无血清悬浮驯化CHO-K1单克隆细胞株的建立及其表达验证  

Establishment and expression verification of serum-free suspension domestication of CHO-K1 monoclonal cell line

作　　者：谢涛 孙青 彭秀娥 张朝 张宽 王翠 刘伯宁 李文宾 姚兵 XIE Tao;SUN Qing;PENG Xiue;ZHANG Chao;ZHANG Kuan;WANG Cui;LIU Boning;LIWenbin;YAO Bing(CSPC Megalith Biopharmaceutical Co.,Ltd.,Shijiazhuang 050000,Hebei Province,China)

机构地区：[1]石药集团巨石生物制药有限公司,河北石家庄050000 [2]石药集团新型药物制剂与辅料国家重点实验室,河北石家庄050000

出　　处：《中国生物制品学杂志》2024年第8期945-951,共7页Chinese Journal of Biologicals

基　　金：中央引导地方科技发展资金项目(科技创新基地项目)(216Z2402G)。

摘　　要：目的通过无血清悬浮驯化CHO-K1细胞,筛选出单克隆源性可追溯、生长状态良好的宿主细胞株,并进行表达验证。方法采用逐步降低血清含量的方法,将贴壁状态的CHO-K1细胞驯化成适应无血清培养基培养的悬浮细胞;通过单细胞接种/成像系统Verified In-Situ Plate Seeding(简称VIPS系统)筛选单克隆,经连续培养综合评估单克隆细胞的倍增时间、结团率、细胞直径等参数,确定候选克隆;将候选克隆分别转染携带绿色及红色荧光蛋白的质粒,根据荧光蛋白表达情况确定1株克隆作为工程细胞株的宿主细胞。结果驯化获得的CHO-K1细胞适应无血清悬浮培养,倍增时间为20~24 h;通过单克隆筛选和产量评估确定的单克隆细胞株单克隆源性良好且可追溯,以0.5×10~6个/mL的细胞密度接种,培养7 d最高细胞密度可达8.27×10~6个/mL,活率不低于80%,平均倍增时间20.31 h,能较好地表达外源基因。结论通过无血清驯化培养,成功获得单克隆源性可追溯、生长状态良好且能够用于蛋白高表达的CHO-K1细胞株。Objective To select a host cell line with traceable monoclonal origin and good growth status by serum-free sus-pension domestication culture of CHO-K1 cells,and verify its expression.Methods Adherent CHO-K1 cells were domesti-cated into suspension cells suitable for serum-free medium by gradually decreasing serum concentration.Monoclonal cells were selected by cell seeding and imaging system Verified In-Situ Plate Seeding(VIPS system for short),and the parame-ters such as doubling time,agglomeration rate and cell diameter of monoclonal cells were comprehensively evaluated in continuous culture to determine the candidate clones.The candidate clones were transfected with plasmids contained green and red fluorescent proteins respectively,and one clone was identified as the final host cell of the engineering cell line according to the expression of fluorescent proteins.Results The obtained CHO-K1 cell lines were adapted to serum-free suspension culture,and the doubling time was 20-24 h.The monoclonal cell line identified by monoclonal screening and yield evaluation had clear monoclonal origin and could be traced back.At an inoculation density of O.5×10°cells/mL,the maximum growth density reached 8.27×10°cells/mL through continuous cultivation of 7 d,the viability was not lower than 80%with the mean doubling time of 20.31 h,and the exogenous gene was effectively expressed.Conclusion Through serum-free domestication culture,CHO-K1 cell line with traceable monoclonal origin,good growth status and high protein expression was successfully obtained.

关 键 词：CHO-K1细胞 无血清培养 细胞驯化 单克隆筛选 外源蛋白表达 

分 类 号：R392.11[医药卫生—免疫学]