MDCK细胞悬浮驯化及细胞系的建立与检定  

Adaptation of MDCK cells to suspension culture and establishment and characterization of cell line

作　　者：阮朝列 刘伯川 雍国胜 施锦丽 鲍晓林 刘瑶 李卫东[1] 周健[1] RUAN Chaolie;LIU Bochuan;YONG Guosheng;SHI Jinli;BAO Xiaolin;LIU Yao;LI Weidong;ZHOU Jian(Institute of Medical Biology,Chinese Academy of Medical Science and Peking Union Medical College,Kunming 650118,Yunnan Province,China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,云南昆明650118

出　　处：《中国生物制品学杂志》2024年第8期921-931,共11页Chinese Journal of Biologicals

基　　金：云南省科技厅科技计划项目(202002AA100009).

摘　　要：目的将贴壁培养的MDCK细胞驯化为悬浮培养,建立悬浮细胞系,并进行检定,以期为细胞基质流感疫苗的研发奠定基础。方法采用摇床适应法将贴壁培养的MDCK(NBL-2)细胞驯化为悬浮培养细胞,命名为MDCK-S细胞,进行18S及物种特异性PCR鉴定。将MDCK-S细胞传代至33代,作为主代细胞库(mastercellbank,MCB);传代至38代,作为工作细胞库(workingcell bank,WCB)。MCB及WCB细胞均进行生物学特征、污染情况、透射电镜、成瘤性和致瘤性、染色体变异性及流感病毒敏感性检测。结果确认MDCK-S细胞为狗源细胞,且未发现突变、缺失等情况,无其他种属DNA污染。MCB及WCB细胞生长状态良好,生长曲线呈“S”型,倍增时间分别为27.78及27.21h;细菌、真菌及支原体检查均为阴性;细胞形态、结构及细胞膜完整,膜上微绒毛较多,表明细胞状态好,不同细胞代次之间差异较小;具有成瘤性,但不具有致瘤性;染色体2n=(78±2)条占比为86%~96.19%,P29~P50代细胞染色体数目保持稳定;对流感病毒较敏感。结论成功将MDCK细胞驯化为悬浮培养,并建立了悬浮细胞系MDCK-S。本研究为细胞基质流感疫苗的研发奠定了基础。Objective To adapt the MDCK cells of adherent culture for suspension culture,establish and characterize a sus-pension cell line,so as to lay a foundation for the development of cell substrate-based influenza vaccines.Methods MDCK(NBL-2)cells of adherent culture were domesticated into suspension culture cells by shaker adaptation method,named MDCK-S cells,and identified by 18S and species-specific PCR.The MDCK-S cells were subcultured to the 33rd passage as the master cell bank(MCB),and passaged to the 38th generation as the working cell bank(WCB).MCB and WCB cells were both detected for biological characteristics,contamination,tumorigenicity and oncogenicity,chromosome variability and influenza virus sensitivity,and observed under transmission electron microscope.Results The MDCK-S cells were confirmed to be dog-derived cells with no mutations and deletions,and there was no DNA contamination of other species.MCB and WCB cells showed good growth status,with“S”-shaped growth curves and doubling time of27.78 and27.21 h,respectively.The cells were negative for bacteria,fungi and mycoplasma.The cell morphology,structure and cell membrane were intact,and there were abundant microvilli on the membrane,indicating that the cell status was good,and there was little difference between different cellgenerations.MDCK-S cells formed epithelial-like subcutaneous tumors,demonstrating its tumorigenicity but not oncogenicity.The proportion of chromosomes with 2 n=78±2 was approximately 86%to 96.19%,and the number of chromosomes remained stable from P29 to P50.In addition,MDCK-S cells were sensitive to influenza virus.Conclusion MDCK cells were successfully domesticated into suspension culture cells,and a suspension cell line MDCK-S was estab-lished in this study,which lays a foundation for the development of cell substrate-based influenza vaccines.

关 键 词：MDCK细胞 悬浮培养 驯化 流感疫苗 

分 类 号：R373.23[医药卫生—病原生物学] Q75[医药卫生—基础医学]