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Basal Medium (basal + medium)
Selected AbstractsLong-term culture of Xenopus presumptive ectoderm in a nutrient-supplemented culture mediumDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5-6 2003Yasuto Fukui Animal cap assay is a useful experimental model for investigating the activity of inducers in amphibian development. This assay has revealed that activin A is a potent mesoderm-inducing factor. However, it has been very difficult to induce highly differentiated tissues such as cartilage in a 3,4 day culture period. It was recently reported that jaw cartilage was induced in vitro in an animal cap that had been cultured for 14 days in Steinberg's solution using the sandwich culture method and activin A. Under these conditions, necrosis was occasionally observed in the explants. In this study, we have achieved long-term animal cap cultures in a nutrient-supplemented culture medium designated RDX. This medium was made by modifying the saline concentration of the RD medium previously developed as a basal medium for the serum-free culture of various kinds of mammalian cells. The explants cultured in RDX grew more vigorously compared with those in Steinberg's solution. RDX medium promoted a wider variety of tissue induction and gene expression in the animal caps than Steinberg's solution, and also increased the frequency of cartilage induction. Therefore, the supplemental nutrients may support and promote the differentiation of cartilage. This long-term culture method using RDX medium is useful for studying the differentiation of tissues or organs such as cartilage in vitro. [source] The effect of genotype and culture medium on somatic embryogenesis and plant regeneration from mature embryos of fourteen apomictic cultivars of buffel grass (Cenchrus ciliaris L.)GRASS & FORAGE SCIENCE, Issue 1 2006E. L. Colomba Abstract Buffel grass (Cenchrus ciliaris L.) is an important apomictic grass used as forage for ruminant livestock. Biotechnological methods provide opportunities for producing new germplasm. Mature embryos of fourteen buffel grass apomictic cultivars (2n = 4x = 36) were used to induce embryogenic callus formation using a basal medium supplemented with 3% sucrose and with the testing of five concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and four concentrations of 6-benzylaminopurine (BAP). The effects of cultivar and culture medium on callus induction and plant regeneration were evaluated. Significant differences were observed among the fourteen cultivars and the five concentrations of 2,4-D (P < 0·01). Values for embryogenic callus production varied from 0 to 86·7. Most cultivars showed the highest level of embryogenic callus production on the medium with the concentration of 3 mg L,1 2,4-D. The addition of different BAP concentrations in combination with 2,4-D in the medium inhibited embryogenic callus growth and did not permit plant regeneration. The data clearly demonstrated that the genotype and concentrations of 2,4-D had significant effects both on the frequency of embryonic callus formation from mature embryos and on the subsequent efficiency of plant regeneration of apomictic cultivars of buffel grass. Cultivars Biloela and Nunbank showed the greatest efficiency in in vitro culture response. [source] Effect of the anti-tumor necrosis factor-, antibody infliximab on the ex vivo mucosal matrix metalloproteinase,proteolytic phenotype in inflammatory bowel diseaseINFLAMMATORY BOWEL DISEASES, Issue 2 2007Martin J. Meijer MSc Abstract Background: Previous studies have shown an upregulation of matrix metalloproteinases (MMPs) in intestinal tissue of patients with inflammatory bowel disease (IBD) and significant clinical improvement after administration of the anti-TNF-, antibody infliximab. The aims of our study were to determine expression and secretion of MMP-1, -2, -3, -9, and their inhibitors TIMP-1, -2 by IBD versus control intestinal mucosa ex vivo and to assess the regulatory capacity by infliximab of the proteolytic phenotype. Methods: Intestinal mucosal explants from 20 IBD and 15 control patients were cultured with or without infliximab and/or the T-cell activator pokeweed mitogen (PWM). Explants and culture supernatants were analyzed for MMPs, TIMPs, and TNF-, protein, activity and/or mRNA levels. All patients were genotyped for functional TNF-,, MMP, and TIMP single nucleotide polymorphism (SNP) loci. Results: Expression of MMP and TIMP protein/activity in basal medium was higher in IBD versus control explants. Dependent on genotype at SNP loci, infliximab downregulated MMP-1, -3, and -9 relative to TIMP-1 and -2 and also decreased MMP-1 and -3 activities, while PWM enhanced these levels, partly counteracted again by infliximab. The expression of MMP-2 relative to TIMP did not change by treatment with infliximab and/or PWM. Conclusions: The high expression of MMPs in patients with IBD suggests a role for these proteinases in the pathogenesis of this disease. Infliximab seems to induce a genotype-associated matrix protective phenotype, which may contribute to the observed therapeutic efficacy of this drug in IBD, particularly at the mucosal surface. (Inflamm Bowel Dis 2007) [source] The effect of carbon and nitrogen sources on bovicin HC5 production by Streptococcus bovis HC5JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2009A.A.T. De Carvalho Abstract Aims:, To investigate the effect of media composition and agroindustrial residues on bovicin HC5 production by Streptococcus bovis HC5. Methods and Results:, Batch cultures of S. bovis HC5 were grown in basal medium containing different carbon and nitrogen sources. The activity of cell-free and cell-associated bovicin HC5 was determined in culture supernatants and acidic extracts obtained from cell pellets, respectively. Streptococcus bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l,1 of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when yeast extract and trypticase (0·5 and 1·0 g l,1, respectively) were added together to the basal medium. Streptococcus bovis HC5 cultures produced bovicin HC5 in cheese whey and sugar cane juice and maximal volumetric productivity was obtained after 12 h of incubation. Conclusions:,Streptococcus bovis HC5 is a versatile lactic acid bacterium that can utilize several carbon and nitrogen sources for bovicin HC5 production. This bacterium could be a useful model to study bacteriocin production in the rumen ecosystem. Significance and Impact of the Study:, The use of agroindustrial residues as carbon sources could have an economical impact on bovicin HC5 production. To our knowledge, this is the first report to show the use of sugar cane juice for bacteriocin production by lactic acid bacteria. [source] In vitro Selection for Fusarium Wilt Resistance in GladiolusJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 5 2008Idrees Ahmad Nasir Abstract Cormels pieces of four Fusarium susceptible Gladiolus cultivars (Friendship, Peter Pears, Victor Borge and Novalux) formed friable calli when cultured in vitro on Murashige and Skoog basal medium containing various concentrations of auxin and cytokinin. The friable calli established cell suspensions. Plantlet regeneration was obtained from the control callus, control cell suspension derived callus and in vitro selected Fusarium oxysporum Schlecht. resistant cell-lines of Friendship. The in vitro cormlets showed 85,95% germination after breaking dormancy of 8 weeks at 4 °C. Cell suspensions of all four Gladiolus cultivars were found to be highly sensitive to fusaric acid. Gradual increase in fusaric acid concentrations to the cell-suspension cultures decreased cell growth considerably. One albino plant was found from the second generation of the in vitro selected cell line of Friendship. The albino plant was found to be highly susceptible to F. oxysporum. The cormlets of all in vitro selected cell lines of Friendship were inoculated with a conidial suspension of the F. oxysporum before planting and were also sprayed with the same spore suspension for further characterization when the height of plants was about 6 cm. The four selected cell lines showed the same response whether or not they were inoculated with conidia of the F. oxysporum. Plantlets of all of the selected cell lines exhibited significant growth as compared with the control after application of conidia of the F. oxysporum. [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 PetrifilmÔ EC method for enumeration of E. coli from soilLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2010A.D. Samarajeewa Abstract Aims:, To evaluate the suitability of commercially available PetrifilmÔ EC plates for enumeration of Escherichia coli from soil. Methods and Results:, A confirmed E. coli strain isolated from liquid swine manure was inoculated into sterilized sandy clay loam and loam soils at the concentrations of 102, 103, 105 CFU g,1 of soil. The efficiency of recovery on PetrifilmÔ EC plates for soils spiked with E. coli was compared with standard membrane filtration techniques on m-FC basal medium supplemented with 3-bromo-4-chloro-5-indoyl-,- d -glucopyranoside (BCIG) and most probable numbers (MPN) techniques in E. coli medium with 4-methylumbelliferyl-,- d -glucuronide (EC-MUG) broth. PetrifilmÔ EC and m-FC (BCIG) methods were then assessed for the ability to recover E. coli from field soils applied with swine manure. No significant differences (P > 0·05) were observed between PetrifilmÔ EC, m-FC (BCIG) and MPN methods for the recovery of E. coli from spiked samples, irrespective of soil type. However, recovery of E. coli from manure-applied field soil samples showed a significant difference (P < 0·05) between the PetrifilmÔ EC method and the m-FC method in enumerating E. coli possibly as a result of false positives on m-FC. Conclusion:, The PetrifilmÔ EC method is suitable for the enumeration of E. coli from soil with a detection limit of 10 CFU g,1 soil. Significance and Impact of the Study:, The commercially available PetrifilmÔ EC method is comparatively low cost, easy to use method for the enumeration of E. coli from soil without the need for further confirmation tests. [source] Influence of nutritional conditions on the mycelial growth and exopolysaccharide production in Paecilomyces sinclairiiLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2002S.W. Kim Aims:,The objective of the study was to optimize the submerged culture conditions for the production of exopolysaccharide from Paecilomyces sinclairii. Methods and Results:,The optimal temperature and initial pH for exopolysaccharide production by Paecilomyces sinclairii in shake flask culture were found to be 30°C and 6·0, respectively. Sucrose (60 g l,1) and corn steep powder (10 g l,1) were the most suitable carbon and nitrogen source for exopolysaccharide production. Conclusions:,Under optimal culture medium, the maximum exopolysaccharide concentration in a 5-l stirred-tank fermenter indicated 7·4 g l,1, which was approximately three times higher than that in basal medium. The maximum specific growth rates (,max) and yield coefficient (YP/S) in the optimal culture medium was 0·16 h,1 and 0·19, respectively. Significance and Impact of the Study:,The optimal culture conditions reported in this article can be widely applied to the processes for submerged cultures of other mushrooms. [source] CHO gene expression profiling in biopharmaceutical process analysis and designBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Jochen Schaub Abstract Increase in both productivity and product yields in biopharmaceutical process development with recombinant protein producing mammalian cells can be mainly attributed to the advancements in cell line development, media, and process optimization. Only recently, genome-scale technologies enable a system-level analysis to elucidate the complex biomolecular basis of protein production in mammalian cells promising an increased process understanding and the deduction of knowledge-based approaches for further process optimization. Here, the use of gene expression profiling for the analysis of a low titer (LT) and high titer (HT) fed batch process using the same IgG producing CHO cell line was investigated. We found that gene expression (i) significantly differed in HT versus LT process conditions due to differences in applied chemically defined, serum-free media, (ii) changed over the time course of the fed batch processes, and that (iii) both metabolic pathways and 14 biological functions such as cellular growth or cell death were affected. Furthermore, detailed analysis of metabolism in a standard process format revealed the potential use of transcriptomics for rational media design as is shown for the case of lipid metabolism where the product titer could be increased by about 20% based on a lipid modified basal medium. The results demonstrate that gene expression profiling can be an important tool for mammalian biopharmaceutical process analysis and optimization. Biotechnol. Bioeng. 2010; 105: 431,438. © 2009 Wiley Periodicals, Inc. [source] Model identification in presence of incomplete information by generalized principal component analysis: Application to the common and differential responses of Escherichia coli to multiple pulse perturbations in continuous, high-biomass density cultureBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Daniel V. Guebel Abstract In a previous report we described a multivariate approach to discriminate between the different response mechanisms operating in Escherichia coli when a steady, continuous culture of these bacteria was perturbed by a glycerol pulse (Guebel et al., 2009, Biotechnol Bioeng 102: 910,922). Herein, we present a procedure to extend this analysis when multiple, spaced pulse perturbations (glycerol, fumarate, acetate, crotonobetaine, hypersaline plus high-glycerol basal medium and crotonobetaine plus hypersaline basal medium) are being assessed. The proposed method allows us to identify not only the common responses among different perturbation conditions, but to recognize the specific response for a given stimulus even when the dynamics of the perturbation is unknown. Components common to all conditions are determined first by Generalized Principal Components Analysis (GPCA) upon a set of covariance matrices. A metrics is then built to quantify the similitude distance. This is based on the degree of variance extraction achieved for each variable along the GPCA deflation processes by the common factors. This permits a cluster analysis, which recognizes several compact sub-sets containing only the most closely related responsive groups. The GPCA is then run again but is restricted to the groups in each sub-set. Finally, after the data have been exhaustively deflated by the common sub-set factors, the resulting residual matrices are used to determine the specific response factors by classical principal component analysis (PCA). The proposed method was validated by comparing its predictions with those obtained when the dynamics of the perturbation was determined. In addition, it showed to have a better performance than the obtained with other multivariate alternatives (e.g., orthogonal contrasts based on direct GPCA, Tucker-3 model, PARAFAC, etc.). Biotechnol. Bioeng. 2009; 104: 785,795 © 2009 Wiley Periodicals, Inc. [source] Perfusion Culture of Hybridoma Cells for Hyperproduction of IgG2a Monoclonal Antibody in a Wave Bioreactor-Perfusion Culture SystemBIOTECHNOLOGY PROGRESS, Issue 1 2007Ya-Jie Tang A novel wave bioreactor-perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based perfusion controller. A polyethylene membrane filter with a pore size of 7 ,m was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 × 105 and 200.5 × 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 × 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L·day) in perfusion culture were much higher than those (i.e., 22.3 × 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L·day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale. [source] | ||||||||||||||||||||||