Mechanism of carbon dioxide inhibition in insect cell culture

昆虫细胞培养中二氧化碳的抑制机制

• 批准号: 0541948
• 负责人: David Murhammer
• 金额: --
• 依托单位: University of Iowa
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0541948&HistoricalAwards=false
• 关键词: Mechanism; carbon; dioxide; inhibition; insect

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.

0541948 Murhammer这项研究将探讨渗透压摩尔浓度、细胞内pH值和氧化应激在未感染和杆状病毒感染的昆虫细胞中CO2抑制作用中的作用。在大型昆虫和哺乳动物细胞生物反应器中，CO2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降低（通常发生在生物反应器体积增加时）和/或细胞密度增加，这种抑制作用变得越来越普遍。然而，二氧化碳的抑制作用背后的机制却知之甚少。迄今为止，机理研究几乎完全集中在渗透压和CO2浓度本身的贡献上。该研究基于两个假设：（1）CO2积累将以浓度依赖性方式抑制昆虫细胞生长，并且这种抑制与细胞内pH相关;（2）CO2积累将抑制杆状病毒感染的昆虫细胞中的产物产生，并且细胞内pH和氧化应激都将起作用。这些假设将通过解决以下具体目标进行检验：（1）确定CO2积累对未感染昆虫细胞的影响，以及（2）确定CO2积累对杆状病毒感染昆虫细胞的影响。相应的实验将涉及监测细胞密度、细胞活力、细胞内pH、蛋白质羰基和脂质氢过氧化物浓度、葡萄糖培养基浓度、O2消耗速率和CO2产生速率作为CO2浓度的函数。蛋白质羰基和脂质氢过氧化物浓度将提供氧化应激的量度，而葡萄糖浓度、O2消耗速率和CO2产生速率将提供细胞代谢的量度。