Jan D C, Petch D A, Huzel N, Butler M
Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
Biotechnol Bioeng. 1997 Apr 20;54(2):153-64. doi: 10.1002/(SICI)1097-0290(19970420)54:2<153::AID-BIT7>3.0.CO;2-K.
The murine B-lymphocyte hybridoma, CC9C10 was grown at steady state under serum-free conditions in continuous culture at dissolved oxygen (DO) concentrations in the range of 10% to 150% of air saturation. Cells could be maintained with this range at high viability in a steady state at a dilution rate of 1 d(-1), although with lower cell concentrations at higher DO. A higher specific antibody production measured at higher DO was matched by a decrease in the viable cell concentration at steady state, so that the volumetric antibody titre was not changed significantly. An attempt to grow cells at 250% of air saturation was unsuccessful but the cells recovered to normal growth once the DO was decreased.There was a requirement for cellular adaptation at each step-wise increase in dissolved oxygen. Adaptation to a DO of 100% was associated with an increase in the specific activities of glutathione peroxidase (x18), glutathione S-transferase (x11) and superoxide dismutase (x6) which are all known antioxidant enzymes. At DO above 100%, the activities of GPX and GST decreased possibly as a result of inactivation by reactive oxygen radicals.The increase in dissolved oxygen concentration caused changes in energy metabolism. The specific rate of glucose uptake increased at higher dissolved oxygen concentrations with a higher proportion of glucose metabolized anaerobically. Short-term radioactive assays showed that the relative flux of glucose through glycolysis and the pentose phosphate pathway increased whereas the flux through the tricarboxylic acid cycle decreased at high DO. Although the specific glutamine utilization rate increased at higher DO, there was no evidence for a change in the pattern of metabolism. This indicates a possible blockage of glycolytic metabolites into the TCA cycle, and is compatible with a previous suggestion that pyruvate dehydrogenase is inhibited by high oxygen concentrations.Analysis of the oxygen uptake rate of cell suspensions at steady state under all conditions showed a pronounced Crabtree effect which was manifest by a decrease (up to 40%) in oxygen consumption on addition of glucose. This indicates that the degree of aerobic metabolism in these cultures is highly sensitive to the glucose concentration.
小鼠B淋巴细胞杂交瘤CC9C10在无血清条件下于连续培养中在溶解氧（DO）浓度为空气饱和度的10%至150%范围内以稳定状态生长。细胞在此范围内可以在稀释率为1 d(-1)的稳定状态下保持高活力，尽管在较高的溶解氧水平下细胞浓度较低。在较高溶解氧水平下测得的更高的特异性抗体产量与稳定状态下活细胞浓度的降低相匹配，因此体积抗体滴度没有显著变化。尝试在空气饱和度的250%下培养细胞未成功，但一旦溶解氧降低，细胞恢复正常生长。在溶解氧的每一步增加时都需要细胞适应。适应100%的溶解氧与谷胱甘肽过氧化物酶（x18）、谷胱甘肽S-转移酶（x11）和超氧化物歧化酶（x6）的比活性增加有关，这些都是已知的抗氧化酶。在溶解氧高于100%时，谷胱甘肽过氧化物酶（GPX）和谷胱甘肽S-转移酶（GST）的活性可能由于活性氧自由基的失活而降低。溶解氧浓度的增加导致能量代谢发生变化。在较高的溶解氧浓度下，葡萄糖摄取的比速率增加，厌氧代谢的葡萄糖比例更高。短期放射性测定表明，在高溶解氧水平下，通过糖酵解和戊糖磷酸途径的葡萄糖相对通量增加，而通过三羧酸循环的通量降低。尽管在较高的溶解氧水平下谷氨酰胺的比利用率增加，但没有证据表明代谢模式发生变化。这表明糖酵解代谢产物可能受阻进入三羧酸循环，这与之前关于丙酮酸脱氢酶被高氧浓度抑制的建议一致。对所有条件下稳定状态的细胞悬液的氧摄取速率分析显示出明显的巴斯德效应，表现为添加葡萄糖后耗氧量降低（高达40%）。这表明这些培养物中的有氧代谢程度对葡萄糖浓度高度敏感。
