Mechanism of carbon dioxide inhibition in insect cell culture

0541948Murhammer建议的研究将解决渗透压、细胞内pH和氧化应激在二氧化碳对未感染和杆状病毒感染的昆虫细胞的S抑制作用中的作用。已经广泛证明，在大型昆虫和哺乳动物细胞生物反应器中，二氧化碳可以积累到抑制浓度。随着气液界面面积与生物反应器体积比的减小(通常发生在生物反应器体积的增加)和/或细胞密度的增加，这种抑制效应变得越来越普遍。然而，二氧化碳S抑制作用背后的机制却知之甚少。到目前为止，机械论研究几乎完全集中在渗透压和二氧化碳浓度本身的贡献上。这项研究基于两个假设：(1)CO2的积累会以浓度依赖的方式抑制昆虫细胞的生长，这种抑制与细胞内的pH有关；(2)在杆状病毒感染的昆虫细胞中，CO2的积累会抑制产物的产生，细胞内的pH和氧化应激都会起作用。这些假说将通过以下具体目标来验证：(1)确定二氧化碳积累对未感染的昆虫细胞的影响，以及(2)确定二氧化碳积累对杆状病毒感染的昆虫细胞的影响。相应的实验将包括监测细胞密度、细胞存活率、细胞内pH、蛋白质羰基和脂质过氧化氢浓度、葡萄糖介质浓度、氧气消耗率和二氧化碳产生率作为二氧化碳浓度的函数。蛋白质羰基和脂质氢过氧化氢浓度将提供氧化应激的衡量标准，而葡萄糖浓度、O2消耗率和CO2产生率将提供细胞代谢的衡量标准。

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.