deZengotita Vivian M, Schmelzer Albert E, Miller William M
Chemical Engineering Department, Northwestern University, Evanston, Illinios 60208-3120, USA.
Biotechnol Bioeng. 2002 Feb 15;77(4):369-80. doi: 10.1002/bit.10176.
CO(2) partial pressure (pCO(2)) in industrial cell culture reactors may reach 150-200 mm Hg, which can significantly inhibit cell growth and recombinant protein production. The inhibitory effects of elevated pCO(2) at constant pH are due to a combination of the increases in pCO(2) and [HCO(-) (3)], per se, and the associated increase in osmolality. To decouple the effects of pCO(2) and osmolality, low-salt basal media have been used to compensate for this associated increase in osmolality. Under control conditions (40 mm Hg-320 mOsm/kg), hybridoma cell growth and metabolism was similar in DMEM:F12 with 2% fetal bovine serum and serum-free HB GRO. In both media, pCO(2) and osmolality made dose-dependent contributions to the inhibition of hybridoma cell growth and synergized to more extensively inhibit growth when combined. Elevated osmolality was associated with increased apoptosis. In contrast, elevated pCO(2) did not increase apoptotic cell death. Specific antibody production also increased with osmolality although not with pCO(2). In an effort to understand the mechanisms through which elevated pCO(2) and osmolality affect hybridoma cells, glucose metabolism, glutamine metabolism, intracellular pH (pHi), and cell size were monitored in batch cultures. Elevated pCO(2) (with or without osmolality compensation) inhibited glycolysis in a dose-dependent fashion in both media. Osmolality had little effect on glycolysis. On the other hand, elevated pCO(2) alone had no effect on glutamine metabolism, whereas elevated osmolality increased glutamine uptake. Hybridoma mean pHi was approximately 0.2 pH units lower than control at 140 mm Hg pCO(2) (with or without osmolality compensation) but further increases in pCO(2) did not further decrease pHi. Osmolality had little effect on pHi. Cell size was smaller than control at elevated pCO(2) at 320 mOsm/kg, and greater than control in hyperosmotic conditions at 40 mm Hg.
工业细胞培养反应器中的二氧化碳分压（pCO₂）可能达到150 - 200毫米汞柱，这会显著抑制细胞生长和重组蛋白的产生。在恒定pH值下，升高的pCO₂的抑制作用是由于pCO₂和[HCO₃⁻]本身的增加以及相关的渗透压升高共同作用的结果。为了区分pCO₂和渗透压的影响，已使用低盐基础培养基来补偿这种相关的渗透压升高。在对照条件下（40毫米汞柱 - 320毫渗摩尔/千克），在含有2%胎牛血清的DMEM:F12和无血清的HB GRO中，杂交瘤细胞的生长和代谢相似。在这两种培养基中，pCO₂和渗透压对杂交瘤细胞生长的抑制作用呈剂量依赖性，并且在联合作用时协同作用以更广泛地抑制生长。渗透压升高与细胞凋亡增加有关。相比之下，升高的pCO₂并未增加凋亡细胞死亡。特异性抗体的产生也随渗透压增加，尽管不随pCO₂增加。为了了解升高的pCO₂和渗透压影响杂交瘤细胞的机制，在分批培养中监测了葡萄糖代谢、谷氨酰胺代谢、细胞内pH（pHi）和细胞大小。升高的pCO₂（有或没有渗透压补偿）在两种培养基中均以剂量依赖性方式抑制糖酵解。渗透压对糖酵解影响很小。另一方面，单独升高的pCO₂对谷氨酰胺代谢没有影响，而升高的渗透压增加了谷氨酰胺的摄取。在pCO₂为140毫米汞柱时（有或没有渗透压补偿），杂交瘤的平均pHi比对照低约0.2个pH单位，但pCO₂的进一步升高并未进一步降低pHi。渗透压对pHi影响很小。在320毫渗摩尔/千克的pCO₂升高时，细胞大小小于对照，而在40毫米汞柱的高渗条件下大于对照。
