阮朝列, 陈洪波, 张泽珩, 雍国胜, 刘伯川, 赵红玲, 陈瑜, 刘双芹, 廖国阳, 李卫东, 周健. 不同类型胰蛋白酶消化Vero细胞和KMB-17细胞的比较[J]. 云南大学学报(自然科学版), 2022, 44(5): 1069-1076. doi: 10.7540/j.ynu.20210184
引用本文: 阮朝列, 陈洪波, 张泽珩, 雍国胜, 刘伯川, 赵红玲, 陈瑜, 刘双芹, 廖国阳, 李卫东, 周健. 不同类型胰蛋白酶消化Vero细胞和KMB-17细胞的比较[J]. 云南大学学报(自然科学版), 2022, 44(5): 1069-1076. doi: 10.7540/j.ynu.20210184
RUAN Chao-lie, CHEN Hong-bo, ZHANG Ze-heng, YONG Guo-sheng, LIU Bo-chuan, ZHAO Hong-ling, CHEN Yu, LIU Shuang-qin, LIAO Guo-yang, LI Wei-dong, ZHOU Jian. Comparison of digestion of Vero cells and KMB-17 cells with different types of digestive solutions[J]. Journal of Yunnan University: Natural Sciences Edition, 2022, 44(5): 1069-1076. DOI: 10.7540/j.ynu.20210184
Citation: RUAN Chao-lie, CHEN Hong-bo, ZHANG Ze-heng, YONG Guo-sheng, LIU Bo-chuan, ZHAO Hong-ling, CHEN Yu, LIU Shuang-qin, LIAO Guo-yang, LI Wei-dong, ZHOU Jian. Comparison of digestion of Vero cells and KMB-17 cells with different types of digestive solutions[J]. Journal of Yunnan University: Natural Sciences Edition, 2022, 44(5): 1069-1076. DOI: 10.7540/j.ynu.20210184

不同类型胰蛋白酶消化Vero细胞和KMB-17细胞的比较

Comparison of digestion of Vero cells and KMB-17 cells with different types of digestive solutions

  • 摘要: 胰蛋白酶(trypsin)是贴壁细胞传代过程中必不可少的工具酶. 研究比较了猪源胰蛋白酶、两种牛源胰蛋白酶和全合成胰蛋白酶TrypLE消化Vero细胞和KMB17细胞的效果,评价在疫苗生产中培养细胞的最佳消化工艺,以及在向伊斯兰国家及人群提供生物制品过程中存在的Halal(清真)验证中关于猪源胰蛋白酶的替代问题. 首先,利用配置好的活力均为1400~1500 U/mL的猪源胰酶、牛源胰酶-1、牛源胰酶-2和全合成胰酶TrypLE试剂分别在37 ℃环境中消化细胞,采用荧光计数仪进行计数及活率检测. 然后,将得到的细胞悬液稀释至2×105 mL−1,接种至6孔板和96孔板中,在培养的不同时间绘制细胞生长曲线和检测细胞增殖及毒性. 在筛选到可替代消化酶之后,进行10次重复实验. 结果显示,Vero细胞消化实验中,猪源胰酶的消化能力相对其他胰酶较好,有显著差异,其中牛源胰酶-2消化细胞后对细胞增殖和毒性影响影响最大. KMB-17细胞消化实验中,4种消化酶的消化能力均无显著差异,消化时间存在显著差异(P<0.05),4种消化酶在48 h之后对KMB-17细胞增殖和毒性影响有明显差异. 猪源胰酶、牛源胰酶-1和TrypLE消化Vero细胞后变异系数(CV)分别为1.08%、1.12%、1.22%;猪源胰酶和牛源胰酶-2消化KMB-17细胞后CV分别为1.15%、1.04%. 为满足全球伊斯兰国家及人群对疫苗及生物制品使用的需求,可使用牛源胰酶-1和TrypLE替代猪源胰酶消化Vero细胞,消化KMB-17可使用牛源胰酶-2替代猪源胰酶.

     

    Abstract: Trypsin is an essential tool enzyme for adherent cell passaging. The effects of porcine trypsin, two kinds of bovine trypsin and fully synthetic trypsin TrypLE on the digestion of Vero cells and KMB17 cells were compared, and the optimal digestion process of cultured cells in vaccine production and the substitution of porcine trypsin in Halal verification in the process of providing biological products to Islamic countries and people were evaluated in this study. First, the cells were digested with porcine derived trypsin, bovine-derived trypsin-1, bovine-derived trypsin-2, and total synthetic trypsin TrypLE reagents with well configured viability units all ranging from 1400 to 1500 U/ml at 37 ℃, respectively, and the fluorescence counter was used for counting and activity detection. Then, the obtained cell suspension was diluted to 2×105 cells/mL and inoculated into 6-well plate and 96 well plate, and cell growth curves were drawn at different times of culture to detect cell proliferation and toxicity. After screening to alternative digestive enzymes, 10 replicate experiments were performed. The results showed that the digestive capacity of porcine derived trypsin was relatively good compared with other pancreatic enzymes in the Vero cell digestion experiment, in which bovine derived trypsin-2 had the greatest effect on cell proliferation and toxicity after digestion of the cells. In the KMB-17 cell digestion experiment, there was no significant difference in the digestive capacity of any of the four digestive enzymes with significant difference in the digestion time (P<0.05), and the effects of the four digestive enzymes on the proliferation and toxicity of KMB-17 cells were significantly different after 48 hours. The CV value of Vero cells digested by porcine trypsin, bovine trypsin-1 and TrypLE were 1.08%, 1.12% and 1.22%, respectively. The CV of KMB-17 cells digested by porcine trypsin and bovine trypsin-2 were 1.15% and 1.04%, respectively. To meet the demand for vaccine and biologic products used in Islamic countries and populations worldwide, bovine derived trypsin-1 and TrypLE can be used instead of porcine derived trypsin to digest Vero cells, and bovine trypsin-2 can be used to replace porcine trypsin to digest KMB-17.

     

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