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Prolonged Culture (prolonged + culture)
Selected AbstractsStrain-dependent viral dynamics and virus-cell interactions in a novel in vitro system supporting the life cycle of blood-borne hepatitis C virus,HEPATOLOGY, Issue 3 2009Hussein Hassan Aly We developed an in vitro system that can be used for the study of the life cycle of a wide variety of blood-borne hepatitis C viruses (HCV) from various patients using a three-dimensional hollow fiber culture system and an immortalized primary human hepatocyte (HuS-E/2) cell line. Unlike the conventional two-dimensional culture, this system not only enhanced the infectivity of blood-borne HCV but also supported its long-term proliferation and the production of infectious virus particles. Both sucrose gradient fractionation and electron microscopy examination showed that the produced virus-like particles are within a similar fraction and size range to those previously reported. Infection with different HCV strains showed strain-dependent different patterns of HCV proliferation and particle production. Fluctuation of virus proliferation and particle production was found during prolonged culture and was found to be associated with change in the major replicating virus strain. Induction of cellular apoptosis was only found when strains of HCV-2a genotype were used for infection. Interferon-alpha stimulation also varied among different strains of HCV-1b genotypes tested in this study. Conclusion: These results suggest that this in vitro infection system can reproduce strain-dependent events reflecting viral dynamics and virus-cell interactions at the early phase of blood-borne HCV infection, and that this system can allow the development of new anti-HCV strategies specific to various HCV strains. (HEPATOLOGY 2009.) [source] Phenotypic and functional comparison of optimum culture conditions for upscaling of bone marrow-derived mesenchymal stem cellsJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 3 2009Rakhi Pal Abstract Human adult bone marrow-derived mesenchymal stem cells (MSCs) are a promising tool in the newly emerging avenue of regenerative medicine. MSCs have already been translated from basic research to clinical transplantation research. However, there is still a lack of consensus on the ideal method of culturing MSCs. Here we have compared different culture conditions of human MSCs with an attempt to preserve their characteristics and multi-lineage differentiation potential. We compare the different basal culture media DMEM-F12, DMEM-high glucose (DMEM-HG), DMEM-low glucose (DMEM-LG), knock-out DMEM (DMEM-KO) and Mesencult® on the proliferation rate, surface markers and differentiation potentials of MSCs. At every fifth passage until the 25th passage, the differentiation potential and the presence of a panel of surface markers was observed, using flow cytometry. We also compared the characteristics of human MSCs when cultured in reduced concentrations of fetal bovine serum (FBS), knockout serum replacement (KO-SR) and human plasma. Data indicate that the presence of serum is essential to sustain and propagate MSCs cultures. The choice of basal medium is equally important so as to preserve their characteristics and multipotent properties even after prolonged culture in vitro. With MSCs emerging as a popular tool for regenerative therapies in incurable diseases, it is essential to be able to obtain a large number of MSCs that continue to preserve their characteristics following passaging. The data reveal the optimum basal medium for prolonged culture of MSCs while retaining their ability to differentiate and hence this may be used for up-scaling to provide sufficient numbers for transplantation. Copyright © 2009 John Wiley & Sons, Ltd. [source] Changes in global histone acetylation pattern in somatic cell nuclei after their transfer into oocytes at different stages of maturationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2008Helena Fulka Abstract In our study, we have examined the pattern of global histone modification changes in somatic cell nuclei after their transfer into mouse oocytes at different stages of maturation or after their parthenogenetic activation. While germinal vesicle (GV) staged immature oocytes are strongly labeled with anti-acetylated histone H3 and H4 antibodies, the signal is absent in both metaphase I and metaphase II oocytes (MI, MII). In contrast, the oocytes of all maturation stages show a presence of trimethylated H3/K4 in their chromatin. When somatic cells were fused to intact or enucleated GV oocytes, both the GV and the somatic cell nucleus showed a very strong signal for all the antibodies used. On the other hand, when somatic cells nuclei that are AcH3 and AcH4 positive before fusion are introduced into either intact or enucleated MI or MII oocytes, their acetylation signal decreased rapidly and was totally absent after a prolonged culture. This was not the case when anti-trimethyl H3/K4 antibody was used. The somatic cell chromatin showed only a slight decrease in the intensity of labeling after its transfer into MI or MII oocytes. This decrease was, however, evident only after a prolonged culture. These results suggest not only a relatively higher stability of the methylation modification but also some difference between the oocyte and somatic chromatin. The ability to deacetylate the chromatin of transferred somatic nuclei disappears rapidly after the oocyte activation. Our results indicate that at least some reprogramming activity appears in the oocyte cytoplasm almost immediately after GV breakdown (GVBD), and that this activity rapidly disappears after the oocyte activation. Mol. Reprod. Dev. 75: 556,564, 2008. © 2007 Wiley-Liss, Inc. [source] Human ovarian surface epithelial cells immortalized with hTERT maintain functional pRb and p53 expressionCELL PROLIFERATION, Issue 5 2007N. F. Li Normal human ovarian surface epithelial (OSE) cells, which are thought to be the origin of most of human ovarian carcinomas, have a very limited lifespan in culture. Establishment of immortalized OSE cell lines has, in the past, required inactivation of pRb and p53 functions. However, this often leads to increased chromosome instability during prolonged culture. Materials and Methods:,In this study, we have used a retroviral infection method to overexpress human telomerase reverse transcriptase (hTERT) gene, in primary normal OSE cells, under optimized culture conditions. Results:,In vitro and in vivo analysis of hTERT-immortalized cell lines confirmed their normal epithelial characteristics. Gene expression profiles and functional analysis of p16INK4A, p15INK4B, pRb and p53 confirmed the presence of their intact functions. Our study suggests that inactivation of pRb and p53 is not necessary for OSE immortalization. Furthermore, down-regulation of p15INK4B in the immortalized cells may indicate a functional role for this protein in them. Conclusion:,These immortal OSE cell lines are likely to be an important tool for studying human OSE biology and carcinogenesis. [source] | |||||||||||||