无血清悬浮培养MDCK细胞系的建立及生物反应器高密度培养

Establishment of MDCK suspension cell line in serum-free medium at high cell density in bioreactors

目的建立无血清悬浮培养MDCK细胞系,分析该悬浮细胞的生长特性、病毒敏感性、代谢动力学特征及生物反应器线性放大的可行性。方法通过逐步降血清和无血清悬浮驯化的方式获得1株MDCK悬浮细胞,检测其病毒敏感性,在1.2 L四联平行生物反应器中分析该悬浮细胞以不同初始接种密度(50×10^(4)、100×10^(4)、150×10^(4)、200×10^(4)个/mL)培养的细胞生长特性、代谢动力学特征和最佳传代时间,并进行5 L到75 L生物反应器的线性放大培养。结果贴壁培养型MDCK细胞以3%低血清稳定培养5代后,将该细胞直接适应无血清培养基获得1株MDCK悬浮细胞(命名为MDCK-XF03)并建库,复苏活力为96.8%;细胞接种不同型别的流感病毒均能较好增殖;以不同初始接种密度培养MDCK-XF03细胞,其最大增殖浓度均达1000×10^(4)个/mL以上,且差异无统计学意义(P>0.05),在培养前48 h均具有较大比生长速率,且差异有统计学意义(P<0.05),此时倍增时间也较短,且差异有统计学意义(P<0.05),MDCK-XF03细胞在84 h之前可充分利用葡萄糖。结论成功获得1株MDCK悬浮细胞MDCK-XF03,选择以200×10^(4)个/mL的初始接种密度进行生物反应器大规模培养,实现了从5 L到75 L的高密度线性放大培养,为流感疫苗的工业化生产提供了细胞基质。

Objective To establish a MDCK cell line in serum-free suspension culture and analyze the growth characteristics,virus sensitivity,metabolic kinetics characteristics and the feasibility of linear amplification in bioreactor.Methods A MDCK suspension cell line was obtained by stepwise serum-low and serum-free suspension domestication,and determined for virus sensitivity.The cell growth characteristics,metabolic kinetics characteristics and optimal time for subculture of the suspension cells were investigated in 1.2 L quadrupled parallel bioreactor,and the cells were subjected to linear scale-up culture in 5 and 75 L bioreactors.Results Adherent MDCK cells were stably cultured at a serum concentration of 3%for five passages and directly adapted to serum-free medium to obtain a MDCK suspension cell strain(named MDCK-XF03)with a resuscitation activity of 96.8%,in which the influenza virus of various types grew well.The maximum proliferation concentrations of MDCK-XF03 cells cultured at different initial inoculating densities were more than 1000×10^(4) cells/mL,which showed no significant difference(P>0.05).High specific growth rates were observed within 48 h after culture,which showed significant difference(P<0.05),while the doubling time was also short and significantly different(P<0.05).MDCK-XF03 cells made full use of glucose within 84 h after culture.Conclusion A MDCK suspension cell line MDCK-XF03 was successfully obtained and cultured in bioreactor at an initial inoculating density of 200×10^(4) cells/mL,which achieved the goal of scale-up culture from 5 L to 75 L bioreactors.It provided a cell matrix for the industrial production of influenza vaccine.