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
谢涛 1孙青 1彭秀娥 1张朝 1张宽 1王翠 1刘伯宁 1李文宾 1姚兵1
作者信息
- 1. 石药集团巨石生物制药有限公司,河北石家庄 050000;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
- 折叠
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 × 106个/mL的细胞密度接种,培养7 d最高细胞密度可达8.27 × 106个/mL,活率不低于80％,平均倍增时间20.31h,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.
Abstract
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 0.5 × 106cells/mL,the maximum growth density reached 8.27 × 106 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Key words
CHO-K1 cells/Serum-free culture/Cell domestication/Monoclonal screening/Exogenous protein expression引用本文复制引用
基金项目
中央引导地方科技发展资金项目(科技创新基地项目)(216Z2402G)
出版年
2024
NETL
NSTL
万方数据