生物反应器外Vero细胞消化扩大培养工艺的研究  

Study of expanded culture process of Vero cells digestion outside the bioreactor

作　　者：陶涛 王玲 徐晓 杨敏 宋远涛 文聪 陈杰杉 王月莉 梁婧 TAO Tao;WANG Ling;XU Xiao;YANG Min;SONG Yuantao;WEN Cong;CHEN Jieshan;WANG Yueli;LIANG Jing(Department of Rabies Vaccine,Wuhan Institute of Biological Products Co.,Ltd.,National Union Vaccine Engineering Technology Research Center,Wuhan 430070,Hubei Province,China)

机构地区：[1]武汉生物制品研究所有限责任公司狂犬病疫苗室,国家联合疫苗工程技术研究中心,湖北武汉430070

出　　处：《微生物学免疫学进展》2023年第4期30-34,共5页Progress In Microbiology and Immunology

摘　　要：目的 探究使用生物反应器外消化Vero细胞的方法,实现细胞从30 L到300 L生物反应器逐级扩大培养的工艺,为生产过程中大规模培养Vero细胞提供一种可能的方法。方法 用生物反应器外消化方法对30 L反应器的Vero细胞进行消化,然后转接扩大至300 L反应器,观察培养工艺中的细胞形态、细胞状态,分析生物反应器外消化Vero细胞工艺应用的关键工艺参数(细胞汇合度、细胞回收率、细胞浓度)变化;通过连续9批Vero细胞放大试验证明罐外细胞消化的有效性和稳定性。结果 Vero细胞经过30 L生物反应器培养后,细胞汇合度为97.5%,细胞浓度为9.83×10^(6)cells/mL,经过生物反应器罐外消化后,分散良好且形态正常,后续300 L生物反应器培养的Vero细胞1 h基本贴壁,92 h后细胞浓度为6.20×10^(6)cells/mL,增殖速度正常。连续共9批试验结果证明罐外细胞消化的细胞回收率均>98.0%;300 L生物反应器培养结束后细胞汇合度均>92.0%,细胞浓度均>6.00×10^(6)cells/mL。结论 使用生物反应器外Vero细胞消化的方法进行30 L到300 L生物反应器放大培养工艺可行、有效且稳定,放大后细胞状态良好且增殖速度正常,适合Vero细胞的大规模生产。Objective To investigate the method of using bioreactor external digestion of Vero cells to realize the process of culturing cells from 30 L to 300 L bioreactor in stepwise expansion,and to provide a possible method for large scale culturing of Vero cells in the production process.Methods Digestion of Vero cells from 30 L reactor by the bioreactor external digestion method,and then transferred to a 300 L bioreactor.Cell morphology and cell state in the culture process were observed,and the changes of key process parameters(cell convergence,cell recovery,cell concentration)in the application of outside bioreactor digestion of Vero cell were analyzed;the effectiveness and stability of out of tank cell digestion were proved through the scale-up test of 9 consecutive batches of Vero cell.Results After Vero cell were cultured in a 30 L bioreactor,Vero cells showed 97.5% cell confluence,and the cell concentration was 9.83×10^(6) cells/mL,and were well dispersed and morphologically normal after bioreactor external digestion,and the Vero cells cultured in the subsequent 300 L bioreactor were basically adhered to the wall for 1 h.The cell concentration was 6.20×10^(6) cells/mL after 92 h.The proliferation rate was normal.The results of nine consecutive batches of experiments demonstrated that the cell recovery rate of extra-tank cell digestion was>98.0%;the cell confluence was>92.0%and the cell concentration was>6.00×10^(6) cells/mL at the end of 300 L bioreactor culture.Conclusion The 30 L to 300 L bioreactor scale-up culture process using bioreactor ex vivo Vero cell digestion is feasible,effective and stable,with good cell status and normal proliferation rate after scale-up,suitable for large-scale production of Vero cells.

关 键 词：VERO细胞 扩大培养 微载体 生物反应器外细胞消化 

分 类 号：R392-33[医药卫生—免疫学]