摘要
目的 比较胰蛋白酶(胰酶)和TrypLE消化Vero细胞的效果,确定细胞工厂培养Vero细胞的最佳消化工艺。方法 10层细胞工厂中Vero细胞生长成致密单层后,分别用(37±1)℃预热的0. 25%胰酶和TrypLE及室温(18~26℃)的TrypLE进行消化,吸取消化后的细胞悬液进行细胞计数,并检测细胞活率。将消化后的细胞悬液接种至生物反应器中继续培养,检测葡萄糖代谢情况,并进行室温TrypLE消化细胞的重复性验证。结果 用(37±1)℃0. 25%胰酶、TrypLE和室温TrypLE消化细胞所获得的平均细胞密度分别为1. 70×10^6、1. 68×10^6、1. 65×10^6个/m L,细胞活率均在95%以上。与0. 25%胰酶相比,使用TrypLE消化的细胞再培养时葡萄糖消耗量较高。10层工厂细胞消化所获细胞数的变异系数(CV)为2. 47%,消化传代后Vero细胞培养阶段葡萄糖代谢量的CV均小于10%。结论TrypLE作用温和,消化时细胞损伤较小,重复性好,细胞工厂规模化培养Vero细胞过程中可利用TrypLE替代胰酶进行细胞传代。
Objective To compare the effects of trypsin and TrypLE in digestion of Vero cells,and optimize the digestion procedure of Vero cells cultured in cell factory.Methods After the Vero cells in 10-layer cell factory grew into a dense monolayer,0.25%trypsin and Tryple,pre-warmed at(37±1)℃,and TrypLE stored at room temperature(18~26)℃were used for digestion the cells respectively.The cell suspension digested was collected for cell count,and the cell viability was calculated.Afterwards,the cell suspension was inoculated into the bioreactor for further culture,and the glucose metabolism was detected daily.The reproducibility of cell digestion with TrypLE was verified at room temperature.Results The densities of cells digested with 0.25%trypsin at(37±1)℃,TrypLE at(37±1)℃and TrypLE at room temperature were 1.70×10^6,1.68×10^6 and 1.65×10^6 cells/mL respectively,while all the cell viability was more than 95%.The glucose consumption in further culture of cells digested with TrypLE was higher than that with 0.25%trypsin.The coefficient of variation(CV)of number of cells obtained by cell digestion in the 10-layer factory was about 2.47%,while that of glucose metabolism in Vero cell culture stage after digestion and passage was less than 10%.Conclusion TrypLE had mild effects,less cell damage and good reproducibility during digestion.In the process of largescale cell culture in cell factory,TrypLE may be used instead of trypsin for cell passage.
作者
张颖
张乐
赵兰英
马轩
邓立强
姜男
代慧慧
张晋
梁宏阳
ZHANG Ying;ZHANG Le;ZHAO Lan-ying;MA Xuan;DENG Li-qiang;JIANG Nan;DAI Hui-hui;ZHANG Jin;LIANG Hong-yang(No.5 Vaccine Workshop,Beijing Institute of Biological Products Co.,Ltd.,Beijing 100176,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2019年第12期1407-1410,共4页
Chinese Journal of Biologicals
