MDCK悬浮细胞制备及流感病毒敏感性研究  

Establishment of a new MDCK suspension cell line and its sensitivity study on influenza virus

作　　者：李自良 王家敏[1] 赵彩红 王美皓[1,2] 李莉 张雪梅 李倬[2] 靳冬武[5] 马忠仁 乔自林[1] LI Zi-liang;WANG Jia-min;ZHAO Cai-hong;WANG Mei-hao;LI li;ZHANG Xue-mei;LI Zhuo;JIN Dong-wu;MA Zhong-ren;QIAO Zi-lin(Gansu Tech Innovation Center of Animal Cell,Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;Science and Engineering College of Northwest University for Nationalities,Lanzhou 730030,China;Jilin Guanjie Biotechnology Co.Ltd,Meihekou 135000,China;Changchun Biological Products Research Institute Co.Ltd.,Changchun 130012,China;Lanzhou MinHai Bio-Engineering Co.Ltd,Lanzhou 730030,China)

机构地区：[1]西北民族大学生物医学研究中心甘肃省动物细胞技术创新中心,兰州730030 [2]西北民族大学生命科学与工程学院,兰州730030 [3]吉林冠界生物技术有限公司,梅河口135000 [4]长春生物制品研究所有限公司,长春130012 [5]兰州民海生物工程有限公司,兰州730010

出　　处：《中国人兽共患病学报》2019年第11期981-988,共8页Chinese Journal of Zoonoses

基　　金：国家科技重大专项(No.2015ZX09102016);西北民族大学动物细胞培养与代谢工程创新团队(No.31920190004);西北民族大学中央高校基本科研业务费专项资金项目(No.31920150029)~~

摘　　要：目的建立无血清悬浮培养MDCK细胞的方法,分析其传代、线性放大过程中的稳定性及增殖流感病毒的敏感性。方法运用逐步降血清悬浮驯化法将贴壁培养型MDCK细胞驯化为无血清全悬浮培养型MDCK细胞;进一步分析其生长动力学特性、连续传代稳定性,在5L生物反应器中扩大培养;接种流感和禽流感病毒,测定不同时间HA效价、CCID50滴度,分析其病毒敏感性。结果成功将ATCC引进的贴壁培养型MDCK细胞驯化为无血清全悬浮培养型MDCK细胞(命名为MDCK-XF02细胞)并冻存;不同接种密度培养MDCK-XF02细胞最大增殖密度均达13.0×10^6 cells/mL以上,且差异无统计学意义(P>0.05);连续传代培养过程中细胞形态和生长状况稳定,比生长速率差异无统计学意义(P>0.05);三个代次的MDCK-XF02细胞以2.0×10^6 cells/mL接种于5 L生物反应器,细胞生长状态良好,倍增时间小于21 h,在批培养中细胞浓度高达(14.57±0.47)×10^6 cells/mL,比生长速率分别为(0.027±0.012)h^-1、(0.028±0.013)h^-1、(0.027±0.013)h^-1(P>0.05);接种甲型流感病毒H1N1和H3N2,HA效价达到(6~7)log2HA/25μL、CCID50为(4.35~4.68)lgCCID50/mL;接种乙型流感病毒B/P和BX-35增殖效果更好,HA效价达到(9~10)log2HA/25μL,CCID50为(6.38~7.31)lgCCID50/mL。接种重组禽流感病毒H5亚型Re-5、Re-6、Re-10均能很好的增殖,HA效价达到(7~9)log2HA/25μL、CCID50为(6.21~6.96)lgCCID50/mL。结论本研究获得一株具有自主知识产权的流感/禽流感病毒敏感的无血清悬浮培养型MDCK-XF02细胞株,实现了生物反应器高密度放大培养,为我国开展流感疫苗研究和生产提供细胞基质,也为其他动物细胞悬浮驯化提供参考。In order to establish a method for serum-free suspension culture of Madin-Daby Canine Kidney Cells(MDCK)cells,its stability during cell passage and linear amplification and the sensitivity of proliferation of influenza virus were analyzed.The adherent MDCK cells were acclimated into serum-free suspended MDCK cells by stepwise serum suspension and acclimation.The growth kinetics and continuous passage stability were further analyzed,and the culture was expanded in a 5L bioreactor.And influenza virus,HA titer and CCID50 titer were measured at different times,and the virus sensitivity was analyzed.The adherent MDCK cells from ATCC were successfully acclimated into serum-free suspension MDCK cells(named MDCK-XF02 cells)and cryopreservation.The maximum proliferation density of MDCK-XF02 cells reached 13.0×10^6 Cells/mL or more,and the difference was not significant(P>0.05);the cell morphology and growth status were stable during continuous subculture,and there was no significant difference in growth rate(P>0.05).The three generations of MDCK-XF02 cells with 2.0×10^6 cells/mL was inoculated into a 5L bioreactor,and the cell growth state was good.The doubling time was less than 21 h.The cell concentration in the batch culture was as high as(14.57±0.47)×10^6 cells/mL,and the specific growth rate was(0.027±0.012)h^-1,(0.028±0.013)h^-1,(0.027±0.013)h^-1 respectively,which showed no significant difference(P>0.05).Inoculated with influenza A virus H1N1 and H3N2,HA titer reached(6-7)log2HA/25μL,CCID50 reached(4.35-4.68)lgCCID50/mL.Inoculation of influenza B virus B/P and BX-35 proliferation effect is better,HA titer reached(9-10)log2HA/25μL,CCID50 reached(6.38-7.31)lgCCID50/mL.Recombinant avian influenza virus H5 subtypes Re-5,Re-6,Re-10 can proliferate well,HA titer reached(7-9)log2HA/25μL,CCID50 reached(6.21-6.96)lgCCID50/mL.This study obtained a strain of serum-free suspension-cultured MDCK-XF02 cells with independent intellectual property rights of influenza/avian influenza virus,which achieved high-density ampl

关 键 词：MDCK细胞 无血清 悬浮驯化 流感病毒 禽流感病毒 

分 类 号：R373.1[医药卫生—病原生物学]