INDUCTION OF APOPTOSIS IN OXYGEN-DEPRIVED CULTURES OF HYBRIDOMA CELLS

It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures. Apoptotic or programmed cell dea th occurs spontaneously in late exponential phase of batch cultures. U ntil lately, no specific triggering factors had been identified. Recen tly, we observed that glutamine, cystine or glucose deprivation induce d apoptosis in both hybridoma and myeloma cell lines whereas accumulat ion of toxic metabolites induced necrotic cell death in these cells. O ther triggering factors such as oxygen deprivation might also be respo nsible for induction of apoptosis. In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone. The mode of cell death was studied b y morphological examination of acridine orange-ethidium bromide staine d cells in a 1.5 L bioreactor culture grown under anoxic conditions fo r 75 hours. Under such conditions, viable cell density levelled off ra pidly and remained constant for 25 hours. After 45 hours of anoxia, ce ll viability had decreased to 30% and the dead cell population was fou nd to be 90% apoptotic. In terms of cellular metabolism, anoxia result ed in an increase in the utilization rates of glucose and arginine, an d in a decrease in the utilization rate of glutamine. The lactate prod uction rate and the yield of lactate on glucose increased significantl y while the MAb production rate decreased. These results demonstrate t hat glycolysis becomes the main source of energy under anoxic conditio ns. Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rate s when reincubated in oxic conditions whereas cells incubated for 22 h ours or more displayed reduced growth rates. Nonetheless, even after 2 2 or 29 h of anoxia, cells reincubated in oxic conditions showed no fu rther progression into apoptosis. Therefore, upon removal of the trigg ering signal, induction of apoptosis ceased.