悬浮驯化Vero细胞制备及其在大流行流感病毒株培养的初步应用  

Preparation of suspension-adapted Vero cells and its preliminary application in culture of pandemic influenza virus strains

作　　者：刘鹏[1] 钏鸿云 阮朝列 陈静[1] 王多义 朱海莲 刘云璨 廖国阳[1] 周健[1] 李卫东[1] LIU Peng;CHUAN Hong-yun;RUAN Chao-lie;CHEN Jing;WANG Duo-yi;ZHU Hai-lian;LIU Yun-can;LIAO Guo-yang;ZHOU Jian;LI Wei-dong(Peking Union Medical College,Institute of Medical Biology,Chinese Academy of Medical Sciences,Kunming,Yunnan 650118,China)

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

出　　处：《中国病毒病杂志》2021年第3期182-186,共5页Chinese Journal of Viral Diseases

基　　金：云南省科技计划项目(2017FB039);中国医学科学院医学与健康科技创新工程(2020-I2M-2-014);中国医学科学院医学科技创新项目(2016-I2M-3-026)。

摘　　要：目的悬浮驯化培养Vero细胞(Vero simian kidney epithelial,Vero),并用于流感病毒H5N1及H7N9 Vero细胞高产重配株的制备。方法使用自配的Vero细胞悬浮培养基(Vero cells suspension media 1,VSM-1)在搅拌瓶中驯化贴壁Vero细胞适应悬浮培养,获得可悬浮培养的Vero细胞。使用工作体积5 ml的50 ml离心管为悬浮培养单元,同时设置相同工作体积的单层贴壁Vero细胞作为对照,以血凝滴度评价流感病毒在悬浮Vero细胞上的复制能力。结果本实验获得的悬浮Vero细胞在1.78×10^(5)细胞/ml的接种密度培养72 h后,细胞密度可达7.45×10^(5)细胞/ml。细胞倍增时间缩短在48 h内,比生长速率(μ)在驯化60~80 d后,维持在(0.016±0.007)h^(-1)。悬浮Vero细胞分别接种H5N1和H7N9后,平均血凝滴度分别为587 HAU/50μl和224 HAU/50μl,均高于贴壁对照组。结论VSM-1可以成功驯化Vero细胞摆脱贴壁依赖,获得适应悬浮培养的Vero细胞。初步探索悬浮Vero细胞生产大流行流感病毒株的能力,可为发酵工艺规模生产大流行流感病毒株疫苗做铺垫。Objective To study Vero cells in suspension culture and the production of high-yielding recombination strains of pandemic influenza virus H5 N1 and H7 N9.Methods Vero cells were adapted to grow in suspension in VSM-1 media,a self-designed suspension medium for Vero cells,and the process of adhered Vero cells to single-cell suspension culture in stirred flask was recorded and obtained the domesticated Vero cells ultimately.Furthermore,a 50 ml centrifuge tube with a working volume of 5 ml was used as a suspension culture unit while adherent Vero cells with the same working volume as the control.The ability of the suspended Vero cells to produce pandemic influenza virus was evaluated by hemagglutination test.Results The maximal cell density of the suspended Vero cells reached 7.45×10^(5)cells/ml 72 h after the inoculation density of 1.78×10^(5)cells/ml.The cell doubling time was reduced within 48 h,and the specific growth rate was maintained at(0.016±0.007)h^(-1)from 60 to 80 days after adaptation.The mean values of the highest hemagglutination titers of H5 N1 and H7 N9 inoculated with suspended Vero were 587 and 224 HAU/50μl,respectively,which were higher than that of the adherent control group.Conclusions VSM-1 can successfully domesticate Vero cells to get rid of anchorage dependence and obtain Vero cells suitable for suspension culture.The potential of suspension Vero cells for the production of pandemic influenza virus is evaluated.

关 键 词：VERO细胞 悬浮培养 流感病毒 大流行 病毒培养 

分 类 号：R373[医药卫生—病原生物学]