CO2分压对贴壁细胞MRC-5连续传代培养的影响

Effect of CO2 Partial Pressure on Subculture of Anchorage-dependent Cells MRC-5

研究培养过程中CO2的使用和pH稳定性对MRC-5细胞增殖、传代培养及产毒水平的影响。采用不同浓度的NaHCO3调节细胞培养液pH值,结合CO2使用,监测细胞培养过程中pH变化情况,以及在连续传代过程中的细胞形态和增殖情况。接种水痘-带状疱疹病毒比较不同状态下细胞的产毒能力。采用碳酸氢盐缓冲体系时,无CO2分压的封闭培养组细胞培养液pH波动较大,在传代培养中观察到细胞形态较差,细胞状态下滑快;细胞连续传代至35-37代;细胞在31代平均产毒水平4.64 LgPFU/mL。有CO2分压的开放组细胞培养液pH相对平稳,在传代培养中细胞活率维持80%以上,细胞状态下滑趋势平缓,可连续传代至41代;31代细胞平均产毒水平4.83Lg PFU/mL。采用碳酸氢盐缓冲体系的细胞培养液匹配合适的CO2分压有助于细胞状态的优化提升。

This work aims to study the effect of pH stability and CO2 employing on the proliferation,subculture and virus production level of MRC-5 for increasing its cells subculture capacity.Preparing cell culture medium with serial pH value by adding NaHCO3 and monitoring the dynamic variation of pH combined with CO2,the cell morphology and proliferation during passages in different condition were evaluated.Furthermore,the cell sensitivity to virus using varicella-zoster virus(VZV)was observed.When cell culture medium with the NaHCO3 as buffer,the pH of culture medium in the sealed group cultured without CO2 partial pressure fluctuated in wide range,correspondingly the cells grew aberrantly and subculture to passage 35-37 was in a significantly weakened trend.The VZV production on passage 31 cells of sealed groups was 4.64 Lg PFU/mL.On the contrary,cells under CO2 partial pressure condition presented a superior state because pH varied gently,and this group of MRC-5 cells could subculture to passage 41 with steady cell viability upon 80%,and the VZV production on the passage 31 was 4.83 Lg PFU/mL.In conclusions,employing NaHCO3 buffer paired with proper CO2 partial pressure is conducive to the optimization and improvement of MRC-5 cells morphologies.