Mechanism of carbon dioxide inhibition in insect cell culture

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0541948MurhammerThe research proposed will address the roles of osmolality, intracellular pH, and oxidative stress in CO2's inhibitory effect in uninfected and baculovirus infected insect cells. It has been widely demonstrated that CO2 can accumulate to inhibitory concentrations in large-scale insect and mammalian cell bioreactors. This inhibitory effect becomes increasingly prevalent as the gas/liquid interfacial area to bioreactor volume ratio decreases (commonly occurs at increasing bioreactor volumes) and/or cell density increases. The mechanism behind CO2's inhibitory effect, however, is poorly understood. To date, mechanistic studies have focused almost exclusively on the contribution of osmolality and CO2 concentration per se. The proposed research is based on two hypotheses: (1) CO2 accumulation will inhibit insect cell growth in a concentration dependent manner and this inhibition will correlate with intracellular pH, and (2) CO2 accumulation will inhibit product production in baculovirus infected insect cells and both intracellular pH and oxidative stress will contribute. These hypotheses will be tested by addressed the following specific aims: (1) determine the effect of CO2 accumulation on uninfected insect cells, and (2) determine the effect of CO2 accumulation on baculovirus infected insect cells. The corresponding experiments will involve monitoring cell density, cell viability, intracellular pH, protein carbonyl and lipid hydroperoxide concentrations, glucose medium concentration, O2 consumption rate, and CO2 production rate as a function of CO2 concentration. The protein carbonyl and lipid hydroperoxide concentrations will provide a measure of oxidative stress, while the glucose concentration, O2 consumption rate, and CO2 production rate will provide a measure of cell metabolism.