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Different Culture Conditions (different + culture_condition)
Selected AbstractsKinetic characterization of vero cell metabolism in a serum-free batch culture processBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Emma Petiot Abstract A global kinetic study of the central metabolism of Vero cells cultivated in a serum-free medium is proposed in the present work. Central metabolism including glycolysis, glutaminolysis, and tricarboxylic acid cycle (TCA) was demonstrated to be saturated by high flow rates of consumption of the two major substrates, glucose, and glutamine. Saturation was reavealed by an accumulation of metabolic intermediates and amino acids, by a high production of lactate needed to balance the redox pathway, and by a low participation of the carbon flow to the TCA cycle supply. Different culture conditions were set up to reduce the central metabolism saturation and to better balance the metabolic flow rates between lactate production and energetic pathways. From these culture conditions, substitutions of glutamine by other carbon sources, which have lower transport rates such as asparagine, or pyruvate in order to shunt the glycolysis pathway, were successful to better balance the central metabolism. As a result, an increase of the cell growth with a concomitant decrease of cell death and a better distribution of the carbon flow between TCA cycle and lactate production occurred. We also demonstrated that glutamine was a major carbon source to supply the TCA cycle in Vero cells and that a reduction of lactate production did not necessary improve the efficiency of the Vero cell metabolism. Thus, to adapt the formulation of the medium to the Vero cell needs, it is important to provide carbon substrates inducing a regulated supply of carbon in the TCA cycle either through the glycolysis or through other pathways such as glutaminolysis. Finally, this study allowed to better understand the Vero cell behavior in serum-free medium which is a valuable help for the implementation of this cell line in serum-free industrial production processes. Biotechnol. Bioeng. 2010;107: 143,153. © 2010 Wiley Periodicals, Inc. [source] An enzyme-linked immunosorbent assay for monitoring of Aspergillus ochraceus growth in coffee powder, chilli powder and poultry feedLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2006S. Anand Abstract Aims:, The work was carried out to develop an immunoassay for estimation of Aspergillus ochraceus biomass on solid substrate. Methods and Results:, An indirect noncompetitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of fungal biomass in food commodities using antibody raised against A. ochraceus mycelial antigen. The sensitivity of the assay was linear in the range of 10,160 ,g fungal biomass per millilitre extract of coffee (R2 = 0·989), poultry feed (R2 = 0·987) and chilli (R2 = 0·989). The growth of A. ochraceus in the food commodities like chilli, coffee beans and poultry feed, under the influence of two levels of moisture (20% and 30%) were monitored by the ELISA. The maximum fungal colonization was observed in poultry feed (9·8 and 11·8 mg g,1) followed by coffee beans (6·8 and 11·3 mg g,1) and chilli (5·1 and 6·3 mg g,1) at 20% and 30% moisture after 20 days of incubation. Similarly the fungus produced maximum ochratoxin A in poultry feed (25 and 120 ,g g,1) followed by coffee beans (8 and 24 ,g g,1) and chilli (0·2 and 0·45 ,g g,1) at 20% and 30% moisture after 20 days of incubation. Conclusions:, The method can be used for quantitative estimation of fungal biomass and comparison of fungal colonization in food substrates varying in composition. Significance and Impact of the Study:, The method can be adapted for studying the fungal colonization in different solid substrates under different culture condition. The method is sensitive to mould colonization of ,0·02% (w/w) and can be used for early detection of specific fungal infestation in food commodities. [source] Co-Cultures of Primary Cells on Self-Supporting Nanoporous Alumina Membranes,ADVANCED ENGINEERING MATERIALS, Issue 7 2010Andreas Hoess Due to their unique properties, self-supporting nanoporous aluminum oxide (alumina) membranes are useful substrates for the indirect co-cultivation of cells. The membrane can act as a physical barrier between different cell types, whereas the cell-to-cell communication is guaranteed by the diffusion of soluble molecules or factors through the pores. With the help of such membranes the mRNA expression of hepatic genes can be induced in human adipose-derived mesenchymal stem cells (hASCs) during an indirect co-cultivation with primary mouse hepatocytes under different culture conditions. This proof of concept shows that such a cultivation approach is beneficial for different issues in the field tissue engineering or cell therapy. [source] Influence of culture conditions on laccase production and isozyme patterns in the white-rot fungus Trametes gallicaJOURNAL OF BASIC MICROBIOLOGY, Issue 3 2005Jia Li Dong Laccase production by the white-rot fungus Trametes gallica was studied, using twelve different media under static or shaking condition. The results indicated that organic nitrogen sources such as tryptone and peptone strongly improved laccase production. The application of an amino acid mixture and a lignin preparation also increased the formation of laccase, which was not observed in the presence of potato extract. Native polyacryl amide gel electrophoresis (PAGE) followed by laccase activity staining using guaiacol as the substrate was performed to analyze the laccase isozyme patterns under the different culture conditions employed. Zymograms revealed a total of twenty different laccase activity bands that appeared in individual patterns, dependent on the respective culture condition applied. This indicates that both the medium composition and the mode of incubation (static or shaking) influenced the laccase isozyme gene expression. This was the first time to report so many laccase isozymes in a fungus. Native PAGE with silver staining showed that laccases were the main protein productions in several media providing a potentially convenient way in purifying laccases from T. gallica. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Extracellular matrix regulates alpha s1-casein gene expression in rabbit primary mammary cells and CCAAT enhancer binding protein (C/EBP) binding activityJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2001Geneviève Jolivet Abstract Previous studies have shown that both the signal transducer and activator of transcription 5 (STAT5) and the CCAAT enhancer binding proteins (C/EBPs) are involved in the regulation of casein gene expression by mammary epithelial cells. Prolactin (Prl) activation of STAT5 is necessary for casein gene expression. The extracellular matrix (ECM) regulates also casein gene expression. Here, we have investigated whether ECM regulates C/EBPs activity in primary rabbit mammary epithelial cells. Isolated primary mammary cells were cultured on plastic or on floating collagen I gel. Prolactin induced ,s 1-casein gene expression when cells were cultured on collagen but not on plastic. It is noteworthy that activated STAT5 was detected in both culture conditions. Several STAT5 isoforms (STAT5a, STAT5b, and other STAT5 related isoforms, some with lower molecular weight than the full-length STAT5a and STAT5b) were detected under the different culture conditions. However, their presence was not related to the expression of ,s 1-casein gene. The binding of nuclear factors to a C/EBP specific binding site and the protein level of C/EBP, differed in cells cultured on plastic or on collagen but these parameters were not modified by Prl. This suggests that C/EBP binding activity was regulated by ECM and not by Prl. Interestingly, these modifications were correlated to the expression of the ,s 1-casein gene. Hence, the activation of the ,s 1-casein gene expression depends on two independent signals, one delivered by Prl via the activation of STAT5, the other delivered by ECM via C/EBP. J. Cell. Biochem. 82:371,386, 2001. © 2001 Wiley-Liss, Inc. [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] Evaluation of the SELDI-TOF MS technique for protein profiling of pancreatic islets exposed to glucose and oleatePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 17 2007Henrik Ortsäter Dr. Abstract The aim of the study was to evaluate the SELDI-TOF MS technique for pancreatic islet research. Mouse islets were cultured at low or high glucose levels in the absence or presence of oleate and characterized by measuring insulin secretion and oxygen tension. Subsequently, the islets were protein profiled. Up to 200 different peaks could be detected in a single experiment with the majority of peaks corresponding to proteins with masses below 30,kDa. By combining different protein arrays, the number of detected peaks could be increased further. The optimal binding of islet proteins was achieved using the anionic exchange array and phosphate buffer (pH,6) when the binding of insulin was low, which allowed other less abundant proteins to be captured. When islets from different culture conditions were profiled and analyzed, in total 25 proteins were found to be oleate/glucose-regulated. An oleate-regulated protein was chosen for identification work, which was conducted by passive elution from SDS-PAGE gels and subsequent in-gel trypsin digestion and MALDI-TOF MS. The protein was identified as peptidyl-prolyl isomerase B (PPI-B). In conclusion, the study demonstrates that SELDI-technique can be used not only to obtain islet protein patterns but is also helpful in the subsequent identification of differentially expressed proteins. [source] Changes in lipid content, fatty acid composition and lipid class composition of eggs and developing larvae (0,40 days old) of cultured common dentex (Dentex dentex Linnaeus 1758)AQUACULTURE NUTRITION, Issue 4 2008G. GIMÉNEZ Abstract Total lipid content, fatty acid (FA) composition and lipid class composition of common dentex eggs spawned at different times and larvae reared under different culture conditions until 40 days posthatch (dph) were analysed to get a general pattern of lipid composition during larval development. Two groups of larvae were kept under starvation to compare their FA composition with that obtained from normally fed larvae. To compare FA use or accumulation during larval development, results were grouped according to the developmental stage of the larvae instead of age in days posthatch. Saturated and monounsaturated FAs decreased along larval development, while polyunsaturated fatty acid (PUFA) content increased. The ratio of docosahexaenoic acid (DHA)/eicosapentaenoic acid shifted from 4 to 5 in early developmental stages to lower than 1 after metamorphosis. Arachidonic acid levels remained constant along larval development. Larvae kept 6 days under starvation consumed most of their n-3 PUFA while conserving the DHA to values at day 0. The results presented here are useful for the design of nutritional experiments, because there were differences detected in terms of lipid and FA composition between developmental stages with higher differences mainly found in first-feeding larvae and early developmental stages. [source] Degradation of an Fc-fusion recombinant protein by host cell proteases: Identification of a CHO cathepsin D proteaseBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009Flavie Robert Abstract A host-cell-related proteolytic activity was identified in a recombinant Fc-fusion protein production process. This report describes the strategy applied to characterize and isolate the enzyme responsible for this degradation by combining cell culture investigation and dedicated analytical tools. After isolation and sequencing of the clipped fragment generated in post-capture material, enzymatic activity was traced in different culture conditions, allowing identification of viable CHO cells as the source of protease. Inhibitors and pH screenings showed that the enzyme belongs to an aspartic protease family and is preferably active at acidic pH. The protease was isolated by purification on a pepstatin A column and characterized as a protein related to cathepsin D. An additional metallo-protease inhibited by EDTA was identified with an optimum activity at neutral pH. This study is an example of how quality and stability of therapeutic recombinant molecules are strongly influenced by cell culture parameters. Biotechnol. Bioeng. 2009; 104: 1132,1141. © 2009 Wiley Periodicals, Inc. [source] Cybernetic Modeling and Regulation of Metabolic Pathways in Multiple Steady States of Hybridoma CellsBIOTECHNOLOGY PROGRESS, Issue 5 2000Maria Jesus Guardia Hybridoma cells utilize a pair of complementary and partially substitutable substrates, glucose and glutamine, for growth. It has been shown that cellular metabolism shifts under different culture conditions. When those cultures at different metabolic states are switched to a continuous mode, they reach different steady states under the same operating conditions. A cybernetic model was constructed to describe the complementary and partial substitutable nature of substrate utilization. The model successfully predicted the metabolic shift and multiple steady-state behavior. The results are consistent with the experimental observation that the history of the culture affects the resulting steady state. [source] | ||||||||||