Cell cycle analysis of Chinese hamster ovary cells stimulated by phosphatidic acid in serum-free culture

When recombinant Chinese hamster ovary cells were treated with pertussis toxin or genistein, not only lysophosphatidic acid (LPA) but also phosphatidic acid (PA) failed to stimulate progression through the cell cycle in serum-free culture, suggesting that PA and LPA induce cell growth through the same signal transduction pathway. Cell cycle analysis also indicates that cell growth promoted by PA results in enhanced protein production.     

Effects of phospholipids on growth of Chinese hamster ovary cells in serum-free media

The effects of phosphatidic acid (PA) and lysophosphatidic acid (LPA) dispersed in serum-free media on the growth of Chinese hamster ovary (CHO) cells were investigated. After cells were incubated in serum-free media supplemented with PA or LPA for 3 d, the cellular growth was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Supplementing PA from egg yolk lecithin to basal synthetic media such as alpha-MEM (minimum essential medium, alpha modification) markedly promoted the growth of CHO cells. PA from egg yolk lecithin also enhanced the cell growth in a serum-free medium containing insulin and transferring. When PA analogues with different acyl chains of C14-18 were compared, PA with unsaturated acyl chains, dioleoyl-PA (C18: 1), was most effective for stimulating the growth of CHO cells. Similar results were obtained when LPA was examined. These results suggest that PA and LPA, especially those with unsaturated acyl chains, are promising growth-promoting supplements for use as constituents in a low-protein serum-free medium.     

Fetal calf serum-free suspension culture of Chinese hamster ovary cells employing fish serum

The effects of heat treatment and concentration of fish serum (FS) on cell growth in a suspension culture of recombinant Chinese hamster ovary (CHO) 1-15₅₀₀ (ATCC CRL-9606) cells were investigated. An increase in FS concentration from 1% to 4% markedly increased cell density. On the other hand, heat treatment of FS showed nearly no effect on cell density.     

Comparative study on delivery of phosphatidic acid to serum-free culture of Chinese hamster ovary cells

Liposomes containing phosphatidic acid (PA) and inclusion complexes of PA with methyl-beta-cyclodextrin (CD) were compared with PA dispersion prepared with a nonionic surfactant, Tween 80, for their effect on the growth of Chinese hamster ovary (CHO) cells in serum-free culture. All of the types of PA preparation were capable of promoting cell growth to almost the same high extent, indicating that the efficacy of PA in stimulating cellular growth may not depend on the preparation method.     

Development of a fed-batch culture process for enhanced production of recombinant human antithrombin by Chinese hamster ovary cells

Antithrombin is a serine protease inhibitor that inactivates several coagulation proteases, primarily thrombin and factor Xa. The Chinese hamster ovary (CHO) cell line transfected with a vector expressing recombinant human antithrombin (rAT) and a selectable marker, glutamine synthetase (GS), was cultivated in a 2-l fed-batch culture process using serum-free, glutamine-free medium. To maximize the rAT yield, effects of culture pH, balanced amino acid feeding, and an increased glutamate concentration on cell metabolism and rAT production were investigated. When cells were grown at pH values of 6.6, 6.8, 7.0, and 7.2, the maximum cell density and maximum lactate concentration decreased with decreasing pH. The highest production level of rAT was obtained at culture pH 6.8 due to the extended culture lifetime. Compared to the imbalanced amino acid feeding at culture pH 6.8, the balanced amino acid feeding increased the amount of rAT activity by 30% as a result of an increased viable cell number. A decrease in the specific glucose consumption rate (q(Glc)) with increasing culture time was observed in all the above-mentioned experiments, while the glucose concentration was maintained above 0.7 g l(-1). In addition, a decrease in the specific rAT production rate (q(rAT)) was observed after the depletion of lactate in the late cultivation stage. Taken together, these results suggest that the reduced availability of cellular energy caused by the decrease in q(Glc) and depletion of lactate led to the decrease in q(rAT). This decrease in q(rAT) was partially prevented by increasing the residual glutamate concentration from 1 mM to 7 mM, thus resulting in an additional 30% increase in the amount of rAT activity. The optimized fed-batch culture process yielded 1.0 g l(-1) rAT at 287 h of cultivation.     

Effect of static pressure on intracellular pH of adhesive Chinese hamster ovary cells

The effect of static pressure on the intracellular pH of the Chinese hamster ovary (CHO) cell line DR1000L4N was investigated. In cultivation of CHO cells at 0.9 MPa, two distinct populations were observed in the histogram of a flow cytometer, while single population was observed in cultivation at 0.1 MPa. The intracellular pH of the major population at 0.9 MPa was markedly lower than that of the single population at 0.1 MPa.     

Differential effects of hydrogen peroxide and ascorbic acid on the aerobic thermosensitivity of yeast cells grown under aerobic and anoxic conditions

We have previously demonstrated that in aerobically‐grown cells of the yeast Saccharomyces cerevisiae, hydrogen peroxide (H₂O₂) increases and ascorbic acid decreases cellular thermosensitivity, as determined by the inducibility of a heat shock (HS)‐reporter gene. In this work, we reveal that the aerobic thermosensitivity of anaerobically‐grown yeast cells also increases in the presence of H₂O₂, albeit differentially between cells with two different lipid profiles. In comparison to aerobically‐grown fermenting cells treated with the same H₂O₂ concentration, both these types of anaerobically‐grown cells were found to be considerably less sensitive to aerobic heat shock and considerably more thermotolerant. Paradoxically, and in contrast to ascorbate‐pretreated aerobically‐grown yeast cells, when anaerobically‐grown cells were heat‐shocked aerobically in the presence of the same ascorbic acid concentration, they exhibited increased thermosensitivity and decreased intrinsic thermotolerance with respect to their untreated counterparts. These findings are discussed with respect to what is currently known about the redox and physiological status of yeast cells grown aerobically and cells reoxygenated following anoxic growth. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of mannoproteins on the growth, gastrointestinal viability, and adherence to Caco-2 cells of lactic acid bacteria

Yeast cell wall (YCW) preparations and yeast mannoprotein extracts have been effective against some enteropathogenic bacteria as Campylobacter jejuni, Escherichia coli, and Salmonella, and they can affect the population of beneficial lactic acid bacteria (LAB). In this work, we studied the effect of a mannoprotein extract on five strains of LAB. This extract was metabolised by the bacteria, enhancing their survival in simulated gastrointestinal juice, and increasing the adherence of Lactobacillus plantarum, L. salivarius, and Enterococcus faecium to Caco-2 cells. Yeast mannoproteins are promising naturally occurring compounds that could be used to enhance LAB intestinal populations and control pathogens.     

Long-chain fatty acid effects on peroxisome proliferator-activated receptor-α-regulated genes in Madin-Darby bovine kidney cells: Optimization of culture conditions using palmitate

Studying long-chain fatty acid (LCFA) effects on gene network expression in bovine cells could provide useful information for future practical applications. An optimized in vitro system that does not require tissue collection or cell isolation could fill a niche in the study of PPARα activity in ruminants. Specific aims were to optimize culture conditions in Madin-Darby bovine kidney (MDBK) cells to achieve maximal mRNA expression of known peroxisome proliferator-activated receptor-α (PPARα) target genes using palmitate (16:0) as a representative LCFA. Variables included length of incubation time, use of albumin-bound (4:1 molar proportion) 16:0 (A16:0), or addition of insulin. A first time-course experiment tested culturing cells in Dulbecco's modified Eagle's medium with 150 μM PPAR ligand Wy-14643 (WY) and A16:0. A second experiment tested the effects of albumin and insulin using 150 μM of 16:0 without albumin or insulin (−Alb/−Ins), 16:0 without albumin plus 5 mg/L of bovine insulin (−Alb/+Ins), A16:0 without insulin (+Alb/−Ins), or a control. A third experiment was a preliminary metabolic characterization of cells and assessed intracellular lipid droplet formation after treatment with 150 μM of 16:0 or an ethanol control. For all experiments, cells were harvested at 0, 6, 12, 18, and 24 h posttreatment. In experiments 1 and 2, mRNA expression was assessed by quantitative PCR of selected PPARα target genes as well as PPARα coactivators (ACOX1, CPT1A, ACADVL, ACSL1, PPARA, PPARGC1A, LPIN1). In experiment 1, there was a linear increase in mRNA expression of CPT1A (∼500%) and ACSL1 (50 to 200%) by 6 h of incubation with both WY and A16:0. The LPIN1 mRNA increased by >100% by 6 h only with A16:0. Further, there was a linear increase in expression of PPARA (∼100%) with A16:0 through 24 h of incubation. In experiment 2, insulin increased, and coupling LCFA with albumin tended to delay the response in expression of CPT1A and ACSL1 to 16:0. Data indicated a toxic effect of 150 μM free 16:0 as assessed by cell counts after 12 h of incubation. In experiment 3, MDBK cells appeared to use glucose and AA as energy sources and were able to secrete triglycerides. In addition, MDBK cells cultured with 150 μM of 16:0 had a substantial uptake of LCFA and synthesized intracellular lipid droplets. Overall, results indicated that a 6-h incubation with free LCFA and addition of insulin was suitable to detect marked effects on mRNA expression of PPARα target genes in MDBK cells.