生物反应器高密度无血清培养全悬浮MDCK细胞和流感病毒工艺的建立  

Development of a process for high-density serum-free culture of fully suspended MDCK cells and influenza virus in bioreactor

作　　者：李乐凯 吴业红 徐军 迟祥 李爽 席莉 张雪梅 郭占龙 LI Le-kai;WU Ye-hong;XU Jun;CHI Xiang;LI Shuang;XI Li;ZHANG Xue-mei;GUO Zhan-long(Changchun Institute of Biological Products Co.,Lid.,Changchun 130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130012

出　　处：《中国生物制品学杂志》2022年第9期1082-1089,共8页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2018ZX09737008-003);吉林省科技发展计划项目(20200301048RQ);吉林省科技创新专项资金(YDZX202022-00004719-1);长春市科技计划项目(18YJ024)。

摘　　要：目的优化生物反应器高密度无血清培养全悬浮MDCK细胞和流感病毒的条件,并放大培养规模,建立MDCK细胞和4种型别流感病毒的培养工艺。方法分别在不同溶氧(DO)、pH、转速、接种密度条件下培养MDCK细胞,每24 h取样计数,优化细胞培养条件后,放大至50 L生物反应器,并接种4种型别流感病毒(H1N1:A/California/7/2009、H3N2:A/Texas/20/2012、BV:B/Brisbane/60/2008、BY:B/Phuket/3073/2013),接毒条件为MOI=10-4,胰酶终浓度为15μg/mL,细胞密度为(1.8~2.2)×10^(6)个/mL,病毒维持液为DMEM,培养温度为甲型34.5℃、乙型33.5℃,检测病毒血凝效价、病毒滴度(CCID_(50))和血凝素(hemagglutinin,HA)含量。结果在5 L及50 L生物反应器中培养MDCK细胞,活细胞密度均可在72 h后达到8.0×10^(6)个/mL,活率在95%以上;H1N1、H3N2、BV、BY的血凝效价分别为7、11、9、9 log2(HAU/25μL),CCID50均达6.5以上,HA含量分别为20、40、35、40μg/mL。结论成功建立了MDCK细胞和流感病毒的无血清全悬浮培养工艺,为细胞基质流感疫苗的研发奠定了基础。Objective To optimize the conditions for high-density serum-free culture of fully suspended MDCK cells and influenza virus in bioreactor,enlarge the culture scale and develop a culture process for MDCK cells and four types of influenza virus.Methods MDCK cells were cultured at various dissolved oxygen(DO)concentrations,pH values,rotation rates and inoculation densities,from which samples were taken for counting every 24 h.The cells were cultured in an enlarged scale under the optimized conditions in 50 L bioreactor and inoculated with four types of influenza virus(H1N1:A/California/7/2009;H3N2:A/Texas/20/2012;BV:B/Brisbane/60/2008;BY:B/Phuket/3073/2013)respectively at a MOI of 10-4,a final pancreatin concentration of 15μg/mL and a cell density of(1.8~2.2)×106cells/mL,using DMEM as maintenance medium,then incubated at a temperature of 34.5℃(for type A)or 33.5℃(for type B),and determined for virus hemagglutination titer,virus titer(CCID50)and hemagglutinin(HA)content.Results Both the viable cell densities of MDCK cells reached 8.0×106cells/mL after culture in 5 L and 50 L bioreactors for 72 h,while the viability rates were more than 95%.The hemagglutination titers of virus of types H1N1,H3N2,BV and BY were 7,11,9 and 9 log2(HAU/25μL)respectively,while all the CCID50were more than 6.5,and the HA contents were 20,40,35 and 40μg/mL respectively.Conclusion The process for serum-free full suspension culture of MDCK cells and influenza virus was successfully developed,which laid a foundation of development of cells-based influenza vaccines.

关 键 词：流感病毒 MDCK细胞 无血清培养 全悬浮培养 

分 类 号：R183.3[医药卫生—流行病学]