MDCK细胞库的建立、检定及其生物学特性

Establishment,control and biological characteristics of MDCK cell bank

目的建立MDCK细胞的主细胞库及工作细胞库,并进行检定,同时评价其生物学特性。方法从欧洲细胞保藏中心(European Collection of Authenticated Cell Culture,ECACC)引进1株MDCK细胞,经传代扩大培养后,分别建立主库及工作细胞库。按照《中国药典》三部(2015版)规定的方法对细胞库进行细胞活力、细胞形态、无菌、支原体及细胞内外源病毒因子检查,并对细胞生长状况、传代稳定性、流感病毒敏感性进行检测。结果建立的MDCK细胞主种子库及工作种子库细胞形态呈不规则的上皮样细胞状,生长状态良好,细胞活率分别为96.0%和95.5%,无菌、支原体及细胞内外源病毒因子检查结果均合格。MDCK细胞生长曲线均呈S型,培养48~96 h进入对数生长期,随后进入平台期。连续传10代48 h细胞活率在93.0%~95.5%之间,较稳定。MDCK细胞对流感病毒较敏感,对B型流感病毒的敏感性高于A型流感病毒。结论建立的主细胞库及工作细胞库各项检测结果均符合《中国药典》规定,具有良好的传代稳定性及流感病毒敏感性。

Objective To establish and control the master and working cell banks of MDCK cells,and evaluate their biological characteristics. Methods A MDCK line was introduced from the European Collection of Cell Cultures(ECACC)and subcultured for expansion,based on which the master and working cell banks were established. The cells from the cell banks were tested for viability,morphology,sterility,mycoplasma,exogenous viral factor according to the Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Meanwhile,the growth,stability during subculture and sensitivity to influenza virus of the cells were evaluated. Results The cells from the established master and working cell banks were irregular epithelial-like cells in good growth state,of which the viabilities were 96. 0% and 95. 5% respectively. The cells were qualified in test for sterility,mycoplasma and exogenous viral factor,of which the growth curves were both in S shapes. The cells entered to the logarithmic growth phase after culture for 48 ~ 96 h then entered to the platform phase.The survival rate of cells after subculture for 10 passages was stable as 93. 0% ~ 95. 5%. MDCK cells were relatively sensitive to influenza virus,of which the sensitivity to influenza type B was higher than that to type A. Conclusion All the test results of the established master and working cell banks met the requirements in Chinese Pharmacopoeia. The cell banks showed good stability during subculture and sensitivity to influenza virus.