed normally into swimming tadpoles. The results demonstrate the feasibility of maintaining vertebrate spermatogenesis in culture and suggest that in vitro analysis of Xenopus spermatogenesis using defined media may provide important insights into the evolution of regulatory mechanisms in spermatogenesis

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

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Corsini Joe

2004-01-01

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

Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

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Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

2014-02-01

Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Immobilization of sericin molecules via amorphous carbon plasma modified-polystyrene dish for serum-free culture

International Nuclear Information System (INIS)

Tunma, Somruthai; Song, Doo-Hoon; Kim, Si-Eun; Kim, Kyoung-Nam; Han, Jeon-Geon; Boonyawan, Dheerawan

2013-01-01

In this study, we focused on sericin hydrolysates, originating from silkworm used in serum-free human bone marrow-derived mesenchymal stem cells (hBM-MSCs) culture. We reported the effect of a covalent linkage between a bioactive protein molecule and polystyrene dish surface via a carbon intermediate layer which can slow down the release rate of protein compounds into the phosphate buffer saline (PBS) solution. Films of amorphous carbon (a-C) and functionalized-carbon were deposited on PS culture dish surfaces by using a DC magnetron sputtering system and RF PECVD system. We found that a-C based-films can increase the hydrophilicity and biocompatibility of polystyrene (PS) dishes, especially a-C films and a-C:N 2 films showed good attachment of hBM-MSCs at 24 h. However, in the case of silica surface (a-C:SiO x films), the cells showed a ragged and unattached boundary resulting from the presence of surface silanol groups. For the UV–vis absorbance, all carbon modified-PS dishes showed a lower release rate of sericin molecules into PBS solution than PS control. This revealed that the functionalized carbon could be enhanced by specific binding properties with given molecules. The carbon-coated PS dishes grafting with sericin protein were used in a serum-free condition. We also found that hBM-MSCs have higher percentage of proliferated cells at day 7 for the modified dishes with carbon films and coated with sericin than the PS control coated with sericin. The physical film properties were measured by atomic force microscopy (AFM), scanning electron microscope (SEM) and contact angle measurement. The presence of -NH 2 groups of sericin compounds on the PS dish was revealed by Fourier transform infrared spectroscopy (FTIR). The stability of covalent bonds of sericin molecules after washing out ungrafted sericin was confirmed by X-ray photoelectron spectroscopy (XPS).

Immobilization of sericin molecules via amorphous carbon plasma modified-polystyrene dish for serum-free culture

Science.gov (United States)

Tunma, Somruthai; Song, Doo-Hoon; Kim, Si-Eun; Kim, Kyoung-Nam; Han, Jeon-Geon; Boonyawan, Dheerawan

2013-10-01

In this study, we focused on sericin hydrolysates, originating from silkworm used in serum-free human bone marrow-derived mesenchymal stem cells (hBM-MSCs) culture. We reported the effect of a covalent linkage between a bioactive protein molecule and polystyrene dish surface via a carbon intermediate layer which can slow down the release rate of protein compounds into the phosphate buffer saline (PBS) solution. Films of amorphous carbon (a-C) and functionalized-carbon were deposited on PS culture dish surfaces by using a DC magnetron sputtering system and RF PECVD system. We found that a-C based-films can increase the hydrophilicity and biocompatibility of polystyrene (PS) dishes, especially a-C films and a-C:N2 films showed good attachment of hBM-MSCs at 24 h. However, in the case of silica surface (a-C:SiOx films), the cells showed a ragged and unattached boundary resulting from the presence of surface silanol groups. For the UV-vis absorbance, all carbon modified-PS dishes showed a lower release rate of sericin molecules into PBS solution than PS control. This revealed that the functionalized carbon could be enhanced by specific binding properties with given molecules. The carbon-coated PS dishes grafting with sericin protein were used in a serum-free condition. We also found that hBM-MSCs have higher percentage of proliferated cells at day 7 for the modified dishes with carbon films and coated with sericin than the PS control coated with sericin. The physical film properties were measured by atomic force microscopy (AFM), scanning electron microscope (SEM) and contact angle measurement. The presence of sbnd NH2 groups of sericin compounds on the PS dish was revealed by Fourier transform infrared spectroscopy (FTIR). The stability of covalent bonds of sericin molecules after washing out ungrafted sericin was confirmed by X-ray photoelectron spectroscopy (XPS).

Development of serum-free media for lepidopteran insect cell lines.

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Agathos, Spiros N

2007-01-01

Lepidopteran insect cell culture technology has progressed to the point of becoming an essential part of one of the most successful eukaryotic expression systems and is increasingly used industrially on a large scale. Therefore, there is a constant need for convenient and low-cost culture media capable of supporting good insect cell growth and ensuring high yield of baculovirus as well as the strong expression of recombinant proteins. Vertebrate sera or invertebrate hemolymph were essential supplements in first-generation insect cell media. These supplements, however, are cumbersome and expensive for routine large-scale culture; thus, their use is now circumvented by substituting the essential growth factors present in these supplements with serum-free substances. Such non-serum supplements are typically of non-animal origin and include protein hydrolysates, lipid emulsions, and specialized substances (e.g., surfactants and shear damage protecting chemicals). These supplements need to complement the defined, synthetic basal medium to ensure that the fundamental nutritional needs of the cells are satisfied. Although there is a significant number of proprietary serum-free and low-protein or protein-free media on the market, the lack of information concerning their detailed composition is a drawback in their adoption for different applications, including their adaptation to the metabolic and kinetic analysis and monitoring of a given insect cell based bioprocess. Hence, there is wide appeal for formulating serum-free media based on a rational assessment of the metabolic requirements of the lepidopteran cells during both the growth and the production phases. Techniques such as statistical experimental design and genetic algorithms adapted to the cellular behavior and the bioreactor operation mode (batch, fed-batch, or perfusion) permit the formulation of versatile serum- and protein-free media. These techniques are illustrated with recent developments of serum-free

Stepwise Differentiation of Pluripotent Stem Cells into Osteoblasts Using Four Small Molecules under Serum-free and Feeder-free Conditions

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Kosuke Kanke

2014-06-01

Full Text Available Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenylpyrido[4′,3′:4,5]thieno[2,3-b]pyridine-2-carboxamide [TH] under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2, a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

Science.gov (United States)

Nollevaux, Géraldine; Devillé, Christelle; El Moualij, Benaïssa; Zorzi, Willy; Deloyer, Patricia; Schneider, Yves-Jacques; Peulen, Olivier; Dandrifosse, Guy

2006-01-01

Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. PMID:16670004

Optimization of a serum-free culture medium for mouse embryonic stem cells using design of experiments (DoE) methodology.

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Knöspel, Fanny; Schindler, Rudolf K; Lübberstedt, Marc; Petzolt, Stephanie; Gerlach, Jörg C; Zeilinger, Katrin

2010-12-01

The in vitro culture behaviour of embryonic stem cells (ESC) is strongly influenced by the culture conditions. Current culture media for expansion of ESC contain some undefined substances. Considering potential clinical translation work with such cells, the use of defined media is desirable. We have used Design of Experiments (DoE) methods to investigate the composition of a serum-free chemically defined culture medium for expansion of mouse embryonic stem cells (mESC). Factor screening analysis according to Plackett-Burman revealed that insulin and leukaemia inhibitory factor (LIF) had a significant positive influence on the proliferation activity of the cells, while zinc and L: -cysteine reduced the cell growth. Further analysis using minimum run resolution IV (MinRes IV) design indicates that following factor adjustment LIF becomes the main factor for the survival and proliferation of mESC. In conclusion, DoE screening assays are applicable to develop and to refine culture media for stem cells and could also be employed to optimize culture media for human embryonic stem cells (hESC).

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum.

Science.gov (United States)

Bruserud, Oystein; Tronstad, Karl Johan; Berge, Rolf

2005-06-01

Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the

Serum-Free Media and the Immunoregulatory Properties of Mesenchymal Stem Cells In Vivo and In Vitro

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Mei Wu

2014-02-01

Full Text Available Background: Mesenchymal stem cells are capable of self-renewal and multi-lineage differentiation. They are used extensively to treat several diseases. Traditionally, mesenchymal stem cells are cultured in serum-containing media, typically supplemented with fetal bovine serum (FBS. However, the variability of FBS is likely to skew experimental results. Although serum-free media used to expand mesenchymal stem cells has facilitated remarkable achievements, immunomodulation of these cells in under serum-free conditions is poorly understood. We hypothesized that mesenchymal stem cells expanded in serum-free media will retain powerful immunoregulatory functions in vitro and in vivo. Design and Methods: Immunosuppressive activity and the immunomodulatory cytokines produced by mesenchymal stem cells in serum-free media were characterized in vitro. Immunomodulation by serum-free mesenchymal stem cell expansion in monocrotaline-induced pulmonary hypertension was explored in vivo. Results: Similar to cells in serum-containing media, mesenchymal stem cells expanded in serum-free media inhibited proliferation and apoptosis of CD4+T cells. They also exhibited strong immunosuppressive activities and secreted high levels of immunomodulatory cytokines such as PGE2, IDO1, COX2, IL-6, and IL-1β, but not HGF. On the other hand, growth of mesenchymal stem cells in serum-free media attenuated pulmonary vascular remodeling and inhibited mRNA expression of proinflammatory cytokines TNF-α, IFN-γ, IL-6, IL-1β, and IL-18. Conclusions: Mesenchymal stem cells in serum-free media maintained powerful immunomodulatory function in vitro and in vivo; serum-free media may replace serum-containing media for basic research and clinical applications.

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21

Directory of Open Access Journals (Sweden)

Schneider Yves-Jacques

2006-05-01

Full Text Available Abstract Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules.

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

Science.gov (United States)

2011-01-01

Background Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C. Results We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Conclusion Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability. PMID:22115125

Immobilization of sericin molecules via amorphous carbon plasma modified-polystyrene dish for serum-free culture

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Tunma, Somruthai [The Graduate School, Chiang Mai University, 239 Huay Kaew Road, Muang District, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics (ThEP), 239 Huay Kaew Road, Muang District, Chiang Mai 50200 (Thailand); Song, Doo-Hoon [Research Center for Orofacial Hard Tissue Regeneration, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Kim, Si-Eun; Kim, Kyoung-Nam [Research Center for Orofacial Hard Tissue Regeneration, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Department and Research Institute of Dental Biomaterials and Bioengineering, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Han, Jeon-Geon [Center for Advanced Plasma Surface Technology, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746 (Korea, Republic of); Boonyawan, Dheerawan [Thailand Center of Excellence in Physics (ThEP), 239 Huay Kaew Road, Muang District, Chiang Mai 50200 (Thailand); Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, 239 Huay Kaew Road, Muang District, Chiang Mai 50200 (Thailand)

2013-10-15

In this study, we focused on sericin hydrolysates, originating from silkworm used in serum-free human bone marrow-derived mesenchymal stem cells (hBM-MSCs) culture. We reported the effect of a covalent linkage between a bioactive protein molecule and polystyrene dish surface via a carbon intermediate layer which can slow down the release rate of protein compounds into the phosphate buffer saline (PBS) solution. Films of amorphous carbon (a-C) and functionalized-carbon were deposited on PS culture dish surfaces by using a DC magnetron sputtering system and RF PECVD system. We found that a-C based-films can increase the hydrophilicity and biocompatibility of polystyrene (PS) dishes, especially a-C films and a-C:N{sub 2} films showed good attachment of hBM-MSCs at 24 h. However, in the case of silica surface (a-C:SiO{sub x} films), the cells showed a ragged and unattached boundary resulting from the presence of surface silanol groups. For the UV–vis absorbance, all carbon modified-PS dishes showed a lower release rate of sericin molecules into PBS solution than PS control. This revealed that the functionalized carbon could be enhanced by specific binding properties with given molecules. The carbon-coated PS dishes grafting with sericin protein were used in a serum-free condition. We also found that hBM-MSCs have higher percentage of proliferated cells at day 7 for the modified dishes with carbon films and coated with sericin than the PS control coated with sericin. The physical film properties were measured by atomic force microscopy (AFM), scanning electron microscope (SEM) and contact angle measurement. The presence of -NH{sub 2} groups of sericin compounds on the PS dish was revealed by Fourier transform infrared spectroscopy (FTIR). The stability of covalent bonds of sericin molecules after washing out ungrafted sericin was confirmed by X-ray photoelectron spectroscopy (XPS).

Influence of serum extraction from the culture medium and of sublethal X-ray irradiation upon microvilli and invaginations of the membrane of Ehrlich ascites tumor cells in monolayer culture

International Nuclear Information System (INIS)

Laudenbach, G.; Pfab, R.; Hess, F.; Schachtschabel, D.O.

1984-01-01

In order to find out modifications of microvilli and invaginations, the cellular surfaces of Ehrlich ascites tumor cells in monolayer culture (basal medium of Eagle + 10% fetal calf serum) were investigated with the aid of electron-microscopic cross-sections. The tumor cells had been cultured without serum 24 hours prior to investigation or irradiated with 2 Gy. Morphometric evaluation after cell culture in a serum-free medium showed a reduced number of microvilli and a diminution of sections of microvilli. As already described before, a reduction of cell proliferation, of the microtubule-microfilament system, and of the endocytosis activity occurs under these serum-free conditions. The number of invaginations (related to a constant membrane part) was reduced by nearly 50% after serum extraction. Similarly to serum extraction, sublethal X-ray irradiation reduced the sections of microvilli, whereas the number of microvilli increased slightly. Contrary to the effect of serum extraction, the irradiated cells showed twice as many invaginations as the non-irradiated control cells. These differences in the surface structures are interpreted as a result of modified growth stimulations (+- serum) and radiogenic reparation processes. (orig.) [de

Defined xenogeneic-free and hypoxic environment provides superior conditions for long-term expansion of human adipose-derived stem cells.

Science.gov (United States)

Yang, Sufang; Pilgaard, Linda; Chase, Lucas G; Boucher, Shayne; Vemuri, Mohan C; Fink, Trine; Zachar, Vladimir

2012-08-01

Development and implementation of therapeutic protocols based on stem cells or tissue-engineered products relies on methods that enable the production of substantial numbers of cells while complying with stringent quality and safety demands. In the current study, we aimed to assess the benefits of maintaining cultures of adipose-derived stem cells (ASCs) in a defined culture system devoid of xenogeneic components (xeno-free) and hypoxia over a 49-day growth period. Our data provide evidence that conditions involving StemPro mesenchymal stem cells serum-free medium (SFM) Xeno-Free and hypoxia (5% oxygen concentration) in the culture atmosphere provide a superior proliferation rate compared to a standard growth environment comprised of alpha-modified Eagle medium (A-MEM) supplemented with fetal calf serum (FCS) and ambient air (20% oxygen concentration) or that of A-MEM supplemented with FCS and hypoxia. Furthermore, a flow cytometric analysis and in vitro differentiation assays confirmed the immunophenotype stability and maintained multipotency of ASCs when expanded under xeno-free conditions and hypoxia. In conclusion, our data demonstrate that growth conditions utilizing a xeno-free and hypoxic environment not only provide an improved environment for the expansion of ASCs, but also set the stage as a culture system with the potential broad spectrum utility for regenerative medicine and tissue engineering applications.

Effect of IL-6 on proliferation and IG production of human EBV-transformed cell lines in serum free culture media

NARCIS (Netherlands)

Jochems, G. J.; Jordens, R.; van Lier, R. A.; Zeijlemaker, W. P.

1990-01-01

To optimalize growth and Ig production of EBV transformed B cells for large scale tissue culture, we analyzed five stable monoclonal EBV-B cell lines for their responsiveness to interleukin (IL)-6 in standard medium with 5% FCS and in several serum-free media. As we previously demonstrated these

Bovine babesiosis: Cattle protected in the field with a frozen vaccine containing Babesia bovis and Babesia bigemina cultured in vitro with a serum-free medium.

Science.gov (United States)

Rojas-Martínez, Carmen; Rodríguez-Vivas, Roger Iván; Millán, Julio Vicente Figueroa; Bautista-Garfias, Carlos Ramón; Castañeda-Arriola, Roberto Omar; Lira-Amaya, José Juan; Urióstegui, Patricia Vargas; Carrasco, Juan José Ojeda; Martínez, Jesús Antonio Álvarez

2018-04-01

An attenuated live vaccine containing Babesia bovis and B. bigemina cultured in vitro with a serum-free medium was assessed for its clinical protection conferred of naïve cattle, under natural tick-challenge in a high endemicity zone to Babesia spp. Three groups of six animals were treated as follows: group I (GI) received a vaccine derived from parasites cultured with a free-serum medium; group II (GII) were immunized with the standard vaccine, with parasites cultured in a medium supplemented with 40% (v/v) bovine serum; and a control group (GIII) inoculated with non-infected bovine erythrocytes. Inocula were administered by IM route. Experimental animals were kept during 23days after vaccination in a cattle farm free of ticks and Babesia spp. Thereafter, cattle were moved to a high endemicity farm for natural exposure to Babesia spp. transmitted by Rhipicephalus microplus ticks. Protection against clinical babesiosis was observed in bovines belonging to GI (100%) and GII (83.33%), while the control animals (GIII) were not protected, and showed severe clinical signs, closely related to babesiosis, were observed for at least three consecutive days during the challenge. These were fever, anemia, which were measured simultaneously, and circulating parasites were detected by optic light microscopy. All cattle showed B. bovis and B. bigemina in stained blood films during the challenge; B. bovis antibody titers were higher than those to B. bigemina in GI and GII, and lower titers were determined in GIII. The protective capacity of the vaccine derived from B. bovis and B. bigemina cultured in vitro in a serum-free medium was demonstrated. Copyright © 2017 Elsevier B.V. All rights reserved.

Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures.

Science.gov (United States)

Mojica-Henshaw, Mariluz P; Jacobson, Pam; Morris, Julie; Kelley, Linda; Pierce, Jan; Boyer, Michael; Reems, Jo-Anna

2013-12-01

Fetal bovine serum (FBS) is commonly used as a serum supplement for culturing human mesenchymal stromal cells (hMSCs). However, human cells grown in FBS, especially for extended periods, risk potential exposure to bovine immunogenic proteins and infectious agents. To address this issue, we investigated the ability of a novel human platelet serum supplement to substitute for FBS in hMSC cultures. Platelet lysate-serum (PL-serum) was converted from platelet lysate-plasma (PL-plasma) that was manufactured from pooled platelet-rich plasma (PRP) apheresis units. Growth factor levels and the number of residual intact platelets in PL-serum and PL-plasma were compared with enzyme-linked immunosorbent assays and flow cytometry, respectively. Proliferation responses of hMSCs cultured in PL-serum, PL-plasma, or FBS were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the immunophenotype of harvested hMSCs was evaluated by flow cytometry and tri-lineage differentiation potential was evaluated by assessing adipogenic, osteogenic and chondrogenic development. Selected growth factor levels in PL-serum were not significantly different from PL-plasma (P > 0.05). hMSC cultures supplemented with PL-serum had comparable growth kinetics to PL-plasma, and hMSC yields were consistently greater than with FBS. hMSCs harvested from cultures supplemented with PL-serum, PL-plasma or FBS had similar cell surface phenotypes and maintained tri-lineage differentiation potential. PL-serum, similar to PL-plasma, can substitute for FBS in hMSC cultures. Use of PL-serum, in contrast to PL-plasma, has an added advantage of not requiring addition of a xenogeneic source of heparin, providing a completely xeno-free culture medium. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Evolution of triiodothyronine nuclear binding sites in hypothalamic serum-free cultures: evidence for their presence in neurons and astrocytes

International Nuclear Information System (INIS)

Puymirat, J.; Faivre-Bauman, A.

1986-01-01

( 125 I)Triiodothyronine (T 3 ) nuclear binding was studied in hypothalamic cultures from fetal mouse grown in serum-free medium. In enriched neuronal cultures, the apparent dissociation constant of the binding does not change with time in vitro (7 x 10 -11 M), but the maximum binding capacity (MBC) doubles between day 7 and day 14 in vitro. We show here for the first time that homologous astrocyte cell cultures, devoid of neurons as checked by tetanus toxin binding, also display T 3 nuclear binding, with the same affinity as neuronal cultures. However, their MBC is 3 times lower than that of neurons after a week in vitro, and increases more quickly thereafter (Author)

The replacement of serum by hormones in cell culture media.

Science.gov (United States)

Sato, G; Hayashi, I

1976-12-01

The replacement of serum by hormones in cell culture media. (Reemplazo del suero por hormonas en el medio de cultivo de células). Arch. Biol. Med. Exper. 10: 120-121, 1976. The serum used in cell culture media can be replaced by a mixture of hormones and some accesory blood factors. The pituitary cell line GH3 can be grown in a medium in which serum is replaced by triiodothyronine, transferrin, parathormone, tyrotrophin releasing hormone and somatomedins. Hela and BHK cell strains can also be grown in serum free medium supplemented with hormones. Each cell type appears to have different hormonal requirements yet it may found that some hormones are required for most cell types.

Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours

NARCIS (Netherlands)

Bate-Eya, Laurel T.; Ebus, Marli E.; Koster, Jan; den Hartog, Ilona J. M.; Zwijnenburg, Danny A.; Schild, Linda; van der Ploeg, Ida; Dolman, M. Emmy M.; Caron, Huib N.; Versteeg, Rogier; Molenaar, Jan J.

2014-01-01

Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of

Specific estrogen-induced cell proliferation of cultured Syrian hamster renal proximal tubular cells in serum-free chemically defined media

International Nuclear Information System (INIS)

Oberley, T.D.; Lauchner, L.J.; Pugh, T.D.; Gonzalez, A.; Goldfarb, S.; Li, S.A.; Li, J.J.

1989-01-01

It has long been recognized that the renal proximal tubular epithelium of the hamster is a bona fide estrogen target tissue. The effect of estrogens on the growth of proximal tubule cell explants and dissociated single cells derived from these explant outgrowths has been studied in culture. Renal tubular cells were grown on a PF-HR-9 basement membrane under serum-free chemically defined culture conditions. At 7-14 days in culture, cell number was enhanced 3-fold in the presence of either 17β-estradiol or diethylstilbestrol. A similar 3-fold increase in cell number was also seen at 1 nM 17β-estradiol in subcultured dissociated single tubular cells derived from hamster renal tubular explant outgrowths at 21 days in culture. Concomitant exposure of tamoxifen at 3-fold molar excess in culture completely abolished the increase in cell number seen with 17β-estradiol. The proliferation effect of estrogens on proximal tubular cell growth appears to be species specific since 17β-estradiol did not alter the growth of either rat or guinea pig proximal tubules in culture. In addition, at 7-10 days in culture in the presence of 17β-estradiol, [ 3 H]thymidine labeling of hamster tubular cells was enhanced 3-fold. These results clearly indicate that estrogens can directly induce primary epithelial cell proliferation at physiologic concentrations and provide strong additional evidence for an important hormonal role in the neoplastic transformation of the hamster kidney

Immunohistochemical expression of stem cell, endothelial cell, and chemosensitivity markers in primary glioma spheroids cultured in serum-containing and serum-free medium

DEFF Research Database (Denmark)

Christensen, Karina; Aaberg-Jessen, Charlotte; Andersen, Claus

2010-01-01

To investigate the influence of serum-free medium (SFM) supplemented with epidermal growth factor and basic fibroblast growth factor compared with conventional serum-containing medium (SCM) on the phenotype of organotypic primary spheroids from seven gliomas.......To investigate the influence of serum-free medium (SFM) supplemented with epidermal growth factor and basic fibroblast growth factor compared with conventional serum-containing medium (SCM) on the phenotype of organotypic primary spheroids from seven gliomas....

Derivation of transgene-free human induced pluripotent stem cells from human peripheral T cells in defined culture conditions.

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Yoshikazu Kishino

Full Text Available Recently, induced pluripotent stem cells (iPSCs were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.

Hematopoietic stem cell cytokines and fibroblast growth factor-2 stimulate human endothelial cell-pericyte tube co-assembly in 3D fibrin matrices under serum-free defined conditions.

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Annie O Smith

Full Text Available We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices that regulates angiogenic events in postnatal life.

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

Science.gov (United States)

Bonazza, Camila; Andrade, Sheila Siqueira; Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J; Girão, Manoel J B C; Oliva, Maria Luiza V; Castro, Rodrigo Aquino

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity.

A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors.

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Akira Niwa

Full Text Available Elucidating the in vitro differentiation of human embryonic stem (ES and induced pluripotent stem (iPS cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose, a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study, we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover, single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new, robust, and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation.

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

Directory of Open Access Journals (Sweden)

Camila Bonazza

Full Text Available Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2 and progesterone (P4 effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation. These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity.

Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell cultures.

Science.gov (United States)

Bieback, Karen

2013-10-01

Mesenchymal stromal cells (MSC) emerged as highly attractive in cell-based regenerative medicine. Initially thought to provide cells capable of differentiation towards mesenchymal cell types (osteoblasts, chondrocytes, adipocytes etc.), by and by potent immunoregulatory and pro-regenerative activities have been discovered, broadening the field of potential applications from bone and cartilage regeneration to wound healing and treatment of autoimmune diseases. Due to the limited frequency in most tissue sources, ex vivo expansion of MSC is required compliant with good manufacturing practice (GMP) guidelines to yield clinically relevant cell doses. Though, still most manufacturing protocols use fetal bovine serum (FBS) as cell culture supplement to isolate and to expand MSC. However, the high lot-to-lot variability as well as risk of contamination and immunization call for xenogenic-free culture conditions. In terms of standardization, chemically defined media appear as the ultimate achievement. Since these media need to maintain all key cellular and therapy-relevant features of MSC, the development of chemically defined media is still - albeit highly investigated - only in its beginning. The current alternatives to FBS rely on human blood-derived components: plasma, serum, umbilical cord blood serum, and platelet derivatives like platelet lysate. Focusing on quality aspects, the latter will be addressed within this review.

Elimination of remaining undifferentiated induced pluripotent stem cells in the process of human cardiac cell sheet fabrication using a methionine-free culture condition.

Science.gov (United States)

Matsuura, Katsuhisa; Kodama, Fumiko; Sugiyama, Kasumi; Shimizu, Tatsuya; Hagiwara, Nobuhisa; Okano, Teruo

2015-03-01

Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture, the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study, we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells, including cardiomyocytes and fibroblasts, using a methionine-free culture condition. When cultured in the methionine-free condition, human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition, gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore, in fabricated cardiac cell sheets, spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination.

Robust and highly-efficient differentiation of functional monocytic cells from human pluripotent stem cells under serum- and feeder cell-free conditions.

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Masakatsu D Yanagimachi

Full Text Available Monocytic lineage cells (monocytes, macrophages and dendritic cells play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3 × 10(6 ± 0.3 × 10(6 floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.

Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

Science.gov (United States)

Pijuan-Galitó, Sara; Tamm, Christoffer; Schuster, Jens; Sobol, Maria; Forsberg, Lars; Merry, Catherine L. R.; Annerén, Cecilia

2016-01-01

Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. PMID:27405751

Serum-Free Media and the Immunoregulatory Properties of Mesenchymal Stem Cells In Vivo and In Vitro

OpenAIRE

Mei Wu; Zhi-Bo Han; Jun Feng Liu; You Wei Wang; Jian Zhong Zhang; Chun Tuan Li; Peng Liang Xin; Zhong Chao Han; Xiong Peng Zhu

2014-01-01

Background: Mesenchymal stem cells are capable of self-renewal and multi-lineage differentiation. They are used extensively to treat several diseases. Traditionally, mesenchymal stem cells are cultured in serum-containing media, typically supplemented with fetal bovine serum (FBS). However, the variability of FBS is likely to skew experimental results. Although serum-free media used to expand mesenchymal stem cells has facilitated remarkable achievements, immunomodulation of these cells in un...

Effects of culture media conditions on production of eggs fertilized in vitro of embryos derived from ovary of high grade Hanwoo

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Jun Young Lee

2016-03-01

Full Text Available Abstract Background This study was investigated the effects of culture media conditions on production of eggs fertilized in vitro of embryos from ovaries of high grade Korean native cow, Hanwoo. Methods The IVMD 101 and IVF 100 were used for in vitro maturation of selected Hanwoo oocytes and In vitro embryo culture after in vitro fertilization, respectively. The IVMD 101 and IVD 101 were used for in vitro culture and completely free of serum. Results The cleavage rates of 2-cell embryos in reference to Hanwoo oocytes were 86.7, 92.9 , and 90.1 % in the control group, IVDM101 medium and IVD101 medium, respectively which indicates that the IVDM101 medium and IVD101 medium may result favorable outcomes. The in vitro development rates of blastocysts were 12.4, 38.4 and 32.4 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. For hatched blastocysts, it was 5.3, 33.9, and 28.6 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. Hence, more favorable results were expected for the hatched blastocysts in which the IVMD101 medium and IVD101 medium were used than the control group. Average cell numbers of blastocysts were 128.3, 165.7, and 163.6 in the groups of TCM-199 + 10 % FBS medium, IVMD 101 medium, and IVD 101 medium, respectively which clearly show that the IVMD 101 and IVD 101 medium consequence significantly higher cell numbers compared to the control group (i.e., TCM-199 + 10 % FBS medium. Pregnancy rate after embryo transfer was 39.6 % when the serum free medium was used which is higher than that of the medium supplemented with serum (32.8 %. In addition, stillbirth rates were 4.9 % in the group of serum free medium whereas it was 13.6 % in the serum supplemented medium (13.6 %. Conclusions Taken altogether, serum free media, the IVMD 101 and IVD 101 represented more favorable results in the embryo development rate of embryos, cell numbers of blastocyst, and

Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours.

Science.gov (United States)

Bate-Eya, Laurel T; Ebus, Marli E; Koster, Jan; den Hartog, Ilona J M; Zwijnenburg, Danny A; Schild, Linda; van der Ploeg, Ida; Dolman, M Emmy M; Caron, Huib N; Versteeg, Rogier; Molenaar, Jan J

2014-02-01

Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of eight newly-derived neuroblastoma TICs from six primary neuroblastoma tumours and two bone marrow metastases. The primary tumours from which these TICs were generated have previously been fully typed by whole genome sequencing (WGS). Array comparative genomic hybridisation (aCGH) analysis showed that TIC lines retained essential characteristics of the primary tumours and exhibited typical neuroblastoma chromosomal aberrations such as MYCN amplification, gain of chromosome 17q and deletion of 1p36. Protein analysis showed expression for neuroblastoma markers MYCN, NCAM, CHGA, DBH and TH while haematopoietic markers CD19 and CD11b were absent. We analysed the growth characteristics and confirmed tumour-forming potential using sphere-forming assays, subcutaneous and orthotopic injection of these cells into immune-compromised mice. Affymetrix mRNA expression profiling of TIC line xenografts showed an expression pattern more closely mimicking primary tumours compared to xenografts from classical cell lines. This establishes that these neuroblastoma TICs cultured under serum-free conditions are relevant and useful neuroblastoma tumour models. Copyright © 2013 Elsevier Ltd. All rights reserved.

Isolation and characterization of Wharton’s jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

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Cardoso Tereza C

2012-05-01

Full Text Available Abstract Background The possibility for isolating bovine mesenchymal multipotent cells (MSCs from fetal adnexa is an interesting prospect because of the potential for these cells to be used for biotechnological applications. Bone marrow and adipose tissue are the most common sources of MSCs derived from adult animals. However, little knowledge exists about the characteristics of these progenitors cells in the bovine species. Traditionally most cell cultures are developed in two dimensional (2D environments. In mammalian tissue, cells connect not only to each other, but also support structures called the extracellular matrix (ECM. The three-dimensional (3D cultures may play a potential role in cell biotechnology, especially in tissue therapy. In this study, bovine-derived umbilical cord Wharton’s jelly (UC-WJ cells were isolated, characterized and maintained under 3D-free serum condition as an alternative of stem cell source for future cell banking. Results Bovine-derived UC-WJ cells, collected individually from 5 different umbilical cords sources, were successfully cultured under serum-free conditions and were capable to support 60 consecutive passages using commercial Stemline® mesenchymal stem cells expansion medium. Moreover, the UC-WJ cells were differentiated into osteocytes, chondrocytes, adipocytes and neural-like cells and cultured separately. Additionally, the genes that are considered important embryonic, POU5F1 and ITSN1, and mesenchymal cell markers, CD105+, CD29+, CD73+ and CD90+ in MSCs were also expressed in five bovine-derived UC-WJ cultures. Morphology of proliferating cells typically appeared fibroblast-like spindle shape presenting the same viability and number. These characteristics were not affected during passages. There were 60 chromosomes at the metaphase, with acrocentric morphology and intense telomerase activity. Moreover, the proliferative capacity of T cells in response to a mitogen stimulus was suppressed when

Optimization of Conditions for In Vitro Culture of the Microphallid Digenean Gynaecotyla adunca

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Jenna West

2014-01-01

Full Text Available In vitro cultivation of digeneans would aid the development of effective treatments and studies of the biology of the parasites. The goal of this study was to optimize culture conditions for the trematode, Gynaecotyla adunca. Metacercariae of the parasite from fiddler crabs, Uca pugnax, excysted in trypsin, were incubated overnight to permit fertilization, and were cultured in different conditions to find those that resulted in maximum worm longevity and egg production. When cultured in media lacking serum, worms lived longer in Hanks balanced salt solution and Dulbecco’s Modified Eagle medium/F-12 (DME/F-12 than in RPMI-1640 but produced the most eggs in DME/F-12. Worm longevity and egg production increased when worms were grown in DME/F-12 supplemented with 20% chicken, horse, or newborn calf serum but the greatest number of eggs was deposited in cultures containing horse or chicken serum. Horse serum was chosen over chicken serum due to the formation of a precipitate in chicken serum. The optimal concentration of horse serum with respect to egg production ranged from 5 to 20%. Infectivity of eggs deposited by worms in culture was tested by feeding eggs to mud snails, Ilyanassa obsoleta. None of these snails produced G. adunca cercariae.

Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

Science.gov (United States)

Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

2016-01-01

As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

Short-term, serum-free, static culture of cord blood-derived CD34+ cells: effects of FLT3-L and MIP-1alpha on in vitro expansion of hematopoietic progenitor cells.

Science.gov (United States)

Capmany, G; Querol, S; Cancelas, J A; García, J

1999-08-01

The use of ex vivo expanded cells has been suggested as a possible means to accelerate the speed of engraftment in cord blood (CB) transplantation. The aim of this study was to fix the optimal condition for the generation of committed progenitors without affecting the stem cell compartment. Analysis of the effects of FLT3-L and MIP-1alpha when combined with SCF, IL-3 and IL-6, in short-term (6 days), serum-free expansion cultures of CB-selected CD34+ cells. An important expansion was obtained that ranged between 8-15 times for CFU-GM, 21-51 times for the BFU-E/CFU-Mix population and 11 to 30 times for CD34+ cells assessed by flow cytometry. From the combinations tested, those in which FLT3-L was present had a significant increase in the expansion of committed progenitors, while the presence of MIP-1alpha had a detrimental effect on the generation of more differentiated cells. However, stem cell candidates assessed by week 5 CAFC assay could be maintained in culture when both MIP-1a and FLT3-L were present (up to 91% recovery). This culture system was also able to expand megakaryocytic precursors as determined by the co-expression of CD34 and CD61 antigens (45-70 times), in spite of the use of cytokines non-specific for the megakaryocytic lineage. The results obtained point to the combination of SCF, IL-3, IL-6, FLT3-L and MIP-1alpha as the best suited for a pre-clinical short-term serum-free static ex vivo expansion protocol of CB CD34+ cells, since it can generate large numbers of committed progenitor cells as well as maintaining week 5 CAFC.

Human autologous serum as a substitute for fetal bovine serum in human Schwann cell culture.

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Parisa Goodarzi

2014-04-01

Full Text Available Nowadays, cell -based and tissue engineered products have opened new horizons in treatment of incurable nervous system disorders. The number of studies on the role of Schwann cells (SC in treating nervous disorders is higher than other cell types. Different protocols have been suggested for isolation and expansion of SC which most of them have used multiple growth factors, mitogens and fetal bovine sera (FBS in culture medium. Because of potential hazards of animal-derived reagents, this study was designed to evaluate the effect of replacing FBS with human autologous serum (HAS on SC's yield and culture parameters. Samples from 10 peripheral nerve biopsies were retrieved and processed under aseptic condition. The isolated cells cultured in FBS (1st group or autologous serum (2nd group. After primary culture the cells were seeded at 10000 cell/cm2 in a 12 wells cell culture plate for each group. At 100% confluency, the cell culture parameters (count, viability, purity and culture duration of 2 groups were compared using paired t-test. The average donors' age was 35.80 (SD=13.35 and except for 1 sample the others cultured successfully. In first group, the averages of cell purity, viability and culture duration were 97% (SD=1.32, 97/33% (SD=1.22 and 11.77 (SD=2.58 days respectively. This parameters were 97.33% (SD=1.00, 97.55% (SD=1.33 and 10.33 days (SD=1.65 in second group. The difference of cell count, purity and viability were not significant between 2 groups (P>0.05. The cells of second group reached to 100% confluency in shorter period of time (P=0.03. The results of this study showed that autologous serum can be a good substitute for FBS in human SC culture. This can reduce the costs and improve the safety of cell product for clinical application.

The influence of metronidazole on free thymidine content of blood serum of irradiated mice

International Nuclear Information System (INIS)

Konov, A.V.; Ryabchenko, N.I.

1986-01-01

The influence of a radiosensitizer, metronidazole, on the free thymidine content of blood serum of irradiated mice was studied in aerobic and hypoxic conditions. A heated metronidazole solution (1 mg/g) was administered intraperitoneally 30 min before irradiation of animals with a dose of 3 Gy. Thymidine concentration in blood serum was determined by the radioimmunological technique. The influence of metronidazole on the level of thymidinemia was only noted in the animals exposed under hypoxic conditions

A defined and xeno-free culture method enabling the establishment of clinical-grade human embryonic, induced pluripotent and adipose stem cells.

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Kristiina Rajala

2010-04-01

Full Text Available The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.Here, we report the development of a fully defined xeno-free medium (RegES, capable of supporting the expansion of human embryonic stem cells (hESC, induced pluripotent stem cells (iPSC and adipose stem cells (ASC. We describe the use of the xeno-free medium in the derivation and long-term (>80 passages culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS, while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific

Determination of thymidine in serum used for cell culture media

International Nuclear Information System (INIS)

Schaer, J.C.; Maurer, U.; Schindler, R.

1978-01-01

Thymidine concentrations in serum used for cell culture media were determined with an assay based on isotope dilution. In this assay, incorporation of (3H)-thymidine into DNA of cultured cells was measured in the presence of 5 and 20% serum as a function of the concentration of unlabeled thymidine added to the medium. Thymidine concentrations were measured using horse serum as well as fetal calf serum in the culture media. Dialysis of serum resulted in a reduction of thymidine levels by factors of at least 10

Tissue culture media supplemented with 10% fetal calf serum contains a castrate level of testosterone.

NARCIS (Netherlands)

Sedelaar, J.P.M.; Isaacs, J.T.

2009-01-01

BACKGROUND: Human prostate cancer cells are routinely maintained in media supplemented with 10% Fetal Calf Serum (FCS) to provide androgen. In the present study, total and free testosterone levels in 10%FCS supplemented tissue culture media were determined and compared to levels in intact and

Feline Neural Progenitor Cells I: Long-Term Expansion under Defined Culture Conditions

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Jing Yang

2012-01-01

Full Text Available Neural progenitor cells (NPCs of feline origin (cNPCs have demonstrated utility in transplantation experiments, yet are difficult to grow in culture beyond the 1 month time frame. Here we use an enriched, serum-free base medium (Ultraculture and report the successful long-term propagation of these cells. Primary cultures were derived from fetal brain tissue and passaged in DMEM/F12-based or Ultraculture-based proliferation media, both in the presence of EGF + bFGF. Cells in standard DMEM/F12-based medium ceased to proliferate by 1-month, whereas the cells in the Ultraculture-based medium continued to grow for at least 5 months (end of study with no evidence of senescence. The Ultraculture-based cultures expressed lower levels of progenitor and lineage-associated markers under proliferation conditions but retained multipotency as evidenced by the ability to differentiate into neurons and glia following growth factor removal in the presence of FBS. Importantly, later passage cNPCs did not develop chromosomal aberrations.

A low percentage of autologous serum can replace bovine serum to engineer human nasal cartilage

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F Wolf

2008-02-01

Full Text Available For the generation of cell-based therapeutic products, it would be preferable to avoid the use of animal-derived components. Our study thus aimed at investigating the possibility to replace foetal bovine serum (FBS with autologous serum (AS for the engineering of cartilage grafts using expanded human nasal chondrocytes (HNC. HNC isolated from 7 donors were expanded in medium containing 10% FBS or AS at different concentrations (2%, 5% and 10% and cultured in pellets using serum-free medium or in Hyaff®-11 meshes using medium containing FBS or AS. Tissue forming capacity was assessed histologically (Safranin O, immunohistochemically (type II collagen and biochemically (glycosaminoglycans -GAG- and DNA. Differences among experimental groups were assessed by Mann Whitney tests. HNC expanded under the different serum conditions proliferated at comparable rates and generated cartilaginous pellets with similar histological appearance and amounts of GAG. Tissues generated by HNC from different donors cultured in Hyaff®-11 had variable quality, but the accumulated GAG amounts were comparable among the different serum conditions. Staining intensity for collagen type II was consistent with GAG deposition. Among the different serum conditions tested, the use of 2% AS resulted in the lowest variability in the GAG contents of generated tissues. In conclusion, a low percentage of AS can replace FBS both during the expansion and differentiation of HNC and reduce the variability in the quality of the resulting engineered cartilage tissues.

Serum triiodothyronine and free triiodothyronine index in normal pregnancy

International Nuclear Information System (INIS)

Karunanidhi, A.; Charles, S.X.; Kanagasabapathy, A.S.

1981-01-01

The thyroid function at various stages of pregnancy and after delivery was assessed by measuring serum total triiodothyronine (T 3 ), T 3 resin uptake (T 3 RU) and free triiodothyronine index (FT 3 I). In first, second and third trimesters of pregnancy, the mean values of serum total T 3 were significantly elevated (P 3 I remained unaltered. During labour, both serum total T 3 and FT 3 I observed during labour and after delivery may be due to the changes in blood oestrogen levels. This study indicates that serum free T 3 concentrations in pregnancy remained unaltered in the presence of elevated serum total T 3 levels. FT 3 I determination would thus appear to be a reliable in vitro thyroid function test for the assessment of thyroid function throughout pregnancy. Serum total T 3 was measured by radioimmunoassay using radioiodinated T 3 ( 125 I-T 3 ). (author)

Optimized human platelet lysate as novel basis for a serum-, xeno-, and additive-free corneal endothelial cell and tissue culture.

Science.gov (United States)

Thieme, Daniel; Reuland, Lynn; Lindl, Toni; Kruse, Friedrich; Fuchsluger, Thomas

2018-02-01

The expansion of donor-derived corneal endothelial cells (ECs) is a promising approach for regenerative therapies in corneal diseases. To achieve the best Good Manufacturing Practice standard the entire cultivation process should be devoid of nonhuman components. However, so far, there is no suitable xeno-free protocol for clinical applications. We therefore introduce a processed variant of a platelet lysate for the use in corneal cell and tissue culture based on a Good Manufacturing Practice-grade thrombocyte concentrate. This processed human platelet lysate (phPL), free of any animal components and of anticoagulants such as heparin with a physiological ionic composition, was used to cultivate corneal ECs in vitro and ex vivo in comparison to standard cultivation with fetal calf serum (FCS). Human donor corneas were cut in quarters while 2 quarters of each cornea were incubated with the respective medium supplement. Three fields of view per quarter were taken into account for the analysis. Evaluation of phPL as a medium supplement in cell culture of immortalized EC showed a superior viability compared with FCS control with reduced cell proliferation. Furthermore, the viability during the expansion of primary cells is significantly (3-fold ±0.5) increased with phPL compared with FCS standard medium. Quartering donor corneas was traumatic for the endothelium and therefore resulted in increased EC loss. Interestingly, however, cultivation of the quartered pieces for 2 weeks in 0.1-mg/ml pHPL in Biochrome I showed a 21 (±10) % EC loss compared with 67 (±12) % EC loss when cultivated in 2% FCS in Biochrome I. The cell culture protocol with pHPL as FCS replacement seems to be superior to the standard FCS protocols with respect to EC survival. It offers a xeno-free and physiological environment for corneal endothelial cells. This alternative cultivation protocol could facilitate the use of EC for human corneal cell therapy. Copyright © 2017 John Wiley & Sons, Ltd.

Free serum thyroxine

International Nuclear Information System (INIS)

Degrossi, O.J.; Altschuler, Noe; Cabrejas, M.L. de; Pinkas, Mirta; Garcia del Rio, Hernan

1982-01-01

The use of radiommunoassay (RIA) tehcniques has increased the diagnosis of thyroid functional alterations. A solid phase RIA method for free thyroxine (FT4) measurement was tested. Serum FT4, Total T4, T3 and TSH were determined by radioimmunoassay in 179 subjects. One hundred twenty two patients were normal (8 to 75 years old); FT4 was 1.42 +- 0.03 ng/100 ml (avg. value and std. error). In 27 cases of thyrotoxicosis the values were 4.66 +- 0.48 ng/100 ml and in 15 cases of hypothyroidism 0.50 +- 0.06 ng/100 ml (statistics probability [es

Normal human serum (HS) prevents oxidant-induced lysis of cultured endothelial cells (ECs)

International Nuclear Information System (INIS)

Callahan, K.S.; Harlan, J.M.

1986-01-01

Most studies demonstrating oxidant lysis of cultured ECs are performed in serum-free media or media containing low concentrations of bovine serum. The authors found that HS protects human and bovine ECs from lysis caused by reagent H 2 O 2 or glucose/glucose oxidase (GO)-generated H 2 O 2 . EC injury was assessed by 51 Cr release, cell detachment, or trypan blue dye exclusion. Protective HS activity was dose-dependent with concentrations greater than or equal to 25% preventing lethal injury. Cytotoxicity at 24 hrs, induced by 20 mU/ml GO, was 90.1 +/- 5.2% without HS vs 1.7 +/- 4.6% with 25% HS present (20 exp). Similar protection was observed with heparinized plasma. Of note, comparable concentrations of bovine serum were devoid of protective activity. Addition of fatty acid-free albumin to the media was also without protective effect. Preliminary characterization showed HS activity was stable to 60 0 C for 30 min, non-dialyzable at 25,000 MW cutoff, and retained in delipidated serum. The HS protection was not merely due to scavenging of exogenous H 2 O 2 as A23187-induced EC lysis was also prevented by HS. Protective activity was not reproduced by purified cerruloplasmin or transferrin. In conclusion, unidentified factor(s) present in HS protect cultured ECs from oxidant-induced lysis. Since endothelium is normally exposed to 100% plasma, the authors suggest that in vitro studies of oxidant-mediated injury be performed in the presence of HS. Factor(s) in HS may play an important role in modulating oxidant-induced vascular injury in vivo

Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

DEFF Research Database (Denmark)

Claesson, Mogens Helweg; Ropke, C

1990-01-01

We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu......We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level...

Selection of osteoprogenitors from the jaw periosteum by a specific animal-free culture medium.

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Dorothea Alexander

Full Text Available The goal of our research work is to establish mesenchymal osteoprogenitors derived from human jaw periosteum for tissue engineering applications in oral and maxillofacial surgery. For future autologous and/or allogeneic transplantations, some issues must be addressed. On the one hand, animal-free culture conditions have yet to be established. On the other hand, attempts should be undertaken to shorten the in vitro culturing process efficiently. The aim of the present study is to compare and analyze the phenotype of osteoprogenitors from the jaw periosteum under normal FCS-containing and animal-free culture conditions. Therefore, we analyzed the proliferation rates of MesenCult-XF medium (MC- in comparison to DMEM-cultured JPCs. Whereas jaw periosteal cells (JPCs show relatively slow proliferation rates and a fibroblastoid shape under DMEM culture conditions, MC-cultured JPCs diminished their cell size significantly and proliferated rapidly. By live-monitoring measurements of adhesion and proliferation, we made an interesting observation: whereas the proliferation of the MSCA-1(+ subpopulation and the unseparated cell fraction were favored by the animal-free culture medium, the proliferation of the MSCA-1(- subpopulation seemed to be repressed under these conditions. The alkaline phosphatase expression pattern showed similar results under both culture conditions. Comparison of the mineralization capacity revealed an earlier formation of calcium-phosphate precipitates under MC culture conditions; however, the mineralization capacity of the DMEM-cultured cells seemed to be higher. We conclude that the tested animal-free medium is suitable for the in vitro expansion and even for the specific selection of osteoprogenitor cells derived from the jaw periosteum.

An improved ultrafiltration method for determining free testosterone in serum

International Nuclear Information System (INIS)

Vlahos, I.; MacMahon, W.; Sgoutas, D.; Bowers, W.; Thompson, J.; Trawick, W.

1982-01-01

In this method, we use the Amicon MPS-1 centrifugal ultrafiltration device and the YMB membrane in measuring free testosterone in serum. Two independent assays are combined: total testosterone and the ultrafiltrable fraction of added [ 3 H]testosterone. The unbound fraction is determined in 0.15-0.5 mL ultrafiltrates of 0.6 to 1 mL of variably diluted serum that has been equilibrated with [ 3 H]testosterone at 37 degrees C. The assay is rapid (less than 1 h), practicable (requires 0.6 mL of serum), and reproducible (CV 3.2% within assay, 3.9% between assays). Accuracy was evaluated as the fraction of free testosterone in the ultrafiltrate of dialyzed serum vs that in a prior dialysate; they were the same confirming the validity of the free testosterone measurement. Samples from ostensibly healthy men and women and from hirsute and pregnant women gave results that agreed with those obtained by equilibrium dialysis. Total testosterone concentrations for normal and hirsute women showed considerable overlap, but data on free testosterone concentrations in these populations were better resolved

Glucose and phosphate modulation of intracellular 45Ca incorporated into pancreatic islets during culture in the absence and presence of serum

International Nuclear Information System (INIS)

Bergsten, P.

1985-01-01

The effects of glucose and phosphate on the intracellular 45 Ca content were measured in β cell-rich pancreatic islets cultured in media containing or lacking serum. Irrespective of the glucose and serum concentrations there were no or very small increments of 45 Ca contents when phosphate was raised from 0.8 to 5.8 mM during culture for 1 day. However, after 7 days of culture in serum-free medium there was a massive accumulation of 45 Ca in the islets in response to the higher phosphate concentration. Glucose markedly reduced, and serum eliminated, the extensive accumulation probably due to increased cell viability. In the cells cultured in the presence of serum, raising the glucose concentration from 1.0 to 5.5 mM resulted in an increased incorporation of 45 Ca. This effect was particularly pronounced after culture for 7 days in 5.8 mM phosphate. A further increase of glucose to 20 mM reduced the 45 Ca content. The results are consistent with the concept that glucose both stimulates 45 Ca uptake into different β-cell pools and degranulates the cell with associated loss of intracellular calcium from the granular calcium pool. (author)

Optimization of chemically defined cell culture media--replacing fetal bovine serum in mammalian in vitro methods

DEFF Research Database (Denmark)

van der Valk, J; Brunner, D; De Smet, K

2010-01-01

with an undefined and variable composition. Defined media supplements are commercially available for some cell types. However, information on the formulation by the companies is often limited and such supplements can therefore not be regarded as completely defined. The development of defined media is difficult......, reproducible and reduce the use of experimental animals. Good cell culture practice (GCCP) is an attempt to develop a common standard for in vitro methods. The implementation of the use of chemically defined media is part of the GCCP. This will decrease the dependence on animal serum, a supplement...... and often takes place in isolation. A workshop was organised in 2009 in Copenhagen to discuss strategies to improve the development and use of serum-free defined media. In this report, the results from the meeting are discussed and the formulation of a basic serum-free medium is suggested. Furthermore...

Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.

Science.gov (United States)

Rodin, Sergey; Antonsson, Liselotte; Hovatta, Outi; Tryggvason, Karl

2014-10-01

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.

Serum free-thyroxine modifications with age and in normal pregnancy

International Nuclear Information System (INIS)

Degrossi, O.J.; Altschuler, N.; Watanabe, T.; Pinkas, M.; Damilano, S.; Garcia del Rio, H.

1982-01-01

The possibility of using radioimmunoassay techniques (RIA) in the assessment of circulating thyroid hormones, total thyroxine (T4) and triiodothyronine (T3) has particularly increases the diagnosis of thyroid diseases. Thyroidal hormones circulate bound to proteins; therefore, variations in the transport capacity of the latter will produce important modifications in the T4 and T3 figures. Only small fractions, less than 0.05% for T4 and than 0.5% for T3, circulate in the free form and are considered as metabolically active forms of both hormones. In order to attain a correct clinical valuation, the rates of the free fractions as well as the total rates of these hormones must be known. Recently, these studies of free hormones are carried out by means of RIA, with the consequent advantages. The variations in serum free thyroxine (FT4) under certain physiogical conditions, such as for different ages and during pregnancy, were particularly studied

Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

International Nuclear Information System (INIS)

Kierszenbaum, A.L.; Crowell, J.A.; Shabanowitz, R.B.; DePhilip, R.M.; Tres, L.L.

1986-01-01

An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates

Multiple free-radical scavenging (MULTIS) capacity in cattle serum.

Science.gov (United States)

Sueishi, Yoshimi; Kamogawa, Erisa; Kimura, Anna; Kitahara, Go; Satoh, Hiroyuki; Asanuma, Taketoshi; Oowada, Shigeru

2017-01-01

Multiple free-radical scavenging (MULTIS) activity in cattle and human sera was evaluated with electron spin resonance spectroscopy. Scavenging rates against six active species, namely hydroxyl radical, superoxide anion, alkoxyl radical, alkylperoxyl radical, methyl radical, and singlet oxygen were quantified. The difference in the electron spin resonance signal intensity in the presence and absence of the serum was converted into the scavenging rates. Comparative MULTIS measurements were made in sera from eight beef cattle, three fetal calves and fifteen healthy human volunteers. Further, we determined the MULTIS value of albumin, the most abundant component in serum. MULTIS values in cattle sera indicated higher scavenging activity against most free radical species tested than human sera. In particular, cattle serum scavenging activities against superoxide and methyl radical were higher than human serum by 2.6 and 3.7 fold, respectively. In cattle serum, albumin appears to play a dominant role in MULTIS activity, but in human serum that is not the case. Previous data indicated that the abundance of uric acid in bovine blood is nearly 80% less than humans; however, this difference does not explain the deviation in MULTIS profile.

Supplements in human islet culture: human serum albumin is inferior to fetal bovine serum.

Science.gov (United States)

Avgoustiniatos, Efstathios S; Scott, William E; Suszynski, Thomas M; Schuurman, Henk-Jan; Nelson, Rebecca A; Rozak, Phillip R; Mueller, Kate R; Balamurugan, A N; Ansite, Jeffrey D; Fraga, Daniel W; Friberg, Andrew S; Wildey, Gina M; Tanaka, Tomohiro; Lyons, Connor A; Sutherland, David E R; Hering, Bernhard J; Papas, Klearchos K

2012-01-01

Culture of human islets before clinical transplantation or distribution for research purposes is standard practice. At the time the Edmonton protocol was introduced, clinical islet manufacturing did not include culture, and human serum albumin (HSA), instead of fetal bovine serum (FBS), was used during other steps of the process to avoid the introduction of xenogeneic material. When culture was subsequently introduced, HSA was also used for medium supplementation instead of FBS, which was typically used for research islet culture. The use of HSA as culture supplement was not evaluated before this implementation. We performed a retrospective analysis of 103 high-purity islet preparations (76 research preparations, all with FBS culture supplementation, and 27 clinical preparations, all with HSA supplementation) for oxygen consumption rate per DNA content (OCR/DNA; a measure of viability) and diabetes reversal rate in diabetic nude mice (a measure of potency). After 2-day culture, research preparations exhibited an average OCR/DNA 51% higher (p < 0.001) and an average diabetes reversal rate 54% higher (p < 0.05) than clinical preparations, despite 87% of the research islet preparations having been derived from research-grade pancreata that are considered of lower quality. In a prospective paired study on islets from eight research preparations, OCR/DNA was, on average, 27% higher with FBS supplementation than that with HSA supplementation (p < 0.05). We conclude that the quality of clinical islet preparations can be improved when culture is performed in media supplemented with serum instead of albumin.

Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production.

Science.gov (United States)

Uchida, Naoya; Demirci, Selami; Haro-Mora, Juan J; Fujita, Atsushi; Raines, Lydia N; Hsieh, Matthew M; Tisdale, John F

2018-06-15

In vitro erythroid differentiation from primary human cells is valuable to develop genetic strategies for hemoglobin disorders. However, current erythroid differentiation methods are encumbered by modest transduction rates and high baseline fetal hemoglobin production. In this study, we sought to improve both genetic modification and hemoglobin production among human erythroid cells in vitro . To model therapeutic strategies, we transduced human CD34 + cells and peripheral blood mononuclear cells (PBMCs) with lentiviral vectors and compared erythropoietin-based erythroid differentiation using fetal-bovine-serum-containing media and serum-free media. We observed more efficient transduction (85%-93%) in serum-free media than serum-containing media (20%-69%), whereas the addition of knockout serum replacement (KSR) was required for serum-free media to promote efficient erythroid differentiation (96%). High-level adult hemoglobin production detectable by electrophoresis was achieved using serum-free media similar to serum-containing media. Importantly, low fetal hemoglobin production was observed in the optimized serum-free media. Using KSR-containing, serum-free erythroid differentiation media, therapeutic adult hemoglobin production was detected at protein levels with β-globin lentiviral transduction in both CD34 + cells and PBMCs from sickle cell disease subjects. Our in vitro erythroid differentiation system provides a practical evaluation platform for adult hemoglobin production among human erythroid cells following genetic manipulation.

Salivary Cortisol Can Replace Free Serum Cortisol Measurements in Patients With Septic Shock

Science.gov (United States)

Orlander, Philip R.

2011-01-01

Background: There is a renewed interest in adrenal function during severe sepsis. Most studies have used total serum cortisol levels; however, only free serum cortisol is biologically active. The aim of this study was to determine the validity of salivary cortisol levels as a surrogate for free serum cortisol levels during septic shock. Methods: Fifty-seven patients with septic shock were studied to determine the correlation between total serum cortisol and salivary cortisol to free serum cortisol levels. Thirty-eight patients were included in the salivary to free serum cortisol correlation. Salivary cortisol level was tested by enzyme immunoassay. Serum total cortisol, free cortisol, and cortisol-binding globulin (CBG) levels were determined by liquid chromatography-mass spectrometry, equilibrium analysis, and radioimmunoassay, respectively. Results: The mean ± SD age was 56.6 ± 18.5 years. Fifty-seven percent were women. APACHE (Acute Physiology and Chronic Health Evaluation) II score median was 26, Simplified Acute Physiology Score II median was 61, and Sequential Organ Failure Assessment median was 13. The correlation between salivary and free serum cortisol levels was 0.79 (95% CI, 0.63-0.89; P cortisol and total serum cortisol levels was 0.86 (95% CI, 0.78-0.92; P cortisol level was 2.27 ± 1.64 μg/dL. The mean ± SD salivary cortisol level was 2.60 ± 2.69 μg/dL. The mean ± SD total serum cortisol level was 21.56 ± 8.71 μg/dL. The mean ± SD CBG level was 23.54 ± 8.33 mg/dL. Conclusions: Salivary cortisol level can be used as a surrogate of free serum cortisol level in patients with septic shock with very good correlation. Salivary cortisol testing is noninvasive, easy to perform, and can be conducted daily. Trial registry: ClinicalTrials.gov; No.: NCT00523198; URL: www.clinicaltrials.gov PMID:21816912

Ultrasensitive radioimmunoassay for direct determination of free triiodothyronine concentration in serum

International Nuclear Information System (INIS)

Weeke, J.; Oerskov, H.

1975-01-01

Free triiodothyronine (T 3 ) in serum has been measured directly in dialysates of serum, using a wick chromatographic radioimmunoassay. Adequate sensitivity was attained by the use of [ 125 I]T 3 with a very high specific activity (2,000 to 3,000μCi/μg). Sera were dialysed against a Krebs-Ringer bicarbonate buffer modified so as to be similar to plasma water. Dialysis took place under carefully controlled circumstances. The influence on the equilibrium of total to free T 3 of temperature, serum dilution and dialysis time was studied. By the present method, free T 3 in serum from groups of subjects including 20 men, 10 women taking oral contraceptives and 20 women with normal menstrual cycles were identical, averaging 5.2 pg/ml. A chromatographic radioimmunoassay of total T 3 using high specific activity [ 125 I]T 3 and a very small test sample is also described. (auth.)

Culture conditions affect photoreactivating enzyme levels in human fibroblasts

International Nuclear Information System (INIS)

Sutherland, B.M.; Oliver, R.

1976-01-01

Photoreactivation of pyrimidine dimers occured under the experimental conditions given in this study, but has not been observed under conditions used by others. Three possible differences were tested in experimental procedures including dimer separation and analysis methods, illumination conditions and cell culture techniques. The methods in this study of dimer separation and analysis indeed measure cis-syn pyrimidine dimers and give results in quantitative agreement with the methods of others. It was found that white light pre-illumination of fibroblasts from the xeroderma pigmentosum line XP12BE or of normal cells does not affect the cellular capacity for dimer photoreactivation. However, the cell culture conditions can affect photoreactivating enzyme levels, and thus cellular dimer photoreactivation capacity. Cells grown in Eagle's minimal essential medium (supplemented with 15% fetal bovine serum) contain very low levels of photoreactivating enzyme and cannot photoreactivate dimers in their DNA; but companion cultures maintained in Dulbecco's modified Eagle's minimal medium do contain photoreactivating enzyme and can reactivate photoreactive cellular dimers

Embryonic Stem Cell Culture Conditions Support Distinct States Associated with Different Developmental Stages and Potency

DEFF Research Database (Denmark)

Martin Gonzalez, Javier; Morgani, Sophie M; Bone, Robert A

2016-01-01

. Conversely, the transcriptome of serum-cultured ESCs correlated with later stages of development (E4.5), at which point embryonic cells are more restricted in their developmental potential. Thus, ESC culture systems are not equivalent, but support cell types that resemble distinct developmental stages. Cells...... derived in one condition can be reprogrammed to another developmental state merely by adaptation to another culture condition....

Differentiation of Dental Pulp Stem Cells into Neuron-Like Cells in Serum-Free Medium

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Shahrul Hisham Zainal Ariffin

2013-01-01

Full Text Available Dental pulp tissue contains dental pulp stem cells (DPSCs. Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146+, Cd166+, and Cd31− in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology.

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Kim, Hyunjeong; Kim, Eosu; Park, Minsun; Lee, Eun; Namkoong, Kee

2013-03-05

One of the most significant barriers towards translational neuropsychiatry would be an unavailability of living brain tissues. Although organotypic brain tissue culture could be a useful alternative enabling observation of temporal changes induced by various drugs in living brain tissues, a proper method to establish a stable organotypic brain slice culture system using adult (rather than neonatal) hippocampus has been still elusive. In this study, we evaluated our simple method using the serum-free culture medium for successful adult organotypic hippocampal slice culture. Several tens of hippocampal slices from a single adult mouse (3-5 months old) were cultured in serum-free versus serum-containing conventional culture medium for 30 days and underwent various experiments to validate the effects of the existence of serum in the culture medium. Neither the excessive regression of neuronal viability nor metabolic deficiency was observed in the serum-free medium culture in contrast to the serum-containing medium culture. Despite such viability, newly generated immature neurons were scarcely detected in the serum-free culture, suggesting that the original neurons in the brain slice persist rather than being replaced by neurogenesis. Key structural features of in vivo neural tissue constituting astrocytes, neural processes, and pre- and post-synapses were also well preserved in the serum-free culture. In conclusion, using the serum-free culture medium, the adult hippocampal slice culture system will serve as a promising ex vivo tool for various fields of neuroscience, especially for studies on aging-related neuropsychiatric disorders or for high throughput screening of potential agents working against such disorders. Copyright © 2012 Elsevier Inc. All rights reserved.

Raman Spectroscopic Analyses of Jaw Periosteal Cell Mineralization

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Eva Brauchle

2017-01-01

Full Text Available To achieve safer patient treatments, serum-free cell culture conditions have to be established for cell therapies. In previous studies, we demonstrated that serum-free culture favored the proliferation of MSCA-1+ osteoprogenitors derived from the jaw periosteum. In this study, the in vitro formation of bone-specific matrix by MSCA-1+ jaw periosteal cells (JPCs, 3 donors was assessed and compared under serum-free and serum-containing media conditions using the marker-free Raman spectroscopy. Based on a standard fluorescence assay, JPCs from one patient were not able to mineralize under serum-containing culture conditions, whereas the other cells showed similar mineralization levels under both conditions. Raman spectra from mineralizing MSCA-1+ JPCs revealed higher levels of hydroxyapatite formation and higher mineral to matrix ratios under serum-free culture conditions. Higher carbonate to phosphate ratios and higher crystallinity in JPCs cultured under serum-containing conditions indicated immature bone formation. Due to reduced collagen production under serum-free conditions, we obtained significant differences in collagen maturity and proline to hydroxyproline ratios compared to serum-free conditions. We conclude that Raman spectroscopy is a useful tool for the assessment and noninvasive monitoring of in vitro mineralization of osteoprogenitor cells. Further studies should extend this knowledge and improve JPC mineralization by optimizing culture conditions.

Multiple free-radical scavenging capacity in serum

Science.gov (United States)

Oowada, Shigeru; Endo, Nobuyuki; Kameya, Hiromi; Shimmei, Masashi; Kotake, Yashige

2012-01-01

We have developed a method to determine serum scavenging-capacity profile against multiple free radical species, namely hydroxyl radical, superoxide radical, alkoxyl radical, alkylperoxyl radical, alkyl radical, and singlet oxygen. This method was applied to a cohort of chronic kidney disease patients. Each free radical species was produced with a common experimental procedure; i.e., uv/visible-light photolysis of free-radical precursor/sensitizer. The decrease in free-radical concentration by the presence of serum was quantified with electron spin resonance spin trapping method, from which the scavenging capacity was calculated. There was a significant capacity change in the disease group (n = 45) as compared with the healthy control group (n = 30). The percent values of disease’s scavenging capacity with respect to control group indicated statistically significant differences in all free-radical species except alkylperoxyl radical, i.e., hydroxyl radical, 73 ± 12% (p = 0.001); superoxide radical, 158 ± 50% (p = 0.001); alkoxyl radical, 121 ± 30% (p = 0.005); alkylperoxyl radical, 123 ± 32% (p>0.1); alkyl radical, 26 ± 14% (p = 0.001); and singlet oxygen, 57 ± 18% (p = 0.001). The scavenging capacity profile was illustrated using a radar chart, clearly demonstrating the characteristic change in the disease group. Although the cause of the scavenging capacity change by the disease state is not completely understood, the profile of multiple radical scavenging capacities may become a useful diagnostic tool. PMID:22962529

Verification of serum reference intervals for free light chains in a local South African population.

Science.gov (United States)

Zemlin, Annalise E; Rensburg, Megan A; Ipp, Hayley; Germishuys, Jurie J; Erasmus, Rajiv T

2013-11-01

Monoclonal serum free light chain measurements are used to follow up and manage patients with monoclonal gammopathies, and abnormal serum free light chain ratios are associated with risk of progression in certain diseases. We aimed to validate the reference intervals in our population. Reference intervals for κ and λ free light chains were established on 120 healthy adults. Creatinine levels were measured to exclude renal dysfunction and serum protein electrophoresis was performed. All creatinine values were within normal limits. After exclusion of subjects with abnormal serum protein electrophoreses, 113 subjects were available for analysis. The 95% reference interval was 6.3-20.6 mg/L for κ free light chains, 8.7-25.9 mg/L for λ free light chains and 0.46-1.23 for free light chain ratio. Most of the values fell within the manufacturer's recommended limits and therefore could be used for our population.

Comparison between human cord blood serum and platelet-rich plasma supplementation for Human Wharton's Jelly Stem Cells and dermal fibroblasts culture

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Hashemi SS

2016-08-01

Full Text Available We carried out a side-by-side comparison of the effects of Human cord blood serum (HcbS versus embryonic PRP on Human Wharton's Jelly Stem Cells(hWMSCand dermal fibroblasts proliferation. Human umbilical cord blood was collected to prepare activated serum (HCS and platelet-rich plasma (CPRP.Wharton's Jelly Stem Cells and dermal fibroblasts were cultured in complete medium with10% CPRP, 10%HCSor 10% fetal bovine serumand control (serum-free media.The efficiency of the protocols was evaluated in terms of the number of adherent cells and their expansion and Cell proliferation. We showed that proliferation of fibroblasts and mesenchymal stem cells in the presence of cord blood serum and platelet-rich plasma significantly more than the control group (p≤0/05. As an alternative to FBS, cord blood serum has been proved as an effective component in cell tissue culture applications and embraced a vast future in clinical applications of regenerative medicine. However, there is still a need to explore the potential of HCS and its safe applications in humanized cell therapy or tissue engineering.

A novel liquid medium for the efficient growth of the salmonid pathogen Piscirickettsia salmonis and optimization of culture conditions.

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Mirtha Henríquez

Full Text Available Piscirickettsia salmonis is the bacterium that causes Piscirickettsiosis, a systemic disease of salmonid fish responsible for significant economic losses within the aquaculture industry worldwide. The growth of the bacterium for vaccine formulation has been traditionally accomplished by infecting eukaryotic cell lines, a process that involves high production costs and is time-consuming. Recent research has demonstrated that it is possible to culture pure P. salmonis in a blood containing (cell-free medium. In the present work we demonstrate the growth of P. salmonis in a liquid medium free from blood and serum components, thus establishing a novel and simplified bacteriological medium. Additionally, the new media reported provides improved growth conditions for P. salmonis, where biomass concentrations of approximately 800 mg cell dry weight L(-1 were obtained, about eight times higher than those reported for the blood containing medium. A 2- level full factorial design was employed to evaluate the significance of the main medium components on cell growth and an optimal temperature range of 23-27°C was determined for the microorganism to grow in the novel liquid media. Therefore, these results represent a breakthrough regarding P. salmonis research in order to optimize pure P. salmonis growth in liquid blood and serum free medium.

Testing of serum atherogenicity in cell cultures: questionable data published

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Sergei V. Jargin

2012-01-01

Full Text Available In a large series of studies was reported that culturing of smooth muscle cells with serum from atherosclerosis patients caused intracellular lipid accumulation, while serum from healthy controls had no such effect. Cultures were used for evaluation of antiatherogenic drugs. Numerous substances were reported to lower serum atherogenicity: statins, trapidil, calcium antagonists, garlic derivatives etc. On the contrary, beta-blockers, phenothiazines and oral hypoglycemics were reported to be pro-atherogenic. Known antiatherogenic agents can influence lipid metabolism and cholesterol synthesis, intestinal absorption or endothelium-related mechanisms. All these targets are absent in cell monocultures. Inflammatory factors, addressed by some antiatherogenic drugs, are also not reproduced. In vivo, relationship between cholesterol uptake by cells and atherogenesis must be inverse rather than direct: in familial hypercholesterolemia, inefficient clearance of LDL-cholesterol by cells predisposes to atherosclerosis. Accordingly, if a pharmacological agent reduces cholesterol uptake by cells in vitro, it should be expected to elevate cholesterol in vivo. Validity of clinical recommendations, based on serum atherogenicity testing in cell monocultures, is therefore questionable. These considerations pertain also to the drugs developed on the basis of the cell culture experiments.

Free Publishing Culture. Sustainable Models?

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Silvia Nanclares Escudero

2013-03-01

Full Text Available As a result of the collective research on the possibilities for publishing production and distribution offered nowadays by the Free Culture scenario, we present here a mapping of symptoms in order to propose a transitory diagnostic of the question: Is it possible to generate an economically sustainable publishing model based on the uses and customs generated and provided by Free Culture? Data, intuitions, experiences and ideas attempt to back up our affirmative answer.

Serum Immunoglobulin Free Light Chain Assessment in IgG4-Related Disease

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Aurélie Grados

2013-01-01

Full Text Available Immunoglobulin free light chains are produced in excess during normal antibody synthesis. Their evaluation is commonly used in case of a monoclonal gammopathy. In polyclonal hypergammaglobulinemia related to the Sjögren syndrome or systemic lupus, erythematosus serum free light chain levels are increased and could correlate with disease activity. We show here that the κ ( and λ ( free light chains and the κ : λ ratio ( are increased in sixteen patients with IgG4-related disease when compared to healthy controls. The increase of κ and λ free light chains probably reflects the marked polyclonal B cell activation of the disease. We could not assess in this small cohort of patients a significative correlation of serum free light chain levels and disease activity or extension.

Increasing synthetic serum substitute (SSS) concentrations in P1 glucose/phosphate-free medium improves implantation rate: a comparative study.

Science.gov (United States)

Ben-Yosef, D; Yovel, I; Schwartz, T; Azem, F; Lessing, J B; Amit, A

2001-11-01

To assess the comparative efficacy of IVF medium (MediCult, with 5.2 mM glucose) and a glucose/phosphate-free medium, P1 (Irvine Scientific), and to investigate the influence of increasing the serum supplementation (synthetic serum substitute; SSS; Irvine Scientific) to P1 on embryo development and implantation. Patients were randomly assigned to IVF medium (Group 1, cycles n = 172) or P1 supplemented with 10% SSS (Group 2, cycles n = 229) according to the medium scheduled for use on the day of oocyte retrieval. Another 555 IVF consequent cycles (Group 3) were performed using increased SSS concentrations (20%) in P1 medium. In this large series of IVF cycles, we herein demonstrate that significantly higher pregnancy and implantation rates were found when embryos were cultured in glucose/phosphate-free medium P1 supplemented with 20% SSS compared to supplementation with the lower SSS concentration and with IVF medium.

Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions.

Science.gov (United States)

Wang, Juan; Hao, Jie; Bai, Donghui; Gu, Qi; Han, Weifang; Wang, Lei; Tan, Yuanqing; Li, Xia; Xue, Ke; Han, Pencheng; Liu, Zhengxin; Jia, Yundan; Wu, Jun; Liu, Lei; Wang, Liu; Li, Wei; Liu, Zhonghua; Zhou, Qi

2015-11-12

Human induced pluripotent stem cells (hiPSCs) are considered as one of the most promising seed cell sources in regenerative medicine. Now hiPSC-based clinical trials are underway. To ensure clinical safety, cells used in clinical trials or therapies should be generated under GMP conditions, and with Xeno-free culture media to avoid possible side effects like immune rejection that induced by the Xeno reagents. However, up to now there are no reports for hiPSC lines developed completely under GMP conditions using Xeno-free reagents. Clinical-grade human foreskin fibroblast (HFF) cells used as feeder cells and parental cells of the clinical-grade hiPSCs were isolated from human foreskin tissues and cultured in Xeno-free media. Clinical-grade hiPSCs were derived by integration-free Sendai virus-based reprogramming kit in Xeno-free pluriton™ reprogramming medium or X medium. Neural cells and cardiomyocytes differentiation were conducted following a series of spatial and temporal specific signals induction according to the corresponding lineage development signals. Biological safety evaluation of the clinical-grade HFF cells and hiPSCs were conducted following the guidance of the "Pharmacopoeia of the People's Republic of China, Edition 2010, Volume III". We have successfully derived several integration-free clinical-grade hiPSC lines under GMP-controlled conditions and with Xeno-free reagents culture media in line with the current guidance of international and national evaluation criteria. As for the source of hiPSCs and feeder cells, biological safety evaluation of the HFF cells have been strictly reviewed by the National Institutes for Food and Drug Control (NIFDC). The hiPSC lines are pluripotent and have passed the safety evaluation. Moreover, one of the randomly selected hiPSC lines was capable of differentiating into functional neural cells and cardiomyocytes in Xeno-free culture media. The clinical-grade hiPSC lines therefore could be valuable sources for

A simplified ultrafiltration method for determination of serum free cortisol

International Nuclear Information System (INIS)

MacMahon, W.; Sgoutas, D.

1983-01-01

The authors describe the suitability of the Amicon MPS-1 centrifugal ultrafiltration device and the YMB membrane for measuring free cortisol in serum. The method combines two independent assays: total cortisol and the ultrafiltrate fraction of added [ 3 H]cortisol. The unbound fraction is determined in 0.25-0.30 ml of ultrafiltrate collected from 0.6 to 1 ml of serum that has been equilibrated with [ 3 H]cortisol at 37 0 C for 20 min. The assay is rapid (less than 1 h), practical (no more than 0.6 ml of serum is necessary) and repeatable (CV: 3.8% within-assay and 12.2% in different assays). Error introduced in free cortisol measurement due to dilution effects in dialysis is systematically defined, and the effect of tracer purity on the ultrafiltration method is examined. Dialyzed sera from normal men and women, from patients with Cushing's disease and adrenal insufficiency, and from pregnant women gave ultrafiltration results that accurately duplicated those obtained by previous dialysis. (Auth.)

A single-cell and feeder-free culture system for monkey embryonic stem cells.

Science.gov (United States)

Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

2014-01-01

Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches.

Science.gov (United States)

Saury, Charlotte; Lardenois, Aurélie; Schleder, Cindy; Leroux, Isabelle; Lieubeau, Blandine; David, Laurent; Charrier, Marine; Guével, Laëtitia; Viau, Sabrina; Delorme, Bruno; Rouger, Karl

2018-05-02

Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL). hMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3'-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL. HS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives. Our

Optimized conditions for primary culture of pituitary cells from the Atlantic cod (Gadus morhua). The importance of osmolality, pCO₂, and pH.

Science.gov (United States)

Hodne, Kjetil; von Krogh, Kristine; Weltzien, Finn-Arne; Sand, Olav; Haug, Trude M

2012-09-01

Protocols for primary cultures of teleost cells are commonly only moderately adjusted from similar protocols for mammalian cells, the main adjustment often being of temperature. Because aquatic habitats are in general colder than mammalian body temperatures and teleosts have gills in direct contact with water, pH and buffer capacity of blood and extracellular fluid are different in fish and mammals. Plasma osmolality is generally higher in marine teleosts than in mammals. Using Atlantic cod (Gadus morhua) as a model, we have optimized these physiological parameters to maintain primary pituitary cells in culture for an extended period without loosing key properties. L-15 medium with adjusted osmolality, adapted to low pCO(2) (3.8mm Hg) and temperature (12°C), and with pH 7.85, maintained the cells in a physiologically sounder state than traditional culture medium, significantly improving cell viability compared to the initial protocol. In the optimized culture medium, resting membrane potential and response to releasing hormone were stable for at least two weeks, and the proportion of cells firing action potentials during spawning season was about seven times higher than in the original culture medium. The cells were moderately more viable when the modified medium was supplemented with newborn calf serum or artificial serum substitute. Compared to serum-free L-15 medium, expression of key genes (lhb, fshb, and gnrhr2a) was better maintained in medium containing SSR, whereas NCS tended to decrease the expression level. Although serum-free medium is adequate for many applications, serum supplement may be preferable for experiments dependent on membrane integrity. Copyright © 2012 Elsevier Inc. All rights reserved.

Optimization of Serum Immunoglobulin Free Light Chain Analysis for Subclassification of Cardiac Amyloidosis.

Science.gov (United States)

Halushka, Marc K; Eng, George; Collins, A Bernard; Judge, Daniel P; Semigran, Marc J; Stone, James R

2015-06-01

Accurate and rapid classification of cardiac amyloidosis is important for patient management. We have optimized the use of serum free light chain kappa and lambda values to differentiate immunoglobulin light chain amyloid (AL) amyloidosis from transthyretin amyloid and amyloid A using 85 cases of tissue-proven cardiac amyloidosis, in which there was direct classification of amyloidosis by mass spectrometry or immunofluorescence. The serum free light chain kappa/lambda ratios were non-overlapping for the three major groups: AL-lambda (0.01-0.41, n = 30), non-AL (0.52-2.7, n = 43), and AL-kappa (6.7-967, n = 12). A kappa/lambda ratio value between 0.5 and 5.0 had 100 % sensitivity and 100 % specificity for distinguishing AL amyloidosis from non-AL amyloidosis. This optimized range for serum light chain kappa/lambda ratio provides extremely robust classification of cardiac amyloidosis. Cases of cardiac amyloidosis in which the serum kappa/lambda free light chain ratio falls close to these new cutoff values may benefit most from direct amyloid subtyping.

Clinical significance of the measurements of serum free thyroxine and free triiodothyronine concentrations

International Nuclear Information System (INIS)

Kubota, Ken; Sasaki, Norio; Takaku, Fumimaro; Uchimura, Hidemasa

1988-01-01

A commercially available ''DPC'' radioimmunoassay kit was used to study the serum concentrations of free triiodothyronine (FT3) and free thyroxine (FT4) in a series of 189 patients with various thyroid diseases and 120 healthy controls. The basal serum concentrations of FT3 and FT4 in normal controls ranged from 0.98 to 2.04 ng/dl and from 1.43 to 3.66 pg/ml, respectively. All untreated patients with Graves' disease had abnormally high FT3 and FT4 values, indicating the discrimination between hyperthyroid and normal subjects. A decreased ratio of FT4 to FT3 was observed in patients managed with antithyroid drugs. In diagnosing hypothyroidism, ''DPC'' FT4 kits were more sensitive than ''DPC'' TT4 and ''Amerlex'' FT4 kits. In the case of non-thyroid diseases, FT4 and FT3 values, as well as total T4 and total T3, were normal or decreased, with the exception of occasionally unknown high values. The interference of thyroxine binding globulin abnormablity was successfully eliminated by using new versions of ''DPC'' kits. (Namekawa, K.)

Serum vitamin D levels in free-ranging koalas (Phascolarctos cinereus).

Science.gov (United States)

Pye, Geoffrey W; Ellis, William; FitzGibbon, Sean; Opitz, Brian; Keener, Laura; Hollis, Bruce W

2013-06-01

Due to climatic conditions in Northern America and Europe, koalas (Phascolarctos cinereus) are often housed indoors. Koala joeys raised in these environments are susceptible to the development of metabolic bone disease due to a lack of exposure to solar ultraviolet radiation to themselves and their dam. As an initial step toward describing vitamin D sufficiency and adequately measuring responses to supplementation, vitamin D values were calculated by using serum collected from 20 free-ranging koalas on St. Bees Island, Queensland, Australia. Vitamin D values ranged from 8.1 to 30.4 pg/ml (18.4 +/- 5.5 pg/ml) for 1, 25-hydroxyvitamin D, and from 1 to 14 nM/L (7.4 +/- 3.0 nM/L) for 25-hydroxyvitamin D. These koala serum vitamin D values are unusually low when compared with eutherian mammals. Although this study was limited in numbers and in the geographically range of the koalas sampled, it does suggest that the koala's requirement for vitamin D is low. Therefore, supplementation to prevent disease may be relatively easy to achieve because low doses will likely meet requirements. Caution should be taken to avoid intoxication if supplementing vitamin D in koalas.

Establishment of feeder-free culture system for human induced pluripotent stem cell on DAS nanocrystalline graphene

Science.gov (United States)

Lee, Hyunah; Nam, Donggyu; Choi, Jae-Kyung; Araúzo-Bravo, Marcos J.; Kwon, Soon-Yong; Zaehres, Holm; Lee, Taehee; Park, Chan Young; Kang, Hyun-Wook; Schöler, Hans R.; Kim, Jeong Beom

2016-02-01

The maintenance of undifferentiated human pluripotent stem cells (hPSC) under xeno-free condition requires the use of human feeder cells or extracellular matrix (ECM) coating. However, human-derived sources may cause human pathogen contamination by viral or non-viral agents to the patients. Here we demonstrate feeder-free and xeno-free culture system for hPSC expansion using diffusion assisted synthesis-grown nanocrystalline graphene (DAS-NG), a synthetic non-biological nanomaterial which completely rule out the concern of human pathogen contamination. DAS-NG exhibited advanced biocompatibilities including surface nanoroughness, oxygen containing functional groups and hydrophilicity. hPSC cultured on DAS-NG could maintain pluripotency in vitro and in vivo, and especially cell adhesion-related gene expression profile was comparable to those of cultured on feeders, while hPSC cultured without DAS-NG differentiated spontaneously with high expression of somatic cell-enriched adhesion genes. This feeder-free and xeno-free culture method using DAS-NG will facilitate the generation of clinical-grade hPSC.

Conditions and limits of serum LH radioimmunoassay in normal, hypophysectomised or castred rats

International Nuclear Information System (INIS)

Andre, M.; Boucher, D.; Thieblot, L.

1976-01-01

Serum LH was measured by radioimmunoassay (NIAMD Kits) free and linked hormones were separated by double antibodies method. Influence of concentration on antibody-hormone complex is studied. Hypophysectomised rats serum does not modify results. The standard (rat LH-RPl) has the same action as serum LH. Rat serum LH contents are measured in normal or castred rats [fr

Elevated serum free thyroxine by thyroxine analog radioimmunoassays in euthyroid patients with familial dysalbuminemic hyperthyroxinemia

International Nuclear Information System (INIS)

Rajatanavin, R.; Fournier, L.; DeCosimo, D.; Abreau, C.; Braverman, L.E.

1982-01-01

A study was done to ascertain whether the serum free T4 measured by free T4 radioimmunoassay kits would, like equilibrium dialysis, be normal in patients with familial dysalbuminemic hyperthyroxinemia. Five free T4 radioimmunoassay kits were used to measure free T4 in serum samples from 19 patients with familial dysalbuminemic hyperthyroxinemia and 20 healthy volunteers. Values (mean +/- SE) for T4, free T4 index, and free T4 (equilibrium dialysis) in these normal subjects and patients with familial dysalbuminemic hyperthyroxinemia, respectively, were as follows: T4, 8.1 +/- 0.2 and 18.3 +/- 0.7 μg/dL; free T4 index, 3.1 +/- 0.1 and 7.3 +/- 0.3 μg/dL; free T4, 1.4 +/- 0.1 and 1.2 +/- 0.1 ng/dL. The following free T4 radioimmunoassay methods were used: antibody coated microfine silica, microencapsulated antibody, two-step antibody-coated tube, and one-step 125 I-T4 analog (2 kits). The present findings in patients with familial dysalbuminemic hyperthyroxinemia and previous observations in ill euthyroid patients suggest that serum free T4 measured by some radioimmunoassay methods must be interpreted with caution in these two clinical situations

Free Speech as a Cultural Value in the United States

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Mauricio J. Alvarez

2018-02-01

Full Text Available Political orientation influences support for free speech, with liberals often reporting greater support for free speech than conservatives. We hypothesized that this effect should be moderated by cultural context: individualist cultures value individual self-expression and self-determination, and collectivist cultures value group harmony and conformity. These different foci should differently influence liberals and conservatives’ support for free speech within these cultures. Two studies evaluated the joint influence of political orientation and cultural context on support for free speech. Study 1, using a multilevel analysis of data from 37 U.S. states (n = 1,001, showed that conservatives report stronger support for free speech in collectivist states, whereas there were no differences between conservatives and liberals in support for free speech in individualist states. Study 2 (n = 90 confirmed this pattern by priming independent and interdependent self-construals in liberals and conservatives. Results demonstrate the importance of cultural context for free speech. Findings suggest that in the U.S. support for free speech might be embraced for different reasons: conservatives’ support for free speech appears to be motivated by a focus on collectively held values favoring free speech, while liberals’ support for free speech might be motivated by a focus on individualist self-expression.

Higher Serum Levels of Free ĸ plus λ Immunoglobulin Light Chains Ameliorate Survival of Hemodialysis Patients

DEFF Research Database (Denmark)

Thilo, Florian; Caspari, Christina; Scholze, Alexandra

2011-01-01

Background/Aims: Impaired immune function is common in patients with chronic renal failure. Now, we determined whether serum levels of free immunoglobulin light chains predict mortality in patients with chronic kidney disease stage 5 on hemodialysis. Methods: We performed a prospective cohort study...... of 160 hemodialysis patients with a median follow-up of 15 months (interquartile range, 3-44 months). Serum levels of free κ and λ immunoglobulin light chains were measured at the start of the study. The primary end point was mortality from any cause. Results: In survivors, median serum levels of free κ...... plus λ immunoglobulin light chains were significantly higher compared with nonsurvivors (p light chains above the median compared with patients with serum levels below the median of 210 mg...

Comparison of serum fractionation methods by data independent label-free proteomics

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D. Baiwir

2015-12-01

Full Text Available Off-line sample prefractionations applied prior to biomarker discovery proteomics are options to enable more protein identifications and detect low-abundance proteins. This work compared five commercial methods efficiency to raw serum analysis using label-free proteomics. The variability of the protein quantities determined for each process was similar to the unprefractionated serum. A 49% increase in protein identifications and 12.2% of reliable quantification were obtained. A 61 times lower limit of protein quantitation was reached compared to protein concentrations observed in raw serum. The concentrations of detected proteins were confronted to estimated reference values.

Can serum free fatty acids assessment predict severe preeclampsia?

African Journals Online (AJOL)

Nermeen Saad El Beltagy

2011-10-20

Oct 20, 2011 ... Methods: Twenty cases with severe preeclampsia (blood pressure P 160/110 after 20th week of ges- tation and ... ing factor with preeclampsia in non-obese pregnant women. ... Preeclampsia (PE) is a common pregnancy disorder that is ... centration of free fatty acids in the serum was measured by an.

Neuroprotection against oxidative stress by serum from heat acclimated rats.

Science.gov (United States)

Beit-Yannai, E; Trembovler, V; Horowitz, M; Lazarovici, P; Kohen, R; Shohami, E

1998-09-25

Exposure of PC12 cells, to 1% serum derived from normothermic (CON) rats resulted in 79% cell death. Sister cultures treated with 1% serum derived from heat acclimated (ACC) rats, were neuroprotected and expressed a significant reduction in cell death. In PC12 cells exposed to a free radical generator causing an oxidative stress, 90% cell death was measured in CON serum treated cultures, while ACC serum treated cultures were neuroprotected. Xanthine oxidase activity and uric acid (UA) levels were lower in ACC serum compared to CON. Addition of UA to both sera abolished the difference in cell viability, and toxicity of ACC serum reached that of CON. These findings suggest a causal relationship between the lower levels of UA in ACC and the neuroprotective effect observed. The present study proposes heat acclimation as an experimental and/or clinical tool for the achievement of neuroprotection.

Comparison of serum hormone levels of captive and free-living maned wolves Chrysocyon brachyurus

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O.B. Maia

2008-02-01

Full Text Available Serum hormone levels were compared between captive and free-living maned wolves and seasonal variations of sex hormones were studied. Blood samples were collected from 16 male and 26 female adult animals from Brazilian zoos, and from 30 male and 24 female free-living adults to determine serum progesterone and testosterone by radioimmunoassay. Serum testosterone concentrations varied (P 0.05. Sixteen captive males showed higher testosterone concentration during winter and spring compared with 30 free-living animals (P < 0.05. Progesterone concentration varied among seasons in 26 captive females (P < 0.05, being higher in autumn (15.3 ± 3.1 ng/mL than in summer (6.6 ± 1.5 ng/mL, winter (5.3 ± 3.1 ng/mL and spring (4.3 ± 0.7 ng/mL. Progesterone concentration of 24 free-living females varied between autumn (17.1 ± 6.0 ng/mL and winter (1.7 ± 0.3 ng/mL (P < 0.05, but we could not obtain data for spring or summer. No difference in progesterone levels was observed between captive and free-living females in autumn and winter.

Free Speech as a Cultural Value in the United States

OpenAIRE

Alvarez, Mauricio J.; Kemmelmeier, Markus

2017-01-01

Political orientation influences support for free speech, with liberals often reporting greater support for free speech than conservatives. We hypothesized that this effect should be moderated by cultural context: individualist cultures value individual self-expression and self-determination, and collectivist cultures value group harmony and conformity. These different foci should differently influence liberals and conservatives’ support for free speech within these cultures. Two studies eval...

Opium consumption is negatively associated with serum prostate-specific antigen (PSA), free PSA, and percentage of free PSA levels.

Science.gov (United States)

Safarinejad, Mohammad Reza; Asgari, Seyyed Alaeddin; Farshi, Alireza; Iravani, Shahrokh; Khoshdel, Alireza; Shekarchi, Babak

2013-01-01

Addiction to opium continues to be a major worldwide medical and social problem. The study addressing the association between opium consumption and serum prostate-specific antigen (PSA) level is lacking. We determined the effects of opium consumption on serum PSA levels in opium-addict men. Our study subjects comprised 438 opium-addict men with a mean age of 52.2 ± 6.4 years (group 1). We compared these men with 446 men who did not indicate current or past opium use (group 2). Serum total PSA (tPSA), free PSA (fPSA), % fPSA, and sex hormones were compared between the 2 groups. The mean serum tPSA level was significantly lower in group 1 (1.05 ng/mL) than in controls (1.45 ng/mL) (P = 0.001). Opium consumption was also associated with lower fPSA (P = 0.001) and % fPSA (P = 0.001). Serum free testosterone level in opium-addict patients (132.5 ± 42 pg/mL) was significantly lower than that in controls (156.2 ± 43 pg/mL) (P = 0.03). However, no significant correlation existed between tPSA and free testosterone levels (r = 0.28, 95% CI, -0.036 to 0.51, P = 0.34). Among the patients with cancer in group 1, 35% were found to have high-grade tumor (Gleason score ≥ 7) compared with 26.7% in group 2 (P = 0.02). Total PSA and fPSA were strongly correlated with duration of opium use (r = -0.06, 95% CI, -0.04 to -0.08, P = 0.0001; and r = -0.05, 95% CI, -0.03 to -0.07, P = 0.0001, respectively). Opium consumption is independently and negatively associated with serum tPSA, fPSA, and % fPSA levels.

Changes in Serum Free Amino Acids and Muscle Fatigue Experienced during a Half-Ironman Triathlon.

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Francisco Areces

Full Text Available The aim of this study was to investigate the relationship between changes in serum free amino acids, muscle fatigue and exercise-induced muscle damage during a half-ironman triathlon. Twenty-six experienced triathletes (age = 37.0 ± 6.8 yr; experience = 7.4 ± 3.0 yr competed in a real half-ironman triathlon in which sector times and total race time were measured by means of chip timing. Before and after the race, a countermovement jump and a maximal isometric force test were performed, and blood samples were withdrawn to measure serum free amino acids concentrations, and serum creatine kinase levels as a blood marker of muscle damage. Total race time was 320 ± 37 min and jump height (-16.3 ± 15.2%, P 20%. However, neither the changes in serum free amino acids nor the tryptophan/BCAA ratio were related muscle fatigue or muscle damage during the race.

Evaluation of performance of bacterial culture of feces and serum ELISA across stages of Johne's disease in cattle using a Bayesian latent class model.

Science.gov (United States)

Espejo, L A; Zagmutt, F J; Groenendaal, H; Muñoz-Zanzi, C; Wells, S J

2015-11-01

The objective of this study was to evaluate the performance of bacterial culture of feces and serum ELISA to correctly identify cows with Mycobacterium avium ssp. paratuberculosis (MAP) at heavy, light, and non-fecal-shedding levels. A total of 29,785 parallel test results from bacterial culture of feces and serum ELISA were collected from 17 dairy herds in Minnesota, Pennsylvania, and Colorado. Samples were obtained from adult cows from dairy herds enrolled for up to 10 yr in the National Johne's Disease Demonstration Herd Project. A Bayesian latent class model was fitted to estimate the probabilities that bacterial culture of feces (using 72-h sedimentation or 30-min centrifugation methods) and serum ELISA results correctly identified cows as high positive, low positive, or negative given that cows were heavy, light, and non-shedders, respectively. The model assumed that no gold standard test was available and conditional independency existed between diagnostic tests. The estimated conditional probabilities that bacterial culture of feces correctly identified heavy shedders, light shedders, and non-shedders were 70.9, 32.0, and 98.5%, respectively. The same values for the serum ELISA were 60.6, 18.7, and 99.5%, respectively. Differences in diagnostic test performance were observed among states. These results improve the interpretation of results from bacterial culture of feces and serum ELISA for detection of MAP and MAP antibody (respectively), which can support on-farm infection control decisions and can be used to evaluate disease-testing strategies, taking into account the accuracy of these tests. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Enhanced engraftment of mesenchymal stem cells in a cutaneous wound model by culture in allogenic species-specific serum and administration in fibrin constructs.

Science.gov (United States)

Gregory, Carl A; Reyes, Emigdio; Whitney, Mandolin J; Spees, Jeffrey L

2006-10-01

Human mesenchymal stem cells (hMSCs), also referred to as multipotent stromal cells, are currently being applied in clinical trials for bone diseases, graft versus host disease, and myocardial infarction. However, the standard growth medium for hMSCs contains 10%-20% fetal calf serum (FCS), and FCS is strongly immunogenic in both rodents and humans. Previously, we reported that by a sensitive fluorescence-based assay, 7-30 mg of internalized FCS is associated with 10(8) hMSCs, a dosage that will probably be needed for most therapies. We also found that a brief culture in medium containing autologous 20% adult human serum (AHS) or autologous 10% AHS supplemented with growth factors (AHS(+)) reduced the contamination by more than 99.9%. We have now extensively characterized the culture conditions and shown that hMSC expansion is possible using heterologous 20% AHS or heterologous 10% AHS(+). The uptake of FCS is an active process that acts to concentrate contamination in the cells even under low serum conditions (2% FCS) but can be actively displaced by incubation of the cells in medium with AHS. Rat MSCs (rMSCs) can be expanded under similar conditions using supplemented heterologous adult rat serum (ARS(+)). After expansion in FCS, a further 8 days of culture with ARS(+) significantly improves the viability of the rMSCs in vivo after encapsulation in fibrin followed by subcutaneous implantation in rats. Our results have the potential to dramatically improve cellular and genetic therapies using hMSCs.

Generation of megakaryocytic progenitors from human embryonic stem cells in a feeder- and serum-free medium.

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Marjorie Pick

Full Text Available BACKGROUND: The production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs towards the megakaryocyte (Mk lineage using a 'spin embryoid body' method in serum-free differentiation medium. METHODOLOGY AND PRINCIPAL FINDINGS: Immunophenotypic analyses of differentiating hESC identified a subpopulation of cells expressing high levels of CD41a that expressed other markers associated with the Mk lineage, including CD110, CD42b and CD61. Differentiated cells were sorted on the basis of their expression of CD41a, CD34 and CD45 and assessed for Mk colony formation, expression of myeloid and Mk genes and ability to endoreplicate DNA. In a collagen-based colony assay, the CD41a⁺ cells sorted from these differentiation cultures produced 100-800 Mk progenitors at day 13 and 25-160 Mk progenitors at day 20 of differentiation per 100,000 cells assayed. Differentiated Mk cells produced platelet-like particles which expressed CD42b and were activated by ADP, similar to platelets generated from precursors in cord blood. These studies were complemented by real time PCR analyses showing that subsets of cells enriched for CD41a⁺ Mk precursors expressed high levels of Mk associated genes such as PF4 and MPL. Conversely, high levels of myeloid and erythroid related transcripts, such as GATA1, TAL1/SCL and PU.1, were detected in sorted fractions containing CD34⁺ and CD45⁺ cells. CONCLUSIONS: We describe a serum- and feeder-free culture system that enabled the generation of Mk progenitors from human embryonic stem cells. These cells formed colonies that included differentiated Mks that fragmented to form platelet-like particles. This protocol represents an important step towards the generation of human platelets for therapeutic use.

Utility of serum periostin and free IgE levels in evaluating responsiveness to omalizumab in patients with severe asthma.

Science.gov (United States)

Tajiri, T; Matsumoto, H; Gon, Y; Ito, R; Hashimoto, S; Izuhara, K; Suzukawa, M; Ohta, K; Ono, J; Ohta, S; Ito, I; Oguma, T; Inoue, H; Iwata, T; Kanemitsu, Y; Nagasaki, T; Niimi, A; Mishima, M

2016-10-01

Omalizumab, a humanized anti-IgE monoclonal antibody, has demonstrated efficacy in patients with severe allergic asthma. However, treatment responses vary widely among individuals. Despite a lack of data, free serum IgE levels following omalizumab treatment have been proposed as a marker of treatment responsiveness. In this prospective, observational study, we assessed the utility of biomarkers of type 2 inflammation in predicting omalizumab treatment responses, as determined by the absence of asthma exacerbation during the first year of treatment. Free serum IgE levels were monitored for 2 years to examine their association with baseline biomarker levels and the number of exacerbations. We enrolled thirty patients who had been treated with omalizumab for at least 1 year, of whom 27 were treated for 2 years. Baseline serum periostin levels and blood eosinophil counts were significantly higher in patients without exacerbations during the first year of treatment than in patients with exacerbations. Baseline serum periostin levels, but not eosinophil counts, were negatively associated with free serum IgE levels after 16 or 32 weeks of treatment. Reduced free serum IgE levels during treatment from those at baseline were associated with reduced exacerbation numbers at 2 years. In 14 patients who continued to have exacerbations during the first year of treatment, exacerbation numbers gradually and significantly decreased over the 2-year study period, with concurrent significant reductions in free serum IgE levels. Baseline serum periostin levels and serum free IgE levels during treatment follow-up may be useful in evaluating responses to omalizumab treatment. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

[Comparison of two types of cell cultures for preparation of sTNFRII-gAD fusion protein].

Science.gov (United States)

Huang, Shigao; Yin, Yuting; Xiong, Chunhui; Wang, Caihong; Lü, Jianxin; Gao, Jimin

2013-01-01

In this study we used two types of cell cultures, i.e., anchorage-dependent basket and full suspension batch cultures of sTNFRII-gAD-expressing CHO cells in the CelliGen 310 bioreactor (7.5 L) to compare their yields in order to optimize the culturing conditions for efficient expression of sTNFRII-gAD fusion protein consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin. The anchorage-dependent basket culture was performed in 4L 10% serum-containing medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 3 days, and then switched to 4 L serum-free LK021 medium to continue the culture for 4 days. The full suspension batch culture was carried out in the 4 L serum-free LK021 medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 7 days. The culturing conditions were monitored in real-time to maintain pH and dissolved oxygen stability through the whole process. The supernatants were collected by centrifuge, and the protein was concentrated through Pellicon flow ultrafiltration system and then purified by DEAE anion exchange. The results showed that the yields of sTNFRII-gAD fusion protein were 8.0 mg/L with 95% purity and 7.5 mg/L with 98% purity in the anchorage-dependent basket and the full suspension batch cultures, respectively. The study provided the framework for the pilot production of sTNFRII-gAD fusion protein.

Diverse Requirements for Microglial Survival, Specification, and Function Revealed by Defined-Medium Cultures.

Science.gov (United States)

Bohlen, Christopher J; Bennett, F Chris; Tucker, Andrew F; Collins, Hannah Y; Mulinyawe, Sara B; Barres, Ben A

2017-05-17

Microglia, the resident macrophages of the CNS, engage in various CNS-specific functions that are critical for development and health. To better study microglia and the properties that distinguish them from other tissue macrophage populations, we have optimized serum-free culture conditions to permit robust survival of highly ramified adult microglia under defined-medium conditions. We find that astrocyte-derived factors prevent microglial death ex vivo and that this activity results from three primary components, CSF-1/IL-34, TGF-β2, and cholesterol. Using microglial cultures that have never been exposed to serum, we demonstrate a dramatic and lasting change in phagocytic capacity after serum exposure. Finally, we find that mature microglia rapidly lose signature gene expression after isolation, and that this loss can be reversed by engrafting cells back into an intact CNS environment. These data indicate that the specialized gene expression profile of mature microglia requires continuous instructive signaling from the intact CNS. Copyright © 2017 Elsevier Inc. All rights reserved.

Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

International Nuclear Information System (INIS)

Walter, M.N.M.; Wright, K.T.; Fuller, H.R.; MacNeil, S.; Johnson, W.E.B.

2010-01-01

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-β1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

Mesenchymal Stromal Cells Cultured in Serum from Heart Failure Patients Are More Resistant to Simulated Chronic and Acute Stress

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Timo Z. Nazari-Shafti

2018-01-01

Full Text Available Despite regulatory issues surrounding the use of animal-derived cell culture supplements, most clinical cardiac cell therapy trials using mesenchymal stromal cells (MSCs still rely on fetal bovine serum (FBS for cell expansion before transplantation. We sought to investigate the effect of human serum from heart failure patients (HFS on cord blood MSCs (CB-MSCs during short-term culture under regular conditions and during simulated acute and chronic stress. Cell survival, proliferation, metabolic activity, and apoptosis were quantified, and gene expression profiles of selected apoptosis and cell cycle regulators were determined. Compared to FBS, HFS and serum from healthy donors (CS showed similar effects by substantially increasing cell survival during chronic and acute stress and by increasing cell yields 5 days after acute stress. Shortly after the termination of acute stress, both HFS and CS resulted in a marked decrease in apoptotic cells. Transcriptome analysis suggested a decrease in TNF-mediated induction of caspases and decreased activation of mitochondrial apoptosis. Our data confirm that human serum from both healthy donors and heart failure patients results in increased cell yields and increased resistance to cellular stress signals. Therefore, we consider autologous serum a valid alternative to FBS in cell-based therapies addressing severe heart disease.

Spinal cord neuronotrophic factors (SCNTFs): I. Bioassay of schwannoma and other conditioned media.

Science.gov (United States)

Longo, F M; Manthorpe, M; Varon, S

1982-02-01

We present a procedure for the dissociation and growth in serum-free defined culture medium of 4-day chick embryo lumbar spinal cord (LC4) neurons. LC4 neurons will not survive for even 24 h without the addition of trophic supplements (putative spinal cord neuronotrophic factors, SCNTFs). Serum-free medium conditioned over chick embryo heart and skeletal muscle, mouse Schwann and rat RN22 Schwannoma cell cultures were found to contain SCNTF activity which could be quantitated using a convenient neuronal survival bioassay. RN22 conditioned medium also contains polyornithine-binding neurite promoting factors (PNPFs) which can be physically separated from SCNTF. When SCNTF and PNPF were presented to LC4 neurons individually or in combination (i) SCNTF, but not PNPF, supported neuronal survival whereas (ii) PNPF, but not SCNTF, induced neurite production. When LC4 neurons were grown in SCNTF alone, nearly all of them exhibited a flattened, circular, 'fried-egg' morphology. The subsequent addition of PNPF caused these cells to extend long neurites with characteristic terminal growth-cone-like structures.

Serum and plasma for total and free anticonvulsant drug analyses: effects on EMIT assays and ultrafiltration devices.

Science.gov (United States)

Godolphin, W; Trepanier, J; Farrell, K

1983-01-01

The suitability of serum and plasma anticoagulated with heparin, EDTA, citrate, or oxalate was assessed for analysis of free and total phenytoin, carbamazepine, and valproic acid. The free fraction was isolated by ultrafiltration through FreeLevel devices (Syva, Palo Alto, CA). Serum, heparin, and EDTA plasma were satisfactory for both free and total phenytoin and carbamazepine. EDTA could not be used for EMIT (Syva) analysis of valproate. Citrate and, to a lesser degree, oxalate cause a significant negative interference in the concentration of these three drugs as measured both by EMIT and gas-liquid chromatography.

Determination of free amino acids of porcine serum responsible for ...

African Journals Online (AJOL)

The 1H NMR spectra of serum metabolites at 600 MHz showed that free amino acids such as alanine, leucine, phenylalanine, and valine were qualitatively higher in the HpHG than in the LpHG. The relative abundance of three amino acids was quantitatively verified by HPLC: Phenylalanine and valine (P<0.01) and leucine ...

Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design

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Lee Gyun

2010-09-01

Full Text Available Abstract Background Serum-containing medium (SCM, which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte cultures. In order to develop a serum-free medium (SFM for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs, a statistical optimization approach based on a fractional factorial method and a response surface method was adopted. A basal medium was prepared by supplementing RPMI1640 medium with insulin, albumin, ferric citrate, ethanolamine, fatty acids, glutamine, sodium pyruvate, 2-mercaptoethanol, 1-thioglycerol, nonessential amino acids, and vitamins. We identified additional positive determinants and their optimal concentrations for cell growth through a statistical analysis. Results From a statistical analysis using the fractional factorial method, cholesterol and polyamine supplement were identified as positive determinants for cell growth. Their optimal concentrations were determined by the response surface method. The maximum viable cell concentration in the developed SFM was enhanced by more than 1.5-fold when compared to that in RPMI1640 supplemented with 10% fetal bovine serum (FBS. Furthermore, a cytotoxicity assay and an enzyme-linked immunospot assay revealed that the effector function of cytotoxic T lymphocytes generated from PBMCs grown in SFM, by stimulation of peptide-presenting dendritic cells, was retained or even better than that in SCM. Conclusions The use of a developed SFM with cholesterol and polyamine supplement for human lymphocyte culture resulted in better growth without loss of cellular function when compared to SCM.

Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design.

Science.gov (United States)

Jeon, Min Kyoung; Lim, Jong-Baeck; Lee, Gyun Min

2010-09-21

Serum-containing medium (SCM), which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte cultures. In order to develop a serum-free medium (SFM) for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs), a statistical optimization approach based on a fractional factorial method and a response surface method was adopted. A basal medium was prepared by supplementing RPMI1640 medium with insulin, albumin, ferric citrate, ethanolamine, fatty acids, glutamine, sodium pyruvate, 2-mercaptoethanol, 1-thioglycerol, nonessential amino acids, and vitamins. We identified additional positive determinants and their optimal concentrations for cell growth through a statistical analysis. From a statistical analysis using the fractional factorial method, cholesterol and polyamine supplement were identified as positive determinants for cell growth. Their optimal concentrations were determined by the response surface method. The maximum viable cell concentration in the developed SFM was enhanced by more than 1.5-fold when compared to that in RPMI1640 supplemented with 10% fetal bovine serum (FBS). Furthermore, a cytotoxicity assay and an enzyme-linked immunospot assay revealed that the effector function of cytotoxic T lymphocytes generated from PBMCs grown in SFM, by stimulation of peptide-presenting dendritic cells, was retained or even better than that in SCM. The use of a developed SFM with cholesterol and polyamine supplement for human lymphocyte culture resulted in better growth without loss of cellular function when compared to SCM.

Quantification of Maternal Serum Cell-Free Fetal DNA in Early-Onset Preeclampsia

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Mulan Ren

2013-04-01

Full Text Available The aim of this study was to determine whether the increased serum cell-free fetal DNA (cffDNA level of gravidas developed into early-onset preeclampsia (EOPE subsequently in the early second trimesters is related to prenatal screening markers. Serum was collected from 1011 gravidas. The level of cffDNA and prenatal screening markers were analyzed in 20 cases with EOPE and 20 controls. All fetuses were male. The maternal serum cffDNA level was assessed by amplification of the Y chromosome specific gene. Correlations between the variables were examined. (Logged cffDNA in EOPE (median, 3.08; interquartile range, 2.93–3.68 was higher than controls (median, 1.79; interquartile range, 1.46–2.53. The increased level of (logged cffDNA was correlated significantly with the increased human chorionic gonadotropin (HCG level (r = 0.628, p < 0.001. Significant reciprocal correlations between cffDNA and babies’ birth weight as well as gestation weeks at delivery were noted (r = −0.516, p = 0.001; r = −0.623, p < 0.001, respectively. The sensitivity and specificity of cffDNA to discriminate between the EOPE cases and the controls were 90% and 85%, respectively. CffDNA is a potential marker for EOPE, which had a significant reciprocal correlation with babies’ birth weight and gestation weeks at delivery. Moreover, it may help in indicating the underlying hypoxic condition in the placenta.

Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts

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Sachchida Nand Pandey

2015-05-01

Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is believed to be caused by aberrant expression of double homeobox 4 (DUX4 due to epigenetic changes of the D4Z4 region at chromosome 4q35. Detecting DUX4 is challenging due to its stochastic expression pattern and low transcription level. In this study, we examined different cDNA synthesis strategies and the sensitivity for DUX4 detection. In addition, we investigated the effects of dexamethasone and knockout serum replacement (KOSR on DUX4 expression in culture. Our data showed that DUX4 was consistently detected in cDNA samples synthesized using Superscript III. The sensitivity of DUX4 detection was higher in the samples synthesized using oligo(dT primers compared to random hexamers. Adding dexamethasone to the culture media significantly suppressed DUX4 expression in immortalized (1.3 fold, p < 0.01 and primary (4.7 fold, p < 0.01 FSHD myoblasts, respectively. Culture medium with KOSR increased DUX4 expression and the response is concentration dependent. The findings suggest that detection strategies and culture conditions should be carefully considered when studying DUX4 in cultured cells.

Serum Levels of Follistatin Are Positively Associated With Serum-Free Thyroxine Levels in Patients With Hyperthyroidism or Euthyroidism

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Tseng, Fen-Yu; Chen, Yen-Ting; Chi, Yu-Chao; Chen, Pei-Lung; Yang, Wei-Shiung

2016-01-01

Abstract Follistatin is a glycoprotein with various biologic functions that plays a role in adipocyte differentiation, muscle stimulation, anti-inflammation, and energy homeostasis. Thyroid hormones influence energy expenditure, glucose, and lipid metabolism. The association between serum follistatin level and thyroid function statuses has seldom been evaluated. The objectives of this study were to compare serum follistatin concentrations in different thyroid function statuses and to evaluate the associations between serum follistatin and free thyroxine (fT4) levels. In this study, 30 patients with hyperthyroidism (HY group) and 30 euthyroid individuals (EU group) were recruited. The patients of HY group were treated with antithyroid regimens as clinically indicated, whereas no medication was given to EU group. The demographic and anthropometric characteristics, biochemical data, serum levels of follistatin, and thyroid function of both groups at baseline and at the 6th month were compared. Data of all patients were pooled for the analysis of the associations between the levels of follistatin and fT4. At baseline, the HY group had significantly higher serum follistatin levels than the EU group (median [Q1, Q3]: 1.81 [1.33, 2.78] vs 1.13 [0.39, 1.45] ng/mL, P hyperthyroidism had higher serum follistatin levels, which decreased after receiving antithyroid treatment. In addition, the serum follistatin concentrations were positively associated with serum fT4 levels in patients with hyperthyroidism or euthyroidism. PMID:26844494

Serum total and free carnitine levels in children with asthma.

Science.gov (United States)

Asilsoy, Suna; Bekem, Ozlem; Karaman, Ozkan; Uzuner, Nevin; Kavukçu, Salih

2009-02-01

Serum carnitine is decreased in recurrent pulmonary infections. We aimed to evaluate serum carnitine levels in asthmatic children. Study group consisted of children with stable asthma and those with acute asthma attacks, while control group included healthy children. Attack severity was determined by the pulmonary score system. Total and free carnitine levels were studied in one blood sample from the control group and stable asthmatics and in two samples from children with acute asthma exacerbation during and after the attack. All the 40 patients in the study group had moderate asthma including 30 with acute attack (13 mild and 17 moderate) and 10 with stable asthma. Carnitine levels were significantly lower in acute attack asthmatics than in the stable asthmatics and controls, while there was no significant difference between the latter two groups. Carnitine levels were not different between asthmatics with mild and moderate attack, and were similar during and after an acute attack. Serum carnitine levels decrease in children with moderate asthma during exacerbation of asthma and shortly thereafter. Further studies are needed to evaluate the effect of carnitine treatment on serum carnitine level.

Serum Iron Protects from Renal Postischemic Injury.

Science.gov (United States)

Vaugier, Céline; Amano, Mariane T; Chemouny, Jonathan M; Dussiot, Michael; Berrou, Claire; Matignon, Marie; Ben Mkaddem, Sanae; Wang, Pamella H M; Fricot, Aurélie; Maciel, Thiago T; Grapton, Damien; Mathieu, Jacques R R; Beaumont, Carole; Peraldi, Marie-Noëlle; Peyssonnaux, Carole; Mesnard, Laurent; Daugas, Eric; Vrtovsnik, François; Monteiro, Renato C; Hermine, Olivier; Ginzburg, Yelena Z; Benhamou, Marc; Camara, Niels O S; Flamant, Martin; Moura, Ivan C

2017-12-01

Renal transplants remain a medical challenge, because the parameters governing allograft outcome are incompletely identified. Here, we investigated the role of serum iron in the sterile inflammation that follows kidney ischemia-reperfusion injury. In a retrospective cohort study of renal allograft recipients ( n =169), increased baseline levels of serum ferritin reliably predicted a positive outcome for allografts, particularly in elderly patients. In mice, systemic iron overload protected against renal ischemia-reperfusion injury-associated sterile inflammation. Furthermore, chronic iron injection in mice prevented macrophage recruitment after inflammatory stimuli. Macrophages cultured in high-iron conditions had reduced responses to Toll-like receptor-2, -3, and -4 agonists, which associated with decreased reactive oxygen species production, increased nuclear localization of the NRF2 transcription factor, increased expression of the NRF2-related antioxidant response genes, and limited NF- κ B and proinflammatory signaling. In macrophage-depleted animals, the infusion of macrophages cultured in high-iron conditions did not reconstitute AKI after ischemia-reperfusion, whereas macrophages cultured in physiologic iron conditions did. These findings identify serum iron as a critical protective factor in renal allograft outcome. Increasing serum iron levels in patients may thus improve prognosis of renal transplants. Copyright © 2017 by the American Society of Nephrology.

Studies of mineralization in tissue culture: optimal conditions for cartilage calcification

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Boskey, A. L.; Stiner, D.; Doty, S. B.; Binderman, I.; Leboy, P.

1992-01-01

The optimal conditions for obtaining a calcified cartilage matrix approximating that which exists in situ were established in a differentiating chick limb bud mesenchymal cell culture system. Using cells from stage 21-24 embryos in a micro-mass culture, at an optimal density of 0.5 million cells/20 microliters spot, the deposition of small crystals of hydroxyapatite on a collagenous matrix and matrix vesicles was detected by day 21 using X-ray diffraction, FT-IR microscopy, and electron microscopy. Optimal media, containing 1.1 mM Ca, 4 mM P, 25 micrograms/ml vitamin C, 0.3 mg/ml glutamine, no Hepes buffer, and 10% fetal bovine serum, produced matrix resembling the calcifying cartilage matrix of fetal chick long bones. Interestingly, higher concentrations of fetal bovine serum had an inhibitory effect on calcification. The cartilage phenotype was confirmed based on the cellular expression of cartilage collagen and proteoglycan mRNAs, the presence of type II and type X collagen, and cartilage type proteoglycan at the light microscopic level, and the presence of chondrocytes and matrix vesicles at the EM level. The system is proposed as a model for evaluating the events in cell mediated cartilage calcification.

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model.

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Lee-Barthel, Ann; Baar, Keith; West, Daniel W D

2017-06-11

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been 'conditioned' by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National

Serum-free microcarrier based production of replication deficient Influenza vaccine candidate virus lacking NS1 using Vero cells

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Yan Mylene L

2011-08-01

Full Text Available Abstract Background Influenza virus is a major health concern that has huge impacts on the human society, and vaccination remains as one of the most effective ways to mitigate this disease. Comparing the two types of commercially available Influenza vaccine, the live attenuated virus vaccine is more cross-reactive and easier to administer than the traditional inactivated vaccines. One promising live attenuated Influenza vaccine that has completed Phase I clinical trial is deltaFLU, a deletion mutant lacking the viral Nonstructural Protein 1 (NS1 gene. As a consequence of this gene deletion, this mutant virus can only propagate effectively in cells with a deficient interferon-mediated antiviral response. To demonstrate the manufacturability of this vaccine candidate, a batch bioreactor production process using adherent Vero cells on microcarriers in commercially available animal-component free, serum-free media is described. Results Five commercially available animal-component free, serum-free media (SFM were evaluated for growth of Vero cells in agitated Cytodex 1 spinner flask microcarrier cultures. EX-CELL Vero SFM achieved the highest cell concentration of 2.6 × 10^6 cells/ml, whereas other SFM achieved about 1.2 × 10^6 cells/ml. Time points for infection between the late exponential and stationary phases of cell growth had no significant effect in the final virus titres. A virus yield of 7.6 Log10 TCID50/ml was achieved using trypsin concentration of 10 μg/ml and MOI of 0.001. The Influenza vaccine production process was scaled up to a 3 liter controlled stirred tank bioreactor to achieve a cell density of 2.7 × 10^6 cells/ml and virus titre of 8.3 Log10 TCID50/ml. Finally, the bioreactor system was tested for the production of the corresponding wild type H1N1 Influenza virus, which is conventionally used in the production of inactivated vaccine. High virus titres of up to 10 Log10 TCID50/ml were achieved. Conclusions We describe for the

Platelet lysate: a replacement for fetal bovine serum in animal cell culture?

OpenAIRE

Johansson, Liselott; Klinth, Jeanna; Holmqvist, Olov; Ohlson, Sten

2003-01-01

A new cell culture supplement, platelet lysate, was evaluated with reference to fetal bovine serum (FBS), an established industrial medium for animal cell culture. Chemical and bacteriological profiles were conducted including the presence of platelet-derived growth factor (PDGF). PDGF was detected in the platelet lysate but it was not present in FBS. The platelet lysate medium demonstrated lack of microorganisms, mycoplasma and endotoxins. The platelet lysate was investigated in culture stud...

Optimization of Pre-transplantation Conditions to Enhance the Efficacy of Mesenchymal Stem Cells

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Haque, Nazmul; Kasim, Noor Hayaty Abu; Rahman, Mohammad Tariqur

2015-01-01

Mesenchymal stem cells (MSCs) are considered a potential tool for cell based regenerative therapy due to their immunomodulatory property, differentiation potentials, trophic activity as well as large donor pool. Poor engraftment and short term survival of transplanted MSCs are recognized as major limitations which were linked to early cellular ageing, loss of chemokine markers during ex vivo expansion, and hyper-immunogenicity to xeno-contaminated MSCs. These problems can be minimized by ex vivo expansion of MSCs in hypoxic culture condition using well defined or xeno-free media i.e., media supplemented with growth factors, human serum or platelet lysate. In addition to ex vivo expansion in hypoxic culture condition using well defined media, this review article describes the potentials of transient adaptation of expanded MSCs in autologous serum supplemented medium prior to transplantation for long term regenerative benefits. Such transient adaptation in autologous serum supplemented medium may help to increase chemokine receptor expression and tissue specific differentiation of ex vivo expanded MSCs, thus would provide long term regenerative benefits. PMID:25678851

Increased serum free tryptophan in patients with diarrhea-predominant irritable bowel syndrome.

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Christmas, David M; Badawy, Abdulla A-B; Hince, Dana; Davies, Simon J C; Probert, Christopher; Creed, Tom; Smithson, John; Afzal, Muhammad; Nutt, David J; Potokar, John P

2010-10-01

Irregularities of serotonin function in irritable bowel syndrome (IBS) may be due to changes in the metabolism of the serotonin precursor l-tryptophan. Dietary alteration of tryptophan intake may impact upon the mood and bowel symptoms of IBS. We hypothesized that diarrhea-predominant irritable bowel syndrome (d-IBS) patients would exhibit an increase in plasma tryptophan due to alterations in tryptophan metabolism. We also hypothesized that a diet low in tryptophan would reverse this change and reduce symptoms. Thirteen patients with d-IBS had fasting serum free and total tryptophan, large neutral amino acids, and 6 kynurenine metabolites measured before and after 2 weeks of a strict dairy-free diet. Baseline tryptophan parameters were compared with an age- and sex-matched control group. Changes in the specific tryptophan parameters before and after dairy-free diet were correlated with symptoms of IBS and mood. Compared with the control group, d-IBS patients at baseline exhibited significantly higher free serum tryptophan (10.5 ± 4.35 vs 4.75 ± 2.43 μmol/L [means ± standard deviation], P = .006) and significantly lower tryptophan dioxygenase and total tryptophan oxidation as measured by the kynurenine to free tryptophan and total kynurenines to free tryptophan ratios (23.37 ± 10.12 vs 55.33 ± 16.02, P < .001 and 49.34 ± 17.84 vs 258.46 ± 98.67, P < .001, respectively). Dairy-free diet did not modulate metabolites of the kynurenine pathway or symptoms. Tryptophan metabolism along the kynurenine pathway is inhibited in d-IBS, and a dairy-free diet does not alter this. Our findings are consistent with possible enhanced serotonin activity in d-IBS. Copyright © 2010 Elsevier Inc. All rights reserved.

Is the color trails culture free?

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Fasfous, Ahmed F; Puente, Antonio E; Pérez-Marfil, María Nieves; Cruz-Quintana, Francisco; Peralta-Ramirez, Isabel; Pérez-García, Miguel

2013-11-01

Increasingly clinical neuropsychology has been addressing the effects of culture on neuropsychological functioning. However, that focus has been on comparing performance on standardized tests across two or more groups, often Hispanic. In this study, Arabic children were tested in Morocco using a "culture-free test," Children's Color Trails. Children of different ages and living in rural and urban centers were tested. The results suggest that the Color Trails Test scores from Arab children differed from U.S. norms available. Furthermore, the location of testing and the age of the child were of significance. The role of culture-specific tests was considered.

A solid phase radioimmunoassay for free triiodothyronine in serum: assay development and validation

International Nuclear Information System (INIS)

Liu Fangyan; Zhou Meiying; Yin Linxiang; Yang Jianzhong; Hua Chenglin

1999-01-01

A solid phase radioimmunoassay for free triiodothyronine in serum was developed based on double-antibody coated tubes. The method was turned out to be reliable with good reproducibility, higher sensitivity and easy performance. The measurable range of FT 3 in serum was 1.2 to 38 pmol/L. The mean coefficients of variation within and between assays were 1.79%∼3.18% and 4.72%∼9.31%, respectively. The FT 3 concentrations in euthyroid serum as determined by this method were 2.8 to 7.8 pmol/L. The FT 3 values determined by this new method correlated well with those measured by a commercial radioimmunoassay (r = 0.853)

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

International Nuclear Information System (INIS)

Vasconcelos, R.B.; Salles, L.P.; Silva, I. Oliveira e; Gulart, L.V.M.; Souza, D.K.; Torres, F.A.G.; Bocca, A.L.; Silva, A.A.M. Rosa e

2013-01-01

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E 2 ) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P 4 ) and E 2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P 4 throughout the culture period; however, P 4 concentration was significantly higher in NDM. In both media, E 2 concentration was increased at 24 h, followed by a decrease at 48 h. The E 2 :P 4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E 2 :P 4 ratio in FWS cultures

Attachment and growth of human keratinocytes in a serum-free environment.

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Gilchrest, B A; Calhoun, J K; Maciag, T

1982-08-01

Using a serum-free system, we have investigated the influence of human fibronectin (HFN) and selected growth factors (GF) on the attachment and growth of normal human keratinocytes in vitro. Single-cell suspensions of keratinocytes from near-confluent primary plates, plated on 5-10 microgram/cm2 HFN, showed approximately 30-40% attachment after 2-24 hours of incubation at 37 degrees C, compared with 4-6% attachment on uncoated platic plates. Percentage of attached cells was independent of seed density, tissue donor age, in vitro culture age, or medium composition, while subsequent cellular proliferation was strongly dependent on these factors. Keratinocytes grown on an adequate HFN matrix in a previously described hormone-supplemented medium (Maciag et al., 1981a) achieved four to eight population doubling over 7-12 days at densities greater than or equal to 104 cell/cm2. Removal of most GF individually from the medium had little or no effect on growth, while removal of epidermal growth factor (EGF) alone reduced growth by 30-35% and removal of bovine brain extract (BE) alone reduced growth by approximately 90%. Conversely, EGF alone in basal medium supported approximately 10% control growth, BE alone supported 30-40% control growth, and the combination of EGF and BE approximately 70%. In addition to its major effect on proliferation in this system, BE was necessary to preserve normal keratinocyte morphology and protein production. These findings expand earlier observations that HFN facilitates keratinocyte attachment in vitro and that a brain-derived extract can exert a major positive influence on cultured keratinocytes.

Regeneration efficiency based on genotype, culture condition and growth regulators of eggplant (Solanum melongena L.

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Md Abdul Muktadir

2016-01-01

Full Text Available Several experiments were carried out to establish an efficient regenerating protocol for cultivated eggplant varieties. Among the five varieties cultured on Murashige and Skoog (MS medium with free plant growth regulator (PGR, Nayantara performed better considering the number of shoots/explant (2.48. Considering explant types and culture conditions, better performance was observed (3.68 shoots/explant when seed germination in the dark was proceeded by bottom hypocotyl segments cultured under dark conditions. A higher rate of shoot regeneration was observed in Nayantara when cultured in Zeatin Riboside (ZR and Thidizuron (TDZ supplemented MS medium. The highest number of shoots per explant was produced on MS medium supplemented with 2.0 mg/L ZR and 0.1 mg/L indole acetic acid (6.65 shoots/explant. Proliferation and elongation of the regenerated shoots were obtained in the MS medium with free PGR. The best rooting performance was observed in MS medium supplemented with 2.0 mg/L indole butyric acid. Plantlets with well developed roots and shoots were successfully transferred to soil.

Quantification of circulating cell-free DNA in the serum of patients with obstructive sleep apnea-hypopnea syndrome.

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Ye, Liang; Ma, Guan-Hua; Chen, Ling; Li, Min; Liu, Jia-Lin; Yang, Kun; Li, Qing-Yun; Li, Ning; Wan, Huan-Ying

2010-12-01

Serum cell-free DNA concentrations have been reported to increase in many acute diseases as well as in some chronic conditions such as cancer and autoimmune diseases. The aim of this study was to examine whether serum DNA concentrations were elevated in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS). The effects of nasal continuous positive airway pressure (nCPAP) on serum DNA were also investigated. One hundred twenty-seven people diagnosed with OSAHS by polysomnography (PSG) were admitted into the OSAHS group, and 52 subjects without OSAHS were recruited for the control group. The OSAHS group was further divided into mild, moderate, and severe OSAHS subgroups based on their apnea-hypopnea index (AHI) during sleep. Ten patients with moderate and severe OSAHS were treated with nCPAP. Serum DNA, interleukin-6 (IL-6), and malonaldehyde (MDA) concentrations were measured and were found to be significantly higher in patients with moderate and severe OSAHS groups than those in the mild OSAHS and control groups (p DNA correlated positively with AHI, oxygen desaturation index (ODI), IL-6, and MDA, and negatively correlated with minimal oxygen saturation (miniSaO(2)) (all p DNA concentrations. After 6 months of nCPAP therapy, serum concentrations of DNA, IL-6, and MDA were significantly decreased (p DNA in patients with OSAHS was positively correlated with disease severity. Serum DNA may become an important parameter for monitoring the severity of OSAHS and effectiveness of therapy.

Changes in serum concentrations of total and free testosterone in young and middle-aged men

International Nuclear Information System (INIS)

Wang Guohong; Xu Ruiji; Zhang Zhongshu

2011-01-01

To determine changes in serum concentrations of total and free testosterone in young and middle-aged men.the healthy men (n=126) were divided 20-29 yr, 30-39 yr and 40-49 yr three groups,their serum levels of total testosterone (T), free testosterone (FT), luteinizing hormone (LH), follicle-stimulating hormone (FSH), sex hormone binding globulin (SHBG) and estradiol (E2) were detected. The results were statically analyzed. The results showed that the levels of serum T and FT was found significantly decreased in 30-39 yr group than in 20-29 yr group (15.06±13 nmol/L vs 20.41±86 nmol/L, P<0.01; 9.36±95 pg/L vs 11.48±88 pg/L, P<0.05; respectively). There were young trends that age-related decline in androgen levels. (authors)

Analysis of relationship between tumor markers and quantification of free DNA in serum of lung cancer patients

International Nuclear Information System (INIS)

Yang Shunfang; Zhang Peiling; Cao Jie; Zeng Jun; Dong Qianggang

2006-01-01

To evaluate the diagnostic value and relationship between five tumor markers (CA19- 9,CA125,CYFRA21-1 ,CEA,NSE) and free DNA in serum for lung cancer detection and try to find a new and more efficient tumor marker, the amounts of CA19-9, CA125, CYFRA21-1, CEA, NSE were determined by RIA and free DNA was determined by the use of quantitative real time PCR amplification of the human epidermal growth factor receptor (EGFR) in 52 lung cancer patients and 8 cases of benign pulmonary disease and 10 healthy controls. The resulls showed that average concentration of free DNA in serum of lung cancer patients, benign pulmo- nary disease and healthy controls was 107.6ng/mL, 76.86ng/mL and 18.8ng/mL, respective- ly. The diagnostic sensitivity, specificity and accuracy of free DNA for lung cancer were 71. 2%, 50% and 68.3%, same as the diagnostic value of combined detection of five tumor markers. The sensitivity, specificity and accuracy of the five tumor markers and free DNA combinend detection for lung cancer were 94.2%, 25% and 85%, respectively. The free DNA in the serum of lung cancer patients may be a new and better tumor marker. (authors)

Bone Marrow Mesenchymal Stem Cells Enhance the Differentiation of Human Switched Memory B Lymphocytes into Plasma Cells in Serum-Free Medium

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Guillaume Bonnaure

2016-01-01

Full Text Available The differentiation of human B lymphocytes into plasma cells is one of the most stirring questions with regard to adaptive immunity. However, the terminal differentiation and survival of plasma cells are still topics with much to be discovered, especially when targeting switched memory B lymphocytes. Plasma cells can migrate to the bone marrow in response to a CXCL12 gradient and survive for several years while secreting antibodies. In this study, we aimed to get closer to niches favoring plasma cell survival. We tested low oxygen concentrations and coculture with mesenchymal stem cells (MSC from human bone marrow. Besides, all cultures were performed using an animal protein-free medium. Overall, our model enables the generation of high proportions of CD38+CD138+CD31+ plasma cells (≥50% when CD40-activated switched memory B lymphocytes were cultured in direct contact with mesenchymal stem cells. In these cultures, the secretion of CXCL12 and TGF-β, usually found in the bone marrow, was linked to the presence of MSC. The level of oxygen appeared less impactful than the contact with MSC. This study shows for the first time that expanded switched memory B lymphocytes can be differentiated into plasma cells using exclusively a serum-free medium.

Treatment of serum with supernatants from cultures of Candida albicans reduces its serum-dependent phagocytosis Tratamento de soro com sobrenadante de cultura de Candida albicans reduz a fagocitose soro-dependente

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Aderbal Antonio dos Santos

2002-01-01

Full Text Available Candida albicans is a potent activator of the complement system, and heat labile opsonins produced by activation of C3 (C3b and iC3b enhance phagocytosis of C. albicans mediated by complement receptors. In this study we treated mouse serum with supernatants from cultures of a protease producer strain of C. albicans and evaluated the ability of this serum to enhance phagocytosis of C. albicans. Cell-free supernatants from cultures of C. albicans were concentrated 5 fold and added to mouse serum for 30 min at 37ºC, before using this serum for opsonization of glutaraldehyde-fixed yeast cells. We observed that normal mouse serum increased about 3 fold the phagocytosis of C. albicans by mice peritoneal macrophages, whereas supernatant-treated serum did not increase phagocytosis. This effect of supernatants on serum was prevented by addition of pepstatin (5 µg/ ml; an inhibitor of C. albicans acid proteases to the medium. Serum treated with supernatants from cultures of a protease-deficient mutant of C. albicans also increased about 3 fold phagocytosis of the yeast. These results suggest that a protease produced by C. albicans causes proteolysis of serum opsonins, thereby reducing the phagocytosis of the yeast.Candida albicans é um potente ativador do sistema complemento, e opsoninas lábeis ao calor produzidas por ativação de C3 (C3b e iC3b aumentam a fagocitose de C. albicans mediada por receptores de complemento. Neste estudo, tratamos o soro de camundongo com sobrenadante de culturas de uma cepa de C. albicans produtora de proteases e avaliamos a capacidade deste soro reduzir a fagocitose de C. albicans. Sobrenadantes livres de células obtidos de cultura de C. albicans foram concentrados 5 vezes e adicionados ao soro de camundongo por 30 minutos a 37ºC, antes deste soro ser usado para opsonização de C. albicans na forma de levedura e fixadas em glutaraldeido. Nós observamos que soro normal aumentou 3 vezes a fagocitose de C. albicans por

Embryogenesis-promoting factors in rat serum.

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Katoh, M; Kimura, R; Shoji, R

1998-06-15

Regarding whole rat embryo cultures in vitro, rat serum as a culture medium is known to support the normal growth of rat embryos in the organogenesis phase. The purpose of the present study was to isolate the embryogenesis-promoting factors from rat serum as a first step in the development of a defined serum-free medium for a whole embryo culture system. Pooled rat serum after heat inactivation was fractionated into three major peaks (frA, containing a region of void volume, frB, and frC) by gel filtration. The 9.5-day rat embryos that were cultivated for 48 hr in essential salt medium containing frB (with a molecular size range of 100-500 kDa) revealed normal growth. Three proteins (27 kDa, 76 kDa, and 190 kDa) that had the embryogenesis-promoting effects were isolated from 3-hr delayed centrifuged rat serum by the ion exchange chromatography. The 76-kDa protein was found to be rat transferrin by immunoblotting. The 27-kDa protein was identified as apo-AI (the major apoprotein of high-density lipoprotein) by immunoblotting. High-density lipoprotein obtained from pooled rat serum by a NaBr density gradient ultracentrifugation was found to have a positive effect on embryogenesis. The 10-kDa protein was also identified as alpha 1-inhibitor 3 by immunoblotting. In addition, the embryogenesis-promoting effect of the fraction containing 27-kDa and 190-kDa proteins declined within a short period of storage at -20 degrees C. This decrease was countered by supplementing its fraction (D-2) with albumin isolated from rat serum. These results in the present study suggest that transferrin, high-density lipoprotein, and alpha 1-inhibitor 3 in rat serum may be embryogenesis-promoting factors, and that albumin appeared to play a role in the embryogenesis of rat embryos in whole embryo cultures.

Hematologic and serum chemistry reference intervals for free-ranging lions (Panthera leo).

Science.gov (United States)

Maas, Miriam; Keet, Dewald F; Nielen, Mirjam

2013-08-01

Hematologic and serum chemistry values are used by veterinarians and wildlife researchers to assess health status and to identify abnormally high or low levels of a particular blood parameter in a target species. For free-ranging lions (Panthera leo) information about these values is scarce. In this study 7 hematologic and 11 serum biochemistry values were evaluated from 485 lions from the Kruger National Park, South Africa. Significant differences between sexes and sub-adult (≤ 36 months) and adult (>36 months) lions were found for most of the blood parameters and separate reference intervals were made for those values. The obtained reference intervals include the means of the various blood parameter values measured in captive lions, except for alkaline phosphatase in the subadult group. These reference intervals can be utilized for free-ranging lions, and may likely also be used as reference intervals for captive lions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Implications of a Culturally Evolved Self for Notions of Free Will

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Lloyd Hawkeye Robertson

2017-10-01

Full Text Available Most schools in psychology have emphasized individual choice despite evidence of genetic and cultural determinism. It is suggested in this paper that the rejection of classical behaviorism by psychology and other humanities flowed from deeply held cultural assumptions about volition and free will. While compatibilists have suggested that notions of free will and determinism are not mutually exclusive, the psychological mechanisms by which such an accommodation could be explained have been inadequately explored. Drawing on research into classical cultures, this paper builds an argument that the notion of free will was adaptive flowing from culturally evolved changes to the self, and that this “evolved self,” containing assumptions of personal volition, continuity, and reason, became benchmarks of what it means to be human. The paper proposes a model of a culturally evolved self that is compatible with understandings of free will and determinism. Implications for therapeutic practice and future research are discussed.

Effects of cultured shrimp (Litopenaeus vannamei consumption on serum lipoproteins of healthy normolipidemic men

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Farzaneh Yousefi

2012-12-01

Full Text Available Background: It has been suggested that moderate shrimp consumption in normolipidemic subjects will not adversely affect the overall lipoprotein profile. Hence, shrimp consumption can be included in “healthy heart" nutritional guidelines. However, the effects of cultured shrimp on serum lipoproteins of normal subjects have not yet investigated. Material and Methods: Twenty-five healthy normolipidemic men who were workers of a shrimp farm in Bushehr province participated in a quasi-experimental study. In a crossover six weeks trial, the effect of three days per week diet (containing 300 g cultured shrimp Litopenaeus vannamei /day on serum lipid profile was compared with a zero-marine baseline diet. Results: After six weeks trial, serum triglyceride and HDL-cholesterol levels were not significantly changed from the baseline levels (p>0.05. However, total cholesterol and LDL-cholesterol levels, total cholesterol to HDL-cholesterol and LDL-cholesterol to HDL-cholesterol ratios were significantly increased (p<0.0001. Conclusion: Moderate cultured shrimp (Litopenaeus vannamei consumption can increase total cholesterol and LDL-cholesterol levels in normolipidemic men. Although a diet containing native shrimp has many benefits for healthy persons, but we do not recommend cultured shrimp in a healthy heart diet for persons with dyslipidemia or cardiovascular diseases.

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

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Naskou, Maria C; Sumner, Scarlett M; Chocallo, Anna; Kemelmakher, Hannah; Thoresen, Merrilee; Copland, Ian; Galipeau, Jacques; Peroni, John F

2018-03-22

Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed

Induction of calcification by serum depletion in cell culture: a model for focal calcification in aortas related to atherosclerosis

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Villar Maria T

2008-01-01

Full Text Available Abstract Background Since aortic calcification has been shown to initiate in the lower zone of well-thickened plaques (LZP adjacent to the aortic media of rabbits fed supplemental cholesterol diets, a restricted supply of serum to vascular cells could play a role in vascular calcification. This study was designed to use a cell culture model to support this hypothesis. Results Rabbit aortic smooth muscle cells were grown to confluence in a culture media containing 10 % fetal bovine serum (FBS. The confluent cells were then exposed to the media for 2 hrs with or without serum at a Ca × P ion product range of 4.5–9.4 mM2. In contrast to the cells cultured in the presence of FBS, confluent cells in its absence displayed marked mineral-positive alizarin red staining and infrared absorption of mineral phosphate. A kinetic parameter C1/2 was used to designate the concentration of serum or its protein constituents needed to reduce the deposition of Ca and P by half. The C1/2 for FBS and rabbit serum was 0.04–0.07 % The C1/2 value for rabbit serum proteins was 13.5 μg/ml corresponding to the protein concentration in 0.06 % of serum. This C1/2 was markedly smaller than 86.2 μg/ml for bovine serum albumin present in 0.37 % serum (p Conclusion The aortic smooth muscle cell culture model suggests that serum depletion may play a role in the initiation of aortic calcification. The serum exhibits remarkable ability to inhibit cell-mediated calcification.

Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies.

Science.gov (United States)

Yang, R; Elankumaran, Y; Hijjawi, N; Ryan, U

2015-06-01

A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages. Copyright © 2015 Elsevier Inc. All rights reserved.

Osteoarthritis treatment using autologous conditioned serum after placebo

NARCIS (Netherlands)

Rutgers, Marijn; Creemers, Laura B; Auw Yang, Kiem Gie; Raijmakers, Natasja J H; Dhert, Wouter J A; Saris, Daniel B F

BACKGROUND AND PURPOSE: Autologous conditioned serum (ACS) is a disease-modifying drug for treatment of knee osteoarthritis, and modest superiority over placebo was reported in an earlier randomized controlled trial (RCT). We hypothesized that when given the opportunity, placebo-treated patients

Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium.

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Alan Yung-Chih Hu

Full Text Available BACKGROUND: Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK cells grown in a serum-free (SF medium microcarrier cell culture system. PRINCIPAL FINDING: The current study has evaluated the performance of cell adaptation switched from serum-containing (SC medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3 × 10(6 cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA units/50 µL and 7.1 ± 0.3 × 10(8 pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera. CONCLUSIONS: The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.

An improved method for the radioimmunoassay of free-thyroxine in serum dialysates

International Nuclear Information System (INIS)

Giles, A.F.

1982-01-01

A convenient, sensitive radioimmunoassay (using 125 I) of free thyroxine in serum dialysates is described. The method utilizes a solid phase separation system (pre-formed double antibody) and a relatively short incubation period (220 min) with a staggered addition of tracer. Blanks were low and consistent. The normal range for non-pregnant euthyroid samples (n = 59) was 11-23 pmol/l. Third trimester pregnancy samples were mostly within the normal range but at the lower end. Patients on T4 replacement showed a much wider variation in free T4 levels with many samples in the hyperthyroid region. Some hypothyroid samples had undetectable free T4 levels and hyperthyroid samples were frequently greater than 80 pmol/l. (author)

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

Energy Technology Data Exchange (ETDEWEB)

Vasconcelos, R.B. [Laboratório de Biotecnologia da Reprodução, Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil); Salles, L.P. [Laboratório de Biologia Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil); Silva, I. Oliveira e; Gulart, L.V.M. [Laboratório de Biotecnologia da Reprodução, Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil); Souza, D.K. [Laboratório de Biotecnologia da Reprodução, Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil); Faculdade de Ceilândia, Universidade de Brasília, Ceilândia, DF (Brazil); Torres, F.A.G. [Laboratório de Biologia Molecular, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil); Bocca, A.L. [Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil); Silva, A.A.M. Rosa e [Laboratório de Biotecnologia da Reprodução, Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF (Brazil)

2013-08-16

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E{sub 2}) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P{sub 4}) and E{sub 2} concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P{sub 4} throughout the culture period; however, P{sub 4} concentration was significantly higher in NDM. In both media, E{sub 2} concentration was increased at 24 h, followed by a decrease at 48 h. The E{sub 2}:P{sub 4} ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E{sub 2}:P{sub 4} ratio in FWS cultures.

Critical evaluation of gamma-irradiated serum used as feeder in the culture and demonstration of putative nanobacteria and calcifying nanoparticles.

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Jan Martel

Full Text Available The culture and demonstration of putative nanobacteria (NB and calcifying nanoparticles (CNP from human and animal tissues has relied primarily on the use of a culture supplement consisting of FBS that had been gamma-irradiated at a dose of 30 kGy (gamma-FBS. The use of gamma-FBS is based on the assumption that this sterilized fluid has been rid entirely of any residual NB/CNP, while it continues to promote the slow growth in culture of NB/CNP from human/animal tissues. We show here that gamma-irradiation (5-50 kGy produces extensive dose-dependent serum protein breakdown as demonstrated through UV and visible light spectrophotometry, fluorometry, Fourier-transformed infrared spectroscopy, and gel electrophoresis. Yet, both gamma-FBS and gamma-irradiated human serum (gamma-HS produce NB/CNP in cell culture conditions that are morphologically and chemically indistinguishable from their normal serum counterparts. Contrary to earlier claims, gamma-FBS does not enhance the formation of NB/CNP from several human body fluids (saliva, urine, ascites, and synovial fluid tested. In the presence of additional precipitating ions, both gamma-irradiated serum (FBS and HS and gamma-irradiated proteins (albumin and fetuin-A retain the inherent dual NB inhibitory and seeding capabilities seen also with their untreated counterparts. By gel electrophoresis, the particles formed from both gamma-FBS and gamma-HS are seen to have assimilated into their scaffold the same smeared protein profiles found in the gamma-irradiated sera. However, their protein compositions as identified by proteomics are virtually identical to those seen with particles formed from untreated serum. Moreover, particles derived from human fluids and cultured in the presence of gamma-FBS contain proteins derived from both gamma-FBS and the human fluid under investigation-a confusing and unprecedented scenario indicating that these particles harbor proteins from both the host tissue and the FBS

Effects of serum on cytotoxicity of nano- and micro-sized ZnO particles

Energy Technology Data Exchange (ETDEWEB)

Hsiao, I-Lun; Huang, Yuh-Jeen, E-mail: yjhuang@mx.nthu.edu.tw [National Tsing Hua University, Department of Biomedical Engineering and Environmental Sciences (China)

2013-09-15

Although an increasing number of in vitro studies are being published regarding the cytotoxicity of nanomaterials, the components of the media for toxicity assays have often varied according to the needs of the scientists. Our aim for this study was to evaluate the influence of serum-in this case, fetal bovine serum-in a cell culture medium on the toxicity of nano-sized (50-70 nm) and micro-sized (<1 {mu}m) ZnO on human lung epithelial cells (A549). The nano- and micro-sized ZnO both exhibited their highest toxicity when exposed to serum-free media, in contrast to exposure in media containing 5 or 10 % serum. This mainly comes not only from the fact that ZnO particles in the serum-free media have a higher dosage-per-cell ratio, which results from large aggregates of particles, rapid sedimentation, absence of protein protection, and lower cell growth rate, but also that extracellular Zn{sup 2+} release contributes to cytotoxicity. Although more extracellular Zn{sup 2+} release was observed in serum-containing media, it did not contribute to nano-ZnO cytotoxicity. Furthermore, non-dissolved particles underwent size-dependent particle agglomeration, resulting in size-dependent toxicity in both serum-containing and serum-free media. A low correlation between cytotoxicity and inflammation endpoints in the serum-free medium suggested that some signaling pathways were changed or induced. Since cell growth, transcription behavior for protein production, and physicochemical properties of ZnO particles all were altered in serum-free media, we recommend the use of a serum-containing medium when evaluating the cytotoxicity of NPs.

A feeder-free culture using autogeneic conditioned medium for undifferentiated growth of human embryonic stem cells: Comparative expression profiles of mRNAs, microRNAs and proteins among different feeders and conditioned media

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Chou Chi-Hsien

2010-10-01

Full Text Available Abstract Background Human embryonic stem (hES cell lines were derived from the inner cell mass of human blastocysts, and were cultured on mouse embryonic fibroblast (MEF feeder to maintain undifferentiated growth, extensive renewal capacity, and pluripotency. The hES-T3 cell line with normal female karyotype was previously used to differentiate into autogeneic fibroblast-like cells (T3HDF as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF for 14 passages. Results A feeder-free culture on Matrigel in hES medium conditioned by the autogeneic feeder cells (T3HDF was established to maintain the undifferentiated growth of hES-T3 cells (T3/CMHDF for 8 passages in this investigation. The gene expression profiles of mRNAs, microRNAs and proteins between the undifferentiated T3/HDF and T3/CMHDF cells were shown to be very similar, and their expression profiles were also found to be similar to those of T3/MEF and T3/CMMEF cells grown on MEF feeder and feeder-free Matrigel in MEF-conditioned medium, respectively. The undifferentiated state of T3/HDF and T3/CMHDF as well as T3/MEF andT3/CMMEF cells was evidenced by the very high expression levels of "stemness" genes and low expression levels of differentiation markers of ectoderm, mesoderm and endoderm in addition to the strong staining of OCT4 and NANOG. Conclusion The T3HDF feeder and T3HDF-conditioned medium were able to support the undifferentiated growth of hES cells, and they would be useful for drug development and toxicity testing in addition to the reduced risks of xenogeneic pathogens when used for medical applications such as cell therapies.

Comparative Serum Fatty Acid Profiles of Captive and Free-Ranging Cheetahs (Acinonyx jubatus) in Namibia.

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Tordiffe, Adrian S W; Wachter, Bettina; Heinrich, Sonja K; Reyers, Fred; Mienie, Lodewyk J

2016-01-01

Cheetahs (Acinonyx jubatus) are highly specialised large felids, currently listed as vulnerable on the IUCN red data list. In captivity, they are known to suffer from a range of chronic non-infectious diseases. Although low heterozygosity and the stress of captivity have been suggested as possible causal factors, recent studies have started to focus on the contribution of potential dietary factors in the pathogenesis of these diseases. Fatty acids are an important component of the diet, not only providing a source of metabolisable energy, but serving other important functions in hormone production, cellular signalling as well as providing structural components in biological membranes. To develop a better understanding of lipid metabolism in cheetahs, we compared the total serum fatty acid profiles of 35 captive cheetahs to those of 43 free-ranging individuals in Namibia using gas chromatography-mass spectrometry. The unsaturated fatty acid concentrations differed most remarkably between the groups, with all of the polyunsaturated and monounsaturated fatty acids, except arachidonic acid and hypogeic acid, detected at significantly lower concentrations in the serum of the free-ranging animals. The influence of age and sex on the individual fatty acid concentrations was less notable. This study represents the first evaluation of the serum fatty acids of free-ranging cheetahs, providing critical information on the normal fatty acid profiles of free-living, healthy individuals of this species. The results raise several important questions about the potential impact of dietary fatty acid composition on the health of cheetahs in captivity.

3D+time acquisitions of 3D cell culture by means of lens-free tomographic microscopy

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Berdeu, Anthony; Laperrousaz, Bastien; Bordy, Thomas; Morales, S.; Gidrol, Xavier; Picollet-D'hahan, Nathalie; Allier, Cédric

2018-02-01

We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multi-angle acquisitions on 3D cell cultures embedded in extracellular matrix (ECM). We developed algorithms based on the Fourier diffraction theorem to perform fully 3D reconstructions of biological samples and we adapted the lens-free microscope to incubator conditions. Here we demonstrate for the first time, 3D+time lens-free acquisitions of 3D cell culture over 8 days directly into the incubator. The 3D reconstructed volume is as large as 5 mm3 and provides a unique way to observe in the same 3D cell culture experiment multiple cell migration strategies. Namely, in a 3D cell culture of prostate epithelial cells embedded within a Matrigel® matrix, we are able to distinguish single cell 'leaders', migration of cell clusters, migration of large aggregates of cells, and also close-gap and large-scale branching. In addition, we observe long-scale 3D deformations of the ECM that modify the geometry of the 3D cell culture. Interestingly, we also observed the opposite, i.e. we found that large aggregates of cells may deform the ECM by generating traction forces over very long distances. In sum we put forward a novel 3D lens-free microscopy tomographic technique to study the single and collective cell migrations, the cell-to-cell interactions and the cell-to-matrix interactions.

Serum lutein response is greater from free lutein than from esterified lutein during 4 weeks of supplementation in healthy adults.

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Norkus, Edward P; Norkus, Katherine L; Dharmarajan, T S; Schierle, Joseph; Schalch, Wolfgang

2010-12-01

Current data suggest great variability in serum response following lutein ingestion from various sources. To compare the relative serum response during supplementation with free lutein (fL) and lutein esters (Le). 72 volunteers (23-52 years; body mass index [BMI] >20 and lutein lutein or 27 mg of lutein ester (equivalent to 13.5 mg free lutein), respectively. Fasting blood was obtained at baseline and after 7, 14, 21, and 28 days of supplementation. Supplements were consumed with standard portions of dry, ready-to-eat cereal and 2% cow's milk. Absolute changes in serum lutein, per mg daily dose, were significantly greater in fL vs. Le after 21 days (p  =  0.0012) and remained so after 28 days (p  =  0.0011) of supplementation. Serum lutein Area Under the Curve [AUC((day 0-28))] response was 17% greater for fL vs. Le (p  =  0.0187). Regression models were used and determined that (1) baseline serum lutein levels and (2) the form of lutein ingested (fL > Le) influence the serum lutein response during supplementation, while subject age, gender, BMI, and serum lipids do not affect serum response. These results suggest that the relative serum lutein response will be significantly greater from supplements containing free lutein than from supplements containing lutein esters. These findings should be useful for future clinical trials exploring the effectiveness of lutein supplementation in the prevention of or protection against age-related macular degeneration and/or cataracts.

Physicochemical characterization of engineered nanoparticles under physiological conditions: effect of culture media components and particle surface coating.

Science.gov (United States)

Fatisson, Julien; Quevedo, Ivan R; Wilkinson, Kevin J; Tufenkji, Nathalie

2012-03-01

The use of engineered nanoparticles (ENPs) in commercial products has increased substantially over the last few years. Some research has been conducted in order to determine whether or not such materials are cytotoxic, but questions remain regarding the role that physiological media and sera constituents play in ENP aggregation or stabilization. In this study, several characterization methods were used to evaluate the particle size and surface potential of 6 ENPs suspended in a number of culture media and in the presence of different culture media constituents. Dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS) were employed for size determinations. Results were interpreted on the basis of ENP surface potentials evaluated from particle electrophoretic mobilities (EPM). Measurements made after 24h of incubation at 37°C showed that the cell culture medium constituents had only moderate impact on the physicochemical properties of the ENP, although incubation in bovine serum albumin destabilized the colloidal system. In contrast, most of the serum proteins increased colloidal stabilization. Moreover, the type of ENP surface modification played a significant role in ENP behavior whereby the complexity of interactions between the ENPs and the medium components generally decreased with increasing complexity of the particle surface. This investigation emphasizes the importance of ENP characterization under conditions that are representative of cell culture media or physiological conditions for improved assessments of nanoparticle cytotoxicity. Copyright © 2011 Elsevier B.V. All rights reserved.

STUDIES ON THE EFFECT OF VARIOUS STERILANTS AND CULTURE CONDITIONS ON IN-VITRO SEED GERMINATION IN TOMATO (SOLANUM LYCOPERSICUM)

OpenAIRE

K.B.Himabindu; M.Shanthi Priya; D.Mohan Reddy; P.Sudhakar; Y.Srinivasulu; M.Reddisekhar; P.Latha; B.Rupesh Kumar Reddy

2012-01-01

Studies on the effectiveness of various sterilants and culture conditions on in-vitro seed germination in tomato (Solanum lycopersicum L.) cv. PKM-1 revealed that among three sterilants used, surface sterilization of seeds with 5 % NaOCl for 20 minutes was found to be more effective resulting in high germination rate and contamination free cultures. Similarly among the different media and culture conditions considered in the present experiment, MS medium without sucrose with dark incubation f...

[Diagnostic significance of serum free DNA human telomerase reverse transcriptase quantitative determination on spinal cord injury].

Science.gov (United States)

Yang, M K; Tang, J; Xiang, Z; Zhang, X; Wang, J; Li, Z; Li, Y; Sheng, W B

2018-02-06

Objective: To investigate the relationship between the content of human telomerase reverse transcriptase (hTERT) and its clinical features in serum free DNA in patients with different degree of spinal cord injury. Methods: From December 2013 to December 2016, inpatients of the Central Hospital of Bazhong, Sichuan Province were enrolledand divided into the experimental group, the disease control group and the negative control group. For the experimental group: 46 patients with spinal cord injury were graded according to the criteria of the American Association of Spinal Cord Injury (ASIA), including 12 cases of grade A, 10 cases of grade B, 10 cases of grade C, 7 cases of grade D and 7 cases of grade E; for the disease control group: 15 patients with spinal fractures (without spinal cord injury) at the same period were included; and for the negative control group: 20 healthy adult volunteers aged 18-50 years were selected.Real-time fluorescence quantitative PCR and immunoblotting were performed to detect the content of hTERT in serum free DNA both in patients and healthy controls and to compare the difference between them. The results of the somatosensory evoked potential (SEP) of all patients were compared and analyzed.The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of hTERT content in serum free DNA in patients with spinal cord injury. Results: Comparison of serum free DNA hTERT content: in the experimental group, the serum free DNA hTERT content of grade A, B, C, D, E was (99.63±8.23), (76.24±4.37), (46.07±5.43), (16.30±0.95) and (15.74±1.12)μg/L, respectively.While it was (15.01±1.39)μg/L in the disease control group and (14.54±1.03)μg/L in the negative control group. The total difference was statistically significant between patients of each group and the control group ( F =857.917, P spinal cord injury has a certain guiding significance for the diagnosis of spinal cord injury and the degree of injury.

Serum Levels Of Free And Total Insulin-Like Growth Factor (IGF)-1 And IGF Binding Protein-3 In Normal And Growth Hormone Deficient Children

International Nuclear Information System (INIS)

Shousha, M.A.; Soliman, S.E.T.; Hafez, M.H.

2006-01-01

Serum levels of total insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) reflect the endogenous GH secretion in healthy children, which makes them good diagnostic markers for screening growth hormone deficiency (GHD) in short children, although some controversy still exists. Only a minor fraction of the total IGF-1 circulates in its free form, which is believed to be the biologically active form. Serum levels of free IGF-1, total IGF-I and IGFBP-3 were measured in 144 healthy children (72 boys and 72 girls, aged from 0 to 16 years) and in 12 pre-pubertal GH deficient (GHD) children to study the correlation between the age and free IGF-1, total IGF-1 and IGFBP-3 levels. In healthy subjects (both sexes), serum free IGF-1, total IGF-1 and IGFBP-3 levels were low in infancy, increasing during puberty and declining thereafter. Free IGF-1 in serum occupied about 0.97-1.45 % of the total IGF-1 values, and the ratios of free IGF-1 to total IGF-1 were significantly increased in the pubertal age groups than in the pre-pubertal age groups. Serum levels of free IGF-1 showed significant positive correlation with those of total IGF-I and IGFBP-3. Serum free IGF-1, total IGF-1 and IGFBP-3 levels in patients with GHD were decreased significantly with increasing the degree of hypopituitarism. These observations suggest that the increase in serum free IGF-1 level during puberty was caused by a dramatic increase in total IGF-1 rather than IGFBP-3. Also, high levels of these hormones may play an important role in pubertal growth spurt and may become a useful tool for diagnosing GHD and predicting growth response to long term GH therapy

Serum levels of free and total insulin-link growth factor (IGF)-1 and (IGF) binding protein-3 in normal and growth hormone deficient children

International Nuclear Information System (INIS)

Shousha, M.A.; Soliman, S.E.T.; Hafez, H.M.

2008-01-01

Serum levels of total insulin-like growth factor- 1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) reflect endogenous GH secretion in healthy children, which makes them good diagnostic markers for screening GH deficiency (GHD) in short children, although some controversy still exists. Only a minor fraction of the total IGF-1 circulates in its free form, which is believed to be the biologically active form. Serum levels of free IGF-1, total IGF-I and IGFBP-3 were measured in 144 healthy children (72 boys and 72 girls, aged from 0 to 16 years) and in 12 prepubertal GH. deficient (GHD) children to study correlation between the age and free IGF-1, total IGF-1 and IGFBP-3 levels. In healthy subjects (both sexes), serum free IGF-1, total IGF-1 and IGFBP-3 levels were low in infancy, increasing during puberty and declining thereafter. Free IGF-1 in serum occupied about 0.97. 1.45 % of the total IGF-1 values, and the ratios of free IGF-1 to total IGF-1 were significantly increased in the pubertal age groups than in the prepubertal age groups. Serum levels of free IGF-1 showed significant positive correlation with those of total IGF-I and IGFBP-3. Serum free IGF-1, total IGF-1 and IGFBP-3 levels in patients with GHD decreased significantly with increasing degree of hypopituitarism. These observations suggest that the increase in serum free IGF-1 level during puberty was caused by a dramatic increase in total IGF-1 rather than IGFBP-3. Also, high levels of these hormones may play an important role in pubertal growth spurt and may become a useful tool for diagnosing GHD and predicting growth response to long term GH therapy

Prognostic Value of Serum Free Light Chain in Multiple Myeloma.

Science.gov (United States)

El Naggar, Amel A; El-Naggar, Mostafa; Mokhamer, El-Hassan; Avad, Mona W

2015-01-01

The measurement of serum free light chain (sFLC) has been shown to be valuable in screening for the presence of plasma cell dyscrasia as well as for baseline prognosis in newly diagnosed patients. The aim of the present work was to study the prognostic value of sFLC in multiple myeloma in relation to other serum biomarkers, response to therapy and survival. Forty five newly diagnosed patients with MM were included in the study. Patients were divided into responders and non-responders groups according to response to therapy. sFLC and serum Amyloid A (SAA) were measured by immunonephelometry. The non-responders group showed a statistically significant higher kappa/lambda or lambda/kappa ratio and higher β2 microglobulin level, but lower albumin level at presentation, as compared to the responders group (P < 0.001). However, no statistically significant difference was detected between the two groups regarding SA A or calcium levels. Comparison between sFLC ratio obtained before and after therapy revealed significant decrease after treatment in the responders group (P = 0.05). Survival was significantly inferior in patients with an FLC ratio of ≥ 2.6 or ≤ 0.56 compared with those with an FLC ratio that was between 0.56 and 2.6 (P = 0.002).

Culture of human cell lines by a pathogen-inactivated human platelet lysate.

Science.gov (United States)

Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

2016-08-01

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

Characteristics of retinol accumulation from serum retinol-binding protein by cultured sertoli cells

International Nuclear Information System (INIS)

Shingleton, J.L.; Skinner, M.K.; Ong, D.E.

1989-01-01

The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoil cells accumulated [ 3 H]retinol in a time- and temperature-dependent manner. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/μg of cellular DNA. Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with [ 3 H]retinol-RBP for [ 3 H]retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free [ 3 H]retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 μM. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. Competition studies indicated, however, that protein recognition is important in the retinol uptake process. RBP, CRBP, and CRBP(II) competed with [ 3 H]retinol-RBP for [ 3 H]retinol accumulation, but free retinol, retinol-bovine serum albumin, and retinol-β-lactoglobulin did not. These studies indicated that Sertoli cell uptake of retinol involved recognition of the retinol-RBP complex at the cell surface with subsequent internalization of retinol, but not RBP

Comparative Serum Fatty Acid Profiles of Captive and Free-Ranging Cheetahs (Acinonyx jubatus) in Namibia

Science.gov (United States)

Wachter, Bettina; Heinrich, Sonja K.; Reyers, Fred; Mienie, Lodewyk J.

2016-01-01

Cheetahs (Acinonyx jubatus) are highly specialised large felids, currently listed as vulnerable on the IUCN red data list. In captivity, they are known to suffer from a range of chronic non-infectious diseases. Although low heterozygosity and the stress of captivity have been suggested as possible causal factors, recent studies have started to focus on the contribution of potential dietary factors in the pathogenesis of these diseases. Fatty acids are an important component of the diet, not only providing a source of metabolisable energy, but serving other important functions in hormone production, cellular signalling as well as providing structural components in biological membranes. To develop a better understanding of lipid metabolism in cheetahs, we compared the total serum fatty acid profiles of 35 captive cheetahs to those of 43 free-ranging individuals in Namibia using gas chromatography-mass spectrometry. The unsaturated fatty acid concentrations differed most remarkably between the groups, with all of the polyunsaturated and monounsaturated fatty acids, except arachidonic acid and hypogeic acid, detected at significantly lower concentrations in the serum of the free-ranging animals. The influence of age and sex on the individual fatty acid concentrations was less notable. This study represents the first evaluation of the serum fatty acids of free-ranging cheetahs, providing critical information on the normal fatty acid profiles of free-living, healthy individuals of this species. The results raise several important questions about the potential impact of dietary fatty acid composition on the health of cheetahs in captivity. PMID:27992457

Free software and free culture in Higher Education: pipelines and workflows for creative purposes

OpenAIRE

Gonçalves, Nelson; Figueiredo, Maria Pacheco

2015-01-01

OpenLab ESEV is a project of the School of Education of the Polytechnic Institute of Viseu (ESEV), Portugal, that aims to promote, foster and support the use of Free/Libre Software and Open Source Software, Open Educational Resources, Free Culture, Free file formats and more flexible copyright licenses for creative and educational purposes in the ESEV's domains of activity (education, arts, media). Most of the OpenLab ESEV activities are related to the teacher education and arts a...

Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

Science.gov (United States)

Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

2017-01-01

Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a

Effects of blood collection conditions on ovarian cancer serum markers.

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Jason D Thorpe

2007-12-01

Full Text Available Evaluating diagnostic and early detection biomarkers requires comparing serum protein concentrations among biosamples ascertained from subjects with and without cancer. Efforts are generally made to standardize blood processing and storage conditions for cases and controls, but blood sample collection conditions cannot be completely controlled. For example, blood samples from cases are often obtained from persons aware of their diagnoses, and collected after fasting or in surgery, whereas blood samples from some controls may be obtained in different conditions, such as a clinic visit. By measuring the effects of differences in collection conditions on three different markers, we investigated the potential of these effects to bias validation studies.We analyzed serum concentrations of three previously studied putative ovarian cancer serum biomarkers-CA 125, Prolactin and MIF-in healthy women, women with ovarian cancer undergoing gynecologic surgery, women undergoing surgery for benign ovary pathology, and women undergoing surgery with pathologically normal ovaries. For women undergoing surgery, a blood sample was collected either in the clinic 1 to 39 days prior to surgery, or on the day of surgery after anesthesia was administered but prior to the surgical procedure, or both. We found that one marker, prolactin, was dramatically affected by collection conditions, while CA 125 and MIF were unaffected. Prolactin levels were not different between case and control groups after accounting for the conditions of sample collection, suggesting that sample ascertainment could explain some or all of the previously reported results about its potential as a biomarker for ovarian cancer.Biomarker validation studies should use standardized collection conditions, use multiple control groups, and/or collect samples from cases prior to influence of diagnosis whenever feasible to detect and correct for potential biases associated with sample collection.

Proposal of a candidate international conventional reference measurement procedure for free thyroxine in serum.

NARCIS (Netherlands)

Thienpont, L.M.; Beastall, G.H.; Christofides, N.D.; Faix, J.D.; Ieiri, T.; Jarrige, V.; Miller, W.G.; Miller, R.; Nelson, J.C.; Ronin, C.; Ross, H.A.; Rottmann, M.; Thijssen, J.H.; Toussaint, B.

2007-01-01

In the present paper the IFCC WG-STFT recommends and provides the rationale to establish metrological traceability of serum free thyroxine (FT4) measurements to a candidate international conventional reference measurement procedure. It is proposed that this procedure be based on equilibrium dialysis

Essential amino acids in the gluten-free diet and serum in relation to depression in patients with celiac disease.

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Nathalie J M van Hees

Full Text Available Celiac disease (CD is associated with an increased risk of major depressive disorder, possibly due to deficiencies in micronutrients in the gluten-free diet. We aimed to investigate whether essential amino acids (i.e., the precursors of serotonin, dopamine and other neurotransmitters are depleted in the diet and serum of CD patients with major depressive disorder.In a cross-sectional study we assessed dietary intake of amino acids and serum levels of amino acids, in 77 CD patients on a gluten-free diet and in 33 healthy controls. Major depressive disorder was assessed with structured interviews (using the Mini International Neuropsychiatric Interview Plus. Dietary intake was assessed using a 203-item food frequency questionnaire.Participants had a mean age of 55 years and 74% were women. The intake of vegetable protein was significantly lower in CD patients than in healthy controls (mean difference of 7.8 g/d; 95% CI: 4.7-10.8, as were serum concentrations of tyrosine, phenylalanine and tryptophan (all p < 0.005. However, within the CD patient group, the presence of major depressive disorder (n = 42 was not associated with intake or serum levels of essential amino acids.Patients with CD on a long-term gluten-free diet, with good adherence, consume significantly less vegetable protein than controls, and their serum levels of several essential amino acids were also lower. Despite its potential adverse effect, intake and serum levels of essential amino acids were not related to major depression.

Nanoparticles of Conjugated Methotrexate-Human Serum Albumin: Preparation and Cytotoxicity Evaluations

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Azade Taheri

2011-01-01

Full Text Available Methotrexate-human serum albumin conjugates were developed by a simple carbodiimide reaction. Methotrexate-human serum albumin conjugates were then crosslinked with 1-ethyl-3-(3-dimethylaminopropyl carbodiimide HCl (EDC to form nanoparticles. The size of nanoparticles determined by laser light scattering and TEM was between 90–150 nm. Nanoparticles were very stable at physiologic conditions (PBS pH 7.4, 37∘C and after incubation with serum. The effect of amount of EDC used for crosslinking on the particle size and free amino groups of nanoparticles was examined. The amount of crosslinker showed no significant effect on the size of nanoparticles but free amino groups of nanoparticles were decreased by increasing the crosslinker. The physicochemical interactions between methotrexate and human serum albumin were investigated by differential scanning calorimetry (DSC. Nanoparticles were more cytotoxic on T47D cells compared to free methotrexate. Moreover, methotrexate-human serum albumin nanoparticles decreased the IC50 value of methotrexate on T47D cells in comparison with free methotrexate.

Nanoparticles of Conjugated Methotrexate-Human Serum Albumin: Preparation and Cytotoxicity Evaluations

International Nuclear Information System (INIS)

Taheri, A.; Atyabi, F.; Nouri, F.S.; Ahadi, F.; Derakhshan, M.A.; Dinarvand, R.; Atyabi, F.; Ghahremani, M.H.; Ostad, S.N.; Dinarvand, R.; Amini, M.; Ghahremani, M.H.; Ostad, S.N.; Mansoori, P.

2011-01-01

Methotrexate-human serum albumin conjugates were developed by a simple carbodiimide reaction. Methotrexate-human serum albumin conjugates were then crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) to form nanoparticles. The size of nanoparticles determined by laser light scattering and TEM was between 90 150 nm. Nanoparticles were very stable at physiologic conditions (PBS pH 7.4, 37 degree C) and after incubation with serum. The effect of amount of EDC used for crosslinking on the particle size and free amino groups of nanoparticles was examined. The amount of cross linker showed no significant effect on the size of nanoparticles but free amino groups of nanoparticles were decreased by increasing the cross linker. The physicochemical interactions between methotrexate and human serum albumin were investigated by differential scanning calorimetry (DSC). Nanoparticles were more cytotoxic on T 47 D cells compared to free methotrexate. Moreover, methotrexate-human serum albumin nanoparticles decreased the C50 value of methotrexate on T 47 D cells in comparison with free methotrexate.

Analysis of Serum Total and Free PSA Using Immunoaffinity Depletion Coupled to SRM: Correlation with Clinical Immunoassay Tests

Science.gov (United States)

Liu, Tao; Hossain, Mahmud; Schepmoes, Athena A.; Fillmore, Thomas L.; Sokoll, Lori J.; Kronewitter, Scott R.; Izmirlian, Grant; Shi, Tujin; Qian, Wei-Jun; Leach, Robin J.; Thompson, Ian M.; Chan, Daniel W.; Smith, Richard D.; Kagan, Jacob; Srivastava, Sudhir; Rodland, Karin D.; Camp, David G.

2012-01-01

Recently, selected reaction monitoring mass spectrometry (SRM-MS) has been more frequently applied to measure low abundance biomarker candidates in tissues and biofluids, owing to its high sensitivity and specificity, simplicity of assay configuration, and exceptional multiplexing capability. In this study, we report for the first time the development of immunoaffinity depletion-based workflows and SRM-MS assays that enable sensitive and accurate quantification of total and free prostate-specific antigen (PSA) in serum without the requirement for specific PSA antibodies. Low ng/mL level detection of both total and free PSA was consistently achieved in both PSA-spiked female serum samples and actual patient serum samples. Moreover, comparison of the results obtained when SRM PSA assays and conventional immunoassays were applied to the same samples showed good correlation in several independent clinical serum sample sets. These results demonstrate that the workflows and SRM assays developed here provide an attractive alternative for reliably measuring candidate biomarkers in human blood, without the need to develop affinity reagents. Furthermore, the simultaneous measurement of multiple biomarkers, including the free and bound forms of PSA, can be performed in a single multiplexed analysis using high-resolution liquid chromatographic separation coupled with SRM-MS. PMID:22846433

Validity of Serum Testosterone, Free Androgen Index, and Calculated Free Testosterone in Women with Suspected Hyperandrogenism

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Manal K. Al Kindi

2012-11-01

Full Text Available Objectives: There are technical limitations for the currently available methods of measuring serum total and free testosteronein females. The study objectives were to evaluate the usefulness of serum total testosterone, sex hormone-binding globulin (SHBG, free androgen index (FAI, and calculated free testosterone (CFT in the assessment of androgen status in women investigated for suspected hyperandrogenism.Methods: This is a case control study that was conducted during the period from 1st May 2011 to 31st October 2011 on 122 patients aged (18-45 years whom were referred to the Clinical Biochemistry Laboratory from the Endocrinology and Gynecology Clinics, Royal Hospital, Oman. Women with no clinical feature or laboratory data indicative of hormonal dysfunction and with midluteal progesterone >30 nmol/L were selected as controls (group 1; n=18. The patients were divided into subgroups based on the clinical/laboratory diagnosis of polycystic ovary syndrome (PCOS [group 2; n=19, hirsutism (group 3; n=18, menstrual disturbances (irregularities or infertility (group 4; n=49, as well as combination of PCOS or hirsutism and menstrual disturbances or infertility (group 5;n=18. Serum total testosterone and SHBG were measured, FAI was calculated as percentage ratio of total testosterone to SHBG values, and CFT was calculated according to Vermeulen equation.Results: There was a statistically significant difference in the mean levels of testosterone, FAI and CFT in each patient group compared with the control group. For diagnosing hyperandrogenism, each indicator was selected at the recommended cut-off: testosterone >3.0 nmol/L, SHBG 5%, and CFT >32 pmol/L. In group 2, 89.5% and 94.7% of the patients had increased FAI and CFT, respectively; compared with 36.4% for increased testosterone. In group 3, 88.9% and 88.9% of the patients had similarly increased FAI and CFT, respectively; compared with 66.7% for testosterone. In group 4, patients had 63.3% and 73

Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine

International Nuclear Information System (INIS)

Lobo, Nazleen C.; Gedye, Craig; Apostoli, Anthony J.; Brown, Kevin R.; Paterson, Joshua; Stickle, Natalie; Robinette, Michael; Fleshner, Neil; Hamilton, Robert J.; Kulkarni, Girish; Zlotta, Alexandre; Evans, Andrew; Finelli, Antonio; Moffat, Jason; Jewett, Michael A. S.; Ailles, Laurie

2016-01-01

Patients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients. ccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media. Established cultures were characterized by genomic verification of mutations present in the primary tumors, expression of renal epithelial markers, and transcriptional profiling. The apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping revealed that the majority of cultures contained normal, not cancer cells. ccRCC characteristically shows biallelic loss of the von Hippel Lindau (VHL) gene, leading to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Purification of cells based on expression of carbonic anhydrase IX (CA9), a cell surface HIF target, followed by culture in FBS enabled establishment of ccRCC cell cultures with an efficiency of >80 %. Culture in serum-free conditions selected for growth of normal renal proximal tubule epithelial cells. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures. The ability

Clonogenic growth of human breast cancer cells co-cultured in direct contact with serum-activated fibroblasts

International Nuclear Information System (INIS)

Samoszuk, Michael; Tan, Jenny; Chorn, Guillaume

2005-01-01

Accumulating evidence suggests that fibroblasts play a pivotal role in promoting the growth of breast cancer cells. The objective of the present study was to characterize and validate an in vitro model of the interaction between small numbers of human breast cancer cells and human fibroblasts. We measured the clonogenic growth of small numbers of human breast cancer cells co-cultured in direct contact with serum-activated, normal human fibroblasts. Using DNA microarrays, we also characterized the gene expression profile of the serum-activated fibroblasts. In order to validate the in vivo relevance of our experiments, we then analyzed clinical samples of metastatic breast cancer for the presence of myofibroblasts expressing α-smooth muscle actin. Clonogenic growth of human breast cancer cells obtained directly from in situ and invasive tumors was dramatically and consistently enhanced when the tumor cells were co-cultured in direct contact with serum-activated fibroblasts. This effect was abolished when the cells were co-cultured in transwells separated by permeable inserts. The fibroblasts in our experimental model exhibited a gene expression signature characteristic of 'serum response' (i.e. myofibroblasts). Immunostaining of human samples of metastatic breast cancer tissue confirmed that myofibroblasts are in direct contact with breast cancer cells. Serum-activated fibroblasts promote the clonogenic growth of human breast cancer cells in vitro through a mechanism that involves direct physical contact between the cells. This model shares many important molecular and phenotypic similarities with the fibroblasts that are naturally found in breast cancers

Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures

NARCIS (Netherlands)

Gonzales, V.K.; Mulder, E.L.W. de; Boer, T. den; Hannink, G.; Tienen, T.G. van; Heerde, W.L. van; Buma, P.

2013-01-01

Concerns over fetal bovine serum (FBS) limit the clinical application of cultured tissue-engineered constructs. Therefore, we investigated if platelet-rich plasma (PRP) can fully replace FBS for meniscus tissue engineering purposes. Human PRP and platelet-poor plasma (PPP) were isolated from three

Plasmodium falciparum in vitro continuous culture conditions: A comparison of parasite susceptibility and tolerance to anti-malarial drugs throughout the asexual intra-erythrocytic life cycle.

Science.gov (United States)

Duffy, Sandra; Avery, Vicky M

2017-12-01

The continuous culture of Plasmodium falciparum is often seen as a means to an end, that end being to probe the biology of the parasite in question, and ultimately for many in the malaria drug discovery arena, to identify means of killing the parasite in order to treat malaria. In vitro continuous culture of Plasmodium falciparum is a fundamental requirement when undertaking malaria research where the primary objectives utilise viable parasites of a desired lifecycle stage. This investigation, and resulting data, compared the impact culturing Plasmodium falciparum long term (4 months) in different environmental conditions had on experimental outcomes and thus conclusions. The example presented here focused specifically on the effect culture conditions had on the in vitro tolerance of Plasmodium falciparum to standard anti-malarial drugs, including artemisinin and lumefantrine. Historical data from an independent experiment for 3D7-ALB (5% O 2 ) was also compared with that obtained from this study. We concluded that parasites cultured for several months in media supplemented with a serum substitute such as Albumax II ® or within hyperoxic conditions (21% O 2 ), demonstrate highly variable responses to artemisinin and lumefantrine but not all anti-malarial drugs, when compared to those cultured in human serum in combination with Albumax II ® under normoxic conditions (5% O 2 ) for the parasite. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Clinical significance of two-step magnetic radioimmunoassay for determining serum free T3 and free T4

International Nuclear Information System (INIS)

Chen Bing

1996-01-01

The concentrations of the serum free T 3 (FT 3 ), free T 4 (FT 4 ), total triodothyronine (TT 3 ), total thyroxine (TT 4 ) and thyrotropin (TSH) are determined for 355 cases of normal persons, pregnant women and various thyropathetic patients. The normal values of FT 3 and FT 4 are 2.0-8.5 pmol/l, and 9.5-26.5 pmol/l, respectively. Neither FT 3 nor FT 4 is affected by the thyroxine combined with globulin (TBG), which is of unique diagnostic value for those with variable TBG (such as pregnant women hyperthyroidism, hypothyroidism, etc.), FT 3 and FT 4 are the most sensitive indices for diagnosis of hyperthyroidism, and hypothyroidism, respectively. In addition, FT 3 and FT 4 can greatly contribute to the observation of curative effectiveness under treatment

The fluctuation of free amino acids in serum during acute ischemic stroke

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Szpetnar Maria

2016-12-01

Full Text Available Currently, little data exists regarding the involvement of free amino acids (AA in the pathogenesis of ischemic stroke (IS. Thus, our objective was to study the degree of the degree of fluctuation of free amino acids level in serum during the acute phase of IS. The study consisted of eighteen patients (female/male: 10/8; age: 73.1 ± 4.1 with acute IS that was confirmed by way of computed tomography, while twelve sex and age matched individuals were assigned as control group. During the study period, the patients did not receive any supplemental amino acids therapy that could affect the obtained results. The venous blood was obtained after >3 hours fasting at two time-points; time-point 1 – at admission to the hospital; time-point 2 – on day 5 from stroke onset. The blood for control purposes was collected only once, and the blood collection at time-point 1 was done before thrombolytic treatment (nine patients. The amino acids were identified using the Amino Acids Analyser (AAA 400 by INGOS Corp., Praha, Czech Republic. Our results revealed a statistically significant increase of glutamate, cystine and methionine on day 1 of stroke, in comparison to control, whereas, proline level was decreased on day 1 of stroke – in comparison to control serum. On comparing day 5 to the initial day of IS, elevation was observed of levels of asparagine, glycine, tyrosine, arginine, threonine, valine, leucine and phenylalanine. It can be said, then, that ischemic stroke induces both essential and nonessential amino acid fluctuations. Moreover, the decrease in proline and glutamine serum level with the simultaneous increase in the concentration of branch chain amino acids, Glu and Thr suggests a violent mobilization of the body’s proteins. Thus, a decrease of Pro and a simultaneous increase of Glu serum level could be considered as a marker of acute IS.

Development of a new mouse palate organ culture system and effect of X-irradiation on palatogenesis

International Nuclear Information System (INIS)

Hiranuma, Hiroko; Jikko, Akitoshi; Maeda, Takashi; Furukawa, Souhei

1999-01-01

On the basis of an already established suspension system of organ culture for mouse palate explants, we developed a new culture system, which has several advantages over the previous methods. We used a 48-well culture plate in which the explants can be cultured individually, and only 300 μl of medium is needed for each well. In order to optimize the culture results, we systematically studied the influence of main ''culture conditions'' such as tilt degree of the culturing palate, rotation speed, and addition of ascorbic acid to the medium. This system allows culturing of palates from day 13.5 of gestation to day 16.5 under serum-free conditions using a chemically defined medium, which resulted in 78% of the palates growing fused. Utilizing this culture system, the direct effect of X-irradiation on palataogesis was analyzed. A 4 Gy dose of X-irradiation was administrated at the beginning of culture period. The incidence of palatal fusion was not significantly different from that of the non-irradiated group. (author)

Detection of atomic scale changes in the free volume void size of three-dimensional colorectal cancer cell culture using positron annihilation lifetime spectroscopy.

Science.gov (United States)

Axpe, Eneko; Lopez-Euba, Tamara; Castellanos-Rubio, Ainara; Merida, David; Garcia, Jose Angel; Plaza-Izurieta, Leticia; Fernandez-Jimenez, Nora; Plazaola, Fernando; Bilbao, Jose Ramon

2014-01-01

Positron annihilation lifetime spectroscopy (PALS) provides a direct measurement of the free volume void sizes in polymers and biological systems. This free volume is critical in explaining and understanding physical and mechanical properties of polymers. Moreover, PALS has been recently proposed as a potential tool in detecting cancer at early stages, probing the differences in the subnanometer scale free volume voids between cancerous/healthy skin samples of the same patient. Despite several investigations on free volume in complex cancerous tissues, no positron annihilation studies of living cancer cell cultures have been reported. We demonstrate that PALS can be applied to the study in human living 3D cell cultures. The technique is also capable to detect atomic scale changes in the size of the free volume voids due to the biological responses to TGF-β. PALS may be developed to characterize the effect of different culture conditions in the free volume voids of cells grown in vitro.

Surface free energy for systems with integrable boundary conditions

International Nuclear Information System (INIS)

Goehmann, Frank; Bortz, Michael; Frahm, Holger

2005-01-01

The surface free energy is the difference between the free energies for a system with open boundary conditions and the same system with periodic boundary conditions. We use the quantum transfer matrix formalism to express the surface free energy in the thermodynamic limit of systems with integrable boundary conditions as a matrix element of certain projection operators. Specializing to the XXZ spin-1/2 chain we introduce a novel 'finite temperature boundary operator' which characterizes the thermodynamical properties of surfaces related to integrable boundary conditions

Viability and proliferation of L929, tumour and hybridoma cells in the culture media containing sericin protein as a supplement or serum substitute.

Science.gov (United States)

Cao, Ting-Ting; Zhang, Yu-Qing

2015-09-01

Cell cultures often require the addition of animal serum and other supplements. In this study, silk sericin, a bioactive protein, recovered from the waste of silk floss production was hydrolysed into three pepsin-degraded sericin peptides with different ranges of molecular mass. Normal animal cells, tumour cells and hybridoma cells were cultured systematically in FBS culture media containing sericin as a supplement or serum substitute. The culture test and microscopic observation of L929 cells showed that the smaller molecular weight of the degraded sericin is most suitable for cell culture. The cell culture results showed that with the degradation of sericin, for normal mouse fibroblast L929 cells, addition of 0.75 % sericin into FBS culture medium yields cell viability that is superior to FBS culture medium alone. When all serum was replaced by sericin, cell viability in the sericin medium could reach about one half of that in FBS medium. When in a medium containing a mixture of FBS: sericin (6:4, v/v), the cell culture effect is about 80 %. For the cultures of four tumour and one hybridoma cells, regardless of the molecular weight range, these degraded sericin peptides could substitute all serum in FBS media. The cell viability and proliferation of these tumour and hybridoma cells are equivalent or superior to that in FBS medium. In other words, cell viability and proliferation of these tumour and hybridoma cells in sericin media are more preferable to serum media. The mechanism of the sericin protein to promote cell growth and proliferation will be further investigated later.

Comparison between human serum and Albuminar-20 (TM) supplement for in-vitro fertilization.

Science.gov (United States)

Staessen, C; Van den Abbeel, E; Carlé, M; Khan, I; Devroey, P; Van Steirteghem, A C

1990-04-01

Patient or fetal cord serum is commonly used as a protein supplement to culture media used in in-vitro fertilization (IVF). To eliminate the variability and possible hazards related to the use of human serum, a well-defined protein supplement, Albuminar-20 (Armour Pharmaceutical Cy) was evaluated as a substitute for serum. Prior to its application in the human, Earle's culture media supplemented with 0.5% (w/v) bovine serum albumin, 8% (v/v) decomplemented patient serum or 2.25% (v/v) Albuminar-20 were compared in a mouse bioassay. For the three different conditions, the percentages of blastocysts formed after 120 h in-vitro culture were respectively 91.2, 85.2 and 87.8% (NS). In the human IVF, a controlled comparison was performed from October to December 1988, between Earle's medium supplemented with patients' serum or Albuminar-20. When oocytes and spermatozoa were cultured in these two media, the fertilization rates were similar, 58.9% in human serum versus 59.4% in Albuminar-20. After further culture, the morphological quality of the cleaved embryos was better in the embryos cultured in Albuminar-20. The higher pregnancy rate in Albuminar-20 was correlated with the better morphological appearance of the embryos and their more advanced cleavage stage at the time of transfer. Therefore, Albuminar-20 can be considered as a suitable protein supplement in human IVF.

Direct, Label-Free, and Rapid Transistor-Based Immunodetection in Whole Serum.

Science.gov (United States)

Gutiérrez-Sanz, Óscar; Andoy, Nesha M; Filipiak, Marcin S; Haustein, Natalie; Tarasov, Alexey

2017-09-22

Transistor-based biosensors fulfill many requirements posed upon transducers for future point-of-care diagnostic devices such as scalable fabrication and label-free and real-time quantification of chemical and biological species with high sensitivity. However, the short Debye screening length in physiological samples (<1 nm) has been a major drawback so far, preventing direct measurements in serum. In this work, we demonstrate how tailoring the sensing surface with short specific biological receptors and a polymer polyethylene glycol (PEG) can strongly enhance the sensor response. In addition, the sensor performance can be dramatically improved if the measurements are performed at elevated temperatures (37 °C instead of 21 °C). With this novel approach, highly sensitive and selective detection of a representative immunosensing parameter-human thyroid-stimulating hormone-is shown over a wide measuring range with subpicomolar detection limits in whole serum. To the best of our knowledge, this is the first demonstration of direct immunodetection in whole serum using transistor-based biosensors, without the need for sample pretreatment, labeling, or washing steps. The presented sensor is low-cost, can be easily integrated into portable diagnostics devices, and offers a competitive performance compared to state-of-the-art central laboratory analyzers.

Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

Science.gov (United States)

Badenes, Sara M.; Fernandes, Tiago G.; Cordeiro, Cláudia S. M.; Boucher, Shayne; Kuninger, David; Vemuri, Mohan C.; Diogo, Maria Margarida; Cabral, Joaquim M. S.

2016-01-01

Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells. PMID:26999816

Changes of Serum Total and Free Testosterone Concentrations in Male Chronic Hemodialysis Patients with Secondary Hyperparathyroidism in Response to Cinacalcet Treatment

Directory of Open Access Journals (Sweden)

Piotr Kuczera

2016-01-01

Full Text Available Background/Aims: Calcium sensing receptor (CaSR is expressed, among others also in testis. Cinacalcet binds to the CaSR, increases sensitivity of CaSR to serum calcium and is used in the treatment of secondary hyperparathyroidism (sHPT in chronic hemodialysis patients (HDP. In most of male HDP, serum testosterone concentration is lower than in healthy males. The aim of this study was to assess the influence of six-month treatment with cinacalcet on the serum total and free testosterone concentration in male HDP with sHPT. Methods: 38 male, hemodialysed CKD patients with sHPT (PTH>300 pg/ml were enrolled into the study. In each patient serum PTH, total testosterone (TT and free testosterone (FT concentrations were assessed before the first dose of cinacalcet and then after 3 and 6 months of treatment. The results are presented as means with 95% confidence interval. Results: In 33 patients who completed the study cinacalcet treatment caused significant decrease of serum PTH from 1143 pg/ml (828 - 1458 pg/ml at the baseline, to 809 pg/ml (487 - 1132pg/ml after 3 month of treatment (p = 0.002, and to 607 pg/ml (281 - 934pg/ml; p Conclusion: Treatment with cinacalcet decreases serum total and free testosterone concentration in male hemodialysed patients with chronic kidney disease and secondary hyperparathyroidism.

Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools.

Science.gov (United States)

Díez, José M; Bauman, Ewa; Gajardo, Rodrigo; Jorquera, Juan I

2015-03-13

Fetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity. SCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation. SCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons. The tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.

Analysis of Serum Total and Free PSA Using Immunoaffinity Depletion Coupled to SRM: Correlation with Clinical Immunoassay Tests

Energy Technology Data Exchange (ETDEWEB)

Liu, Tao; Hossain, Mahmud; Schepmoes, Athena A.; Fillmore, Thomas L.; Sokoll, Lori J.; Kronewitter, Scott R.; Izmirlian, Grant; Shi, Tujin; Qian, Weijun; Leach, Robin; Thompson, Ian M.; Chan, Daniel W.; Smith, Richard D.; Kagan, Jacob; Srinivastava, Sudhir; Rodland, Karin D.; Camp, David G.

2012-08-03

Sandwich immunoassay is the standard technique used in clinical labs for quantifying protein biomarkers for disease detection, monitoring and therapeutic intervention. Albeit highly sensitive, the development of a specific immunoassay is rather time-consuming and associated with extremely high cost due to the requirement for paired immunoaffinity reagents of high specificity. Recently, mass spectrometry-based methods, specifically selected reaction monitoring mass spectrometry (SRM-MS), have been increasingly applied to measure low abundance biomarker candidates in tissue and biofluids, owing to high sensitivity and specificity, simplicity of assay configuration, and great multiplexing capability. In this study, we report for the first time the development of immunoaffinity depletion-based workflows and SRM-MS assays that enable sensitive and accurate quantification of total and free prostate-specific antigen (PSA) in serum without the requirement for specific PSA antibodies. With stable isotope dilution and external calibration, low ng/mL level detection of both total and free PSA was consistently achieved in both PSA-spiked female serum samples and actual patient serum samples. Moreover, comparison of the results obtained when SRM PSA assays and conventional immunoassays were applied to the same samples showed very good correlation (R2 values ranging from 0.90 to 0.99) in several independent clinical serum sample sets, including a set of 33 samples assayed in a blinded test. These results demonstrate that the workflows and SRM assays developed here provide an attractive alternative for reliably measuring total and free PSA in human blood. Furthermore, simultaneous measurement of free and total PSA and many other biomarkers can be performed in a single analysis using high-resolution liquid chromatographic separation coupled with SRM-MS.

Formation of proteoglycan and collagen-rich scaffold-free stiff cartilaginous tissue using two-step culture methods with combinations of growth factors.

Science.gov (United States)

Miyazaki, Tatsuya; Miyauchi, Satoshi; Matsuzaka, Satoshi; Yamagishi, Chie; Kobayashi, Kohei

2010-05-01

Tissue-engineered cartilage may be expected to serve as an alternative to autologous chondrocyte transplantation treatment. Several methods for producing cartilaginous tissue have been reported. In this study, we describe the production of scaffold-free stiff cartilaginous tissue of pig and human, using allogeneic serum and growth factors. The tissue was formed in a mold using chondrocytes recovered from alginate bead culture and maintained in a medium with transforming growth factor-beta and several other additives. In the case of porcine tissue, the tear strength of the tissue and the contents of proteoglycan (PG) and collagen per unit of DNA increased dose-dependently with transforming growth factor-beta. The length of culture was significantly and positively correlated with thickness, tear strength, and PG and collagen contents. Tear strength showed positive high correlations with both PG and collagen contents. A positive correlation was also seen between PG content and collagen content. Similar results were obtained with human cartilaginous tissue formed from chondrocytes expanded in monolayer culture. Further, an in vivo pilot study using pig articular cartilage defect model demonstrated that the cartilaginous tissue was well integrated with surrounding tissue at 13 weeks after the implantation. In conclusion, we successfully produced implantable scaffold-free stiff cartilaginous tissue, which characterized high PG and collagen contents.

Application of a nitrocellulose immunoassay for quantitation of proteins secreted in cultured media

International Nuclear Information System (INIS)

LaDuca, F.M.; Dang, C.V.; Bell, W.R.

1986-01-01

A macro immunoassay was developed to quantitate proteins (antigens) secreted in the culture media of primary rat hepatocytes. Dilutions of protein standards and undiluted spent culture media were applied to numbered sheets of nitrocellulose (NC) paper by vacuum filtration (in volumes up to 1 ml) through a specially designed macrofiltration apparatus constructed of plexiglas. Sequential incubation of the NC with bovine serum albumin blocking buffer, monospecific antibody, and 125 I Protein A enabled quantitation of protein concentration by determination of NC bound radioactivity. Linear and reproducible standard curves were obtained with fibrinogen, albumin, transferrin, and haptoglobin. A high degree of coefficient of correlation between radioactivity (cmp) and protein concentration was found. Intra- and inter-test reproducibility was excellent. By using monospecific antibodies, single proteins (i.e., fibrinogen), as low as 32 ng/ml, could be quantified in heterogeneous protein mixtures and in spent culture media. The assay was sensitive to the difference of fibrinogen secretion under nonstimulatory (serum-free hormonally define medium, SFHD) and stimulatory (SFHD plus hydrocortisone) culture conditions. The procedure and techniques described are applicable to the quantitation of any protein in a suitable buffer

Effects of age and health on the euthyroid reference ranges for serum free thyroxine and free triiodothyronine

International Nuclear Information System (INIS)

Midgley, J.E.M.

1985-01-01

Age-related trends in serum free thyroxine (FT 4 ) and free triiodothyronine (FT 3 ) concentrations were measured in 7248 euthyroid subjects (age-range 3 months to 106 years). 5700 were patients referred to hospitals for investigation of suspected thyroid dysfunction, but who were diagnosed euthyroid. 1548 were healthy blood donors (age-range 18-63 years) with no indication of thyroid dysfunction. FT 4 concentrations were little affected by the age, the sex or the state of health of the subjects in either group. Serum FT 3 concentrations were significantly affected by both age and health factors. The upper limit of the euthyroid reference range for young subjects up to 15 years was about 20% higher (10.4 pmol/l) than for adult subjects older than 25 years (8.8 pmol/l). The change in the upper limits typical of young subjects to that typical of adults occurred steadily over the decade 15-25 years. After this age, little further change occurred, especially in healthy subjects. Additionally, the lower limit of the euthyroid range for FT 3 was extended by the inclusion in the reference group of patients referred to hospitals. Compared with the lower limit of the FT 3 range for healthy subjects (5 pmol/l), the corresponding limit for referred subjects (young or adult) was 3.5-3.8 pmol/l. Broadening of the FT 3 reference range was probably brought about by a significant number of patients in the hospital-referred group with the 'low-T 3 syndrome' of mild non-thyroidal illness. Accordingly, FT 3 was inferior to FT 4 in the discrimination of hypothyroidism, as FT 4 was unaffected by this phenomenon. Effects of age and non-thyroidal illness on serum FT 3 concentrations require great care when selecting subjects for a laboratory euthyroid reference range typical of the routine workload. Constraints on the choice of subjects for FT 4 reference ranges are less stringent. (orig.) [de

Microfluidic perfusion culture of human induced pluripotent stem cells under fully defined culture conditions.

Science.gov (United States)

Yoshimitsu, Ryosuke; Hattori, Koji; Sugiura, Shinji; Kondo, Yuki; Yamada, Rotaro; Tachikawa, Saoko; Satoh, Taku; Kurisaki, Akira; Ohnuma, Kiyoshi; Asashima, Makoto; Kanamori, Toshiyuki

2014-05-01

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for drug screening. For this application, self-renewal or differentiation of the cells is required, and undefined factors in the culture conditions are not desirable. Microfluidic perfusion culture allows the production of small volume cultures with precisely controlled microenvironments, and is applicable to high-throughput cellular environment screening. Here, we developed a microfluidic perfusion culture system for hiPSCs that uses a microchamber array chip under defined extracellular matrix (ECM) and culture medium conditions. By screening various ECMs we determined that fibronectin and laminin are appropriate for microfluidic devices made out of the most popular material, polydimethylsiloxane (PDMS). We found that the growth rate of hiPSCs under pressure-driven perfusion culture conditions was higher than under static culture conditions in the microchamber array. We applied our new system to self-renewal and differentiation cultures of hiPSCs, and immunocytochemical analysis showed that the state of the hiPSCs was successfully controlled. The effects of three antitumor drugs on hiPSCs were comparable between microchamber array and 96-well plates. We believe that our system will be a platform technology for future large-scale screening of fully defined conditions for differentiation cultures on integrated microfluidic devices. © 2013 Wiley Periodicals, Inc.

[The value of serum free light chain in differential diagnosis of monoclonal gammopathy of renal significance].

Science.gov (United States)

Li, C; Wen, Y B; Li, H; Su, W; Li, J; Cai, J F; Chen, L M; Li, X M; Li, X W

2017-08-08

Objective: To investigate the value of serum free light chain (FLC) in differential diagnosis of monoclonal gammopathy of renal significance (MGRS). Methods: Forty-nine hospitalized patients who underwent renal biopsy in Peking Union Medical College Hospital between January 2013 and December 2015 were included. Monoclonal gammopathy was detected by serum protein electrophoresis (SPE), serum immunofixation electrophoresis (IFE), urine IFE and serum FLC. All patients were classified as MGRS ( n =32) and monoclonal gammopathy of undetermined significance (MGUS) ( n =17). Results: Renal lesions in MGRS subgroup included light chain amyloidosis ( n =24, 75.0%), light chain deposition disease ( n =7, 21.9%), and fibrillary glomerulopathy ( n =1, 3.1%). Renal diseases in MGUS subgroup included membranous nephropathy ( n =10), focal segmental glomerulosclerosi (FSGS) ( n =3), diabetic glomerulopathy ( n =1), Henoch-Schonlein purpura nephritis ( n =1), anti-GBM disease concurrent with membranous nephropathy ( n =1) and glomerulomegaly ( n =1). Positive number of SPE, serum IFE, urine IFE and abnormal number of serum FLC ratio in MGRS subgroup were 12, 16, 23 and 30, respectively. Positive number of SPE, serum IFE, urine IFE and abnormal number of serum FLC ratio in MGUS subgroup were 11, 17, 6 and 3, respectively. MGRS and MGUS subgroups differed significantly in positive rate of serum IFE ( P value for MGRS, which was helpful for differential diagnosis of patients who had contraindication to renal biopsy.

Serum free light chain reference values: a critical approach.

Science.gov (United States)

Altinier, Sara; Seguso, Mara; Zaninotto, Martina; Varagnolo, Mariacristina; Adami, Fausto; Angeli, Paolo; Plebani, Mario

2013-05-01

The clinical usefulness of serum free light chain (FLC) measurement in the management of patients with plasma cell proliferative disorders has been reported in several papers, and most clinical studies use the reference ranges declared by the manufacturer. The aim of the present study was to evaluate the reproducibility of FLCs immunoassay and to validate the reference range, before introducing it in routine setting. Internal quality control materials and a pool of fresh serum samples were used to evaluate imprecision; 162 fresh sera from healthy blood donors were analyzed to evaluate the reference range for FLCs. In order to verify the κ/λ FLC ratio, 43 sera from patients with polyclonal hypergammaglobulinemia were tested. The FLC immunoassay was performed using a nephelometer with the Freelite reagents. The imprecision studies performed using a serum pool tested with two different lots of reagents showed a mean CV of 16.09% for κFLC and of 16.72% for λFLC. Lower CV%s and different mean values were found by calculating the results from each specific lot separately, while different results were obtained using the control materials provided by the manufacturer. In reference subjects, the 2.5-97.5th percentiles were found to be 4.52-22.33 and 4.84-21.88mg/L for κFLC and λFLC, respectively. The range for κ/λ ratio (0.65-2.36) was validated with the values obtained from subjects with polyclonal hypergammaglobulinemia. In retesting 15 samples from blood donor subjects with a different lot of reagents, mean bias percentages of 17.60 for κFLC and 15.26 for λFLC were obtained. These findings confirm the lot-to-lot variability of the FLC assays also in the measurement of polyclonal light chains, as well as the need to carefully validate the reference values. Published by Elsevier Inc.

Serum amyloid A isoforms in serum and synovial fluid from spontaneously diseased dogs with joint diseases or other conditions

DEFF Research Database (Denmark)

Kjelgaard-Hansen, Mads Jens; Christensen, Michelle B.; Lee, Marcel Huisung

2007-01-01

Serum amyloid A (SAA) is a major acute phase protein in dogs. However, knowledge of qualitative properties of canine SAA and extent of its synthesis in extrahepatic tissues is limited. The aim of the study was to investigate expression of different SAA isoforms in serum and synovial fluid...... in samples obtained from dogs (n = 16) suffering from different inflammatory or non-inflammatory conditions, which were either related or unrelated to joints. Expression of SAA isoforms was visualized by denaturing isoelectric focusing and Western blotting. Serum amyloid A was present in serum from all dogs...... with systemic inflammatory activity, and up to four major isoforms with apparent isoelectric points between 6.1 and 7.9 were identified. In synovial fluid from inflamed joints one or more highly alkaline SAA isoforms (with apparent isoelectric points above 9.3) were identified, with data suggesting local...

Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrick™ design, serum-free virus production and microcarrier-based cultivation of CV-1 cells.

Science.gov (United States)

Liu, Shuchang; Ruban, Ludmila; Wang, Yaohe; Zhou, Yuhong; Nesbeth, Darren N

2017-02-01

Vaccinia virus (VACV) is an established vector for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. The CV-1 cell line can be used for VACV propagation and has been used extensively with the CRISPR/Cas9 system for making precise edits of the VACV genome. Here we take first steps toward establishing a scalable synthetic biology platform for VACV production with CV-1 cells featuring standardised biological tools and serum free cell cultivation. We propose a new BioBrick™ plasmid backbone format for inserting transgenes into VACV. We then test the performance of CV-1 cells in propagation of a conventional recombinant Lister strain VACV, VACVL-15 RFP, in a serum-free process. CV-1 cells grown in 5% foetal bovine serum (FBS) Dulbecco's Modified Eagle Medium (DMEM) were adapted to growth in OptiPRO and VP-SFM brands of serum-free media. Specific growth rates of 0.047 h -1 and 0.044 h -1 were observed for cells adapted to OptiPRO and VP-SFM respectively, compared to 0.035 h -1 in 5% FBS DMEM. Cells adapted to OptiPRO and to 5% FBS DMEM achieved recovery ratios of over 96%, an indication of their robustness to cryopreservation. Cells adapted to VP-SFM showed a recovery ratio of 82%. Virus productivity in static culture, measured as plaque forming units (PFU) per propagator cell, was 75 PFU/cell for cells in 5% FBS DMEM. VP-SFM and OptiPRO adaptation increased VACV production to 150 PFU/cell and 350 PFU/cell respectively. Boosted PFU/cell from OptiPRO-adapted cells persisted when 5% FBS DMEM or OptiPRO medium was observed during the infection step and when titre was measured using cells adapted to 5% FBS DMEM or OptiPRO medium. Finally, OptiPRO-adapted CV-1 cells were successfully cultivated using Cytodex-1 microcarriers to inform future scale up studies.

Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrick™ design, serum-free virus production and microcarrier-based cultivation of CV-1 cells

Directory of Open Access Journals (Sweden)

Shuchang Liu

2017-02-01

Full Text Available Vaccinia virus (VACV is an established vector for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. The CV-1 cell line can be used for VACV propagation and has been used extensively with the CRISPR/Cas9 system for making precise edits of the VACV genome. Here we take first steps toward establishing a scalable synthetic biology platform for VACV production with CV-1 cells featuring standardised biological tools and serum free cell cultivation. We propose a new BioBrick™ plasmid backbone format for inserting transgenes into VACV. We then test the performance of CV-1 cells in propagation of a conventional recombinant Lister strain VACV, VACVL-15 RFP, in a serum-free process. CV-1 cells grown in 5% foetal bovine serum (FBS Dulbecco’s Modified Eagle Medium (DMEM were adapted to growth in OptiPRO and VP-SFM brands of serum-free media. Specific growth rates of 0.047 h−1 and 0.044 h−1 were observed for cells adapted to OptiPRO and VP-SFM respectively, compared to 0.035 h−1 in 5% FBS DMEM. Cells adapted to OptiPRO and to 5% FBS DMEM achieved recovery ratios of over 96%, an indication of their robustness to cryopreservation. Cells adapted to VP-SFM showed a recovery ratio of 82%. Virus productivity in static culture, measured as plaque forming units (PFU per propagator cell, was 75 PFU/cell for cells in 5% FBS DMEM. VP-SFM and OptiPRO adaptation increased VACV production to 150 PFU/cell and 350 PFU/cell respectively. Boosted PFU/cell from OptiPRO-adapted cells persisted when 5% FBS DMEM or OptiPRO medium was observed during the infection step and when titre was measured using cells adapted to 5% FBS DMEM or OptiPRO medium. Finally, OptiPRO-adapted CV-1 cells were successfully cultivated using Cytodex-1 microcarriers to inform future scale up studies.

Adhesion of cultured human endothelial cells onto methacrylate polymers with varying surface wettability and charge

NARCIS (Netherlands)

van Wachem, P.B.; Hogt, A.H.; Beugeling, T.; Feijen, Jan; Bantjes, A.; Detmers, J.P.; van Aken, W.G.

1987-01-01

The adhesion of human endothelial cells (HEC) onto a series of well-characterized methacrylate polymer surfaces with varying wettabilities and surface charges was studied either in serum-containing (CMS) or in serum-free (CM) culture medium. HEC adhesion in CMS onto (co)polymers * of hydroxyethyl

Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?

Science.gov (United States)

Burnouf, Thierry; Strunk, Dirk; Koh, Mickey B C; Schallmoser, Katharina

2016-01-01

The essential physiological role of platelets in wound healing and tissue repair builds the rationale for the use of human platelet derivatives in regenerative medicine. Abundant growth factors and cytokines stored in platelet granules can be naturally released by thrombin activation and clotting or artificially by freeze/thaw-mediated platelet lysis, sonication or chemical treatment. Human platelet lysate prepared by the various release strategies has been established as a suitable alternative to fetal bovine serum as culture medium supplement, enabling efficient propagation of human cells under animal serum-free conditions for a multiplicity of applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet derived products for inducing human cell proliferation and differentiation has also uncovered a considerable variability of human platelet lysate preparations which limits comparability of results. The main variations discussed herein encompass aspects of donor selection, preparation of the starting material, the possibility for pooling in plasma or additive solution, the implementation of pathogen inactivation and consideration of ABO blood groups, all of which can influence applicability. This review outlines the current knowledge about human platelet lysate as a powerful additive for human cell propagation and highlights its role as a prevailing supplement for human cell culture capable to replace animal serum in a growing spectrum of applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.

Science.gov (United States)

Valtink, Monika; Donath, Patricia; Engelmann, Katrin; Knels, Lilla

2016-02-01

To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system. The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS). The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system. The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

RNA synthesis in primary cultures of adult rat hepatocytes

International Nuclear Information System (INIS)

Fugassa, E.; Gallo, G.; Voci, A.; Cordone, A.

1983-01-01

The ability of hepatocyte monolayers to synthesize RNA was investigated by measuring [3H]orotic acid incorporation into RNA and the total nuclear RNA polymerase activity as a function of the time in culture. The results demonstrate that primary cultures of hepatocytes maintained in a chemically defined serum- and hormone-free medium are able to synthesize RNA actively. This ability increases within the first 2 d of culture, despite the concomitant decrease in [3H]orotic acid uptake, and decreases only after 3 d. Factors such as serum, insulin, and dexamethasone, known to improve maintenance of functional hepatocytes, markedly stimulate the uptake of labeled precursor without apparently affecting the rate of RNA synthesis by cultured cells. It is suggested that the culture of adult rat hepatocytes provides a useful experimental model for the studies of hormonal regulation of transcription in liver

The comparison of free androgen index and serum free testosterone levels in women with hirsutism or polycystic ovary syndrome

Directory of Open Access Journals (Sweden)

M. Metin Yıldırımkaya

2011-06-01

Full Text Available In many laboratories free testosterone can not be measured, so that free androgen index is suggested instead. The aim of this study was to compare free androgen index and serum free testosterone levels measured by radioimmunoassay in women with hirsutism or polycystic ovary syndrome.Materials and methods: Totally 94 women referred to the polyclinics of Ankara Numune Hospital were retrospectively included. Three patient groups were composed; 55 of hirsutism, 20 of polycystic ovary syndrome and 19 of both hirsutism and polycystic ovary syndrome. Total testosterone and sex hormone binding globuline levels were measured by chemiluminescence method and free testosterone levels were measured by radioimmunoassay. Free androgen index was calculated from total testosterone and sex hormone binding globuline.Results: There was a significant positive correlation between free testosterone and free androgen index in patients with hirsutism, in patients with polycystic ovary syndrome, in patients with hirsutism and polycystic ovary syndrome, and in total patient group [r(hirsutism=0,597, r(PCOS=0,617, r(hirsutism and PCOS=0,779, r(total patient group=0,649, P<0,01].Receiver operating characteristics curves were drawn to assess the diagnostic power of parameters for all patient groups [For hirsutism (n=55 auROC (FT=0,431 auROC (FAI=0,485] [For PCOS (n=20 auROC (FT=0,431 auROC (FAI=0,359] [For hirsutism and PCOS (n=19 auROC (FT=0,676 auROC (FAI=0,669]. In our study, free testosterone and free androgen index were found useful to diagnose ‘hirsutism and polycystic ovary syndrome’ but not others.Conclusion: Free androgen index can be used instead of free testosterone in hirsutism and polycystic ovary syndrome for diagnosis. J Clin Exp Invest 2011;2(2:152-6

PRGF exerts more potent proliferative and anti-inflammatory effects than autologous serum on a cell culture inflammatory model.

Science.gov (United States)

Anitua, E; Muruzabal, F; de la Fuente, M; Riestra, A; Merayo-Lloves, J; Orive, G

2016-10-01

Ocular graft versus host disease (oGVHD) is part of a systemic inflammatory disease that usually affects ocular surface tissues manifesting as a dry eye syndrome. Current treatments provide unsatisfactory results. Blood-derived products, like plasma rich in growth factors (PRGF) emerge as a potential therapy for this disease. The purpose of this study was to evaluate the tissue regeneration and anti-inflammatory capability of PRGF, an autologous platelet enriched plasma eye-drop, compared to autologous serum (AS) obtained from oGVHD patients on ocular surface cells cultured in a pro-inflammatory environment. PRGF and AS were obtained from four GVHD patients. Cell proliferation and inflammation markers, intercellular adhesion molecule-1 (ICAM-1) and cyclooxygenase-2 (COX-2), were measured in corneal and conjunctival fibroblastic cells cultured under pro-inflammatory conditions and after treatment with PRGF or AS eye drops. Moreover, cell proliferation increased after treatment with PRGF and AS, though this enhancement in the case of keratocytes was significantly higher with PRGF. PRGF eye drops showed a significant reduction of both inflammatory markers with respect to the initial inflammatory situation and to the AS treatment. Our results concluded that PRGF exerts more potent regenerative and anti-inflammatory effects than autologous serum on ocular surface fibroblasts treated with pro-inflammatory IL-1β and TNFα. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cytoarchitecture in cultured rat neocortex explants

NARCIS (Netherlands)

de Jong, B. M.; Ruijter, J. M.; Romijn, H. J.

1988-01-01

Neocortex explants obtained from 6-day-old rat pups and cultured in a serum-free medium from 5 hr to 13 days in vitro (DIV) show preservation of cytoarchitectural characteristics. Major changes in the size of the explants and their layers occur during the first 2 DIV. A radial arrangement of neurons

Pedagogical Conditions of Future Philologists’ Research Culture Formation

Directory of Open Access Journals (Sweden)

Marina Trufkina

2014-08-01

Full Text Available The article deals with the problem of - the pedagogical conditions- and it discloses the give phenomenon. In the following work there are outlined three kinds of pedagogical conditions that determine the formation of the future philologist's research culture and it also gives their detailed analysis. The urgency of the paper is determined by progressive methods of contemporary higher education. The aim of the work is to analyse pedagogical conditions that contribute to the research culture formation. The outlook of our investigations is connected with the detailed analysis of the Ŗresearch cultureŗ phenomenon, its components and pedagogical conditions contributing to its development.

Serum TBG and T4 concentration in non-thyroidal diseases

International Nuclear Information System (INIS)

Sasaki, Y.; Tobari, C.; Sekita, N.; Onodera, Y.; Asazu, M.; Someya, K.

1983-01-01

Routinely available radioassay kits have recently enabled the measurement of serum concentrations of thyroxine binding globulin (TBG) and thyroxine (T 4 ), both total (TT 4 ) and free (FT 4 ) in various disease conditions. Serum TBG and T 4 level were measured in variety of non-thyroidal diseases, of which significance was evaluated in comparison with that in thyroidal diseases. Abnormal serum TBG concentrations in various non-thyroidal diseases and pregnancy result in abnormal serum TT 4 levels, which may cause difficulty in differentiation of these conditions from hyper- or hypothyroidal states. Serum FT 4 levels give better indicator than TT 4 , though the difference among RIA kits are considerably large. However, measurement of serum FT 4 levels alone is not sufficient to distinguish non-thyroidal disease from thyroidal diseases with abnormal thyroidal function. The differentiation has to be based on the combination of clinical findings and results of multiple thyroidal function tests

Serum Free Light Chains in Neoplastic Monoclonal Gammopathies: Relative Under-Detection of Lambda Dominant Kappa/Lambda Ratio, and Underproduction of Free Lambda Light Chains, as Compared to Kappa Light Chains, in Patients With Neoplastic Monoclonal Gammopathies.

Science.gov (United States)

Lee, Won Sok; Singh, Gurmukh

2018-07-01

Quantitative evaluation of serum free light chains is recommended for the work up of monoclonal gammopathies. Immunoglobulin light chains are generally produced in excess of heavy chains. In patients with monoclonal gammopathy, κ/λ ratio is abnormal less frequently with lambda chain lesions. This study was undertaken to ascertain if the levels of overproduction of the two light chain types and their detection rates are different in patients with neoplastic monoclonal gammopathies. Results of serum protein electrophoresis (SPEP), serum protein immunofixation electrophoresis (SIFE), urine protein electrophoresis (UPEP), urine protein immunofixation electrophoresis (UIFE), and serum free light chain assay (SFLCA) in patients with monoclonal gammopathies were examined retrospectively. The κ/λ ratios were appropriately abnormal more often in kappa chain lesions. Ratios of κ/λ were normal in about 25% of patients with lambda chain lesions in whom free homogenous lambda light chains were detectable in urine. An illustrative case suggests underproduction of free lambda light chains, in some instances. The lower prevalence of lambda dominant κ/λ ratio in lesions with lambda light chains is estimated to be due to relative under-detection of lambda dominant κ/λ ratio in about 25% of the patients and because lambda chains are not produced in as much excess of heavy chains as are kappa chains, in about 5% of the patients. The results question the medical necessity and clinical usefulness of the serum free light chain assay. UPEP/UIFE is under-utilized.

The Effect of Calcusol™ to the Plasma Free Radical and Serum Creatinin in Mus Musculus Nephrolithiasis Model

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A’liyatur Rosyidah

2013-09-01

Full Text Available Calcusol™ is a traditional medicine (jamu made from Tempuyung (Sonchus arvensis extract and is usually used for cure kidney stone disease. However, there has not been any studies which investigate the its mechanism. The aim of this study is to know the effect of Calcusol™ to the plasma free radical and serum creatinin of Mus musculus model for renal calcium-oxalate accumulation. This study is carried out by administration of Porang (Amorphophallus muelleri tuber flour for 3 months with the doses 6 mg/100g BW to induce renal calcium oxalate accumulation and Calcusol™ treatment for 7 days with the doses of 3.3mg/g BW. Group I was used as a control group. Group II was only given porang every day for 3 months. Group III was only given Calcusol™ for 7 days. Group IV were given porang for 3 months then given Calcusol™ for 7 days. Group V were given porang and Calcusol™ simultaneously for 3 months. Porang and CalcusolTM is administrated orally. Blood was collected from the tail of the animal for serum creatinin test and plasma free radical test using TBARS method. The data was analyzed using ANOVA followed by Tukey HSD to compare the means employing SPSS 16.0 for windows. The result of the research shows that the treatment Calcusol™ on mice model for renal calcium oxalate accumulation. The content of MDA at group I, group II, group III, group IV and group V, respectivelyis 0.81±0.5 mg/ml; 2.63±0.8 mg/ml; 0.56±0.5 mg/ml; 2.09±0.9 mg/ml and 0.17±0.17 mg/ml. The content of serum creatinine are 0.15±0.04 mg/dL; 0.13±0.03 mg/dL; 0.12±0.08 mg/dL; 0.11±0.016 mg/dL and 0.14±0.015 mg/dL at group I, group II, group III, Group IV, and group V respectively. This indicates that Calcusol™ decreases plasma free radical production during renal stone formation, while serum creatinin reduced but not significantly changed. Calcusol™ has an effect to decrease free radical during renalstone formation inmice model for calcium oxalate accumulation

The influence of parenteral nitrogen feeding on free amino acid composition of blood serum and hepatic tissue of irradiated rats

International Nuclear Information System (INIS)

Mil'ko, V.I.; Kirichenko, A.V.; Chalaya, L.A.

1985-01-01

A considerable change in the free am ino acid composition of blood serum and hepatic tissued was noted on the 7th and 14th days following total-body X-irradiation of rats with a dose of 2.9 Gy. The total free amino acid content of blood serum increased and that of hepatic tissue decreased by 85% (on an average) as compared to the intact controls. Quantitative changes in the content of individual amino acids were analysed. Polyamine injected enterally for 7 days and parenterally for 3 days after irradiation a the elimination of the postirradiation changes in the amino acid balance

Effects of cell suspension and cell·free culture filtrate of Pseudomonas aeruginosa in the control of root rot-root kont disease complex of tomato (Lycopersicon esculentum Mill.

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I. A. Siddiqui

2013-12-01

Full Text Available The plant growth-promoting rhizobacterium Pseudomonas aeruginosa strain IE-6 was tested for antagonistic activity towards Meloidogyne javanica, the root-knot nematode and soilbome root-infecting fungi viz., Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani under laboratory and greenhouse conditions. Cell-free culture filtrate of the bacterium caused significant reduction in egg hatching of M.javanica and inhibited radial growth of fungi in vitro. Cell-free culture filtrate also caused lyses in mycelium of F.solani. Under greenhouse conditions, soil drenches with the aqueous cell suspension or cell-free culture resulted in a considerable reduction in nematode population densities in soil and subsequent root-knot development due to M.javanica. In addition to nematode control, rhizobacterium application also inhibited root-infection caused by soilborne root~infecting fungi with significant enhancement of growth of tomato seedlings.

Micro-bead injection spectroscopy for label-free automated determination of immunoglobulin G in human serum.

Science.gov (United States)

Ramos, Inês I; Magalhães, Luís M; Barreiros, Luisa; Reis, Salette; Lima, José L F C; Segundo, Marcela A

2018-01-01

Immunoglobulin G (IgG) represents the major fraction of antibodies in healthy adult human serum, and deviations from physiological levels are a generic marker of disease corresponding to different pathologies. Therefore, screening methods for IgG evaluation are a valuable aid to diagnostics. The present work proposes a rapid, automatic, and miniaturized method based on UV-vis micro-bead injection spectroscopy (μ-BIS) for the real-time determination of human serum IgG with label-free detection. Relying on attachment of IgG in rec-protein G immobilized in Sepharose 4B, a bioaffinity column is automatically assembled, where IgG is selectively retained and determined by on-column optical density measurement. A "dilution-and-shoot" approach (50 to 200 times) was implemented without further sample treatment because interferences were flushed out of the column upon sample loading, with minimization of carryover and cross-contamination by automatically discarding the sorbent (0.2 mg) after each determination. No interference from human serum albumin at 60 mg mL -1 in undiluted sample was found. The method allowed IgG determination in the range 100-300 μg mL -1 (corresponding to 5.0-60 mg mL -1 in undiluted samples), with a detection limit of 33 μg mL -1 (1.7 mg mL -1 for samples, dilution factor of 50). RSD values were time-to-result decreased from several hours to times, showing the potential of the proposed approach as a point-of-care method. Graphical abstract Micro-Bead Injection Spectroscopy method for real time, automated and label-free determination of total serum human Immunoglobulin G (IgG). The method was designed for Lab-on-Valve (LOV) platforms using a miniaturised protein G bioaffinity separative approach. IgG are separated from serum matrix components upon quantification with low non-specific binding in less than 5 min.

Agency Beliefs Over Time and Across Cultures: Free Will Beliefs Predict Higher Job Satisfaction

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Feldman, Gilad; Farh, Jiing-Lih; Wong, Kin Fai Ellick

2017-01-01

In three studies, we examined the relationship between free will beliefs and job satisfaction over time and across cultures. Study 1 examined 252 Taiwanese real-estate agents over a 3-months period. Study 2 examined job satisfaction for 137 American workers on an online labor market over a 6-months period. Study 3 extended to a large sample of 14,062 employees from 16 countries and examined country-level moderators. We found a consistent positive relationship between the belief in free will and job satisfaction. The relationship was above and beyond other agency constructs (Study 2), mediated by perceived autonomy (Studies 2-3), and stronger in countries with a higher national endorsement of the belief in free will (Study 3). We conclude that free-will beliefs predict outcomes over time and across cultures beyond other agency constructs. We call for more cross-cultural and longitudinal studies examining free-will beliefs as predictors of real-life outcomes. PMID:29191084

The impact of hypoxemia on serum total and free prostate-specific antigen levels in patients with chronic obstructive pulmonary disease.

Science.gov (United States)

Ozge, Cengiz; Bozlu, Murat; Ozgur, Eylem Sercan; Tek, Mesut; Tunckiran, Ahmet; Muslu, Necati; Ilvan, Ahmet

2015-05-01

Prostate-specific antigen (PSA) is the most important biochemical marker in the diagnosis and follow-up of patients with prostate cancer. In recent years, a relationship between PSA levels and hypoxic conditions has been described. However, no study has investigated the PSA levels in patients with chronic obstructive pulmonary disease (COPD). The aim of the present study was to investigate the impact of hypoxemia on serum total (tPSA) and free PSA (fPSA) levels in patients with COPD. Between January 2010 and January 2014, 95 male patients who hospitalized for acute exacerbations of COPD and 80 control subjects were enrolled in the study. Serum tPSA and fPSA levels and f/tPSA ratios were determined in all patients on the first day of hospitalization (exacerbation) and 7 days after the treatment (stable state). Statistical analysis included paired t test and Mann-Whitney U test. No statistically significant differences were found between COPD and control groups with regard to the baseline characteristics, except for smoking status. The levels of serum tPSA and fPSA during exacerbation of COPD were significantly higher than the levels of the stable period (p 0.05). Hypoxemia during acute exacerbation of COPD can cause a rise in serum tPSA and fPSA levels, but f/tPSA ratio is not affected. Acute exacerbation of COPD may be added to list of the events in which PSA measurements must be interpreted with caution.

Differentially expressed genes: OCT-4, SOX2, STAT3, CDH1 and CDH2, in cultured mesenchymal stem cells challenged with serum of women with endometriosis

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Ehab Salama

2018-06-01

Full Text Available Endometriosis is a common chronic gynecological disorder defined as the presence of ectopic functional endometrial tissues, outside uterine cavity, primarily on the pelvic peritoneum and the ovaries. Several studies revealed a correlation between aberrant stem-cell activity in the endometrium and endometriosis. Yet the molecular and cellular behaviors of mesnchymal stem cells in development of endometriosis are hampered by lack of invitro experiments. Our aim was to explore morphological and molecular changes associated with mesenchymal stem cells (MSCs exposition to serum derived from women with severe endometriosis. Two cell cultures of MSCs isolated from endometrial tissues of two endometriosis-free women. Each cell culture was treated individually with the serum of women with endometriosis (experimental group/n = 7, and serum of women without endometriosis (control group/ n = 4 for 14 days. Quantitative Real-Time PCR was performed later to reveal expression of OCT-4, CDH1 and CDH2, STAT3 and SOX2 genes. Morphologically, cells showed no significant changes. However from molecular point of view, we found increased expression in OCT-4, CDH1 and CDH2. For STAT3 and SOX2 we did not find a significant difference. This study shows that endometriosis serum induced molecular changes in human endometrial MSCs (EnMSCs that might be related to altered cell behavior which may be a step in differentiation that may be completed invivo by other factors to complete the process of transition. Further researches are needed for optimization to reach differentiation. Keywords: Endometriosis, Mesnchymal stem cells, OCT-4, SOX2, STAT3, E-cadherin, N-cadherin

Changes during hibernation in different phospholipid and free and esterified cholesterol serum levels in black bears

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Chauhan, V.; Sheikh, A.; Chauhan, A.; Tsiouris, J.; Malik, M.; Vaughan, M.

2002-01-01

During hibernation, fat is known to be the preferred source of energy. A detailed analysis of different phospholipids, as well as free and esterified cholesterol, was conducted to investigate lipid abnormalities during hibernation. The levels of total phospholipids and total cholesterol in the serum of black bears were found to increase significantly in hibernation as compared with the active state. Both free and esterified cholesterol were increased in the hibernating state in comparison with the active state (P biologie mole??culaire. All rights reserved.

Culture conditions tailored to the cell of origin are critical for maintaining native properties and tumorigenicity of glioma cells.

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Ledur, Pítia F; Liu, Chong; He, Hua; Harris, Alexandra R; Minussi, Darlan C; Zhou, Hai-Yan; Shaffrey, Mark E; Asthagiri, Ashok; Lopes, Maria Beatriz S; Schiff, David; Lu, Yi-Cheng; Mandell, James W; Lenz, Guido; Zong, Hui

2016-10-01

Cell culture plays a pivotal role in cancer research. However, culture-induced changes in biological properties of tumor cells profoundly affect research reproducibility and translational potential. Establishing culture conditions tailored to the cancer cell of origin could resolve this problem. For glioma research, it has been previously shown that replacing serum with defined growth factors for neural stem cells (NSCs) greatly improved the retention of gene expression profile and tumorigenicity. However, among all molecular subtypes of glioma, our laboratory and others have previously shown that the oligodendrocyte precursor cell (OPC) rather than the NSC serves as the cell of origin for the proneural subtype, raising questions regarding the suitability of NSC-tailored media for culturing proneural glioma cells. OPC-originated mouse glioma cells were cultured in conditions for normal OPCs or NSCs, respectively, for multiple passages. Gene expression profiles, morphologies, tumorigenicity, and drug responsiveness of cultured cells were examined in comparison with freshly isolated tumor cells. OPC media-cultured glioma cells maintained tumorigenicity, gene expression profiles, and morphologies similar to freshly isolated tumor cells. In contrast, NSC-media cultured glioma cells gradually lost their OPC features and most tumor-initiating ability and acquired heightened sensitivity to temozolomide. To improve experimental reproducibility and translational potential of glioma research, it is important to identify the cell of origin, and subsequently apply this knowledge to establish culture conditions that allow the retention of native properties of tumor cells. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Serum free amino acid concentration in hepatic lipidosis of dairy cows in the periparturient period.

Science.gov (United States)

Shibano, Ken-ichi; Kawamura, Seiichi

2006-04-01

Blood samples were taken from eight multiparous cows at a dairy farm on eight occasions between the prepartum period and peak lactation to study the serum concentrations of amino acids and biochemical constituents. The cows were classified as having either severe hepatic lipidosis (HL) or non-hepatic lipidosis (non-HL) according to their clinical condition after calving and changes in serum biochemical parameters. The serum concentrations of beta-hydroxybutyric acid were higher in the HL group than in the non-HL group (ANOVA: phepatic lipidosis.

Create, share and learn. Experiences with free software, free culture and collaboration in formal and non formal education in Colombia

OpenAIRE

Medina Cardona, Luis Fernando

2012-01-01

New media technologies, specially software have been of great impact in modern society.The combination of computer/software/networks as creative machines is present in everyday life. This article is focused in this interactions specially from the perspective of free open source software (FOSS). In doing so, the influence of its values is traced from the original hacker culture ethics to the free software four freedoms, showing a software explained culture as in the software studies discipline...

Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Skelton, David; Goodyear, Abbey [Florida State University, Department of Chemistry and Biochemistry (United States); Ni, DaQun; Walton, Wendy J.; Rolle, Myron; Hare, Joan T. [Florida State University, Institute of Molecular Biophysics (United States); Logan, Timothy M., E-mail: tlogan@fsu.ed [Florida State University, Department of Chemistry and Biochemistry (United States)

2010-10-15

NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-{sup 13}C-glucose and {sup 15}N-glutamate as labeled precursors. This study suggests that uniformly {sup 15}N,{sup 13}C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

International Nuclear Information System (INIS)

Skelton, David; Goodyear, Abbey; Ni, DaQun; Walton, Wendy J.; Rolle, Myron; Hare, Joan T.; Logan, Timothy M.

2010-01-01

NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U- 13 C-glucose and 15 N-glutamate as labeled precursors. This study suggests that uniformly 15 N, 13 C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals.

Science.gov (United States)

Butler, Michael

2005-08-01

There has been a rapid increase in the number and demand for approved biopharmaceuticals produced from animal cell culture processes over the last few years. In part, this has been due to the efficacy of several humanized monoclonal antibodies that are required at large doses for therapeutic use. There have also been several identifiable advances in animal cell technology that has enabled efficient biomanufacture of these products. Gene vector systems allow high specific protein expression and some minimize the undesirable process of gene silencing that may occur in prolonged culture. Characterization of cellular metabolism and physiology has enabled the design of fed-batch and perfusion bioreactor processes that has allowed a significant improvement in product yield, some of which are now approaching 5 g/L. Many of these processes are now being designed in serum-free and animal-component-free media to ensure that products are not contaminated with the adventitious agents found in bovine serum. There are several areas that can be identified that could lead to further improvement in cell culture systems. This includes the down-regulation of apoptosis to enable prolonged cell survival under potentially adverse conditions. The characterization of the critical parameters of glycosylation should enable process control to reduce the heterogeneity of glycoforms so that production processes are consistent. Further improvement may also be made by the identification of glycoforms with enhanced biological activity to enhance clinical efficacy. The ability to produce the ever-increasing number of biopharmaceuticals by animal cell culture is dependent on sufficient bioreactor capacity in the industry. A recent shortfall in available worldwide culture capacity has encouraged commercial activity in contract manufacturing operations. However, some analysts indicate that this still may not be enough and that future manufacturing demand may exceed production capacity as the number

Diagnostic significance of the serum thyroid hormone indicies in various thyroid diseases

International Nuclear Information System (INIS)

Han, B.H.; Ko, S.M.; Yoon, S.R.; Ro, H.K.

1980-01-01

In an attempt to evaluate the diagnostic significance of the serum thyroid hormones in various thyroid function states, the author measured serum T 3 uptake, serum T 3 , serum T 4 , serum free T 4 and free T 4 index in 27 cases of normal subjects, 11 cases of hypothyroidism, 152 cases of euthyroidism and 81 cases of hyperthyroidism by the radioimmunoassay method. The results were as follows: 1) The ranges of serum thyroid hormones in normal subjects were, serum T 3 uptake; 27.4-42.1%, serum T 3 ; 93-245 ng/dl, serum T 4 ; 4.08-12.9 ng/dl and serum free T 4 ; 0.57-1.53 ng/dl (M+-2 S.D.). 2) Free T 4 index and serum T 4 show relatively high diagnostic value in euthyroidism group, and serum T 3 and T 4 in hypothyroidism group, while serum T 3 , free T 4 and T 4 show relatively high diagnostic value in hyperthyroidism group. 3) There were significant correlation between free T 4 index and serum T 4 (r=0.68) and between free T 4 index and serum free T 4 (r=0.67) in hyperthyroidism group. (author)

Differentiating Graves' disease from subacute thyroiditis using ratio of serum free triiodothyronine to free thyroxine.

Science.gov (United States)

Sriphrapradang, Chutintorn; Bhasipol, Adikan

2016-09-01

The measurement of free thyroid hormone, instead of the total form, is more commonly used in current practice. We aimed to evaluate the usefulness of the ratio of serum free triiodothyronine (FT3, pg/mL) to free thyroxine (FT4, ng/dL) for differentiating Graves' disease from subacute thyroiditis. Medical records of thyrotoxic patients aged >15 years who had measurement of FT3, FT4 and thyrotropin on the first diagnosis of thyrotoxicosis before initiating treatment were retrospectively reviewed. Data were collected from all clinics, and were not limited to the endocrine clinic. Pregnant women were excluded. A total of 548 patients (468 with Graves' disease, 40 with subacute thyroiditis and 40 with toxic adenoma/multinodular goiter) were recruited. Mean age was 43.9 ± 15.4 years. Most were female 434 (79.2%), and goiter was present in 55.3%. Prevalence of T3-toxicosis and T4-toxicosis were 5.6% and 6.6%, respectively. Mean FT3/FT4 ratios were 4.62 ± 2 (10(-2) pg/ng) in patients with Graves' disease and 2.73 ± 0.5 in subacute thyroiditis. The area under the ROC curve of the FT3/FT4 ratio for diagnosis of Graves' disease was 0.83 (95%CI, 0.76-0.91). Cutoff level of this ratio >4.4 offered sensitivity of 47.2% and specificity of 92.8%. FT3/FT4 ratio of >4.4 (10(-2) pg/ng) may help in differentiating the cause of thyrotoxicosis.

Optimal growth conditions for Isochrysis galbana

Energy Technology Data Exchange (ETDEWEB)

Kaplan, D; Cohen, Z; Abeliovich, A

1986-01-01

Environmental and nutritional growth conditions of the unicellular microalga Isochrysis galbana were studied under laboratory conditions. The information obtained was used for cultivating the alga in outdoor miniponds. Outdoor cultures stayed monoalgal and free of predators as long as the temperature did not fall below 19 degrees C and the rate of dilution did not exceed 40% of the culture's volume. Isochrysis galbana grown in outdoor cultures provided lipid concentrations of 24-28% of ash free dry matter. 12 references.

Weights, hematology and serum chemistry of seven species of free-ranging tropical pelagic seabirds

Science.gov (United States)

Work, Thierry M.

1996-01-01

I established reference values for weight, hematology, and serum chemistry for seven species of free-ranging Hawaiian tropical pelagic seabirds comprising three orders (Procellariiformes, Pelecaniformes, Charadriiformes) and six families (Procellariidae, Phaethontidae, Diomedeidae, Sulidae, Fregatidae, and Laridae). Species examined included 84 Hawaiian dark-rumped petrels (Pterodoma phaeopygia), 90 wedge-tailed shearwaters (Puffinus pacificus), 151 Laysan albatrosses (Diomedea immutabilis), 69 red-footed boobies (Sula sula), 154 red-tailed tropicbirds (Phaeton rubricauda), 90 great frigatebirds (Fregata minor), and 72 sooty terns (Sterna fuscata). Hematocrit, total plasma solids, total and differential white cell counts, serum glucose, calcium, phosphorus, uric acid, total protein, albumin, globulin, aspartate aminotransferase and creatinine phosphokinase were analyzed. Among and within species, hematology and chemistry values varied with age, sex, season, and island of collection. Despite this variation, order-wide trends were observed.

Serum Removal from Culture Induces Growth Arrest, Ploidy Alteration, Decrease in Infectivity and Differential Expression of Crucial Genes in Leishmania infantum Promastigotes.

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Pedro J Alcolea

Full Text Available Leishmania infantum is one of the species responsible for visceral leishmaniasis. This species is distributed basically in the Mediterranean basin. A recent outbreak in humans has been reported in Spain. Axenic cultures are performed for most procedures with Leishmania spp. promastigotes. This model is stable and reproducible and mimics the conditions of the gut of the sand fly host, which is the natural environment of promastigote development. Culture media are undefined because they contain mammalian serum, which is a rich source of complex lipids and proteins. Serum deprivation slows down the growth kinetics and therefore, yield in biomass. In fact, we have confirmed that the growth rate decreases, as well as infectivity. Ploidy is also affected. Regarding the transcriptome, a high-throughput approach has revealed a low differential expression rate but important differentially regulated genes. The most remarkable profiles are: up-regulation of the GINS Psf3, the fatty acyl-CoA synthase (FAS1, the glyoxylase I (GLO1, the hydrophilic surface protein B (HASPB, the methylmalonyl-CoA epimerase (MMCE and an amastin gene; and down-regulation of the gPEPCK and the arginase. Implications for metabolic adaptations, differentiation and infectivity are discussed herein.

Serum Removal from Culture Induces Growth Arrest, Ploidy Alteration, Decrease in Infectivity and Differential Expression of Crucial Genes in Leishmania infantum Promastigotes.

Science.gov (United States)

Alcolea, Pedro J; Alonso, Ana; Moreno-Izquierdo, Miguel A; Degayón, María A; Moreno, Inmaculada; Larraga, Vicente

2016-01-01

Leishmania infantum is one of the species responsible for visceral leishmaniasis. This species is distributed basically in the Mediterranean basin. A recent outbreak in humans has been reported in Spain. Axenic cultures are performed for most procedures with Leishmania spp. promastigotes. This model is stable and reproducible and mimics the conditions of the gut of the sand fly host, which is the natural environment of promastigote development. Culture media are undefined because they contain mammalian serum, which is a rich source of complex lipids and proteins. Serum deprivation slows down the growth kinetics and therefore, yield in biomass. In fact, we have confirmed that the growth rate decreases, as well as infectivity. Ploidy is also affected. Regarding the transcriptome, a high-throughput approach has revealed a low differential expression rate but important differentially regulated genes. The most remarkable profiles are: up-regulation of the GINS Psf3, the fatty acyl-CoA synthase (FAS1), the glyoxylase I (GLO1), the hydrophilic surface protein B (HASPB), the methylmalonyl-CoA epimerase (MMCE) and an amastin gene; and down-regulation of the gPEPCK and the arginase. Implications for metabolic adaptations, differentiation and infectivity are discussed herein.

Comparison of Different Cytokine Conditions Reveals Resveratrol as a New Molecule for Ex Vivo Cultivation of Cord Blood-Derived Hematopoietic Stem Cells.

Science.gov (United States)

Heinz, Niels; Ehrnström, Birgitta; Schambach, Axel; Schwarzer, Adrian; Modlich, Ute; Schiedlmeier, Bernhard

2015-09-01

Human cord blood (CB)-derived hematopoietic stem cells (HSCs) are an interesting source for HSC transplantation. However, the number of collected CB-HSCs is often too low for one transplantation; therefore, ex vivo expansion of CB-HSCs is desirable. Current expansion protocols are based on the use of cytokine combinations, including insulin-like growth factor-binding protein 2 (IGFBP2) and angiopoietin-like proteins, or combinations with "small molecules" such as stemregenin-1. The aim of our project was to compare the potential of different CB-HSC expansion strategies side-by-side by phenotypical analysis in vitro and serial engraftment properties in NOD/SCID/IL2rg-/- (NSG) immunodeficient mice. We further identified resveratrol, a naturally occurring polyphenol, as a new, alternative small molecule combined with cytokines to facilitate serum-free ex vivo expansion of human CB-HSCs. The cultivation in resveratrol preserved the CB-HSC phenotype in vitro most efficiently and was ∼2 times more potent than commonly used cytokine conditions (including stem cell factor, thrombopoietin, Fms-related tyrosine kinase 3 ligand, interleukin-6) and the recently established serum-free culture, including IGFBP2 and angiopoietin-like 5. Serial transplantation studies further confirmed resveratrol to support robust multilineage engraftment in primary and secondary NSG recipients. Therefore, our work proposes resveratrol as a new small molecule for improved ex vivo culture and modification of human HSCs based on an efficient ex vivo propagation of the HSC fate. Human cord blood (CB)-derived hematopoietic stem cells (HSCs) are an important source for HSC transplantations but restricted in their usage because of their low numbers. In gene therapy, modifications of HSCs relies on their ex vivo modification without losing their stemness properties. Therefore, ex vivo cultivation and expansion of CB-HSCs is important for their effective application in HSC transplantation and gene

Coupling the Gaussian Free Fields with Free and with Zero Boundary Conditions via Common Level Lines

Science.gov (United States)

Qian, Wei; Werner, Wendelin

2018-06-01

We point out a new simple way to couple the Gaussian Free Field (GFF) with free boundary conditions in a two-dimensional domain with the GFF with zero boundary conditions in the same domain: Starting from the latter, one just has to sample at random all the signs of the height gaps on its boundary-touching zero-level lines (these signs are alternating for the zero-boundary GFF) in order to obtain a free boundary GFF. Constructions and couplings of the free boundary GFF and its level lines via soups of reflected Brownian loops and their clusters are also discussed. Such considerations show for instance that in a domain with an axis of symmetry, if one looks at the overlay of a single usual Conformal Loop Ensemble CLE3 with its own symmetric image, one obtains the CLE4-type collection of level lines of a GFF with mixed zero/free boundary conditions in the half-domain.

A comparison of serum amyloid A (SAA) synthesis with that of the pentraxins: Serum amyloid P (SAP) and C-reactive protein (CRP)

International Nuclear Information System (INIS)

Tatsuta, E.; Shirahama, T.; Sipe, J.D.; Skinner, M.

1986-01-01

Serum amyloid A (SAA) and serum amyloid P (SAP) were detected in cultures of hepatocytes which had been isolated from normal CBA/J mice by the collagenase perfusion technique. SAP production in 24 h cultures was more resistant than SAA and total protein synthesis to inhibition by actinomycin D, but was more sensitive to inhibition by 48 h. However, the production of SAP was more sensitive to cycloheximide than SAA and total protein throughout the 48 hr incubation period. SAP and SAA levels in the culture media were suppressed by treatment of liver cells with 10 -6 M of colchicine for 48 h. Inhibition of SAP production by colchicine was the same regardless of culture condition, but the effect of colchicine on SAA synthesis varied according to the presence of serum of monokine. These observations also support the concept that the two amyloid proteins are produced under different regulatory mechanisms. When C-reactive protein (CRP) was not detected in the sera of patients with severe chronic liver diseases, the SAA levels were very low. When CRP was detected, SAA values were within the normal range. Thus, in order to produce SAA, liver cells in these patients not only were viable but also maintained their specialized function

Interrelationships of serum testosterone and free testosterone index with FFM and strength in aging men.

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Roy, Tracey Ann; Blackman, Marc R; Harman, S Mitchell; Tobin, Jordan D; Schrager, Matthew; Metter, E Jeffery

2002-08-01

Muscle mass and strength losses during aging may be associated with declining levels of serum testosterone (T) in men. Few studies have shown a direct relationship between T and muscle mass and strength. Subjects were 262 men, aged 24-90 yr, from the Baltimore Longitudinal Study of Aging, who had T and sex hormone-binding globulin sex hormone-binding globulin (SHBG) measurements, from which the free T index (FTI) was calculated (T/SHBG) from serum samples collected longitudinally since 1963, total body fat mass and arm and leg fat-free mass (FFM) by dual-energy X-ray absorptiometry and arm and leg strength by dynanomometry. Mixed-effects models estimated T and FTI at the time of mass and strength measurements. Age, total body fat, arm and leg FFM, T, and FTI were significantly associated with concentric and eccentric strength. FTI, not T, was modestly, but directly, related to arm and leg strength after fat, arm and leg FFM, height, and age were accounted for and indirectly through body mass. FTI is a better predictor of arm and leg strength than T in aging men.

Determination of free cisplatin in medium by differential pulse polarography after ultrasound and cisplatin treatment of a cancer cell culture

International Nuclear Information System (INIS)

Bernard, Vladan; Skorpikova, Jirina; Mornstein, Vojtech; Fojt, Lukas

2011-01-01

The in vitro study was carried out for detection of the cisplatin in free form and in culture medium, depending on various conditions of sonodynamic human ovarian cancer cells A2780 treatment by differential pulse polarography (DPP). For sonodynamic treatment, we used cisplatin alone and combined cisplatin/ultrasound treatments. The ultrasound exposure intensity of 1.0 and 2.0 Wcm 2 in far field for incubation periods 1, 24 and 48 h was used. The parameters of DPP measurements were - 1 s drop time, 5 mV.s -1 voltage scan rate, 50 mV modulation amplitude and negative scanning direction; platinum wire served as counter electrode and Ag|AgCl|3 M KCI as reference electrode. The results showed the dependence of free platinum quantities in culture medium on incubation time and treatment protocol. We found difference in concentration of free cisplatin between conventional application of cisplatin and sonodynamic treatment. The sonodynamic combined treatment of cisplatin and ultrasound field showed a higher cisplatin content in the culture medium than cisplatin treatment alone; a difference of 20% was observed for incubation time 48 h. The results also showed the influence of a time sequence of ultrasound and cytostatics in the sonodynamic treatment. The highest amount of free cisplatin in the solution was found for primary application of cisplatin and the subsequent ultrasound exposure. The quantity of free cisplatin increased with time, namely for time intervals 1-24 h. There was no difference between the DPP signal of cisplatin in reaction mixture containing cells in small quantities and micro-filtered mixture without cells. Thus, the DPP method is suitable for the detection and quantification of free cisplatin in the culture medium of cell suspension. Ultrasound field can be important factor during cytostatic therapy. (author)

Phylogenetically distinct bacteria involve extensive dechlorination of aroclor 1260 in sediment-free cultures.

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Shanquan Wang

Full Text Available Microbial reductive dechlorination of the persistent polychlorinated biphenyls (PCBs is attracting much attention in cleanup of the contaminated environment. Nevertheless, most PCB dechlorinating cultures require presence of sediment or sediment substitutes to maintain their dechlorination activities which hinders subsequent bacterial enrichment and isolation processes. The information on enriching sediment-free PCB dechlorinating cultures is still limited. In this study, 18 microcosms established with soils and sediments were screened for their dechlorination activities on a PCB mixture - Aroclor 1260. After one year of incubation, 10 out of 18 microcosms showed significant PCB dechlorination with distinct dechlorination patterns (e.g., Process H, N and T classified based on profiles of PCB congeners loss and new congeners formation. Through serial transfers in defined medium, six sediment-free PCB dechlorinating cultures (i.e., CW-4, CG-1, CG-3, CG-4, CG-5 and SG-1 were obtained without amending any sediment or sediment-substitutes. PCB dechlorination Process H was the most frequently observed dechlorination pattern, which was found in four sediment-free cultures (CW-4, CG-3, CG-4 and SG-1. Sediment-free culture CG-5 showed the most extensive PCB dechlorination among the six cultures, which was mediated by Process N, resulting in the accumulation of penta- (e.g., 236-24-CB and tetra-chlorobiphenyls (tetra-CBs (e.g., 24-24-CB, 24-25-CB, 24-26-CB and 25-26-CB via dechlorinating 30.44% hepta-CBs and 59.12% hexa-CBs after three months of incubation. For culture CG-1, dechlorinators mainly attacked double flanked meta-chlorines and partially ortho-chlorines, which might represent a novel dechlorination pattern. Phylogenetic analysis showed distinct affiliation of PCB dechlorinators in the microcosms, including Dehalogenimonas and Dehalococcoides species. This study broadens our knowledge in microbial reductive dechlorination of PCBs, and provides

Nutritional manipulation of primate retinas, I: effects of lutein or zeaxanthin supplements on serum and macular pigment in xanthophyll-free rhesus monkeys.

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Neuringer, Martha; Sandstrom, Marita M; Johnson, Elizabeth J; Snodderly, D Max

2004-09-01

The xanthophylls lutein (L) and zeaxanthin (Z) are the primary components of macular pigment (MP) and may protect the macula from age-related degeneration (AMD). In this study, L or Z was fed to rhesus monkeys reared on xanthophyll-free diets to follow the accumulation of serum carotenoids and MP over time. Eighteen rhesus monkeys were fed xanthophyll-free semipurified diets from birth until 7 to 16 years. The diets of six were then supplemented with pure L and six with pure Z at 3.9 micromol/kg per day (2.2 mg/kg per day) for 24 to 56 weeks. At baseline and 4- to 12-week intervals during supplementation, serum carotenoids were measured by HPLC, and MP density was estimated by two-wavelength reflectometry. Serum carotenoids and MP were also measured in monkeys fed a stock diet. Monkeys fed xanthophyll-free diets had no L or Z in serum and no detectable MP. During supplementation, serum L or Z increased rapidly over the first 4 weeks and from 16 weeks onward maintained similar levels, both several times higher than in stock-diet-fed monkeys. The central peak of MP optical density increased to a relatively steady level by 24 to 32 weeks in both L- and Z-fed groups. Rhesus monkeys fed a stock diet had lower blood concentrations of L than those found in humans and other nonhuman primates. Rhesus monkeys respond to either dietary L or Z supplementation with increases in serum xanthophylls and MP, even after life-long xanthophyll deficiency. These animals provide a potential model to study mechanisms of protection from AMD. Copyright Association for Research in Vision and Ophthalmology

Serum complexed and free prostate-specific antigen (PSA) for the diagnosis of the polycystic ovarian syndrome (PCOS).

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Diamandis, Eleftherios P; Stanczyk, Frank Z; Wheeler, Sarah; Mathew, Anu; Stengelin, Martin; Nikolenko, Galina; Glezer, Eli N; Brown, Marshall D; Zheng, Yingye; Chen, Yen-Hao; Wu, Hsiao-Li; Azziz, Ricardo

2017-10-26

Polycystic ovarian syndrome (PCOS) is a common cause of reproductive and metabolic dysfunction. We hypothesized that serum prostate-specific antigen (PSA) may constitute a new biomarker for hyperandrogenism in PCOS. We conducted a cross-sectional study of 45 women with PCOS and 40 controls. Serum from these women was analyzed for androgenic steroids and for complexed PSA (cPSA) and free PSA (fPSA) with a novel fifth- generation assay with a sensitivity of ~10 fg/mL for cPSA and 140 fg/mL for fPSA. cPSA and fPSA levels were about three times higher in PCOS compared to controls. However, in PCOS, cPSA and fPSA did not differ according to waist-to-hip ratio, Ferriman-Gallwey score, or degree of hyperandrogenemia or oligo-ovulation. In PCOS and control women, serum cPSA and fPSA levels were highly correlated with each other, and with free and total testosterone levels, but not with other hormones. Adjusting for age, body mass index (BMI) and race, cPSA was significantly associated with PCOS, with an odds ratio (OR) of 5.67 (95% confidence interval [CI]: 1.86, 22.0). The OR of PCOS for fPSA was 7.04 (95% CI: 1.65, 40.4). A multivariate model that included age, BMI, race and cPSA yielded an area-under-the-receiver-operating-characteristic curve of 0.89. Serum cPSA and fPSA are novel biomarkers for hyperandrogenism in PCOS and may have value for disease diagnosis.

Human platelet lysate is a successful alternative serum supplement for propagation of monocyte-derived dendritic cells.

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Švajger, Urban

2017-04-01

Clinical protocols for dendritic cell (DC) generation from monocytes require the use of animal serum-free supplements. Serum-free media can also require up to 1% of serum supplementation. In addition, recommendations based on the 3Rs (Refinement, Reduction, Replacement) principle also recommend the use of non-animal sera in in vitro studies. The aim of this study was to explore the potential use of platelet lysate (PL) for generation of optimally differentiated DCs from monocytes. Cells were isolated from buffy coats from healthy volunteers using immunomagnetic selection. DCs were differentiated in RPMI1640 supplemented with either 10% fetal bovine serum (FBS), 10% AB serum or 10% PL with the addition of granulocyte monocyte colony stimulating factor and interleukin-4. Generated DCs were assessed for their morphology, viability, endocytotic capacity, surface phenotype (immature, mature and tolerogenic DCs) and activation of important signaling pathways. DC function was evaluated on the basis of their allostimulatory capacity, cytokine profile and ability to induce different T-helper subsets. DCs generated with PL displayed normal viability, morphology and endocytotic capacity. Their differentiation and maturation phenotype was comparable to FBS-cultured DCs. They showed functional plasticity and up-regulated tolerogenic markers in response to their environment. PL-cultured mature DCs displayed unhindered allostimulatory potential and the capacity to induce Th1 responses. The use of PL allowed for activation of crucial signaling proteins associated with DC differentiation and maturation. This study demonstrates for the first time that human PL represents a successful alternative to FBS in differentiation of DCs from monocytes. DCs display the major phenotypic and functional characteristics compared with existing culture protocols. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Non-Mulberry and Mulberry Silk Protein Sericins as Potential Media Supplement for Animal Cell Culture

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Neety Sahu

2016-01-01

Full Text Available Silk protein sericins, in the recent years, find application in cosmetics and pharmaceuticals and as biomaterials. We investigate the potential of sericin, extracted from both mulberry Bombyx mori and different non-mulberry sources, namely, tropical tasar, Antheraea mylitta; muga, Antheraea assama; and eri, Samia ricini, as growth supplement in serum-free culture medium. Sericin supplemented media containing different concentrations of sericins from the different species are examined for attachment, growth, proliferation, and morphology of fibrosarcoma cells. The optimum sericin supplementation seems to vary with the source of sericins. The results indicate that all the sericins promote the growth of L929 cells in serum-free culture media; however, S. ricini sericin seems to promote better growth of cells amongst other non-mulberry sericins.

Bipotential precursors of putative fibrous astrocytes and oligodendrocytes in rat cerebellar cultures express distinct surface features and neuron-like γ-aminobutyric acid transport

International Nuclear Information System (INIS)

Levi, G.; Gallo, V.; Ciotti, T.

1986-01-01

When postnatal rat cerebellar cells were cultured in a chemically defined, serum-free medium, the only type of astrocyte present was unable to accumulate γ-[ 3 H]aminobutyric acid (GABA), did not express surface antigens recognized by two monoclonal antibodies, A2B5 and LB1, and showed minimal proliferation. In these cultures, nonneuronal A2B5 + , LB1 + stellate cells exhibiting neuron-like [ 3 H]GABA uptake formed cell colonies of increasing size and were GFAP - . After about one week of culturing, the A2B5 + , LB1 + , GABA-uptake positive cell groups became galactocerebroside (GalCer) positive. Immunocytolysis of the A2B5 + cells at 3 and 4 days in vitro prevented the appearance of the A2B5 + , LB1 + , GABA-uptake positive cell colonies, and also of the GalCer + cell groups. If 10% (vol/vol) fetal calf serum was added to 6-day cultures, the A2B5 + , LB1 + , GABA-uptake positive cell groups expressed GFAP and not GalCer. If the serum was added to the cultures 2 days after lysing the A2B5 + cells, only A2B5 - , LB1 - , GABA-uptake negative astrocytes proliferated. It is concluded that the putative fibrous astrocytes previously described in serum-containing cultures derive from bipotential precursors that differentiate into oligodendrocytes (GalCer + ) in serum-free medium or into astrocytes (GFAP + ) in the presence of serum, while the epithelioid A2B5 - , LB1 - , GABA-uptake negative astrocytes originate from a different precursor not yet identified

The presence of serum alters the properties of iron oxide nanoparticles and lowers their accumulation by cultured brain astrocytes

International Nuclear Information System (INIS)

Geppert, Mark; Petters, Charlotte; Thiel, Karsten; Dringen, Ralf

2013-01-01

Iron oxide nanoparticles (IONPs) are considered for various diagnostic and therapeutic applications. Such particles are able to cross the blood–brain barrier and are taken up into brain cells. To test whether serum components affect the properties of IONPs and/or their uptake into brain cells, we have incubated dimercaptosuccinate-coated magnetic IONPs without and with fetal calf serum (FCS) and have exposed cultured brain astrocytes with IONPs in the absence or presence of FCS. Incubation with FCS caused a concentration-dependent increase in the average hydrodynamic diameter of the particles and of their zeta-potential. In the presence of 10 % FCS, the diameter of the IONPs increased from 57 ± 2 to 107 ± 6 nm and the zeta-potential of the particles from −22 ± 5 to −9 ± 1 mV. FCS affected also strongly the uptake of IONPs by cultured astrocytes. The efficient time- and temperature-dependent cellular accumulation of IONPs was lowered with increasing concentration of FCS by up to 90 %. In addition, in the absence of serum, endocytosis inhibitors did not alter the IONP accumulation by astrocytes, while chlorpromazine or wortmannin lowered significantly the accumulation of IONPs in the presence of FCS, suggesting that clathrin-mediated endocytosis and macropinocytosis are involved in astrocytic IONP uptake from serum-containing medium. These data demonstrate that the presence of FCS strongly affects the properties of IONPs as well as their accumulation by cultured brain cells.

Serum thyroid stimulating hormone, total and free T4 during the neonatal period: Establishing regional reference intervals

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Sara Sheikhbahaei

2014-01-01

Full Text Available Context: Congenital hypothyroidism (CH, the most common etiology of preventable mental retardation in children, is estimated to be more prevalent among Asian population. Aims: Since thyroid function tests (TFTs varied among different ages and geographical regions, in this study, the neonatal thyroid reference intervals in a healthy neonatal population is determined for the first time in Iran. Settings and Design: A cross-sectional study performed on 246 healthy term newborns aged between 2 days and 1 month. Materials and Methods: Blood samples were obtained by venipuncture from all subjects. The median, 2.5 th , 5 th , 95 th , and 97.5 th percentile of serum thyroid-stimulating hormone (TSH, as well as the total and free T4 were assessed among different age groups. Statistical Analysis Used: Predictive Analytics Software (PASW Statistics 18 was used for the analysis. Results: Serum TSH, total and free T4 concentration peaked in 5 th to 7 th days of life, continued over 2 weeks, then decreased and started reaching to adult reference range. A significant negative correlation between age and serum concentration of TSH (P = 0.02, total T4 (P = 0.01 and free T4 (P = 0.01 was found. Conclusion: This study yielded fairly different values for TFTs compared compared values found in other countries and also different from values reported for laboratory kits we used. These differences were assumed to be due to variations in ethnicity, age, and laboratory methods used. Due to the lack of international standardization, conducting multicenter studies helps in making a more precise evaluation of thyroid status in neonates.

Enhancement of Human Prolactin Synthesis by Sodium Butyrate Addition to Serum-Free CHO Cell Culture

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Herbert Rodrigues Goulart

2010-01-01

Full Text Available Sodium butyrate (NaBu has been used as a productivity enhancer for the synthesis of recombinant proteins in Chinese hamster ovary (CHO cells. Thus, the influence of NaBu on the production of recombinant human prolactin (hPRL from CHO cells was investigated for the first time. CHO cell cultures were submitted to a treatment with different concentrations of NaBu (0.25 to 4 mM. Quantitative and qualitative analyses by reverse-phase high-performance liquid chromatography (RP-HPLC and Western blot or SDS-PAGE, carried out directly on CHO-conditioned medium, showed that the highest hPRL expression was obtained with 1 mM NaBu. In vitro biological assays based on noble rat lymphoma (Nb2 and mouse pro-B lymphoma (Ba/F3-LLP cells were carried out on purified hPRL. Its bioactivity in the presence of NaBu was not apparently different from that of the First International Reference Reagent of recombinant hPRL (WHO 97/714. Our results show that NaBu increased the synthesis of recombinant hPRL in CHO cells, apparently without compromising either its structure or function.

Calorimetric and spectroscopic properties of small globular proteins (bovine serum albumin, hemoglobin) after free radical generation

International Nuclear Information System (INIS)

Farkas, N.; Belagyi, J.; Lorinczy, D.

2003-01-01

Mild oxidation of -SH-containing proteins (serum albumin, hemoglobin) by Ce(IV)-ions in the presence of the spin trap phenyl-tert-butylnitrone (PBN) resulted in the appearance of strongly immobilized nitroxide free radicals which evidences the formation of thiyl radicals on the thiol site of the proteins. In hydroxyl free radical generating system a fraction of strongly immobilized nitroxide radicals was also detected in these proteins, which implies that the oxidation of a fraction of the thiol groups was also involved in the free radical reaction. According to the differential scanning calorimetry (DSC) experiments the melting processes of the proteins were calorimetrically irreversible, therefore the two-state kinetic model was used to evaluate the experiments. The results support the view that site-specific interaction of SH-containing proteins with hydroxyl and thiyl free radicals is able to modify the internal dynamics of proteins and affect the conformation of large molecules

Effects of different culture conditions (photoautotrophic ...

African Journals Online (AJOL)

Effects of different culture conditions (photoautotrophic, photomixotrophic) and the auxin indole-butyric acid on the in vitro acclimatization of papaya ( Carica papaya L. var. Red Maradol) plants using zeolite as support.

Changes in responsiveness of rat tracheal epithelial cells to growth factors during preneoplastic transformation in cell culture

International Nuclear Information System (INIS)

Thomassen, D.G.

1988-01-01

Preneoplastic rat tracheal epithelial (RTE) cell lines require fewer growth factors for clonal proliferation in culture than normal cells. Serum-free media missing various combinations of growth factors (e.g., cholera toxin, serum albumin, epidermal growth factor, hydrocortisone) required for proliferation of normal, but not preneoplastic, RTE cells can be used to select for carcinogen-induced preneoplastic variants having an increased proliferative potential in culture. These results suggest that reductions in growth factor requirements are primary events in the carcinogenic process. (author)

The relationship between serum total testosterone and free testosterone levels with serum hemoglobin and hematocrit levels: a study in 1221 men.

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Shin, Yu Seob; You, Jae Hyung; Cha, Jai Seong; Park, Jong Kwan

2016-12-01

To investigate the relationship between serum total testosterone (TT) and free testosterone (FT) levels in men with anemia. We reviewed the records of 1221 subjects between March 2009 and December 2014. All the subjects' blood samples were drawn for TT and FT assays. Their serum hemoglobin (Hb) and serum hematocrit (Hct) levels were measured. The primary objective of our study was to investigate the association between TT and FT levels with Hb and Hct levels. The mean age was 59.82 ± 12.71 years. The mean TT and FT levels were 4.54 ± 2.02 ng/mL and 10.63 ± 3.69 pg/mL, respectively. The mean Hb and Hct levels were 14.72 ± 1.34 g/dL and 43.11 ± 3.75%, respectively. Subjects with low TT (<2.35 ng/mL) had low Hb and Hct levels (p < 0.001, p < 0.001, respectively). TT was positively associated with FT, Hb, and Hct. TT and FT levels were significantly lower in older men. Subjects with low TT and FT levels had low Hb and Hct levels. This suggests that TT and FT play a significant role in erythropoiesis. Testosterone replacement therapy may be effective in men with hypogonadism to reduce the incidence of anemia.

Effects of culture conditions on acetic acid production by bacteria ...

African Journals Online (AJOL)

SARAH

2015-11-30

Nov 30, 2015 ... acid under certain culture conditions similar to cocoa fermentation stress. However ... Keywords: Acetic acid bacteria, acetic acid production, Cocoa fermentation, culture conditions ..... American Society Microbiology Press, pp.

Complete human serum maintains viability and chondrogenic potential of human synovial stem cells: suitable conditions for transplantation.

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Mizuno, Mitsuru; Katano, Hisako; Otabe, Koji; Komori, Keiichiro; Kohno, Yuji; Fujii, Shizuka; Ozeki, Nobutake; Horie, Masafumi; Tsuji, Kunikazu; Koga, Hideyuki; Muneta, Takeshi; Sekiya, Ichiro

2017-06-13

In our clinical practice, we perform transplantations of autologous synovial mesenchymal stem cells (MSCs) for cartilage and meniscus regenerative medicine. One of the most important issues to ensuring clinical efficacy involves the transport of synovial MSCs from the processing facility to the clinic. Complete human serum (100% human serum) is an attractive candidate material in which to suspend synovial MSCs for their preservation during transport. The purpose of this study was to investigate whether complete human serum maintained MSC viability and chondrogenic potential and to examine the optimal temperature conditions for the preservation of human synovial MSCs. Human synovium was harvested from the knees of 14 donors with osteoarthritis during total knee arthroplasty. Passage 2 synovial MSCs were suspended at 2 million cells/100 μL in Ringer's solution or complete human serum at 4, 13, and 37 °C for 48 h. These cells were analyzed for live cell rates, cell surface marker expression, metabolic activity, proliferation, and adipogenic, calcification, and chondrogenic differentiation potentials before and after preservation. After preservation, synovial MSCs maintained higher live cell rates in human serum than in Ringer's solution at 4 and 13 °C. Synovial MSCs preserved in human serum at 4 and 13 °C also maintained high ratios of propidium iodide - and annexin V - cells. MSC surface marker expression was not altered in cells preserved at 4 and 13 °C. The metabolic activities of cells preserved in human serum at 4 and 13 °C was maintained, while significantly reduced in other conditions. Replated MSCs retained their proliferation ability when preserved in human serum at 4 and 13 °C. Adipogenesis and calcification potential could be observed in cells preserved in each condition, whereas chondrogenic potential was retained only in cells preserved in human serum at 4 and 13 °C. The viability and chondrogenic potential of synovial MSCs were

Two different serum-free media and osmolality effect upon human 293 cell growth and adenovirus production.

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Ferreira, Tiago B; Ferreira, Ana L; Carrondo, Manuel J T; Alves, Paula M

2005-11-01

Adenoviruses are promising vectors for gene therapy and vaccination protocols. Consequently, the market demands for adenovirus are increasing, driving the search for new methodologies for large-scale production of concentrated vectors with warranted purity and efficacy, in a cost-effective way. Nevertheless, the production of adenovirus is currently limited by the so-called 'cell density effect', i.e. a drop in cell specific productivity concomitant with increased cell concentration at infection. Of two different serum-free culture media (CD293 and EX-Cell), evaluated for their effect on human 293 cells growth and adenovirus production at cell densities higher than 1x10(6) cells/ml, EX-Cell proved the better medium for cell growth. Although adenovirus production was equivalent in both media when the infection was performed at 1x10(6) cells/ml, at 3x10(6) cells/ml CD293 was the better. This result related to the high ammonia content in EX-Cell medium at the highest cell concentration at infection. Besides this, the large-scale production of these vectors at high cell densities often requires re-feed strategies, which increase medium osmolality. While a negative effect on cell growth was observed with increasing osmolalities, adenovirus productivity was only affected for osmolalities higher than 430 mOsm.

Comparison of real-time reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis.

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Doenges, Stephanie J; Weber, Karin; Dorsch, Roswitha; Fux, Robert; Hartmann, Katrin

2017-04-01

Objectives Diagnosis of feline infectious peritonitis (FIP) remains challenging, especially in cats without effusions. The objective of this study was to evaluate the sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) detecting feline coronavirus (FCoV) RNA in peripheral blood mononuclear cells (PBMCs) and serum in comparison with the same real-time RT-PCR in cell-free body cavity effusion. Methods This prospective case-control study included 92 cats. Forty-three cats had a definitive diagnosis of FIP, established either by histopathological examination (n = 28) or by positive immunofluorescence staining of FCoV antigen in macrophages of effusions (n = 11), or by both methods (n = 4). Forty-nine control cats had other diseases but similar clinical signs. Real-time RT-PCR was performed on PBMCs of 37 cats (21 cats with FIP, 16 controls), on serum of 51 cats (26 cats with FIP, 25 controls) and on cell-free body cavity effusion of 69 cats (36 cats with FIP, 33 controls). Sensitivity, specificity, positive and negative predictive value, including 95% confidence intervals (CI), were calculated. Results Real-time RT-PCR of PBMCs, serum and cell-free body cavity effusion showed a specificity of 100% (95% CI 79.4-100% in PBMCs, 86.3-100% in serum, 89.4-100% in cell-free body cavity effusion) and a sensitivity of 28.6% (95% CI 11.3-52.2%) in PBMCs, 15.4% (95% CI 4.4-34.9%) in serum and 88.9% (95% CI 73.9-96.9%) in cell-free body cavity effusion to diagnose FIP. Conclusions and relevance Although it is known that RT-PCR can often provide false-positive results in healthy cats, this real-time RT-PCR was shown to be a specific tool for the diagnosis of FIP when applied in a clinical setting. Sensitivity in cell-free body cavity effusion was high but low in PBMCs and serum. PBMC samples showed a higher sensitivity than serum samples, and are therefore a better choice if no effusion is present.

Effect of operating conditions in production of diagnostic Salmonella Enteritidis O-antigen-specific monoclonal antibody in different bioreactor systems.

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Ayyildiz-Tamis, Duygu; Nalbantsoy, Ayse; Elibol, Murat; Deliloglu-Gurhan, Saime Ismet

2014-01-01

In this study, different cultivation systems such as roller bottles (RB), 5-L stirred-tank bioreactor (STR), and disposable bioreactors were used to cultivate hybridoma for lab-scale production of Salmonella Enteritidis O-antigen-specific monoclonal antibody (MAb). Hybridoma cell line was cultivated in either serum-containing or serum-free medium (SFM) culture conditions. In STR, MAb production scaled up to 4 L, and production capabilities of the cells were also evaluated in different featured production systems. Moreover, the growth parameters of the cells in all production systems such as glucose consumption, lactate and ammonia production, and also MAb productivities were determined. Collected supernatants from the reactors were concentrated by a cross-flow filtration system. In conclusion, cells were not adapted to SFM in RB and STR. Therefore, less MAb titer in both STR and RB systems with SFM was observed compared to the cultures containing fetal bovine serum-supplemented medium. A higher MAb titer was gained in the membrane-aerated system compared to those in STR and RB. Although the highest MAb titer was obtained in the static membrane bioreactor system, the highest productivity was obtained in STR operated in semicontinuous mode with overlay aeration.

Defined three-dimensional culture conditions mediate efficient induction of definitive endoderm lineage from human umbilical cord Wharton's jelly mesenchymal stem cells.

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Al Madhoun, Ashraf; Ali, Hamad; AlKandari, Sarah; Atizado, Valerie Lopez; Akhter, Nadeem; Al-Mulla, Fahd; Atari, Maher

2016-11-16

Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are gaining increasing interest as an alternative source of stem cells for regenerative medicine applications. Definitive endoderm (DE) specification is a prerequisite for the development of vital organs such as liver and pancreas. Hence, efficient induction of the DE lineage from stem cells is crucial for subsequent generation of clinically relevant cell types. Here we present a defined 3D differentiation protocol of WJ-MSCs into DE cells. WJ-MSCs were cultured in suspension to generate spheroids, about 1500 cells each, for 7 days. The serum-free differentiation media contained specific growth factors, cytokines, and small molecules that specifically regulate signaling pathways including sonic hedgehog, bone morphogenetic protein, Activin/Wnt, and Notch. We obtained more than 85 % DE cells as shown with FACS analysis using antibodies directed against the DE marker CXCR4. In addition, biochemical and molecular analysis of bona-fide DE markers revealed a time-course induction of Sox17, CXCR4, and FoxA2. Focused PCR-based array also indicated a specific induction into the DE lineage. In this study, we report an efficient serum-free protocol to differentiate WJ-MSCs into DE cells utilizing 3D spheroid formation. Our approach might aid in the development of new protocols to obtain DE-derivative lineages including liver-like and pancreatic insulin-producing cells.

Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs

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Chen, Yen-Ming; Chen, Li-Hua; Li, Meng-Pei; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Ling, Qing-Dong; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Chang, Yung; Benelli, Giovanni; Murugan, Kadarkarai; Umezawa, Akihiro

2017-01-01

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells. PMID:28332572

A thin-layer liquid culture technique for the growth of Helicobacter pylori.

Science.gov (United States)

Joo, Jung-Soo; Park, Kyung-Chul; Song, Jae-Young; Kim, Dong-Hyun; Lee, Kyung-Ja; Kwon, Young-Cheol; Kim, Jung-Min; Kim, Kyung-Mi; Youn, Hee-Shang; Kang, Hyung-Lyun; Baik, Seung-Chul; Lee, Woo-Kon; Cho, Myung-Je; Rhee, Kwang-Ho

2010-08-01

Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin-layer liquid culture technique for the growth of H. pylori. A thin-layer liquid culture system was established by adding liquid media to a 90-mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI-1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum-free RPMI-1640 supported the growth of H. pylori when supplemented with dimethyl-beta-cyclodextrin (200 microg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD(600) with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD(600) contained 1.3 +/- 0.1 x 10(9 )CFU/mL. gamma-Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin-layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. Thin-layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.

Stimulation of DNA synthesis in cultured rat alveolar type II cells

International Nuclear Information System (INIS)

Leslie, C.C.; McCormick-Shannon, K.; Robinson, P.C.; Mason, R.J.

1985-01-01

Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated 3 H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of 3 H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture

Effects of cell culture and laboratory conditions on type 2 dengue virus infectivity.

Science.gov (United States)

Manning, J S; Collins, J K

1979-01-01

The stability of type 2 dengue virus to exposure to a variety of laboratory conditions was determined. Suckling mouse brain passage virus was adapted for growth in BHK-21 cells, and plaque assays were performed using a tragacanth gum overlay. A three- to fourfold increase in plaque size could be obtained if monolayers were subconfluent at time of inoculation. Incubation of virus for 24 h at 37 degrees C, pH 6.5, or in buffer containing 1 mM ethylenediaminetetraacetate considerably reduced virus infectivity as compared with virus incubated for the same period at 4 degrees C, pH 8.0, or in buffer with or without 1 mM CaCl2 and 1 mM MgCl2. Multiple freezing and thawing of virus tissue culture medium containing 10% fetal calf serum did not reduce virus infectivity. Images PMID:41848

Serum Dried Samples to Detect Dengue Antibodies: A Field Study

Directory of Open Access Journals (Sweden)

Angelica Maldonado-Rodríguez

2017-01-01

Full Text Available Background. Dried blood and serum samples are useful resources for detecting antiviral antibodies. The conditions for elution of the sample need to be optimized for each disease. Dengue is a widespread disease in Mexico which requires continuous surveillance. In this study, we standardized and validated a protocol for the specific detection of dengue antibodies from dried serum spots (DSSs. Methods. Paired serum and DSS samples from 66 suspected cases of dengue were collected in a clinic in Veracruz, Mexico. Samples were sent to our laboratory, where the conditions for optimal elution of DSSs were established. The presence of anti-dengue antibodies was determined in the paired samples. Results. DSS elution conditions were standardized as follows: 1 h at 4°C in 200 µl of DNase-, RNase-, and protease-free PBS (1x. The optimal volume of DSS eluate to be used in the IgG assay was 40 µl. Sensitivity of 94%, specificity of 93.3%, and kappa concordance of 0.87 were obtained when comparing the antidengue reactivity between DSSs and serum samples. Conclusion. DSS samples are useful for detecting anti-dengue IgG antibodies in the field.

Comparative studies of types 1 and 2 herpes simplex virus infection of cultured normal keratinocytes.

OpenAIRE

Su, S J; Wu, H H; Lin, Y H; Lin, H Y

1995-01-01

AIMS--To investigate the differences in biological properties, multiplication patterns, and cytopathic effects between type 1 and type 2 herpes simplex virus (HSV) through the replication of HSV in cultured normal human keratinocytes. METHODS--Keratinocytes were obtained from surgical specimens of normal gingiva, cervix, trunk skin, and newborn foreskin. They were cultured in serum free, chemically defined, culture medium and infected with a pool of HSV collected from clinical specimens. RESU...

Platelet lysate as a serum replacement for skin cell culture on biomimetic PCL nanofibers.

Science.gov (United States)

Sovkova, Vera; Vocetkova, Karolina; Rampichova, Michala; Mickova, Andrea; Buzgo, Matej; Lukasova, Vera; Dankova, Jana; Filova, Eva; Necas, Alois; Amler, Evzen

2018-06-01

Platelets are a popular source of native growth factors for tissue engineering applications. The aim of the study was to verify the use of platelet lysate as a fetal bovine serum (FBS) replacement for skin cell culture. The cytokine content of the platelet lysate was characterized using the Bio-Plex system. The cells (fibroblasts, melanocytes, and keratinocytes) were cultured on PCL nanofibrous scaffolds to mimic their natural microenvironment. The cytokine content of the platelet lysate was determined, and to the cells, a medium containing platelet lysate or platelet lysate in combination with FBS was added. The results showed that 7% (v/v) platelet lysate was sufficient to supplement 10% (v/v) FBS in the culture of fibroblasts and keratinocytes. The combination of platelet lysate and FBS had a rather inhibitory effect on fibroblasts, in contrary to keratinocytes, where the effect was synergic. Platelet lysate did not sufficiently promote proliferation in melanocytes; however, the combination of FBS and platelet lysate yielded a better outcome and resulted in bipolar morphology of the cultured melanocytes. The data indicated that platelet lysate improved cell proliferation and metabolic activity and may be used as an additive to the cell culture media.

Cultured rat and purified human Pneumocystis carinii stimulate intra- but not extracellular free radical production in human neutrophils

DEFF Research Database (Denmark)

Jensen, T; Aliouat, E M; Lundgren, B

1998-01-01

The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide...... generation, hardly any free radical production was observed after stimulation with cultured rat-derived P. carinii. A chemiluminescence technique, which separately measured intra- and extracellular free radical production, was subsequently employed to differentiate the free radical generation....... It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils, 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intracellular response of superoxide production, and 3) opsonized P. carinii, purified from human lung also...

Correlation of serum androgens and pituitary hormone levels with serum PSA less than 2.5 ng/ml.

Science.gov (United States)

Sofikerim, Mustafa; Oruç, Ozgür; Eskicorapci, Sadettin; Guliyev, Fuat; Ozen, Haluk

2007-07-27

The aim of this clinical study was to determine whether there is a relationship between total serum testosterone, free testosterone, FSH (Follicle-Stimulating Hormone), LH (Luteinizing Hormone) and serum prostate specific antigen (PSA) levels. We postulated that such a correlation existed then the use of hormone specific reference ranges might enhance the usefullness of PSA concentrations 40 years of age visiting our urology outpatient clinics. PSA was correlated to age (r = 0.23, p = 0.019), but there none between serum testosterone and age. No significant correlation was noted between testosterone or free testosterone and serum PSA levels, and none between serum FSH or LH and PSA. In age specific reference groups (41-49; 50-59; 60-69 years), we found no significant correlation between PSA and hormone concentrations. In this population of eugonadal men with serum PSA values less than 2.5 ng/ml, serum androgens and pituitary hormones do not appear to correlate with serum PSA.

Examining the sources of variability in cell culture media used for biopharmaceutical production.

Science.gov (United States)

McGillicuddy, Nicola; Floris, Patrick; Albrecht, Simone; Bones, Jonathan

2018-01-01

Raw materials, in particular cell culture media, represent a significant source of variability to biopharmaceutical manufacturing processes that can detrimentally affect cellular growth, viability and specific productivity or alter the quality profile of the expressed therapeutic protein. The continual expansion of the biopharmaceutical industry is creating an increasing demand on the production and supply chain consistency for cell culture media, especially as companies embrace intensive continuous processing. Here, we provide a historical perspective regarding the transition from serum containing to serum-free media, the development of chemically-defined cell culture media for biopharmaceutical production using industrial scale bioprocesses and review production mechanisms for liquid and powder culture media. An overview and critique of analytical approaches used for the characterisation of cell culture media and the identification of root causes of variability are also provided, including in-depth liquid phase separations, mass spectrometry and spectroscopic methods.

Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

Science.gov (United States)

Sidney, Laura E; Branch, Matthew J; Dua, Harminder S; Hopkinson, Andrew

2015-12-01

The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Acute phase protein concentrations in serum and milk from healthy cows, cows with clinical mastitis and cows with extramammary inflammatory conditions

DEFF Research Database (Denmark)

Nielsen, B.H.; Jacobsen, S.; Andersen, P.H.

2004-01-01

The concentrations of the two acute phase proteins, serum amyloid A and haptoglobin, in serum and milk were compared in 10 cows with clinical mastitis, 11 cows with extramammary inflammatory conditions and 10 clinically healthy control cows. The concentrations of both acute phase proteins were...... higher in the serum and milk of the cows with mastitis than in the cows in the other two groups. Four of the cows with extramammary inflammatory conditions had serum amyloid A concentrations in serum above 100 mug/ml, but negligible concentrations in milk, indicating that a pathogen must be present...

Human skin condition and its associations with nutrient concentrations in serum and diet

NARCIS (Netherlands)

Boelsma, E.; Vijver, L.P.L. van de; Goldbohm, R.A.; Klöpping-Ketelaars, I.A.A.; Hendriks, H.F.J.; Roza, L.

2003-01-01

Background: Nutritional factors exert promising actions on the skin, but only scant information is available on the modulating effects of physiologic concentrations of nutrients on the skin condition of humans. Objective: The objective was to evaluate whether nutrient concentrations in serum and

Optimization of culture conditions of Streptomyces rochei (MTCC ...

African Journals Online (AJOL)

Fermentation and culture conditions were studied in shaken-flask culture to induce the production of greater amounts of antimicrobial metabolites by Streptomyces rochei (10109). Antimicrobial metabolite production started after 48 h incubation and reached its optimum level at 20% inoculum size at 120 h, at which point the ...

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support

Directory of Open Access Journals (Sweden)

Anita Muraglia

2017-11-01

Full Text Available Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i an heparin-free human platelet lysate (PL devoid of serum or plasma components (v-PL and (ii an heparin-free human serum derived from plasma devoid of PL components (Pl-s and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment, but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79 regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype.

Influence of cell type and cell culture media on the propagation of foot-and-mouth disease virus with regard to vaccine quality.

Science.gov (United States)

Dill, Veronika; Hoffmann, Bernd; Zimmer, Aline; Beer, Martin; Eschbaumer, Michael

2018-03-16

Suspension culture of BHK cells allows large-scale virus propagation and cost-efficient vaccine production, while the shift to animal-component-free cell culture media without serum is beneficial for the quality and downstream processing of the product. Foot-and-mouth disease virus is still endemic in many parts of the world and high-quality vaccines are essential for the eradication of this highly contagious and economically devastating disease. Changes to the viral genome sequence during passaging in an adherent and a suspension cell culture system were compared and the impact of amino acid substitutions on receptor tropism, antigenicity and particle stability was examined. Virus production in suspension cells in animal-component-free media and in serum-containing media as well as in adherent cells in serum-containing media was compared. Infection kinetics were determined and the yield of intact viral particles was estimated in all systems using sucrose density gradient centrifugation. Capsid protein sequence alterations were serotype-specific, but varied between cell lines. But The A 24 -2P virus variant had expanded its receptor tropism, but virus neutralization tests found no changes in the antigenic profile in comparison to the original viruses. There were no differences in viral titer between a suspension and an adherent cell culture system, independent of the type of media used. Also, the usage of a serum-free suspension culture system promoted viral growth and allowed an earlier harvest. For serotype O isolates, no differences were seen in the yield of 146S particles. Serotype A preparations revealed a decreased yield of 146S particles in suspension cells independent of the culture media. The selective pressure of the available surface receptors in different cell culture systems may be responsible for alterations in the capsid coding sequence of culture-grown virus. Important vaccine potency characteristics such as viral titer and the neutralization

Growth of microbial mixed cultures under anaerobic, alkaline conditions

International Nuclear Information System (INIS)

Wenk, M.

1993-09-01

Cement and concrete are the most important engineered barrier materials in a repository for low- and intermediate-level waste and thus represent the most significant component of the total disposal inventory. Based on the chemical composition of the concrete used in the repository and the groundwater fluxes in the modelled host rock, it is to be expected that the pH in the near vicinity of the repository could exceed a value of 10.5 for more than a million years. The groundwater in the repository environment also has a limited carbon concentration. Since microorganisms will be present in a repository and can even find suitable living conditions within the waste itself, investigations were carried out in order to establish the extent to which microbial activity is possible under the extreme conditions of the repository near-field. For the investigations, alkalophilic cultures were enriched from samples from alkaline habitats and from Valanginian Marl. Anaerobic bacteria with fermentative, sulfate-reducing and methanogenic metabolism were selected. The growth and activity of the mixed cultures were studied under alkaline conditions and the dependence on pH and carbon concentration determined. All the mixed cultures investigated are alkalophilic. The optimum growth range for the cultures is between pH 9.0 and pH 10.0. The activity limit for the fermentative mixed culture is at pH 12, for the sulfate-reducers at pH 11 and for the methanogens at pH 10.5. Given the limited supply of carbon, the mixed cultures can only grow under slightly alkaline conditions. Only the fermentative cultures are capable of surviving with limited carbon supply at pH 13. (author) 24 figs., 18 tabs., 101 refs

Tooth Tissue Engineering: The Importance of Blood Products as a Supplement in Tissue Culture Medium for Human Pulp Dental Stem Cells.

Science.gov (United States)

Pisciolaro, Ricardo Luiz; Duailibi, Monica Talarico; Novo, Neil Ferreira; Juliano, Yara; Pallos, Debora; Yelick, Pamela Crotty; Vacanti, Joseph Phillip; Ferreira, Lydia Masako; Duailibi, Silvio Eduardo

2015-11-01

One of the goals in using cells for tissue engineering (TE) and cell therapy consists of optimizing the medium for cell culture. The present study compares three different blood product supplements for improved cell proliferation and protection against DNA damage in cultured human dental pulp stem cells for tooth TE applications. Human cells from dental pulp were first characterized as adult stem cells (ectomesenchymal mixed origin) by flow cytometry. Next, four different cell culture conditions were tested: I, supplement-free; II, supplemented with fetal bovine serum; III, allogeneic human serum; and IV, autologous human serum. Cultured cells were then characterized for cell proliferation, mineralized nodule formation, and colony-forming units (CFU) capability. After 28 days in culture, the comet assay was performed to assess possible damage in cellular DNA. Our results revealed that Protocol IV achieved higher cell proliferation than Protocol I (p = 0.0112). Protocols II and III resulted in higher cell proliferation than Protocol I, but no statistical differences were found relative to Protocol IV. The comet assay revealed less cell damage in cells cultured using Protocol IV as compared to Protocols II and III. The damage percentage observed on Protocol II was significantly higher than all other protocols. CFUs capability was highest using Protocol IV (p = 0.0018) and III, respectively, and the highest degree of mineralization was observed using Protocol IV as compared to Protocols II and III. Protocol IV resulted in significantly improved cell proliferation, and no cell damage was observed. These results demonstrate that human blood product supplements can be used as feasible supplements for culturing adult human dental stem cells.

Free insulin-like growth factor I serum levels in 1430 healthy children and adults, and its diagnostic value in patients suspected of growth hormone deficiency

DEFF Research Database (Denmark)

Juul, A; Holm, K; Kastrup, K W

1997-01-01

fraction of the total IGF-I circulates in its free form, which is believed to be the biologically active form. However, our knowledge of the clinical or physiological value of determination of free IGF-I in serum is limited at present. In adults, the diagnostic value of total IGF-I and IGFBP-3......, commercially available immunoradiometric assay (Diagnostic Systems Laboratories) to establish valid normative data for this analysis. We studied the diagnostic value of free IGF-I in relation to total IGF-I and IGFBP-3 determinations in adults who were suspected of GHD. A GH provocative test, using oral...... determinations in patients suspected of GHD has only been reported in a few studies, whereas no previous reports on the diagnostic value of free IGF-I levels in adults suspected of GHD exist. Serum levels of free IGF-I were determined in 1430 healthy children, adolescents, and adults by a newly developed...

Free insulin-like growth factor I serum levels in 1430 healthy children and adults, and its diagnostic value in patients suspected of growth hormone deficiency

DEFF Research Database (Denmark)

Juul, A; Holm, K; Kastrup, K W

1997-01-01

fraction of the total IGF-I circulates in its free form, which is believed to be the biologically active form. However, our knowledge of the clinical or physiological value of determination of free IGF-I in serum is limited at present. In adults, the diagnostic value of total IGF-I and IGFBP-3...... determinations in patients suspected of GHD has only been reported in a few studies, whereas no previous reports on the diagnostic value of free IGF-I levels in adults suspected of GHD exist. Serum levels of free IGF-I were determined in 1430 healthy children, adolescents, and adults by a newly developed......, commercially available immunoradiometric assay (Diagnostic Systems Laboratories) to establish valid normative data for this analysis. We studied the diagnostic value of free IGF-I in relation to total IGF-I and IGFBP-3 determinations in adults who were suspected of GHD. A GH provocative test, using oral...

Cell-free DNA in a three-dimensional spheroid cell culture model

DEFF Research Database (Denmark)

Aucamp, Janine; Calitz, Carlemi; Bronkhorst, Abel J.

2017-01-01

Background Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo...... cultures can serve as effective, simplified in vivo-simulating “closed-circuit” models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability. Biological...... significance 3D cell cultures can be used to translate “closed-circuit” in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth...

Influence of starter culture on total free aminoacids concentration during ripening of Krk cheese

Directory of Open Access Journals (Sweden)

Biljana Radeljević

2013-03-01

Full Text Available The aim of this study was to determine the influence of microbial (commercial starter culture on concentration of total free amino groups (amino acids in cheeses in different ripening stages. Free amino groups were determined by reaction with ninhydrin with cadmium (Cd in the water soluble cheese extract, and were expressed as the concentration of leucine in cheese dry matter. Changes in concentration of total free amino acids during cheese ripening (0th, 30th, 60th, 90th and 120th day were monitored. In water soluble extracts of cheese, the presence of free NH2 groups in all ripening stages was detected, which means smaller peptides and amino acids, whose concentration significantly (P<0.01 increased during ripening. Cheeses produced with and without microbial culture resulted in statistically significant differences (P<0.01 in content amino acids free on the 90th and 120th day of ripening. Cd - ninhydrin method was found to be suitable for cheese ripening monitoring, as well as for determination of the differences in mature characteristics of cheeses, depending on the production process.

Free radical scavenging activity and secondary metabolites from in vitro cultures of Sanicula graveolens.

Science.gov (United States)

Cheel, José; Schmeda-Hirschmann, Guillermo; Jordan, Miguel; Theoduloz, Cristina; Rodríguez, Jaime A; Gerth, André; Wilken, Dirk

2007-01-01

An in vitro propagation system was developed to obtain shoot and root cultures from the Andean spice Sanicula graveolens (Apiaceae). Propagation of shoots, roots and plantlets was achieved by the temporary immersion system. The free radical scavenging effect of the methanol/water (7:3 v/v) extracts was determined by the discoloration of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). Total phenolic, flavonoid, chlorogenic acid (CA) and quercetin 3-O-glucoside content in the samples was assessed by spectrophotometry and DAD-HPLC analysis, respectively. On a dry weight basis, the crude extracts showed total phenolic values ranging from 3.57 to 6.93%, with highest content for the root culture sample. Total flavonoid content ranged from 1.23 to 2.23% and was lower for the root culture. Chlorogenic acid and neochlorogenic acid were identified by TLC in all samples. Highest free radical scavenging effect was observed for the root culture which also presented the highest CA content. Two of the shoot culture samples, with similar IC50 values in the DPPH discoloration assay, also presented close quercetin-3-O-glucoside content.

Metabolite profiling of microfluidic cell culture conditions for droplet based screening

DEFF Research Database (Denmark)

Björk, Sara M.; Sjoström, Staffan L.; Svahn, Helene Andersson

2015-01-01

We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets......, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast...... limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format....

Diagnostic value of serum free PSA and the ratio of free to total PSA in the diagnosis of prostate cancer

International Nuclear Information System (INIS)

Qiu Ningyan; Zhang Jingxin; Wu Jinchang; Gong Yiming; Li Huiping

2005-01-01

In order to evaluate the value of free prostate specific antigen (FPSA) and F/T PSA ratio in differential diagnosis of benign prostate hyperplasia (BPH) from prostate cancer (PC), serum FPSA and TPSA levels were measured in 85 patients with PC, 97 BPH and 89 healthy volunteers by chemiluminescence enzyme immunoassay (CLEIA), and the ratio of F/T PSA was calculated. The results showed that serum FPSA and TPSA levels were increased in healthy volunteers of 41-88 years old and were significantly higher in healthy volunteers of 61-88 years old than that in 20-40 gear old (P 10.0 μg/L were 65.0%, 30.9% and 4.1%, respectively, while they were 5.9%, 20.0% and 74.1% in PC patients (P<0.01). When the TPSA value was between 4.0-10.0 μg/L and the ratio of F/T PSA was at 0.10 and below, the probability of PC was larger(88.9%). But the ratio of F/T PSA was at 0.25 and above, the probability of PC was smaller(6.20%). Serum FPSA and TPSA both increased with age in healthy volunteers of 41-88 years old and were positively correlated with age. There were about 30.9% of BPH and 20.0% of PC patients with overlapping of TPSA level. Our conclusion is that the F/T PSA ratio can significantly enhance the specificity for PC diagnosis, especially when the TPSA is within the diagnostic gray zone. (authors)

Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

Energy Technology Data Exchange (ETDEWEB)

Fani, Nesa [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Ziadlou, Reihane [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Shahhoseini, Maryam, E-mail: m.shahhoseini@royaninstitute.org [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Baghaban Eslaminejad, Mohamadreza, E-mail: eslami@royaninstitute.org [Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of)

2016-06-10

Mesenchymal stem cells (MSCs) derived from bone marrow (BM) represents a useful source of adult stem cells for cell therapy and tissue engineering. MSCs are present at a low frequency in the BM; therefore expansion is necessary before performing clinical studies. Fetal bovine serum (FBS) as a nutritional supplement for in vitro culture of MSCs is a suitable additive for human cell culture, but not regarding subsequent use of these cells for clinical treatment of human patients due to the risk of viral and prion transmission as well as xenogeneic immune responses after transplantation. Recently, autologous serum (AS) has been as a supplement to replace FBS in culture medium. We compared the effect of FBS versus AS on the histone modification pattern of MSCs through in vitro osteogenesis and adipogenesis. Differentiation of stem cells under various serum conditions to a committed state involves global changes in epigenetic patterns that are critically determined by chromatin modifications. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR showed significant changes in the acetylation and methylation patterns in lysine 9 (Lys9) of histone H3 on the regulatory regions of stemness (Nanog, Sox2, Rex1), osteogenic (Runx2, Oc, Sp7) and adipogenic (Ppar-γ, Lpl, adiponectin) marker genes in undifferentiated MSCs, FBS and AS. All epigenetic changes occurred in a serum dependent manner which resulted in higher expression level of stemness genes in undifferentiated MSCs compared to differentiated MSCs and increased expression levels of osteogenic genes in AS compared to FBS. Adipogenic genes showed greater expression in FBS compared to AS. These findings have demonstrated the epigenetic influence of serum culture conditions on differentiation potential of MSCs, which suggest that AS is possibly more efficient serum for osteogenic differentiation of MSCs in cell therapy purposes. - Highlights: • Bone marrow derived MSC could proliferate in AS as well as in FBS

Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

International Nuclear Information System (INIS)

Fani, Nesa; Ziadlou, Reihane; Shahhoseini, Maryam; Baghaban Eslaminejad, Mohamadreza

2016-01-01

Mesenchymal stem cells (MSCs) derived from bone marrow (BM) represents a useful source of adult stem cells for cell therapy and tissue engineering. MSCs are present at a low frequency in the BM; therefore expansion is necessary before performing clinical studies. Fetal bovine serum (FBS) as a nutritional supplement for in vitro culture of MSCs is a suitable additive for human cell culture, but not regarding subsequent use of these cells for clinical treatment of human patients due to the risk of viral and prion transmission as well as xenogeneic immune responses after transplantation. Recently, autologous serum (AS) has been as a supplement to replace FBS in culture medium. We compared the effect of FBS versus AS on the histone modification pattern of MSCs through in vitro osteogenesis and adipogenesis. Differentiation of stem cells under various serum conditions to a committed state involves global changes in epigenetic patterns that are critically determined by chromatin modifications. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR showed significant changes in the acetylation and methylation patterns in lysine 9 (Lys9) of histone H3 on the regulatory regions of stemness (Nanog, Sox2, Rex1), osteogenic (Runx2, Oc, Sp7) and adipogenic (Ppar-γ, Lpl, adiponectin) marker genes in undifferentiated MSCs, FBS and AS. All epigenetic changes occurred in a serum dependent manner which resulted in higher expression level of stemness genes in undifferentiated MSCs compared to differentiated MSCs and increased expression levels of osteogenic genes in AS compared to FBS. Adipogenic genes showed greater expression in FBS compared to AS. These findings have demonstrated the epigenetic influence of serum culture conditions on differentiation potential of MSCs, which suggest that AS is possibly more efficient serum for osteogenic differentiation of MSCs in cell therapy purposes. - Highlights: • Bone marrow derived MSC could proliferate in AS as well as in FBS

Divergence-Free Wavelets on the Hypercube : General Boundary Conditions

NARCIS (Netherlands)

Stevenson, R.

2016-01-01

On the n-dimensional hypercube, for given k∈N, wavelet Riesz bases are constructed for the subspace of divergence-free vector fields of the Sobolev space Hk((0,1)n)n with general homogeneous Dirichlet boundary conditions, including slip or no-slip boundary conditions. Both primal and suitable dual

Serum Free Light Chains

Science.gov (United States)

... lab's website in order to provide you with background information about the test(s) you had performed. You will need to return ... the free light chains have a much shorter half-life (3-5 hours) than ... cell disorders, the test is also used for assessing response and minimal ...

Development of humanized culture medium with plant-derived serum replacement for human pluripotent stem cells

Czech Academy of Sciences Publication Activity Database

Kunová, M.; Matulka, K.; Eiselleová, L.; Trčková, P.; Hampl, Aleš; Dvořák, Petr

2010-01-01

Roč. 21, - (2010), s. 676-686 ISSN 1472-6483 Grant - others:GA MŠk(CZ) LC06077; EC FP6(XE) LSHG-CT-2006-018739 Program:LC Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50390703 Keywords : animal protein-free culture * high-density culture * human embryonic stem cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.285, year: 2010

Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

Directory of Open Access Journals (Sweden)

Christian Niehage

Full Text Available Multipotent mesenchymal stromal cells (MSCs are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2% or high (10% serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention.

Media Compositions for Three-Dimensional Mammalian Tissue Growth under Microgravity Culture Conditions

Science.gov (United States)

Goodwin, Thomas J. (Inventor)

1998-01-01

Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue.The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

Media Compositions for Three Dimensional Mammalian Tissue Growth Under Microgravity Culture Conditions

Science.gov (United States)

Goodwin, Thomas J. (Inventor)

1998-01-01

Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

Batch culture of Azotobacter vinelandii under oxygen limitation conditionS

Energy Technology Data Exchange (ETDEWEB)

Camacho Rubio, F.; Martinez Nieto, L.; Fernandez Serrano, M.; Jimenez Moleon, M.C. [Departamento de Ingenieria Quimica, Universidad de Granada, Granada (Spain)

1996-12-01

The batch culture of Azotobacter vinealandii on glucose under nitrogen-fixing conditions, seeking oxygen limitation conditions, has been studied in order to use it as a Biological Test System for the experimental study of oxygen transfer enhancement methods in aerobic fermenters. overall kinetic parameters for exponential growth and for linear growth (under oxygen limitation) have been determined. It was noted an appreciable influence of the oxygen transfer rate on glucose and oxygen uptake, which seems to be due to alginate production, excreted as a nitrogenase protection mechanisms. (Author) 12 refs.

Humanized medium (h7H) allows long-term primary follicular thyroid cultures from human normal thyroid, benign neoplasm, and cancer.

Science.gov (United States)

Bravo, Susana B; Garcia-Rendueles, Maria E R; Garcia-Rendueles, Angela R; Rodrigues, Joana S; Perez-Romero, Sihara; Garcia-Lavandeira, Montserrat; Suarez-Fariña, Maria; Barreiro, Francisco; Czarnocka, Barbara; Senra, Ana; Lareu, Maria V; Rodriguez-Garcia, Javier; Cameselle-Teijeiro, Jose; Alvarez, Clara V

2013-06-01

Mechanisms of thyroid physiology and cancer are principally studied in follicular cell lines. However, human thyroid cancer lines were found to be heavily contaminated by other sources, and only one supposedly normal-thyroid cell line, immortalized with SV40 antigen, is available. In primary culture, human follicular cultures lose their phenotype after passage. We hypothesized that the loss of the thyroid phenotype could be related to culture conditions in which human cells are grown in medium optimized for rodent culture, including hormones with marked differences in its affinity for the relevant rodent/human receptor. The objective of the study was to define conditions that allow the proliferation of primary human follicular thyrocytes for many passages without losing phenotype. Concentrations of hormones, transferrin, iodine, oligoelements, antioxidants, metabolites, and ethanol were adjusted within normal homeostatic human serum ranges. Single cultures were identified by short tandem repeats. Human-rodent interspecies contamination was assessed. We defined an humanized 7 homeostatic additives medium enabling growth of human thyroid cultures for more than 20 passages maintaining thyrocyte phenotype. Thyrocytes proliferated and were grouped as follicle-like structures; expressed Na+/I- symporter, pendrin, cytokeratins, thyroglobulin, and thyroperoxidase showed iodine-uptake and secreted thyroglobulin and free T3. Using these conditions, we generated a bank of thyroid tumors in culture from normal thyroids, Grave's hyperplasias, benign neoplasms (goiter, adenomas), and carcinomas. Using appropriate culture conditions is essential for phenotype maintenance in human thyrocytes. The bank of thyroid tumors in culture generated under humanized humanized 7 homeostatic additives culture conditions will provide a much-needed tool to compare similarly growing cells from normal vs pathological origins and thus to elucidate the molecular basis of thyroid disease.

Technology Change And Working Conditions – A Cultural Perspective

DEFF Research Database (Denmark)

Sørensen, Ole Henning

2004-01-01

When technology change improves working conditions, the success is often attributed to skilful change agents. When it is not, the blame is on “resistance to change” and “resilient cultures”. How can these failures be understood differently? A cultural perspective on technology change might be a way...... to facilitate technology change processes that lead to improved working conditions. The research based project described here has developed a special homepage that explains how this might be achieved. The homepage is targeted at working life professionals. The homepage presents theoretical explanations...... of the concept of organizational culture, a model for analysis and several practical case stories. This paper explains how the project tries to reach a broad spectrum of professionals in order to facilitate their use of a cultural perspective. It also discusses the ethical consequences of the cultural...

Effects of a honeybee sting on the serum free amino acid profile in humans.

Directory of Open Access Journals (Sweden)

Jan Matysiak

Full Text Available The aim of this study was to assess the response to a honeybee venom by analyzing serum levels of 34 free amino acids. Another goal of this study was to apply complex analytic-bioinformatic-clinical strategy based on up-to-date achievements of mass spectrometry in metabolomic profiling. The amino acid profiles were determined using hybrid triple quadrupole/linear ion trap mass spectrometer coupled with a liquid chromatography instrument. Serum samples were collected from 27 beekeepers within 3 hours after they were stung and after a minimum of 6 weeks following the last sting. The differences in amino acid profiles were evaluated using MetaboAnalyst and ROCCET web portals. Chemometric tests showed statistically significant differences in the levels of L-glutamine (Gln, L-glutamic acid (Glu, L-methionine (Met and 3-methyl-L-histidine (3MHis between the two analyzed groups of serum samples. Gln and Glu appeared to be the most important metabolites for distinguishing the beekeepers tested shortly after a bee sting from those tested at least 6 weeks later. The role of some amino acids in the response of an organism to the honeybee sting was also discussed. This study indicated that proposed methodology may allow to identify the individuals just after the sting and those who were stung at least 6 weeks earlier. The results we obtained will contribute to better understanding of the human body response to the honeybee sting.

Culture conditions defining glioblastoma cells behavior: what is the impact for novel discoveries?

Science.gov (United States)

Ledur, Pítia Flores; Onzi, Giovana Ravizzoni; Zong, Hui; Lenz, Guido

2017-09-15

In cancer research, the use of established cell lines has gradually been replaced by primary cell cultures due to their better representation of in vivo cancer cell behaviors. However, a major challenge with primary culture involves the finding of growth conditions that minimize alterations in the biological state of the cells. To ensure reproducibility and translational potentials for research findings, culture conditions need to be chosen so that the cell population in culture best mimics tumor cells in vivo . Glioblastoma (GBM) is one of the most aggressive and heterogeneous tumor types and the GBM research field would certainly benefit from culture conditions that could maintain the original plethora of phenotype of the cells. Here, we review culture media and supplementation options for GBM cultures, the rationale behind their use, and how much those choices affect drug-screening outcomes. We provide an overview of 120 papers that use primary GBM cultures and discuss the current predominant conditions. We also show important primary research data indicating that "mis-cultured" glioma cells can acquire unnatural drug sensitivity, which would have devastating effects for clinical translations. Finally, we propose the concurrent test of four culture conditions to minimize the loss of cell coverage in culture.

Simplifying the human serum proteome for discriminating patients with bipolar disorder of other psychiatry conditions.

Science.gov (United States)

de Jesus, Jemmyson Romário; Galazzi, Rodrigo Moretto; de Lima, Tatiani Brenelli; Banzato, Cláudio Eduardo Muller; de Almeida Lima E Silva, Luiz Fernando; de Rosalmeida Dantas, Clarissa; Gozzo, Fábio Cézar; Arruda, Marco Aurélio Zezzi

2017-12-01

An exploratory analysis using proteomic strategies in blood serum of patients with bipolar disorder (BD), and with other psychiatric conditions such as Schizophrenia (SCZ), can provide a better understanding of this disorder, as well as their discrimination based on their proteomic profile. The proteomic profile of blood serum samples obtained from patients with BD using lithium or other drugs (N=14), healthy controls, including non-family (HCNF; N=3) and family (HCF; N=9), patients with schizophrenia (SCZ; N=23), and patients using lithium for other psychiatric conditions (OD; N=4) were compared. Four methods for simplifying the serum samples proteome were evaluated for both removing the most abundant proteins and for enriching those of lower-abundance: protein depletion with acetonitrile (ACN), dithiothreitol (DTT), sequential depletion using DTT and ACN, and protein equalization using commercial ProteoMiner® kit (PM). For proteomic evaluation, 2-D DIGE and nanoLC-MS/MS analysis were employed. PM method was the best strategy for removing proteins of high abundance. Through 2-D DIGE gel image comparison, 37 protein spots were found differentially abundant (p<0.05, Student's t-test), which exhibited ≥2.0-fold change of the average value of normalized spot intensities in the serum of SCZ, BD and OD patients compared to subject controls (HCF and HCNF). From these spots detected, 13 different proteins were identified: ApoA1, ApoE, ApoC3, ApoA4, Samp, SerpinA1, TTR, IgK, Alb, VTN, TR, C4A and C4B. Proteomic analysis allowed the discrimination of patients with BD from patients with other mental disorders, such as SCZ. The findings in this exploratory study may also contribute for better understanding the pathophysiology of these disorders and finding potential serum biomarkers for these conditions. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Free Port of Vladivostok: Development Conditions, Prospects, Risks

Directory of Open Access Journals (Sweden)

Elena Viktorovna Krasova

2015-12-01

Full Text Available One of the priority directions of Russia’s state policy with regard to territories’ development is the development of the Far Eastern region. The free port of Vladivostok is one of the key projects that are currently being implemented in the southern part of Primorsky Krai and they focus on boosting the socio-economic development of Vladivostok city, Primorsky Krai and the Far Eastern region as a whole. The goal of this research is to consider the conceptual issues of the free port of Vladivostok, to outline the conditions and prospects of its operation and identify risks associated with its future development. The article defines the modern concept of “free port”, characterizes historical experience of the “free port” regime in Vladivostok in the end of the 19th century, substantiates the strategic importance of using the free port of Vladivostok as a tool of intensive growth of Russian Far Eastern territories. The article also discloses the essence of the free port of Vladivostok as a means to promote free entrepreneurship, which is used to increase freight traffic through the port of Vladivostok, increase foreign trade, attract new investments, create new industries and develop the port’s infrastructure. The article provides a review of the main tax benefits and perspectives of the free port of Vladivostok in terms of the gross regional product formation. Analyzing the mechanisms of effective functioning of the free port of Vladivostok, the authors identify the short-term (mid-term and long-term priorities and operational risks for the free port of Vladivostok. The article also touches upon some issues concerning the response of some foreign countries to the creation of the free port of Vladivostok

Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

Science.gov (United States)

Saha, Krishanu; Mei, Ying; Reisterer, Colin M; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C; Alexander, Morgan R; Langer, Robert; Anderson, Daniel G; Jaenisch, Rudolf

2011-11-15

The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

Multiplexed activity-based protein profiling of the human pathogen Aspergillus fumigatus reveals large functional changes upon exposure to human serum.

Science.gov (United States)

Wiedner, Susan D; Burnum, Kristin E; Pederson, LeeAnna M; Anderson, Lindsey N; Fortuin, Suereta; Chauvigné-Hines, Lacie M; Shukla, Anil K; Ansong, Charles; Panisko, Ellen A; Smith, Richard D; Wright, Aaron T

2012-09-28

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli.

Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum*

Science.gov (United States)

Wiedner, Susan D.; Burnum, Kristin E.; Pederson, LeeAnna M.; Anderson, Lindsey N.; Fortuin, Suereta; Chauvigné-Hines, Lacie M.; Shukla, Anil K.; Ansong, Charles; Panisko, Ellen A.; Smith, Richard D.; Wright, Aaron T.

2012-01-01

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli. PMID:22865858

Characterization of a plasminogen activator from human melanoma cells cultured in vitro

International Nuclear Information System (INIS)

Heussen, C.

1982-08-01

This thesis describes the work that have been done on the isolation and characterization of a plasminogen activator, Mel-PA, that is released by human melanoma cells cultured in vitro. This enzyme was compared to the urinary plasminogen activator, urokinase. The human melanoma cell line released large amounts of Mel-PA into the surrounding medium when cultured under serum-free conditions. These cells released only one type of plasminogen activator. A technique was developed in which plasminogen activators were seperated electrophoretically and detected in polyacrylamide gel slabs. Mel-PA was concentrated and partially purified by affinity chromatography on benzamidine-sepharose. A study of the distribution of plasminogen activators in tissues and body fluids showed that all mammals examined had two immunochemically distinct plasminogen activators that corresponded, in their distribution, to the urokinase-like and Mel-PA like enzymes of man. A comparitive study of the kinetic behaviour of Mel-PA and urokinase showed numerous differences between the catalytic activities of these two enzymes

Chitosan-catechol: a writable bioink under serum culture media.

Science.gov (United States)

Lee, Daiheon; Park, Joseph P; Koh, Mi-Young; Kim, Pureum; Lee, Junhee; Shin, Mikyung; Lee, Haeshin

2018-05-01

Mussel-inspired adhesive coatings on biomedical devices have attracted significant interest due to their unique properties such as substrate independency and high efficiency. The key molecules for mussel-inspired adhesive coatings are catechol and amine groups. Along with the understanding of catechol chemistry, chitosan-catechol has also been developed as a representative mussel-inpired adhesive polymer that contains catechol and amine groups for adhesiveness. Herein, we demonstrated the direct writability of chitosan-catechol as a bioink for 3D printing, one of the additive techniques. The use of chitosan-catechol bioink results in the formation of 3D constructs in normal culture media via rapid complexation of this bioink with serum proteins; in addition, the metal/catechol combination containing tiny amounts of vanadyl ions, in which the ratio of metal to catechol is 0.0005, dramatically enhances the mechanical strength and printability of the cell-encapsulated inks, showing a cell viability of approximately 90%. These findings for mussel-inspired bioinks will be a promising way to design a biocompatible 3D bioink cross-linked without any external stimuli.

Screening of culture condition for xylanase production by ...

African Journals Online (AJOL)

The study demonstrated not only the importance of the nature of the substrate in obtaining a system resistant to catabolic repression, but also the importance of the culture conditions for biosynthesis of this enzyme. T. viride showed a high potential for xylanase production under the conditions presented in these assays.

Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis

Directory of Open Access Journals (Sweden)

Amanda Piccoli Frasson

2012-03-01

Full Text Available Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum. The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

A knowledge-based theory of rising scores on "culture-free" tests.

Science.gov (United States)

Fox, Mark C; Mitchum, Ainsley L

2013-08-01

Secular gains in intelligence test scores have perplexed researchers since they were documented by Flynn (1984, 1987). Gains are most pronounced on abstract, so-called culture-free tests, prompting Flynn (2007) to attribute them to problem-solving skills availed by scientifically advanced cultures. We propose that recent-born individuals have adopted an approach to analogy that enables them to infer higher level relations requiring roles that are not intrinsic to the objects that constitute initial representations of items. This proposal is translated into item-specific predictions about differences between cohorts in pass rates and item-response patterns on the Raven's Matrices (Flynn, 1987), a seemingly culture-free test that registers the largest Flynn effect. Consistent with predictions, archival data reveal that individuals born around 1940 are less able to map objects at higher levels of relational abstraction than individuals born around 1990. Polytomous Rasch models verify predicted violations of measurement invariance, as raw scores are found to underestimate the number of analogical rules inferred by members of the earlier cohort relative to members of the later cohort who achieve the same overall score. The work provides a plausible cognitive account of the Flynn effect, furthers understanding of the cognition of matrix reasoning, and underscores the need to consider how test-takers select item responses. PsycINFO Database Record (c) 2013 APA, all rights reserved.

Serum Thyroxine to Thyroxine-Binding Globulin Ratio in Pregnancy and Newborn

International Nuclear Information System (INIS)

Kim, Ji Yeul

1982-01-01

To evaluate the diagnostic value of the ratio of serum thyroxine(T 4 ) /thyroxine-binding globulin (TBG) for the thyroid status in pregnancy and newborn serum thyroxine, TBG, triiodothyronine, and free thyroxine levels were radioimmunoassayed in normal pregnant women at each of the trimesters, and the calculated serum T 4 /TBG ratios were compared with other parameters such as T 3 /TBG ratio and free T 4 /TBG ratio and free T 4 /TBG ratio. Serum T 4 levels were elevated with the proportionate increase in TBG levels during pregnancy, leading to the nearly constant value of serum T 4 /TBG ratios as in normal non-pregnant controls. In contrast, serum T 3 /TBG and free T 4 /TBG ratios varied considerably during pregnancy. In newborn, T 4 levels were nearly not changed with compared non-pregnant control value and TBG levels were elevated. The results indicate that serum T 4 /TBG ratio is a better parameter than others in evaluating the thyroid status during pregnancy and but newborn is a no better.

Fasting induces the generation of serum thyronine-binding globulin in Zucker rats

International Nuclear Information System (INIS)

Young, R.A.; Rajatanavin, R.; Moring, A.F.; Braverman, L.E.

1985-01-01

Five-month-old lean and obese Zucker rats were fasted for up to 7 days (lean rats) or 28 days (obese rats), and serum total and free T4 and T3 concentrations, percent free T4 and T3 by equilibrium dialysis, and the binding of [ 125 I] T4 to serum proteins by gel electrophoresis were measured. In the lean rats, a 4- or 7-day fast resulted in significant decreases in serum total and free T4 and T3 concentrations. There was a decrease in the percent free T3 after 7 days of starvation. In contrast, a 4- or 7-day fast did not alter any of these variables in the obese rats. However, after 14 or more days of starvation, serum total T4 and T3 concentrations increased, and the percent free T4 and T3 decreased, resulting in no change in the serum free T4 or T3 concentrations in the obese rats. The percent of [ 125 I]T4 bound to serum thyronine-binding globulin increased and the percent bound to thyronine-binding prealbumin decreased with the duration of the fast in both the lean and obese rats. The increase in serum thyronine-binding globulin binding of T4 can explain the increase in serum total T4 and T3 concentrations, the decrease in percent free T4 and T3, and the normal free hormone concentration in the long term fasted obese rats. The findings in the lean rats appear to be due to a combination of the known central hypothyroidism that occurs during 4-7 days of fasting and the fasting-induced changes in T4 binding in serum. Changes in T4 and T3 binding in serum during fasting in the rat must be considered when the effects of fasting on serum concentrations of the thyroid hormones, thyroid hormone kinetics, and the peripheral action of the thyroid hormones are evaluated

Equine dendritic cells generated with horse serum have enhanced functionality in comparison to dendritic cells generated with fetal bovine serum.

Science.gov (United States)

Ziegler, Anja; Everett, Helen; Hamza, Eman; Garbani, Mattia; Gerber, Vinzenz; Marti, Eliane; Steinbach, Falko

2016-11-15

Dendritic cells are professional antigen-presenting cells that play an essential role in the initiation and modulation of T cell responses. They have been studied widely for their potential clinical applications, but for clinical use to be successful, alternatives to xenogeneic substances like fetal bovine serum (FBS) in cell culture need to be found. Protocols for the generation of dendritic cells ex vivo from monocytes are well established for several species, including horses. Currently, the gold standard protocol for generating dendritic cells from monocytes across various species relies upon a combination of GM-CSF and IL-4 added to cell culture medium which is supplemented with FBS. The aim of this study was to substitute FBS with heterologous horse serum. For this purpose, equine monocyte-derived dendritic cells (eqMoDC) were generated in the presence of horse serum or FBS and analysed for the effect on morphology, phenotype and immunological properties. Changes in the expression of phenotypic markers (CD14, CD86, CD206) were assessed during dendritic cell maturation by flow cytometry. To obtain a more complete picture of the eqMoDC differentiation and assess possible differences between FBS- and horse serum-driven cultures, a transcriptomic microarray analysis was performed. Lastly, immature eqMoDC were primed with a primary antigen (ovalbumin) or a recall antigen (tetanus toxoid) and, after maturation, were co-cultured with freshly isolated autologous CD5 + T lymphocytes to assess their T cell stimulatory capacity. The microarray analysis demonstrated that eqMoDC generated with horse serum were indistinguishable from those generated with FBS. However, eqMoDC incubated with horse serum-supplemented medium exhibited a more characteristic dendritic cell morphology during differentiation from monocytes. A significant increase in cell viability was also observed in eqMoDC cultured with horse serum. Furthermore, eqMoDC generated in the presence of horse serum

Modification and standardization of the culture of early postimplantation embryos for toxicological studies

Energy Technology Data Exchange (ETDEWEB)

Klug, S.; Lewandowski, C.; Neubert, D.

1985-12-01

The method of culturing ''whole'' rat embryos (days 9.5-11.5 of gestation, i.e. at the early stage of organogenesis) as modified and standardized in our laboratory is presented. We have succeeded in using bovine serum as culture medium instead of rat serum as recommended in the original procedure. Experimental conditions are described for obtaining reproducible results. An improved scoring system was developed which, in connection with a computerized documentation, greatly facilitates the evaluation of the data.

Radioimmunological determination of reverse triiodo thyronine in unextracted serum and serum dialysates

International Nuclear Information System (INIS)

Laurberg, P.; Weeke, J.

1977-01-01

A sensitive radioimmunoassay for measurements of 3,3,5-triiodothyronine (reverse T3,rT3) in small amounts of unextracted serum is described. The interference of rT3 binding proteins in serum was precluded by addition of 8-anilinol-naphthalene sulphonic acid (ANS). The cross reaction of T4 with the rT3 anti-serum varied with the concentration of T4 in the samples. At 50 per cent inhibition of I 125 rT3 binding, the T4 cross reaction was 0.075% (mol/mol). All values were corrected for T4 cross reaction. By the present method total rT3 averaged 0.37 nmol/l in thirty-two normal subjects. Higher values (0.81-1.98 nmol/l) were obtained in seventeen thyrotoxic patients, while the rT3 was very low (<=0.046 nmol/l) in ten patients with severe primary hypothyroidism. A modification of the total rT3 assay was used for measurements of rT3 in serum dialysates (free rT3). The sensitivity was 0.46 pmol/l. This sensitivity did not allow detection of free rT3 in all normal subjects. (Auth.)

Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

Science.gov (United States)

Maruyama, Junya; Matsunaga, Tamihide; Yamaori, Satoshi; Sakamoto, Sakae; Kamada, Noboru; Nakamura, Katsunori; Kikuchi, Shinji; Ohmori, Shigeru

2013-01-01

We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.

Utility of free prostate specific antigen serum level and its related parameters in the diagnosis of prostate cancer

Directory of Open Access Journals (Sweden)

Azmi A Haroun

2011-01-01

Full Text Available We evaluated the role of free prostate specific antigen (f-PSA serum level and its related parameters in detecting prostate cancer. This retrospective study was conducted between January 2006 and March 2008. Trans-rectal ultrasound guided prostate biopsy was performed for 107 patients who had total PSA (t-PSA level of either >4 ng/mL with or without palpable nodule or ≤4 ng/mL with palpable nodule on digital rectal examination. The perfor-mance measurements for f-PSA, percent free PSA (%f-PSA and free PSA density (f-PSAD were determined and compared with those for t-PSA and total PSA density (t-PSAD. Descriptive statistics for all variables of interest were calculated, and receiver operating characteristic curves were generated. Nine patients (8.4% had normal histology, 69 patients (64.4% had benign disease and 29 patients (27.1% had prostate cancer. The performance of f-PSA in PCa detection was better than other evaluated parameters. The largest area under the curve for patients in the gray area (t-PSA range 4.1-10 ng/mL was for f-PSA, with a value of 0.64 and a sensitivity and specificity of 44% and 87%, respectively. For %f-PSA, these values were 0.59, 63% and 62%, respectively. For patients with a t-PSA level of 10.1-20 ng/mL, they were 0.68, 67%, and 81%, respectively, for f-PSA, and 0.64, 67%, and 76%, respectively, for %f-PSA. In conclusion, f-PSA serum levels performed better than free to total PSA ratio and t-PSA for prostate cancer screening. It is of clinical value which could affect the biopsy decision avoiding unnecessary interventions.

Sensitive and selective culture medium for detection of environmental Clostridium difficile isolates without requirement for anaerobic culture conditions.

Science.gov (United States)

Cadnum, Jennifer L; Hurless, Kelly N; Deshpande, Abhishek; Nerandzic, Michelle M; Kundrapu, Sirisha; Donskey, Curtis J

2014-09-01

Effective and easy-to-use methods for detecting Clostridium difficile spore contamination would be useful for identifying environmental reservoirs and monitoring the effectiveness of room disinfection. Culture-based detection methods are sensitive for detecting C. difficile, but their utility is limited due to the requirement of anaerobic culture conditions and microbiological expertise. We developed a low-cost selective broth medium containing thioglycolic acid and l-cystine, termed C. difficile brucella broth with thioglycolic acid and l-cystine (CDBB-TC), for the detection of C. difficile from environmental specimens under aerobic culture conditions. The sensitivity and specificity of CDBB-TC (under aerobic culture conditions) were compared to those of CDBB (under anaerobic culture conditions) for the recovery of C. difficile from swabs collected from hospital room surfaces. CDBB-TC was significantly more sensitive than CDBB for recovering environmental C. difficile (36/41 [88%] versus 21/41 [51%], respectively; P = 0.006). C. difficile latex agglutination, an enzyme immunoassay for toxins A and B or glutamate dehydrogenase, and a PCR for toxin B genes were all effective as confirmatory tests. For 477 total environmental cultures, the specificity of CDBB-TC versus that of CDBB based upon false-positive yellow-color development of the medium without recovery of C. difficile was 100% (0 false-positive results) versus 96% (18 false-positive results), respectively. False-positive cultures for CDBB were attributable to the growth of anaerobic non-C. difficile organisms that did not grow in CDBB-TC. Our results suggest that CDBB-TC provides a sensitive and selective medium for the recovery of C. difficile organisms from environmental samples, without the need for anaerobic culture conditions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Estimation of Radiation Limit from a Huygens' Box under Non-Free-Space Conditions

DEFF Research Database (Denmark)

Franek, Ondrej; Sørensen, Morten; Bonev, Ivan Bonev

2013-01-01

The recently studied Huygens' box method has difficulties when radiation of an electronic module is to be determined under non-free-space conditions, i.e. with an enclosure. We propose an estimate on radiation limit under such conditions based only on the Huygens' box data from free...

Tumour-cytolytic human monocyte-derived macrophages: a simple and efficient method for the generation and long-term cultivation as non-adherent cells in a serum-free medium.

Science.gov (United States)

Streck, R J; Hurley, E L; Epstein, D A; Pauly, J L

1992-01-01

We report a simple and efficient culture procedure for the generation of tumour-cytolytic human monocyte-derived macrophages (MAC). In this method, normal human peripheral blood mononuclear cells, isolated using a conventional Ficoll-Hypaque density gradient procedure, are cultured as a heterogenous leukocyte population in Teflon or other hydrophobic cultureware, in a commercially available serum-free culture medium (M-SFM) that has been formulated specifically for the cultivation and ex vivo stimulation of human monocytes and MAC, and in the absence of exogenous mitogens, antigens, cytokines or other stimulants. This procedure features a negative-selection technique that takes advantage of the differential survival of blood leukocytes. Using the prescribed in vitro conditions, lymphocytes survived relatively poorly, whereas monocytes differentiated in the absence of exogenous stimulants into mature tumour-cytolytic MAC. The MAC were present as non-adherent, single cells that expressed good viability (greater than 95%) for a prolonged period (greater than 60 days). When compared to conventional procedures for generating MAC, the prescribed technique is thought to offer several important advantages in that it: (a) eliminates the tedious and cumbersome monocyte isolation procedures, thus providing a significant savings not only in time and money but also in eliminating repetitive cell manipulations that have often been associated with damage to monocyte morphology and/or function; (b) reduces the loss of monocyte subsets that are not recovered during specific isolation procedures; (c) facilitates harvesting a single cell, non-adherent suspension of immunocompetent MAC suitable for various examinations including analyses defining MAC morphology, cytochemistry, phenotype and function; and (d) eliminates variability and artifacts associated with different sera that are utilised frequently as medium supplements. The utility of the prescribed method is illustrated by the

Correlation between Serum Aldosterone Level and Hearing Condition of Elderly Patients Referred to Otolaryngology Services of Hamadan, Western Iran

Directory of Open Access Journals (Sweden)

Dr. Farhad Farahani

2010-06-01

Full Text Available Background and Aim: Recently, more attention was paid to the direct protective effect of aldosterone against hearing impairment in elderly patients. The aim of this study was determination of possible correlation between serum aldosterone level and hearing condition of elderly patients that referred to the Otolaryngology services of Hamadan in 2005-2006.Methods: In this case control study 54 (27 males,27 females persons above 60 years old were evaluated. They contained twenty eight cases with normal hearing and 26 cases with presbycusis. Persons with any abnormal biochemical finding or history of conditions that predispose them to the sensorineural hearing loss (SNHL were excluded. In both groups serum level of sodium, potassium and aldosterone were measured and hearing condition evaluated by puretone, speech and immitance audiometry.Results: Statistical relationship between serum aldostrone level and hearing condition, sex, configuration of audiogram and speech discrimination score (SDS were not significant. In addition, no significant relationship between sodium and potassium levels with hearing condition was found (p>0.05.Conclusion: This study could not confirm protective effect of aldostrone against presbycusis. This discrepancy may originate from epidemiologic differences, laboratory errors or small sample size.

Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system.

Science.gov (United States)

McLaughlin, M; Albertini, D F; Wallace, W H B; Anderson, R A; Telfer, E E

2018-03-01

Can complete oocyte development be achieved from human ovarian tissue containing primordial/unilaminar follicles and grown in vitro in a multi-step culture to meiotic maturation demonstrated by the formation of polar bodies and a Metaphase II spindle? Development of human oocytes from primordial/unilaminar stages to resumption of meiosis (Metaphase II) and emission of a polar body was achieved within a serum free multi-step culture system. Complete development of oocytes in vitro has been achieved in mouse, where in vitro grown (IVG) oocytes from primordial follicles have resulted in the production of live offspring. Human oocytes have been grown in vitro from the secondary/multi-laminar stage to obtain fully grown oocytes capable of meiotic maturation. However, there are no reports of a culture system supporting complete growth from the earliest stages of human follicle development through to Metaphase II. Ovarian cortical biopsies were obtained with informed consent from women undergoing elective caesarean section (mean age: 30.7 ± 1.7; range: 25-39 years, n = 10). Laboratory setting. Ovarian biopsies were dissected into thin strips, and after removal of growing follicles were cultured in serum free medium for 8 days (Step 1). At the end of this period secondary/multi-laminar follicles were dissected from the strips and intact follicles 100-150 μm in diameter were selected for further culture. Isolated follicles were cultured individually in serum free medium in the presence of 100 ng/ml of human recombinant Activin A (Step 2). Individual follicles were monitored and after 8 days, cumulus oocyte complexes (COCs) were retrieved by gentle pressure on the cultured follicles. Complexes with complete cumulus and adherent mural granulosa cells were selected and cultured in the presence of Activin A and FSH on membranes for a further 4 days (Step 3). At the end of Step 3, complexes containing oocytes >100 μm diameter were selected for IVM in SAGE medium (Step 4) then

Erythroid differentiation and commitment in rat erythroleukemia cells with hypertonic culture conditions.

OpenAIRE

Yamaguchi, Y; Kluge, N; Ostertag, W; Furusawa, M

1981-01-01

Cell cultures of 7,12-dimethylbenz[a]anthracene-induced rat erythroleukemia can be stimulated to synthesize hemoglobin when cultured in hypertonic media. During hypertonic treatment the intracellular osmotic conditions immediately readjust to those of the extracellular medium. None of the Friend virus-induced mouse erythroleukemia cell lines was inducible for differentiation with the same hypertonic culture conditions used for rat cells. Earliest commitment to erythroid terminal differentiati...

Statistical optimization of cultural conditions by response surface ...

African Journals Online (AJOL)

STORAGESEVER

2009-08-04

Aug 4, 2009 ... Full Length Research Paper. Statistical optimization of cultural conditions by response surface methodology for phenol degradation by a novel ... Phenol is a hydrocarbon compound that is highly toxic, ... Microorganism.

A portable anaerobic microbioreactor reveals optimum growth conditions for the methanogen Methanosaeta concilii.

Science.gov (United States)

Steinhaus, Benjamin; Garcia, Marcelo L; Shen, Amy Q; Angenent, Largus T

2007-03-01

Conventional studies of the optimum growth conditions for methanogens (methane-producing, obligate anaerobic archaea) are typically conducted with serum bottles or bioreactors. The use of microfluidics to culture methanogens allows direct microscopic observations of the time-integrated response of growth. Here, we developed a microbioreactor (microBR) with approximately 1-microl microchannels to study some optimum growth conditions for the methanogen Methanosaeta concilii. The microBR is contained in an anaerobic chamber specifically designed to place it directly onto an inverted light microscope stage while maintaining a N2-CO2 environment. The methanogen was cultured for months inside microchannels of different widths. Channel width was manipulated to create various fluid velocities, allowing the direct study of the behavior and responses of M. concilii to various shear stresses and revealing an optimum shear level of approximately 20 to 35 microPa. Gradients in a single microchannel were then used to find an optimum pH level of 7.6 and an optimum total NH4-N concentration of less than 1,100 mg/liter (<47 mg/liter as free NH3-N) for M. concilii under conditions of the previously determined ideal shear stress and pH and at a temperature of 35 degrees C.

Serum-dependent effects of tamoxifen and cannabinoids upon C6 glioma cell viability.

Science.gov (United States)

Jacobsson, S O; Rongård, E; Stridh, M; Tiger, G; Fowler, C J

2000-12-15

In the present study, the effects of the combination of tamoxifen ((Z)-2[p-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylamine citrate) and three cannabinoids (Delta(9)-tetrahydrocannabinol [Delta(9)-THC], cannabidiol, and anandamide [AEA]) upon the viability of C6 rat glioma cells was assessed at different incubation times and using different culturing concentrations of foetal bovine serum (FBS). Consistent with previous data for human glioblastoma cells, the tamoxifen sensitivity of the cells was increased as the FBS content of the culture medium was reduced from 10 to 0.4 and 0%. The cells expressed protein kinase C alpha and calmodulin (the concentration of which did not change significantly as the FBS concentration was reduced), but did not express estrogen receptors. Delta(9)-THC and cannabidiol, but not AEA, produced a modest reduction in cell viability after 6 days of incubation in serum-free medium, whereas no effects were seen in 10% FBS-containing medium. There was no observed synergy between the effects of tamoxifen and the cannabinoids upon cell viability.

Isolation of intracellular parasites (Plasmodium falciparum) from culture using free-flow electrophoresis: separation of the free parasites according to stages.

Science.gov (United States)

Heidrich, H G; Mrema, J E; Vander Jagt, D L; Reyes, P; Rieckmann, K H

1982-06-01

Parasitized human erythrocytes were concentrated from continuous cultures of Plasmodium falciparum from 5-7% up to 80-95% using Plasmagel. After aggregation of the cells with phythemagglutinin, the aggregated erythrocytes were fragmented by passing them, with minimal force, through successive nylon filters of decreasing pore size (100 microns-3 microns). The mixture of liberated, free parasites, intact erythrocytes and erythrocyte membrane vesicles was separated using free-flow electrophoresis. Most of the fractions containing free parasites did not show contamination with erythrocyte constituents as determined by light and electron microscopy, polyacrylamide gel electrophoresis, and enzymatic analysis. In addition, the various stages of free parasites of Plasmodium falciparum exhibited different electrical surface charges. Rings and trophozoites were highly negatively charged whereas schizonts and, in particular, merozoites showed low negative charges. Thus, the various stages could be isolated separate from each other.

[Detection of leptospira by culture of vitreous humor and detection of antibodies against leptospira in vitreous humor and serum of 225 horses with equine recurrent uveitis].

Science.gov (United States)

Dorrego-Keiter, Elisa; Tóth, József; Dikker, Lieke; Sielhorst, Jutta; Schusser, Gerald Fritz

2016-01-01

In the ongoing discussion regarding the aetiopathogenesis of equine recurrent uveitis (ERU) it was the aim of the present study to elucidate the relationship of leptospira infection and ERU. In a population of 225 horses leptospira were examined in vitreous humor by culture and leptospira antibody were detected in vitreous humor and serum samples. Preoperative serum samples were collected from 221/225 ERU patients of different age, gender and breed. Undiluted vitreous humor was aseptically taken from 198/225 patients that underwent pars plana vitrectomy at the beginning of surgery and from 27/225 patients' eyeball after enucleation: Serum and vitreous humor were tested for specific leptospiral antibodies by microscopic agglutination test (MAT). Furthermore, vitreous humor was examined by culture. 20 patients which were euthanized due to a live-threatening disease other than ERU served as a control group. A total of 127/221 (57.5%) horses had serum antibodies (≥ 1:100). Most frequently antibodies against L. interrogans serovar Grippotyphosa were detected (79/127), followed by L. interrogans serovar lcterohaemorrhagiae (34/127) and L. interrogans serovar Bratislava (29/127). Only 79/225 horses (35.1%) had leptospiral antibodies in vitreous humor, in which L. interrogans serovar Grippotyphosa (67/79) was identified most frequently followed by L. interrogans serovar Pomona (18/79) and L. interrogans serovar lcterohaemorrhagiae (8/79) which was identified as single or multiple reaction. Isolation of leptospira from vitreous humor was positive in 34/212 horses (16%). 10/20 control horses had a positive antibody titer against leptospira in serum and 2/20 horses in vitreous humor, whereas there was no leptospira detected in culture. The result of 84% negative cultures from vitreous humor of 212 ERU patients is decisive for the diagnosis and therapy of ERU.

Boundary conditions for free surface inlet and outlet problems

KAUST Repository

Taroni, M.; Breward, C. J. W.; Howell, P. D.; Oliver, J. M.

2012-01-01

We investigate and compare the boundary conditions that are to be applied to free-surface problems involving inlet and outlets of Newtonian fluid, typically found in coating processes. The flux of fluid is a priori known at an inlet, but unknown

Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

NARCIS (Netherlands)

Naaijkens, B.A.; Niessen, H.W.M.; Prins, H.J.; Krijnen, P.A.J.; Kokhuis, T.J.A.; de Jong, N.; van Hinsbergh, V.W.M.; Kamp, O.; Helder, M.N.; Musters, R.J.P.; van Dijk, A.; Juffermans, L.J.M.

2012-01-01

Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically,

Assessment of genetic and epigenetic changes in virus-free garlic (Allium sativum L.) plants obtained by meristem culture followed by in vitro propagation.

Science.gov (United States)

Gimenez, Magalí Diana; Yañez-Santos, Anahí Mara; Paz, Rosalía Cristina; Quiroga, Mariana Paola; Marfil, Carlos Federico; Conci, Vilma Cecilia; García-Lampasona, Sandra Claudia

2016-01-01

This is the first report assessing epigenetic variation in garlic. High genetic and epigenetic polymorphism during in vitro culture was detected.Sequencing of MSAP fragments revealed homology with ESTs. Garlic (Allium sativum) is a worldwide crop of economic importance susceptible to viral infections that can cause significant yield losses. Meristem tissue culture is the most employed method to sanitize elite cultivars.Often the virus-free garlic plants obtained are multiplied in vitro (micro propagation). However, it was reported that micro-propagation frequently produces somaclonal variation at the phenotypic level, which is an undesirable trait when breeders are seeking to maintain varietal stability. We employed amplification fragment length polymorphism and methylation sensitive amplified polymorphism (MSAP) methodologies to assess genetic and epigenetic modifications in two culture systems: virus-free plants obtained by meristem culture followed by in vitro multiplication and field culture. Our results suggest that garlic exhibits genetic and epigenetic polymorphism under field growing conditions. However, during in vitro culture system both kinds of polymorphisms intensify indicating that this system induces somaclonal variation. Furthermore, while genetic changes accumulated along the time of in vitro culture, epigenetic polymorphism reached the major variation at 6 months and then stabilize, being demethylation and CG methylation the principal conversions.Cloning and sequencing differentially methylated MSAP fragments allowed us to identify coding and unknown sequences of A. sativum, including sequences belonging to LTR Gypsy retrotransposons. Together, our results highlight that main changes occur in the initial 6 months of micro propagation. For the best of our knowledge, this is the first report on epigenetic assessment in garlic.

Optimization of culture conditions to support long-term self-renewal of buffalo (Bubalus bubalis) embryonic stem cell-like cells.

Science.gov (United States)

Sharma, Ruchi; George, Aman; Kamble, Nitin Manchindra; Singh, Karn Pratap; Chauhan, Manmohan Singh; Singla, Suresh Kumar; Manik, Radhey Sham; Palta, Prabhat

2011-12-01

A culture system capable of sustaining self-renewal of buffalo embryonic stem (ES) cell-like cells in an undifferentiated state over a long period of time was developed. Inner cell masses were seeded on KO-DMEM+15% KO-serum replacer on buffalo fetal fibroblast feeder layer. Supplementation of culture medium with 5 ng/mL FGF-2 and 1000 IU/mL mLIF gave the highest (p<0.05) rate of primary colony formation. The ES cell-like cells' colony survival rate and increase in colony size were highest (p<0.05) following supplementation with FGF-2 and LIF compared to other groups examined. FGF-2 supplementation affected the quantitative expression of NANOG, SOX-2, ACTIVIN A, BMP 4, and TGFβ1, but not OCT4 and GREMLIN. Supplementation with SU5402, an FGFR inhibitor (≥20 μM) increased (p<0.05) the percentage of colonies that differentiated. FGFR1-3 and ERK1, K-RAS, E-RAS, and SHP-2, key signaling intermediates of FGF signaling, were detected in ES cell-like cells. Under culture conditions described, three ES cell lines were derived that, to date, have been maintained for 135, 95, and 85 passages for over 27, 19, and 17 months, respectively, whereas under other conditions examined, ES cell-like cells did not survive beyond passage 10. The ES cell-like cells were regularly monitored for expression of pluripotency markers and their potency to form embryoid bodies.

[Preliminary Study of Lonicera hypoglauca on Germination Conditions of Sand Culture Seeds and Sterilization Method of Sand Culture Seedling Sterilization].

Science.gov (United States)

Tan, Mu-xiu; Zeng, Wen-wen; Wei, Peng-xiao; Mo, Qiao-cheng; Pu, Zu-ning; Cen, Xiu-fen; Shi, Feng-hua

2015-05-01

To explore the germination conditions of Lonicera hypoglauca sand culture seeds and the effects of sand culture seedlings sterilization. 0.1% HgCl2 with different sterilization time, different illumination time and temperature culture condition were adopted to study the germination conditions of sand culture seeds. Different sterilization treatments and different hardening-seedling days were used to test the sterilization effect of sand culture seedlings. The sterilization effect of the combination of 75% ethanol 30 s + 0.1% HgCl2 5 min on Lonicera hypoglauca seeds was the optimum,with the average pollution rate of 15.56%, and the average germination rate reached 51.11%. The combination of varied temperature-room temperature under light for 12 h/d was the best, with the average germination rate peaked at 75.49%, and the average germination potential reached 68.36%. The treatment of detergent liquor scrub-tap water wash on the part above the hypocotyl, which was sand cultured under the opening condition and had no root, showed the best sterilization effect, with the average pollution rate was zero, and the average survival rate peaked at 100.00%. The sterilization effect of sand culture seedlings, which was disinfected after cleaning by detergent liquor scrub-tap water wash after hardening-seeding for 30 days, was the best, with the average pollution rate of 50.00%, and the average survival rate of 100.00%. The best sterilization effect is the combination of 75% ethanol 30 s + 0.1% HgCl2 5 min; Lighting for 12 h/d of varied temperature-room temperature is regarded as the optimum culture condition. The treatment of detergent liquor scrub-tap water wash treatment on the part above the hypocotyl,which is sand cultured under the opening condition and had no root, shows the best sterilization effect. For the sand culture seedlings, before inoculated in subculture medium, should be hardening-seedling for some days and sterilized after detergent liquor scrub-tap water wash.

Long-term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes.

Science.gov (United States)

Suyatno; Kitamura, Yuka; Ikeda, Shuntaro; Minami, Naojiro; Yamada, Masayasu; Imai, Hiroshi

2018-03-01

Undifferentiated spermatogonia eventually differentiate in the testis to produce haploid sperm. Within this cell population, there is a small number of spermatogonial stem cells (SSCs). SSCs are rare cells in the testis, and their cellular characteristics are poorly understood. Establishment of undifferentiated cell line would provide an indispensable tool for studying their biological nature and spermiogenesis/spermatogenesis in vitro. However, there have been few reports on the long-term culture of undifferentiated spermatogonia in species other than rodents. Here, we report the derivation and long-term in vitro culture of undifferentiated spermatogonia cell lines from immature and adult bovine testes. Cell lines from immature testes were maintained in serum-free culture conditions in the presence of glial-cell-line-derived neurotropic factor (GDNF) and bovine leukemia inhibitory factor (bLIF). These cell lines have embryonic stem (ES)-like cell morphology, express pluripotent-stem-cell-specific and germ-cell-specific markers at the protein and mRNA levels, and contributed to the inner cell mass (ICM) of embryos in the blastocyst stage. Meanwhile, cell lines established from adult testes were maintained in low-serum media in the presence of 6-bromoindirubin-3'-oxime (BIO). These cell lines have characteristics resembling those of previously reported male mouse germ cell lines as confirmed by their botryoidally aggregated morphology, as well as the expression of germ-cell-specific markers and pluripotent stem cell markers. These findings could be useful for the development of long-term culture of undifferentiated spermatogonia, which could aid in conservation of species and improvement of livestock production through genome editing technology. © 2018 Wiley Periodicals, Inc.

A comparison of the metabolism of the abortifacient compounds from Ponderosa pine needles in conditioned versus naive cattle.

Science.gov (United States)

Welch, K D; Gardner, D R; Pfister, J A; Panter, K E; Zieglar, J; Hall, J O

2012-12-01

Isocupressic acid (ICA) is the abortifacient compound in ponderosa pine (Pinus ponderosa L.) needles, which can cause late-term abortions in cattle (Bos taurus). However, cattle rapidly metabolize ICA to agathic acid (AGA) and subsequent metabolites. When pine needles are dosed orally to cattle, no ICA is detected in their serum, whereas AGA is readily detected. Recent research has demonstrated that AGA is also an abortifacient compound in cattle. The observation has been made that when cattle are dosed with labdane acids for an extended time, the concentration of AGA in serum increases for 1 to 2 d but then decreases to baseline after 5 to 6 d even though they are still being dosed twice daily. Therefore, in this study we investigated whether cattle conditioned to pine needles metabolize ICA, and its metabolites, faster than naïve cattle. Agathic acid was readily detected in the serum of naïve cattle fed ponderosa pine needles, whereas very little AGA was detected in the serum of cattle conditioned to pine needles. We also compared the metabolism of ICA in vitro using rumen cultures from pine-needle-conditioned and naïve cattle. In the rumen cultures from conditioned cattle, AGA concentrations were dramatically less than rumen cultures from naïve cattle. Thus, an adaptation occurs to cattle conditioned to pine needles such that the metabolism AGA by the rumen microflora is altered.

Hydraulic Darrieus turbines efficiency for free fluid flow conditions versus power farms conditions

Energy Technology Data Exchange (ETDEWEB)

Antheaume, Sylvain [Electricite de France, Recherche et Developpement, Laboratoire National d' Hydraulique et Environnement, 6 Quai Watier, 78400 Chatou (France); Maitre, Thierry; Achard, Jean-Luc [Laboratoire des Ecoulements Geophysiques et Industriels, BP 53, 38041 Grenoble (France)

2008-10-15

The present study deals with the efficiency of cross flow water current turbine for free stream conditions versus power farm conditions. In the first part, a single turbine for free fluid flow conditions is considered. The simulations are carried out with a new in house code which couples a Navier-Stokes computation of the outer flow field with a description of the inner flow field around the turbine. The latter is based on experimental results of a Darrieus wind turbine in an unbounded domain. This code is applied for the description of a hydraulic turbine. In the second part, the interest of piling up several turbines on the same axis of rotation to make a tower is investigated. Not only is it profitable because only one alternator is needed but the simulations demonstrate the advantage of the tower configuration for the efficiency. The tower is then inserted into a cluster of several lined up towers which makes a barge. Simulations show that the average barge efficiency rises as the distance between towers is decreased and as the number of towers is increased within the row. Thereby, the efficiency of a single isolated turbine is greatly increased when set both into a tower and into a cluster of several towers corresponding to possible power farm arrangements. (author)

Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

NARCIS (Netherlands)

B. Naaijkens (Benno); H.W.M. Niessen (Hans ); H.-J. Prins (H.); P.A.J. Krijnen (Paul); T.J.A. Kokhuis (Tom); N. de Jong (Nico); V.W.M. van Hinsbergh (Victor); O. Kamp (Otto); K. Helder MScN (Onno); R.J.P. Musters (René); A. van Dijk (Annemieke); L.J.M. Juffermans (Lynda)

2012-01-01

textabstractAdipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically,

Baseline growth and reproductive parameters in Lymnaea stagnalis for OECD test guideline development: optimization of diets and culturing conditions

DEFF Research Database (Denmark)

Holbech, Henrik; Hutchinson, Tom

laboratories in Denmark, Germany and the UK for the OECD pre-validation work to date. Laboratory cultures of L. stagnalis are traditionally fed fresh (preferably organic) lettuce; however, interrupted supplies of fresh lettuce in some countries in 2011 highlighted a potential problem for the draft OECD test...... of a mollusc reproduction test guideline. An ad hoc mollusc expert group has been formed in Europe to validate methods that can meet this need. Currently, a key species for use in this context is the freshwater gastropod Lymnaea stagnalis. An important aspect of this work is to first develop a specific...... pathogen free defined strain of L. stagnalis and second to establish a historical database of growth and reproductive rates under defined culturing conditions. A mass culture of the RENILYS® strain of L. stagnalis have been established at INRA (France) since 2002 and has been distributed to research...

Studies on Using Cattle and Sheep Hydatid Cyst Fluid Instead of the Fetal Calf Serum in Leishmania Culture

Directory of Open Access Journals (Sweden)

Hossein Rezvan

2013-12-01

Full Text Available Background: Leishmania is a single cell parasite causing leishmaniasis, which is a common disease between humans and animals. Due to the importance of in-vitro culture of the parasite in leishmania research, developing new methods for in-vitro cultivation of the parasite has always been a goal for leishmania researchers. The main objective of7T 5T7Tthis study was to use sheep and bovine hydatid cyst fluids as alternatives for fetal calf serum (FCS in leishmania in-vitro5T culture5T. Materials and Methods: 7TA total of 5T7T1 million leishmania promastigotes were added to 4 flasks as follow5T7T. A f5T7Tlask containing DMEM medium with 105T7T% 5T7Tfetal bovine serum5T7T, a f5T7Tlask containing DMEM and 10% sheep hydatid cyst fluid5T7T, a f5T7Tlask containing DMEM medium with 105T7T% 5T7Tbovine hydatid cyst fluid and a5T7T f5T7Tlask containing DMEM medium alone. After 2, 45T7T, 5T7T7, 95T7T, 11, 5T7T21 and 24 days, the number of parasites were counted and compared5T7T. Results: The result of this study showed that, DMEM medium enriched with 10% sheep hydatid cyst fluid in 168 hours and medium enriched with 10% bovine hydatid cyst fluid in 96 hours can act as a good alternative for fetal bovine serum in the culture Leishmania major. Conclusion: 5TThe results showed that sheep and bovine hydatid cyst fluid can be used as alternatives to FCS for dense cultivation of leishmania. The results also showed that5T, 5Tthe growth of promastigotes in medium enriched with bovine cyst fluid is more rapid than the medium enriched with sheep5T c5Tyst fluid5T in5T the beginning of cultivation.

Enhancement of excision-repair efficiency by conditioned medium from density-inhibited cultures in V79 Chinese hamster cells

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Nakano, S.

1979-01-01

Conditioned medium from density-inhibited V79 Chinese hamster cell cultures, given as a post-treatment to UV-irradiated homologous cells, was demonstrated to reduce the lethal action of ultraviolet light by temporarily blocking DNA replication. Since the increased survival was not affected by various nontoxic concentrations of caffeine, such protective effect would be attributable to the prolonged intervention of excision repair before DNA replication during the post-treatment period. The influence of conditioned medium on the UV-induced mutation at the ouabain-resistance locus was also examined and a significant decrease in mutation frequecy was noted. The observed reduction in killing and mutation as a result of post-incubation in conditioned medium, which delays DNA replication, would be interpreted as evidence that conditioned medium provides a longer period of time for an error-free excision-repair process, leaving lesion in DNA available for error-prone post-replication repair. (Auth.)

The Correlation between Serum and Peritoneal Fluid CA125 The Correlation between Serum and Peritoneal Fluid CA125

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Saghar Salehpour

2009-01-01

Full Text Available Background: Despite a high prevalence of endometriosis, there still exist many challenges indiagnosing the disease. This study aims to evaluate non-invasive and practical diagnostic methodsby measuring serum and peritoneal fluid CA 125 levels in patients with endometriosis. A secondaryaim is to determine the correlation between these markers with the stage of disease as well as therelationship of the two markers with each other.Materials and Methods: This is a cross-sectional study of 60 women who underwent laparoscopyfor benign conditions. Based on laparoscopic findings and biopsy results, patients were divided to twogroups; one group included patients with pelvic endometriosis (35 patients and the second enrolledpatients free from endometriosis (25 patients. Serum and peritoneal fluid specimens were provided at thetime of laparoscopy and CA125 levels were then assessed by electrochemiluminescence immunoassay.Results: Mean serum and peritoneal fluid CA125 levels were significantly higher in women withendometriosis as compared to the control group (26.42 ± 24.34 IU/ml versus 12.64 ± 6.87 IU/mlin serum and 2203.54 + 993.19 IU/ml versus 1583.42 ± 912.51 IU/ml in peritoneal fluid, p<0.05.CA 125 levels also varied proportionally with the stage of endometriosis; but showed a significantdifference only in higher stages of the disease, both in serum and peritoneal fluid. We calculatedthe cut-off value suggesting a diagnosis of pelvic endometriosis as 14.70 IU/ml for serum and1286.5 IU/ml for peritoneal fluid CA125. A linear correlation between CA 125 levels in serum andperitoneal fluid in patients with pelvic endometriosis has also been observed.Conclusion: Serum and peritoneal fluid CA 125 levels are simple and non-surgical tools fordiagnosing and staging pelvic endometriosis. These markers are of greater diagnostic value inhigher stages of the disease.

Effects of endothelial removal and regeneration on smooth muscle glycosaminoglycan synthesis and growth in rat carotid artery in organ culture

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Merrilees, M.J.; Scott, L.J.

1985-01-01

Segments of rat carotid artery were maintained in serum-free and serum-supplemented media with endothelium both present and substantially removed by air drying. At intervals of 3, 7, and 14 days the synthesis of glycosaminoglycan across the vessel walls was determined by autoradiographic detection of incorporated [ 3 H]glucosamine. In control carotids the typical pattern of incorporation was 40% of label in the intima, consisting of endothelium and subendothelial matrix, 23, 13, and 15% in the three medial layers (M1, M2, M3, respectively), and 9% in the adventitia. During the first week in culture the proportion, and often the amount, of label in M1 increased significantly. Following air drying labeling decreased markedly in M1 but often increased in M2 and M3. By 14 days residual endothelial cells had regenerated, and the pattern of incorporation in the medial layers beneath this new endothelium was the same as for the controls with a high level of labeling in M1. In areas free of endothelium incorporation in M1 remained at a low level. Digestion with chondroitinase ABC and Streptomyces hyaluronidase showed that the changes in M1-labeling levels were due to changes in the amounts of both hyaluronic acid and sulfated glycosaminoglycan, whereas pulse and continuous labeling studies showed that the different labeling levels for the various layers and conditions were due to different rates of synthesis and not degradation. Carotids were also labeled with [ 3 H]thymidine. Control and regenerating endothelia were active in serum- free and serum-supplemented media and had similar mitotic indices. Indices for smooth muscle cells in M1, however, were generally very low and were not affected by the presence or absence of endothelium

Label-free LC-MSe in tissue and serum reveals protein networks underlying differences between benign and malignant serous ovarian tumors.

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Wouter Wegdam

Full Text Available PURPOSE: To identify proteins and (molecular/biological pathways associated with differences between benign and malignant epithelial ovarian tumors. EXPERIMENTAL PROCEDURES: Serum of six patients with a serous adenocarcinoma of the ovary was collected before treatment, with a control group consisting of six matched patients with a serous cystadenoma. In addition to the serum, homogeneous regions of cells exhibiting uniform histology were isolated from benign and cancerous tissue by laser microdissection. We subsequently employed label-free liquid chromatography tandem mass spectrometry (LC-MSe to identify proteins in these serum and tissues samples. Analyses of differential expression between samples were performed using Bioconductor packages and in-house scripts in the statistical software package R. Hierarchical clustering and pathway enrichment analyses were performed, as well as network enrichment and interactome analysis using MetaCore. RESULTS: In total, we identified 20 and 71 proteins that were significantly differentially expressed between benign and malignant serum and tissue samples, respectively. The differentially expressed protein sets in serum and tissue largely differed with only 2 proteins in common. MetaCore network analysis, however inferred GCR-alpha and Sp1 as common transcriptional regulators. Interactome analysis highlighted 14-3-3 zeta/delta, 14-3-3 beta/alpha, Alpha-actinin 4, HSP60, and PCBP1 as critical proteins in the tumor proteome signature based on their relative overconnectivity. The data have been deposited to the ProteomeXchange with identifier PXD001084. DISCUSSION: Our analysis identified proteins with both novel and previously known associations to ovarian cancer biology. Despite the small overlap between differentially expressed protein sets in serum and tissue, APOA1 and Serotransferrin were significantly lower expressed in both serum and cancer tissue samples, suggesting a tissue-derived effect in serum

Evaluation of serum and milk ELISAs for paratuberculosis in Danish dairy cattle

DEFF Research Database (Denmark)

Klausen, Joan; Huda, A.; Ekeroth, Lars

2003-01-01

concurrently from six dairy herds infected with MAP and from two dairy herds without history of infection with MAP. A cut-off value of 7 OD% was used in the ELISAs. At this cut-off value, all six culture-positive herds were positive in the serum ELISA but one was negative in the milk ELISA. All six culture......-positive herds were positive in the CFT. In the two culture-negative herds, the serum and the milk ELISA deemed all serum samples negative at this cut-off value, whereas four serum samples from one of these herds were positive in the CFT. The highest cut-off value enabling the milk ELISA to record all six...... culture-positive herds as positive was 4 OD%. The highest cut-off value enabling the serum ELISA to record all six culture-positive herds as positive was 17 OD%. Individual-sample relative sensitivities of the ELISAs ranged from 49 to 64% and relative specificities were 80-96% at the cut-off values of 4...

The shear-free condition and constant-mean-curvature hyperboloidal initial data

International Nuclear Information System (INIS)

Allen, Paul T; Allen, Iva Stavrov; Isenberg, James; Lee, John M

2016-01-01

We consider the Einstein–Maxwell-fluid constraint equations, and make use of the conformal method to construct and parametrize constant-mean-curvature hyperboloidal initial data sets that satisfy the shear-free condition. This condition is known to be necessary in order that a spacetime development admit a regular conformal boundary at future null infinity; see (Andersson and Chruściel 1994 Commun. Math. Phys. 161 533–68). We work with initial data sets in a variety of regularity classes, primarily considering those data sets whose geometries are weakly asymptotically hyperbolic , as defined in (Allen et al 2015 arXiv:1506.03399). These metrics are C 1,1 conformally compact, but not necessarily C 2 conformally compact. In order to ensure that the data sets we construct are indeed shear-free, we make use of the conformally covariant traceless Hessian introduced in (Allen et al 2015 arXiv:1506.03399). We furthermore construct a class of initial data sets with weakly asymptotically hyerbolic metrics that may be only C 0,1 conformally compact; these data sets are insufficiently regular to make sense of the shear-free condition. (paper)

Dynamic quantitative analysis of adherent cell cultures by means of lens-free video microscopy

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Allier, C.; Vincent, R.; Navarro, F.; Menneteau, M.; Ghenim, L.; Gidrol, X.; Bordy, T.; Hervé, L.; Cioni, O.; Bardin, S.; Bornens, M.; Usson, Y.; Morales, S.

2018-02-01

We present our implementation of lens-free video microscopy setup for the monitoring of adherent cell cultures. We use a multi-wavelength LED illumination together with a dedicated holographic reconstruction algorithm that allows for an efficient removal of twin images from the reconstructed phase image for densities up to those of confluent cell cultures (>500 cells/mm2). We thereby demonstrate that lens-free video microscopy, with a large field of view ( 30 mm2) can enable us to capture the images of thousands of cells simultaneously and directly inside the incubator. It is then possible to trace and quantify single cells along several cell cycles. We thus prove that lens-free microscopy is a quantitative phase imaging technique enabling estimation of several metrics at the single cell level as a function of time, for example the area, dry mass, maximum thickness, major axis length and aspect ratio of each cell. Combined with cell tracking, it is then possible to extract important parameters such as the initial cell dry mass (just after cell division), the final cell dry mass (just before cell division), the average cell growth rate, and the cell cycle duration. As an example, we discuss the monitoring of a HeLa cell cultures which provided us with a data-set featuring more than 10 000 cell cycle tracks and more than 2x106 cell morphological measurements in a single time-lapse.

Stem cell recovering effect of copper-free GHK in skin.

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Choi, Hye-Ryung; Kang, Youn-A; Ryoo, Sun-Jong; Shin, Jung-Won; Na, Jung-Im; Huh, Chang-Hun; Park, Kyoung-Chan

2012-11-01

The peptide Gly-His-Lys (GHK) is a naturally occurring copper(II)-chelating motifs in human serum and cerebrospinal fluid. In industry, GHK (with or without copper) is used to make hair and skin care products. Copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. We also reported that copper-GHK promotes the survival of basal stem cells in the skin. However, the effects of copper-free GHK (GHK) have not been investigated well. In this study, the effects of GHK were studied using cultured normal human keratinocytes and skin equivalent (SE) models. In monolayer cultured keratinocytes, GHK increased the proliferation of keratinocytes. When GHK was added during the culture of SE models, the basal cells became more cuboidal than control model. In addition, there was linear and intense staining of α6 and β1 integrin along the basement membrane. The number of p63 and proliferating cell nuclear antigen positive cells was also significantly increased in GHK-treated SEs than in control SEs. Western blot and slide culture experiment showed that GHK increased the expression of integrin by keratinocytes. All these results showed that GHK increased the stemness and proliferative potential of epidermal basal cells, which is associated with increased expression of integrin. In conclusion, copper-free GHK showed similar effects with copper-GHK. Thus, it can be said that copper-free GHK can be used in industry to obtain the effects of copper-GHK in vivo. Further study is necessary to explore the relationship between copper-free GHK and copper-GHK. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

Modulation of cultured porcine granulosa cell responsiveness to follicle stimulating hormone and epidermal growth factor

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Buck, P.A.

1986-01-01

Ovarian follicular development is dependent upon the coordinated growth and differentiation of the granulosa cells which line the follicle. Follicle stimulating hormone (FSH) induces granulosa cell differentiation both in vivo and in vitro. Epidermal growth factor (EGF) stimulates granulosa cell proliferation in vitro. The interaction of these two effectors upon selected parameters of growth and differentiation was examined in monolayer cultures of porcine granulose cells. Analysis of the EGF receptor by /sup 125/I-EGF binding revealed that the receptor was of high affinity with an apparent dissociation constant of 4-6 x 10/sup -10/ M. The average number of receptors per cell varied with the state of differentiation both in vivo and in vitro; highly differentiated cells bound two-fold less /sup 125/I-EGF and this effect was at least partially induced by FSH in vitro. EGF receptor function was examined by assessing EGF effects on cell number and /sup 3/H-thymidine incorporation. EGF stimulated thymidine incorporation in both serum-free and serum-supplemented culture, but only in serum-supplemented conditions was cell number increased. EGF receptor function was inversely related to the state of differentiation and was attenuated by FSH. The FSH receptor was examined by /sup 125/I-FSH binding. EGF increased FSH receptor number, and lowered the affinity of the receptor. The function of these receptors was assessed by /sup 125/I-hCG binding and progesterone radioimmunoassay. If EGF was present continuously in the cultures. FSH receptor function was attenuated regardless of FSH receptor number. A preliminary effort to examine the mechanism of this interaction was performed by analyzing hormonally controlled protein synthesis with /sup 35/S-methionine labeling, SDS polyacrylamide gel electrophoresis and fluorography. FSH promoted the expression of a 27,000 dalton protein. This effect was attenuated by EGF.

[Influence of liquid or solid culture conditions on the volatile components of mycelia of Isariacateinannulata].

Science.gov (United States)

Zhang, Delong; Wang, Xiaodong; Lu, Ruili; Li, Kangle; Hu, Fenglin

2011-12-01

To determine the volatile components of mycelia of Isaria cateinannulata cultured under different culture conditions, and to analyze the relationships between the culture conditions and volatile metabolites. Mycelia were cultured in solid plates with SDAY medium and liquid shake flasks with SDY medium. The culture conditions were at 25 degrees C and 8 days. Volatile components in the mycelia of I. cateinannulata were extracted with simultaneous distillation extraction and analyzed by gas chromatography-mass spectrometry. Alkenes, alkanes, heterocyclic and polycyclic aromatic hydrocarbons (PAH) were existed abundantly both in the mycelia of liquid and solid cultures, but the kinds and relative concentrations of the volatile components in mycelia of liquid and solid cultures were very different. Forty-one compounds were identified from the mycelia of solid culture and 32 compounds were identified from the mycelia of liquid culture. Esters, quinones and oximes were only found in solid cultured mycelia whereas carboxylic acids were only discovered in the mycelia of liquid culture. At the same time, mycelia of liquid culture contained much more phenols. The most abundant compounds in mycelia of liquid and solid cultures were hydrocarbons. The volatile extracts of solid cultured mycelia contained 57.6% alkenes and 9.19% alkanes. The volatile extracts of liquid cultured mycelia contained 7.85% alkenes and 22.4% alkanes. Liquid or solid culture conditions influenced the volatile components of mycelia of I. cateinannulata.

[Characterization of epithelial primary culture from human conjunctiva].

Science.gov (United States)

Rivas, L; Blázquez, A; Muñoz-Negrete, F J; López, S; Rebolleda, G; Domínguez, F; Pérez-Esteban, A

2014-01-01

To evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules. One-hundred and forty-eight human conjunctiva explants were cultured in CnT50(®) supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37°C, 5% CO2 and 95% HR, for 3 weeks. The typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative). Conjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50(®) supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

Antioxidant flavonoids bind human serum albumin

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Kanakis, C. D.; Tarantilis, P. A.; Polissiou, M. G.; Diamantoglou, S.; Tajmir-Riahi, H. A.

2006-10-01

Human serum albumin (HSA) is a principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. Flavonoids are powerful antioxidants and prevent DNA damage. The antioxidative protections are related to their binding modes to DNA duplex and complexation with free radicals in vivo. However, flavonoids are known to inhibit the activities of several enzymes such as calcium phospholipid-dependent protein kinase, tyrosine protein kinase from rat lung, phosphorylase kinase, phosphatidylinositol 3-kinase and DNA topoisomerases that exhibit the importance of flavonoid-protein interaction. This study was designed to examine the interaction of human serum albumin (HSA) with quercetin (que), kaempferol (kae) and delphinidin (del) in aqueous solution at physiological conditions, using constant protein concentration of 0.25 mM (final) and various drug contents of 1 μM-1 mM. FTIR and UV-vis spectroscopic methods were used to determine the polyphenolic binding mode, the binding constant and the effects of flavonoid complexation on protein secondary structure. The spectroscopic results showed that flavonoids are located along the polypeptide chains through H-bonding interactions with overall affinity constant of Kque = 1.4 × 10 4 M -1, Kkae = 2.6 × 10 5 M -1 and Kdel = 4.71 × 10 5 M -1. The protein secondary structure showed no alterations at low pigment concentration (1 μM), whereas at high flavonoid content (1 mM), major reduction of α-helix from 55% (free HSA) to 42-46% and increase of β-sheet from 15% (free HSA) to 17-19% and β-anti from 7% (free HSA) to 10-20% occurred in the flavonoid-HSA adducts. The major reduction of HSA α-helix is indicative of a partial protein unfolding upon flavonoid interaction.

Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells

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Mangum, Lauren H.; Stone, Randolph; Wrice, Nicole L.; Larson, David A.; Florell, Kyle F.; Christy, Barbara A.; Herzig, Maryanne C.; Cap, Andrew P.

2017-01-01

Stem cells derived from the subcutaneous adipose tissue of debrided burned skin represent an appealing source of adipose-derived stem cells (ASCs) for regenerative medicine. Traditional tissue culture uses fetal bovine serum (FBS), which complicates utilization of ASCs in human medicine. Human platelet lysate (hPL) is one potential xeno-free, alternative supplement for use in ASC culture. In this study, adipogenic and osteogenic differentiation in media supplemented with 10% FBS or 10% hPL was compared in human ASCs derived from abdominoplasty (HAP) or from adipose associated with debrided burned skin (BH). Most (95–99%) cells cultured in FBS were stained positive for CD73, CD90, CD105, and CD142. FBS supplementation was associated with increased triglyceride content and expression of adipogenic genes. Culture in hPL significantly decreased surface staining of CD105 by 31% and 48% and CD142 by 27% and 35% in HAP and BH, respectively (p < 0.05). Culture of BH-ASCs in hPL also increased expression of markers of osteogenesis and increased ALP activity. These data indicate that application of ASCs for wound healing may be influenced by ASC source as well as culture conditions used to expand them. As such, these factors must be taken into consideration before ASCs are used for regenerative purposes. PMID:29138638

Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells

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Lauren H. Mangum

2017-01-01

Full Text Available Stem cells derived from the subcutaneous adipose tissue of debrided burned skin represent an appealing source of adipose-derived stem cells (ASCs for regenerative medicine. Traditional tissue culture uses fetal bovine serum (FBS, which complicates utilization of ASCs in human medicine. Human platelet lysate (hPL is one potential xeno-free, alternative supplement for use in ASC culture. In this study, adipogenic and osteogenic differentiation in media supplemented with 10% FBS or 10% hPL was compared in human ASCs derived from abdominoplasty (HAP or from adipose associated with debrided burned skin (BH. Most (95–99% cells cultured in FBS were stained positive for CD73, CD90, CD105, and CD142. FBS supplementation was associated with increased triglyceride content and expression of adipogenic genes. Culture in hPL significantly decreased surface staining of CD105 by 31% and 48% and CD142 by 27% and 35% in HAP and BH, respectively (p<0.05. Culture of BH-ASCs in hPL also increased expression of markers of osteogenesis and increased ALP activity. These data indicate that application of ASCs for wound healing may be influenced by ASC source as well as culture conditions used to expand them. As such, these factors must be taken into consideration before ASCs are used for regenerative purposes.

[Role of phosphoinositide 3 kinase/protein kinase B signal pathway in monocyte-endothelial adhesion induced by serum of rats with electrical burn].

Science.gov (United States)

Ruan, Qiongfang; Zhao, Chaoli; Ye, Ziqing; Zhang, Weidong; Xie, Qionghui; Xie, Weiguo

2014-06-01

To observe the change in phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signal pathway in monocytes as induced by serum of rats with electrical burn, and to explore the effects of PI3K/Akt pathway on monocyte-endothelial cell adhesion. Sixty-four SD rats of clean grade were inflicted with electrical burn for the collection of serum of rats with electrical burn; another group of twenty-four SD rats were used to obtain normal serum without treatment. (1) Human monocyte line THP-1 was routinely cultured. The THP-1 cells in logarithmic phase were divided into normal serum group (resuspended in RPMI 1640 medium with 20% normal rat serum) and burn serum group (resuspended with RPMI 1640 medium with 20% serum of rats with electrical burn) according to the random number table, with 6 wells in each group. Morphology of THP-1 cells in normal serum group was observed at post culture hour (PCH) 24, and that in burn serum group at PCH 3, 6, 24. The contents of TNF-α in culture supernatant were determined by double-antibody sandwich ELISA at the corresponding time point in each group. The state of Akt activation was determined by Western blotting at PCH 3, 6, 24. (2) Another portion of THP-1 cells were divided into 4 groups according to the random number table, with 6 wells in each group. Cells in normal serum group and burn serum group were given with the same culture condition as above; cells in normal serum+inhibitor group and burn serum+inhibitor group were cultured with the same culture conditions as in the former two groups correspondingly with addition of 100 nmol/L wortmannin in the nutrient solution. At PCH 3 and 6, THP-1 cells were added into the well with a monolayer of endothelial cell line EA.hy926 to observe the monocyte-endothelial cell adhesion. Data were processed with one-way analysis of variance and LSD- t test. (1) In normal serum group, THP-1 cells showed growth in suspension, with uniform shape at PCH 24. In burn serum group, the cell shape became

Association of PSA, free-PSA and testosteron levels in serum of patients with benign prostate hyperplasia (BPH) and prostate cancer

International Nuclear Information System (INIS)

Wiwin Mailana; Kristina Dwi P; Sri Insani WW; Puji Widayati

2015-01-01

Prostate cancer screening can be done by measuring the concentration levels of PSA, free-PSA and testosterone in serum that examined with radioimmunoassay (RIA). A total of 30 patients of 45-81 years old had enrolled in this study and were taken their venous blood. The aim of research is to know the relationship between PSA and testosterone free-PSA with BPH and prostate cancer. Results showed that there was no correlation between age with BPH and prostate cancer (p = 0.06), but there is a relationship between PSA with BPH and prostate cancer (p = 0.002), the relationship between free-PSA with BPH and prostate cancer (p = 0.001). No correlation was found between PSA ratio with BPH and prostate cancer as well as the absence of a relationship between testosterone with BPH and prostate cancer (p = 0.924). (author)

Serum adiponectin level in obese and non-obese COPD patients during acute exacerbation and stable conditions

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Magdy Mohammad Omar

2014-04-01

Conclusion: Serum adiponectin was significantly higher in obese and nonobese COPD than controls, the rising is more during exacerbation than stable condition and more in non obese than obese COPD and non significant correlation between changes in adiponectin and ventilatory functions was found.

Attempts at establishing the culture conditions for Lemna minor L.

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M. Krzychowska

2015-01-01

Full Text Available The influence of the concentration, composition and pH of the substrate as well as of light intensity on the growth and vegetative propagation of Lemna minor L. was investigated. The media of Hutner, Hoagland and Pirson and Seidel were used. At first the experiments were carried out under unsterile conditions. Later sterilized duckweed was cultured in aseptic conditions. The dry matter was determined. Surface area increment and an increase in the number of fronds were evaluated by the planimetric method. For total protein determination in Lemna minor L. from unsterile and sterile cultures Lowry's method was used.

Production of virus-free orchid Cymbidium aloifolium (L.) Sw. by various tissue culture techniques.

Science.gov (United States)

Pradhan, Shreeti; Regmi, Tripti; Ranjit, Mukunda; Pant, Bijaya

2016-10-01

Orchids are affected by many viruses resulting in poor growth, yield and quality, and an overall decline in population. Cymbidium mosaic virus (CymMV) is one of the common orchid viruses found in Cymbidium species but it infects different orchid genera. In this study Cymbidium aloifolium was propagated in vitro using MS medium at different strength (1.0, ½, and ¼) with or without 0.5 mg/l BAP (6-benzylaminopurine) and 0.5 mg/l NAA (Naphthalene acetic acid). To provide disease-free planting material, source plant for in vitro propagation needs to be screened for pathogenic viruses. In the present study, in vivo -grown source (mother) plants and tissue culture-derived plants of C. aloifolium were tested for CymMV virus using Double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). All the tissue cultured plants were found to be 100% virus-free whereas the in vivo grown source plants were highly affected by CymMV virus (83.33%). The virus-free in vitro plantlets were multiplied in large scale and then acclimatized on earthen pot containing a mixture of cocopeat, litter and clay in the ratio of 3:2:1. Eighty five percent of acclimatized plantlets survived making this method an efficient mass production system for high quality virus-free C. aloifolium for commercial floriculture and germplasm preservation.

Production of virus-free orchid Cymbidium aloifolium (L. Sw. by various tissue culture techniques

Directory of Open Access Journals (Sweden)

Shreeti Pradhan

2016-10-01

Full Text Available Orchids are affected by many viruses resulting in poor growth, yield and quality, and an overall decline in population. Cymbidium mosaic virus (CymMV is one of the common orchid viruses found in Cymbidium species but it infects different orchid genera. In this study Cymbidium aloifolium was propagated in vitro using MS medium at different strength (1.0, ½, and ¼ with or without 0.5 mg/l BAP (6-benzylaminopurine and 0.5 mg/l NAA (Naphthalene acetic acid. To provide disease-free planting material, source plant for in vitro propagation needs to be screened for pathogenic viruses. In the present study, in vivo-grown source (mother plants and tissue culture-derived plants of C. aloifolium were tested for CymMV virus using Double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA. All the tissue cultured plants were found to be 100% virus-free whereas the in vivo grown source plants were highly affected by CymMV virus (83.33%. The virus-free in vitro plantlets were multiplied in large scale and then acclimatized on earthen pot containing a mixture of cocopeat, litter and clay in the ratio of 3:2:1. Eighty five percent of acclimatized plantlets survived making this method an efficient mass production system for high quality virus-free C. aloifolium for commercial floriculture and germplasm preservation. Keywords: Biological sciences, Plant biology

Internal structures of scaffold-free 3D cell cultures visualized by synchrotron radiation-based micro-computed tomography

Science.gov (United States)

Saldamli, Belma; Herzen, Julia; Beckmann, Felix; Tübel, Jutta; Schauwecker, Johannes; Burgkart, Rainer; Jürgens, Philipp; Zeilhofer, Hans-Florian; Sader, Robert; Müller, Bert

2008-08-01

Recently the importance of the third dimension in cell biology has been better understood, resulting in a re-orientation towards three-dimensional (3D) cultivation. Yet adequate tools for their morphological characterization have to be established. Synchrotron radiation-based micro computed tomography (SRμCT) allows visualizing such biological systems with almost isotropic micrometer resolution, non-destructively. We have applied SRμCT for studying the internal morphology of human osteoblast-derived, scaffold-free 3D cultures, termed histoids. Primary human osteoblasts, isolated from femoral neck spongy bone, were grown as 2D culture in non-mineralizing osteogenic medium until a rather thick, multi-cellular membrane was formed. This delicate system was intentionally released to randomly fold itself. The folded cell cultures were grown to histoids of cubic milli- or centimeter size in various combinations of mineralizing and non-mineralizing osteogenic medium for a total period of minimum 56 weeks. The SRμCT-measurements were performed in the absorption contrast mode at the beamlines BW 2 and W 2 (HASYLAB at DESY, Hamburg, Germany), operated by the GKSS-Research Center. To investigate the entire volume of interest several scans were performed under identical conditions and registered to obtain one single dataset of each sample. The histoids grown under different conditions exhibit similar external morphology of globular or ovoid shape. The SRμCT-examination revealed the distinctly different morphological structures inside the histoids. One obtains details of the histoids that permit to identify and select the most promising slices for subsequent histological characterization.

Acute phase protein concentrations in serum and milk from healthy cows, cows with clinical mastitis and cows with extramammary inflammatory conditions

NARCIS (Netherlands)

Nielsen, B.H.; Jacobsen, S.; Andersen, P.H.; Niewold, T.A.; Heegaard, P.M.H.

2004-01-01

The concentrations of the two acute phase proteins, serum amyloid A and haptoglobin, in serum and milk were compared in 10 cows with clinical mastitis, 11 cows with extramammary inflammatory conditions and 10 clinically healthy control cows. The concentrations of both acute phase proteins were

Influence of flow conditions and matrix coatings on growth and differentiation of three-dimensionally cultured rat hepatocytes.

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Fiegel, Henning C; Havers, Joerg; Kneser, Ulrich; Smith, Molly K; Moeller, Tim; Kluth, Dietrich; Mooney, David J; Rogiers, Xavier; Kaufmann, Peter M

2004-01-01

Maintenance of liver-specific function of hepatocytes in culture is still difficult. Improved culture conditions may enhance the cell growth and function of cultured cells. We investigated the effect of three-dimensional culture under flow conditions, and the influence of surface modifications in hepatocyte cultures. Hepatocytes were harvested from Lewis rats. Cells were cultured on three-dimensional polymeric poly-lactic-co-glycolic acid (PLGA) matrices in static culture, or in a pulsatile flow-bioreactor system. Different surface modifications of matrices were investigated: coating with collagen I, collagen IV, laminin, or fibronectin; or uncoated matrix. Hepatocyte numbers, DNA content, and albumin secretion rate were assessed over the observation period. Culture under flow condition significantly enhanced cell numbers. An additional improvement of this effect was observed, when matrix coating was used. Cellular function also showed a significant increase (4- to 5-fold) under flow conditions when compared with static culture. Our data showed that culture under flow conditions improves cell number, and strongly enhances cellular function. Matrix modification by coating with extracellular matrix showed overall an additive stimulatory effect. Our conclusion is that combining three-dimensional culture under flow conditions and using matrix modification significantly improves culture conditions and is therefore attractive for the development of successful culture systems for hepatocytes.

Silver nanoparticle protein corona composition in cell culture media.

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Shannahan, Jonathan H; Lai, Xianyin; Ke, Pu Chun; Podila, Ramakrishna; Brown, Jared M; Witzmann, Frank A

2013-01-01

The potential applications of nanomaterials as drug delivery systems and in other products continue to expand. Upon introduction into physiological environments and driven by energetics, nanomaterials readily associate proteins forming a protein corona (PC) on their surface. This PC influences the nanomaterial's surface characteristics and may impact their interaction with cells. To determine the biological impact of nanomaterial exposure as well as nanotherapeutic applications, it is necessary to understand PC formation. Utilizing a label-free mass spectrometry-based proteomics approach, we examined the composition of the PC for a set of four silver nanoparticles (AgNPs) including citrate-stabilized and polyvinlypyrrolidone-stabilized (PVP) colloidal silver (20 or 110 nm diameter). To simulate cell culture conditions, AgNPs were incubated for 1 h in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, washed, coronal proteins solubilized, and proteins identified and quantified by label-free LC-MS/MS. To determine which attributes influence PC formation, the AgNPs were characterized in both water and cell culture media with 10% FBS. All AgNPs associated a common subset of 11 proteins including albumin, apolipoproteins, keratins, and other serum proteins. 110 nm citrate- and PVP-stabilized AgNPs were found to bind the greatest number of proteins (79 and 85 respectively) compared to 20 nm citrate- and PVP-stabilized AgNPs (45 and 48 respectively), suggesting a difference in PC formation based on surface curvature. While no relationships were found for other protein parameters (isoelectric point or aliphatic index), the PC on 20 nm AgNPs (PVP and citrate) consisted of more hydrophobic proteins compared to 110 nm AgNPs implying that this class of proteins are more receptive to curvature-induced folding and crowding in exchange for an increased hydration in the aqueous environment. These observations demonstrate the significance of electrostatic

Silver nanoparticle protein corona composition in cell culture media.

Directory of Open Access Journals (Sweden)

Jonathan H Shannahan

Full Text Available The potential applications of nanomaterials as drug delivery systems and in other products continue to expand. Upon introduction into physiological environments and driven by energetics, nanomaterials readily associate proteins forming a protein corona (PC on their surface. This PC influences the nanomaterial's surface characteristics and may impact their interaction with cells. To determine the biological impact of nanomaterial exposure as well as nanotherapeutic applications, it is necessary to understand PC formation. Utilizing a label-free mass spectrometry-based proteomics approach, we examined the composition of the PC for a set of four silver nanoparticles (AgNPs including citrate-stabilized and polyvinlypyrrolidone-stabilized (PVP colloidal silver (20 or 110 nm diameter. To simulate cell culture conditions, AgNPs were incubated for 1 h in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, washed, coronal proteins solubilized, and proteins identified and quantified by label-free LC-MS/MS. To determine which attributes influence PC formation, the AgNPs were characterized in both water and cell culture media with 10% FBS. All AgNPs associated a common subset of 11 proteins including albumin, apolipoproteins, keratins, and other serum proteins. 110 nm citrate- and PVP-stabilized AgNPs were found to bind the greatest number of proteins (79 and 85 respectively compared to 20 nm citrate- and PVP-stabilized AgNPs (45 and 48 respectively, suggesting a difference in PC formation based on surface curvature. While no relationships were found for other protein parameters (isoelectric point or aliphatic index, the PC on 20 nm AgNPs (PVP and citrate consisted of more hydrophobic proteins compared to 110 nm AgNPs implying that this class of proteins are more receptive to curvature-induced folding and crowding in exchange for an increased hydration in the aqueous environment. These observations demonstrate the significance of

Cultured Cortical Neurons Can Perform Blind Source Separation According to the Free-Energy Principle

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Isomura, Takuya; Kotani, Kiyoshi; Jimbo, Yasuhiko

2015-01-01

Blind source separation is the computation underlying the cocktail party effect––a partygoer can distinguish a particular talker’s voice from the ambient noise. Early studies indicated that the brain might use blind source separation as a signal processing strategy for sensory perception and numerous mathematical models have been proposed; however, it remains unclear how the neural networks extract particular sources from a complex mixture of inputs. We discovered that neurons in cultures of dissociated rat cortical cells could learn to represent particular sources while filtering out other signals. Specifically, the distinct classes of neurons in the culture learned to respond to the distinct sources after repeating training stimulation. Moreover, the neural network structures changed to reduce free energy, as predicted by the free-energy principle, a candidate unified theory of learning and memory, and by Jaynes’ principle of maximum entropy. This implicit learning can only be explained by some form of Hebbian plasticity. These results are the first in vitro (as opposed to in silico) demonstration of neural networks performing blind source separation, and the first formal demonstration of neuronal self-organization under the free energy principle. PMID:26690814

Cultured Cortical Neurons Can Perform Blind Source Separation According to the Free-Energy Principle.

Directory of Open Access Journals (Sweden)

Takuya Isomura

2015-12-01

Full Text Available Blind source separation is the computation underlying the cocktail party effect--a partygoer can distinguish a particular talker's voice from the ambient noise. Early studies indicated that the brain might use blind source separation as a signal processing strategy for sensory perception and numerous mathematical models have been proposed; however, it remains unclear how the neural networks extract particular sources from a complex mixture of inputs. We discovered that neurons in cultures of dissociated rat cortical cells could learn to represent particular sources while filtering out other signals. Specifically, the distinct classes of neurons in the culture learned to respond to the distinct sources after repeating training stimulation. Moreover, the neural network structures changed to reduce free energy, as predicted by the free-energy principle, a candidate unified theory of learning and memory, and by Jaynes' principle of maximum entropy. This implicit learning can only be explained by some form of Hebbian plasticity. These results are the first in vitro (as opposed to in silico demonstration of neural networks performing blind source separation, and the first formal demonstration of neuronal self-organization under the free energy principle.

Characterization of cryopreserved primary human corneal endothelial cells cultured in human serum-supplemented media

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Lucas Monferrari Monteiro Vianna

2016-02-01

Full Text Available ABSTRACT Purpose: To compare cryopreserved human corneal endothelial cells (HCECs grown in human serum-supplemented media (HS-SM with cryopreserved HCECs grown in fetal bovine serum-supplemented media (FBS-SM. Methods: Three pairs of human corneas from donors aged 8, 28, and 31 years were obtained from the eye bank. From each pair, one cornea was used to start a HCEC culture using HS-SM; the other cornea was grown in FBS-SM. On reaching confluence, the six cell populations were frozen using 10% dimethyl sulfoxidecontaining medium. Thawed cells grown in HS-SM were compared with those grown in FBS-SM with respect to morphology, growth curves, immunohistochemistry, real time-reverse transcriptase polymerase chain reaction (RT-PCR for endothelial cell markers, and detachment time. Results: No difference in morphology was observed for cells grown in the two media before or after cryopreservation. By growth curves, cell counts after thawing were similar in both media, with a slight trend toward higher cell counts in FBS-SM. Cells grown in both the media demonstrated a similar expression of endothelial cell markers when assessed by immunohistochemistry, although HCEC marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM as assessed by RT-PCR. With FBS-SM, there was a tendency of longer detachment time and lower cell passages. Conclusions: HS-SM was similar to FBS-SM for cryopreservation of cultured HCECs as assessed by analysis of cell morphology, proliferation, and protein expression, although marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM. Detachment time was longer with FBS-SM and in lower passages.

Effects of egg white protein supplementation on muscle strength and serum free amino acid concentrations.

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Hida, Azumi; Hasegawa, Yuko; Mekata, Yuko; Usuda, Mika; Masuda, Yasunobu; Kawano, Hitoshi; Kawano, Yukari

2012-10-19

The aim of this study was to evaluate the effects of egg white protein compared to carbohydrate intake prior to exercise on fat free mass (FFM), one repetition maximum (1RM) muscle strength and blood biochemistry in female athletes. Thirty healthy female collegiate athletes were recruited for this study and matched by sport type, body fat percentage and 1RM leg curl muscle strength. Participants were randomly divided into two groups: protein group (15.0 g egg white protein; 75 kcal) and carbohydrate group (17.5 g maltodextrin, 78 kcal). Supplements were administered daily at the same time in a double-blind manner prior to training during an 8-week period. Measurements were performed before and after the 8-week regimen. The mean dietary energy intake did not change throughout the study period. FFM and 1RM assessments (i.e., leg curl, leg extension, squat, and bench press) increased in both groups. Furthermore, serum urea and serum citrulline levels after the 8-week regimen increased significantly only in the protein group. Our findings indicated that compared to the carbohydrate supplement, the protein supplement was associated with some changes in protein metabolites but not with changes in body composition or muscle strength.

Effects of Egg White Protein Supplementation on Muscle Strength and Serum Free Amino Acid Concentrations

Directory of Open Access Journals (Sweden)

Yukari Kawano

2012-10-01

Full Text Available The aim of this study was to evaluate the effects of egg white protein compared to carbohydrate intake prior to exercise on fat free mass (FFM, one repetition maximum (1RM muscle strength and blood biochemistry in female athletes. Thirty healthy female collegiate athletes were recruited for this study and matched by sport type, body fat percentage and 1RM leg curl muscle strength. Participants were randomly divided into two groups: protein group (15.0 g egg white protein; 75 kcal and carbohydrate group (17.5 g maltodextrin, 78 kcal. Supplements were administered daily at the same time in a double-blind manner prior to training during an 8-week period. Measurements were performed before and after the 8-week regimen. The mean dietary energy intake did not change throughout the study period. FFM and 1RM assessments (i.e., leg curl, leg extension, squat, and bench press increased in both groups. Furthermore, serum urea and serum citrulline levels after the 8-week regimen increased significantly only in the protein group. Our findings indicated that compared to the carbohydrate supplement, the protein supplement was associated with some changes in protein metabolites but not with changes in body composition or muscle strength.

Effects of Egg White Protein Supplementation on Muscle Strength and Serum Free Amino Acid Concentrations

Science.gov (United States)

Hida, Azumi; Hasegawa, Yuko; Mekata, Yuko; Usuda, Mika; Masuda, Yasunobu; Kawano, Hitoshi; Kawano, Yukari

2012-01-01

The aim of this study was to evaluate the effects of egg white protein compared to carbohydrate intake prior to exercise on fat free mass (FFM), one repetition maximum (1RM) muscle strength and blood biochemistry in female athletes. Thirty healthy female collegiate athletes were recruited for this study and matched by sport type, body fat percentage and 1RM leg curl muscle strength. Participants were randomly divided into two groups: protein group (15.0 g egg white protein; 75 kcal) and carbohydrate group (17.5 g maltodextrin, 78 kcal). Supplements were administered daily at the same time in a double-blind manner prior to training during an 8-week period. Measurements were performed before and after the 8-week regimen. The mean dietary energy intake did not change throughout the study period. FFM and 1RM assessments (i.e., leg curl, leg extension, squat, and bench press) increased in both groups. Furthermore, serum urea and serum citrulline levels after the 8-week regimen increased significantly only in the protein group. Our findings indicated that compared to the carbohydrate supplement, the protein supplement was associated with some changes in protein metabolites but not with changes in body composition or muscle strength. PMID:23201768

The concentration of free amino acids in blood serum of dairy cows with primary ketosis.

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Marczuk, J; Brodzki, P; Brodzki, A; Kurek, Ł

2018-03-01

Ketosis is a common condition found in the initial stages of lactation in high-yielding dairy cows. The major cause of ketosis is a negative energy balance. During the energy deficiency, proteolysis processes develop parallel to lipolysis. During proteolysis, muscle tissue can be used as a source of amino acid. To date, the participation of amino acids in gluconeogenesis (glucogenic amino acids) and ketogenesis (ketogenic amino acids) has not been determined in detail. This paper presents the study on determination of the parameters of protein and free amino acid metabolism in blood serum of dairy cows with primary ketosis compared to healthy cows. This study contributes to better understanding of the role of amino acids in pathogenesis of ketosis. A total of 30 cows, divided into two groups: experimental (15 cows with ketosis) and control (15 healthy cows), were included in the study. The concentrations of glucose, β-hydroxybutyrate, total protein, albumin, urea, and free amino acids were determined in peripheral blood. Statistically significantly higher concentrations of glutamine, glutamic acid, isoleucine (p≤0.001), and tyrosine (p≤0.05) were found in cows with primary ketosis compared to healthy cows. Significant decrease in the concentrations of asparagine, histidine, methionine, and serine (p≤0.001), alanine, leucine, lysine and proline (p≤0.05) was observed. Significant increase of total ketogenic and glucogenic amino acids (p≤0.05), and an increased ratio of total ketogenic and glucogenic amino acids to total amino acids (p≤0.001) were noted in cows with ketosis. In our study, the changes, in particular observed in amino acid concentration in cows with primary ketosis, indicate its intensive use in both ketogenesis and gluconeogenesis processes. Therefore, a detailed understanding of the role that amino acids play in gluconeogenesis and ketogenesis will improve ketosis diagnostics and monitoring the course of a ketosis episode. Perhaps, the

Defined Conditions for the Isolation and Expansion of Basal Prostate Progenitor Cells of Mouse and Human Origin