单克隆接种/成像设备VIPS在CHO工程细胞株单克隆筛选中的应用  

Application of monoclonal inoculation/imaging equipment VIPS in monoclonal screening of CHO engineered cell lines

作　　者：谢涛 彭秀娥 孙青 张朝 陈文烨 张宽 XIE Tao;PENG Xiue;SUN Qing;ZHANG Chao;CHEN Wenye;ZHANG Kuan(CSPC Megalith Biopharmaceutical Co.,Ltd.,State Key Lab of Novel Pharmaceutical Preparations and Excipients,Shijiazhuang 050000,Hebei Province,China)

机构地区：[1]石药集团巨石生物制药有限公司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》2023年第12期1497-1502,共6页Chinese Journal of Biologicals

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

摘　　要：目的探讨应用单克隆接种/成像设备VIPS(Verified In-situ Plate Seeding)建立CHO工程细胞株单克隆筛选的方法。方法采用VIPS将稳定转染目的基因的细胞池Cell pool 1分别于含不同组合(共18种组合,编号1~18)及体积(100和200μL/孔)培养基的96孔板中进行单细胞接种,并进行单克隆源性追踪拍照,根据图像结果,计算单克隆接种率、克隆形成率、单克隆增殖速率,评价单克隆源性图像效果。通过稳定转染目的基因的细胞池Cell pool 2、Cell pool 3、Cell pool 4验证该方法的适用性。结果最适单克隆培养基为MediumⅡ,培养基体积为100μL/孔。最佳工艺条件下,Cell pool 1的单克隆接种率为80%,克隆形成率为83%,增殖速率较快,单克隆源性图像清晰。Cell pool 2、Cell pool 3、Cell pool 4在最佳工艺条件下,单克隆接种率均值为78%,克隆形成率均值为67%,增殖速率略有差异,细胞分裂过程图像均清晰。结论采用VIPS建立的CHO工程细胞株单克隆筛选方法可提高CHO工程细胞株的克隆形成率,且能够提供充分的单克隆源性证明。Objective To develop a method for monoclonal screening of CHO engineered cell lines using Verified In-situ Plate Seeding(VIPS).Methods Cell pool 1 stably transfected with target gene was inoculated by using monoclonal inoculation/imaging equipment(VIPS)in 96-well plates containing culture medium of different combinations(18 combinations,numbered 1~18)and volumes(100 and 200μL/well).Monoclonal origin tracing pictures were taken,according to which,the monoclonal inoculation rate,clone formation rate and monoclonal proliferation rate were calculated and the image effects of monoclonal origin were evaluated.The applicability of this method was verified by cell pool 2,cell pool 3 and cell pool 4 stably transfected with target gene.Results The optimum monoclonal medium was MediumⅡ,and the volume of medium was 100μL/well.Under the optimum process conditions,the monoclonal inoculation rate of Cell pool 1 was 80%,the clone formation rate was 83%,the proliferation rate was fast,and the monoclonal origin image was clear.While for Cell pool2,Cell pool 3 and Cell pool 4,the average rate of monoclonal inoculation was 78%,the average rate of clone formation was 67%,the proliferation rate was slightly different and the image of cell division process was clear under the optimum process conditions.Conclusion In this study,the monoclonal screening method of CHO engineered cell lines developed using VIPS can improve the clone formation rate of CHO engineered cell lines and provide sufficient proof of monoclonal origin.

关 键 词：单克隆接种/成像设备VIPS CHO细胞 工程细胞株 单克隆筛选 单克隆源性 

分 类 号：Q2-33[生物学—细胞生物学]