机械搅拌式生物反应器悬浮培养293T细胞生产慢病毒的工艺

Producing lentivirus by suspension culture of 293T cells in a stirred bioreactor

采用L型大泡通气系统机械搅拌式生物反应器培养293T悬浮细胞和规模化生产慢病毒载体,通过4种方法驯化293T悬浮细胞,对其慢病毒包装能力进行验证,并建立种子扩增链,在5.5 L工作体积生物反应器中绘制悬浮细胞生长曲线和包装慢病毒。结果表明,采用体积分数50%的293 CD05 Medium无血清基础培养基和体积分数50%的SMM293-TⅡ培养基可以快速驯化293T细胞,成功得到野生型且具有慢病毒包装能力的悬浮细胞系。悬浮细胞在机械搅拌式生物反应器培养,2 d细胞密度可达1.5×106个·mL^(-1)。添加质粒复合物和补料培养基,培养4 d可以收获6.5×107 TU·mL^(-1)滴度的慢病毒粗产品,相比细胞工厂的滴度提高34倍。

293T suspensions cells were cultured in a type L big bubble-stirred bioreactors for large-scale lentivirus vectors production in the study.Four approaches were utilized for the acclimation of 293T suspensions cells.After verification of the lentivirus packaging ability,adapted 293T cells were expanded in flask,drawing a growth curve and packaging lentivirus into a stirred bioreactor with 5.5 L work volume.The results show under the medium(half with 293 CD05 Medium and half with SMM293-TⅡ)the transition of 293T from adherent cells to suspensions cells was successful,and also retained the capacity for lentivirus vectors production.Using stirred bioreactor,the cell densities of 293T suspensions cells reached values of up to 1.5×106 cell·mL^(-1) for 2 days.After this point,plasmid complex and feeding were added to the cultures.Cultivation for 4 days in bioreactor enabled production of 6.5×107 TU·mL^(-1) coarse lentivirus,representing a 34-fold improvement compared to cell factory.