不同培养基和胰蛋白酶对Vero细胞培养及消化效果的比较  

Comparison of culturing and detachment effects by different media and trypsins for Vero cells

作　　者：陈红 周刘一帆 杨彪 刘玉晴 张久威 李庆亮 冯冬扬 白萱 姜志军 董犇 阮波 徐葛林 段凯 Chen Hong;Zhou-Liu Yifan;Yang Biao;Liu Yuqing;Zhang Jiuwei;Li Qingliang;Feng Dongyang;Bai Xuan;Jiang Zhijun;Dong Ben;Ruan Bo;Xu Gelin;Duan Kai(Department of Rotavirus Vaccine,Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,China;National Engineering Technology Research Center of Combination Vaccines,Wuhan 430207,China)

机构地区：[1]武汉生物制品研究所有限责任公司轮状病毒疫苗室,430207 [2]国家联合疫苗工程技术研究中心,武汉430207

出　　处：《国际生物制品学杂志》2020年第3期116-119,共4页International Journal of Biologicals

基　　金：湖北省技术创新专项(重大项目)(2018ACA120)。

摘　　要：目的比较不同公司生产的无血清培养基对病毒性疫苗生产用Vero细胞培养效果,及基因工程胰蛋白酶的细胞消化效果,以判断是否适用。方法实验组用VirusPro Vero-A培养基进行Vero细胞的培养,用Trpzyme消化细胞。对照组用VP-SFM培养基进行细胞培养,用TrypLE Select消化细胞。两组Vero细胞以相同密度接种在T175培养瓶中,以相同的培养条件和传代方法在T175瓶和细胞工厂中各培养3代。培养期间观察细胞的上清液、形态以及汇合度,消化后检测细胞活率并计算细胞收获量。采用配对t检验比较两组消化液的pH值、总消化时长、37℃消化孵育时长、细胞活率以及细胞收获量。结果两组细胞生长状态均良好。配对t检验显示,实验组消化液的pH值(6.99)、总消化时长(17.28 min)和37℃消化孵育时长(6.93 min)均值均大于对照组消化液(分别为6.75、12.34 min、3.30 min),细胞活率(94.79%)及细胞收获量(T175瓶:3.91×107个;细胞工厂:1.90×109个)均值均高于对照组的(分别为90.20%、3.33×107个、1.26×109个),且差异有统计学意义(t值分别为9.17、3.46、2.98、2.31和4.38,P值均<0.05)。结论实验组无血清培养基培养效果较好,实验组基因工程胰蛋白酶细胞消化液温和、细胞损伤小,均适用于Vero细胞。Objective To compare serum-free culture media and genetic engineered trypsins from different companies to determine their suitability for Vero cells used in viral vaccine production.Methods Vero cells in test group were cultured with VirusPro Vero-A medium and trypsinized with Trpzyme,while Vero cells in control group were cultured with VP-SFM medium and trypsinized with TrypLE Select.These 2 groups of Vero cells were seeded on T175 flasks at same concentration and cultured under same conditions for 3 passages in T175 flasks and cell factories,respectively.The supernatant appearance,morphology,and confluence of cells were observed.Cell viability and harvest cell numbers of each T175 flask and cell factory were calculated after each trypsinization.The pH of trypsinization solution,total trypsinization duration,and incubating duration at 37℃were measured and compared using paired t test.Results Both groups of cells grew well.The average pH value(6.99),total trypsinization duration(17.28 min),incubating duration at 37℃(6.93 min),cell viability(94.79%)and cell harvest number(T175 flask:3.91×107,cell factory:1.90×109)in test group were higher than those in control group(6.75,12.34 min,3.30 min,90.20%,3.33×107 and 1.26×109,respectively)and the differences were statistically significant(t values were 9.17,3.46,2.98,2.31 and 4.38,respectively,all P values were<0.05).Conclusions The serum-free medium of test group results in better cell culture,and the genetic engineered trypsin of test group is mild and less damaging to cell,indicating that they are suitable for Vero cell culture.

关 键 词：细胞培养技术 VERO细胞 培养基 无血清 胰蛋白酶 

分 类 号：Q813.11[生物学—生物工程]