Vero细胞生物反应器微载体无血清培养甲型流感病毒  

Serum⁃free culture of influenza A virus in Vero cells by microcarriers in bioreactor

作　　者：钏鸿云 阮朝列 莫明和[2] 王静 王多义 朱莲海 刘云璨 廖国阳[1] 周健[1] CHUAN Hong-yun;RUAN Chao-lie;MO Ming-he;WANG Jing;WANG Duo-yi;ZHU Lian-hai;LIU Yun-can;LIAO Guo-yang;ZHOU Jian(Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Kunming 650118,Yunnan Province,China;不详)

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 [2]云南大学省部共建云南生物资源与利用国家重点实验室,云南昆明650091

出　　处：《中国生物制品学杂志》2022年第11期1388-1391,共4页Chinese Journal of Biologicals

基　　金：云南省自然科学基金(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：目的采用Vero细胞生物反应器微载体无血清培养甲型流感病毒。方法将Vero细胞分别采用无血清和有血清培养基于生物反应器(pH 7.2,温度37℃,溶氧量50%,转数60 r/min)中培养24 h,均用无血清培养基进行灌流培养至7 d,进行计数,并按MOI=0.1接种H1N1型流感病毒,加入终浓度为1μg/mL的TPCK-胰酶,于生物反应器(pH 7.8,温度33.0℃,溶氧量25%,转数60 r/min)中培养18、22、42、48、66 h,取样,检测血凝效价。结果Vero细胞经无血清和有血清培养基培养7 d的细胞数分别为(133.4±2.0)×104和(193.8±1.3)×104个/mL,接种H1N1流感病毒66 h后血凝效价几何平均数分别为1∶388和1∶675,前者细胞数及病毒血凝效价几何平均数均显著低于后者(t分别为7.068和4.332,P均<0.05)。结论采用Vero细胞通过生物反应器微载体无血清培养H1N1型流感病毒的血凝效价可满足流感病毒裂解疫苗制备的要求。Objective To culture influenza A virus in Vero cells by microcarriers in bioreactor.Methods Vero cells were cultured with serum-free and serum-containing media in a bioreactor(pH 7.2,temperature 37℃,dissolved oxygen 50%,revolution 60 r/min)respectively for 24 h,then subjected to perfusion culture in serum-free medium for 7 d and counted,inoculated with H1N1 influenza virus at a MOI of 0.1,added with TPCK pancreatin to a final concentration of 1μg/mL,and further cultured in a bioreactor(pH 7.8,temperature 33.0℃,dissolved oxygen 25%,revolution 60 r/min)for 18,22,42,48 and 66 h respectively,from which samples were taken and determined for hemagglutination titer.Results The Vero cell counts after culture in serum-free and serum-containing media for 7 d were(133.4±2.0)×104 and(193.8±1.3)×104 cells/mL,while the geometric means of hemagglutination titer 66 h after inoculation with H1N1 influenza virus were 1∶388 and 1∶675,respectively.The cell count and geometric mean of hemagglutination titer of virus of Vero cells cultured in serum-free media were significantly lower than those of Vero cells cultured in serum-containing media(t=7.068 and 4.332,each P<0.05)Conclusion The hemagglutination titer of H1N1 influenza virus cultured in Vero cells by microcarriers in serum-free medium in bioreactor met the requirements for preparation of influenza virus split vaccine.

关 键 词：流感病毒 无血清培养 微载体 VERO细胞 

分 类 号：R373.1[医药卫生—病原生物学]