THE EFFECT OF DISSOLVED-OXYGEN ON THE METABOLIC PROFILE OF A MURINE HYBRIDOMA GROWN IN SERUM-FREE MEDIUM IN CONTINUOUS-CULTURE

The murine B-lymphocyte hybridoma, CC9C10 was grown at steady state un der serum-free conditions in continuous culture at dissolved oxygen (D O) concentrations in the range of 10% to 150% of air saturation; Cells could be maintained within this range at high viability in a steady s tate at a dilution rate of 1 d(-1), although with lower cell concentra tions at higher DO. A higher specific antibody production measured at higher DO was matched by a decrease in the viable cell concentration a t 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 ste p-wise increase in dissolved oxygen. Adaptation to a DO of 100% was as sociated with an increase in the specific activities of glutathione pe roxidase (x18), glutathione S-transferase (x11) and superoxide dismuta se (x6) which are all known antioxidant enzymes. At DO above 100%, the activities of GPX and GST decreased possibly as a result of inactivat ion by reactive oxygen radicals. The increase in dissolved oxygen conc entration caused changes in energy metabolism. The specific rate of gl ucose uptake increased at higher dissolved oxygen concentrations with a higher proportion of glucose metabolized anaerobically. Short-term r adioactive assays showed that the relative flux of glucose through gly colysis and the pentose phosphate pathway increased whereas the flux t hrough 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 upta ke rate of cell suspensions at steady state under all conditions showe d a pronounced Crabtree effect which was manifest by a decrease (up to 40%) in oxygen consumption on addition of glucose. This indicates tha t the degree of aerobic metabolism in these cultures is highly sensiti ve to the glucose concentration. (C) 1997 John Wiley & Sons, Inc.