人二倍体细胞(2BS株)细胞库的建立及生物学特性鉴定
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摘要
目的建立人二倍体细胞(2BS株)主细胞库和工作细胞库。方法将人二倍体细胞(2BS株)扩大培养后冻存,建立主细胞库和工作细胞库,采用台盼蓝染色计数细胞,计算细胞活力,并进行染色体、外源病毒因子、微生物污染、致瘤性的检测及鉴别试验。将全面检定合格的工作细胞库细胞分别接种至T75一次性培养瓶、3 L玻璃转瓶和10层细胞工厂,观察细胞形态;将水痘病毒分别按0.010 5、0.012 8、0.018 8 MOI接种至T75一次性培养瓶、3 L玻璃转瓶和10层细胞工厂上的单层细胞,收获病毒原液,检测病毒滴度。结果主细胞库细胞活力为94.1%,工作细胞库细胞活力为94.4%;1 000个处于分裂相时期的细胞的核型分析结果均在《中国药典》三部(2010版)人二倍体细胞染色体分析标准的规定范围内;细胞上清液和裂解液中人外源病毒因子检测结果均为阴性,符合《中国药典》三部(2010版)规程要求;细胞均贴壁,成纤维细胞形态,种属鉴别为人细胞B型;细胞库未被细菌、真菌、病毒及支原体污染,也未出现致瘤性。建立的细胞库在3种培养器皿中均可在工艺要求时间内生长为单层,单位面积活细胞浓度在(2.01~2.59)×105个/cm2之间,收获的病毒滴度在5.0~5.25 lg PFU/ml之间,符合成都生物制品研究所有限责任公司《水痘减毒活疫苗制造及检定规程》要求。结论建立了人二倍体细胞(2BS株)主细胞库和工作细胞库,为2BS细胞应用于水痘减毒活疫苗的研发和生产提供细胞资源和理论依据。
Objective To establish the Master Cell Bank(MCB) and Working Cell Bank(WCB) of human diploid cell(2BS strain). Methods Human diploid cells(2BS strain)were cultured in a large scale and stored lyophilized to establish the master and working cell banks. The cells in established cell banks were counted by trypan blue staining, calculated for cell activity,and tested for chromosome,adventitious viral agents,identity,microorgan contamination and tumorigenicity. The cells from working cell bank qualified in overall control tests were inoculated to disposable T75 culture flask,3 L glass flask and 10-layer cell factory,then observed for morphology and counted,onto which varicella virus was inoculated at MOIs of0. 010 5,0. 012 8 and 0. 018 8. The bulk was harvested and determined for virus titer. Results The cell activities of MCB and WCB were 94. 1% and 94. 4% respectively. The result of karyotyping of 1 000 cells at mitotic phase was within the specified range of requirements for chromosome analysis of human diploid cells in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition). Both the determination results of human adventitious agents in supernatant and lysate of cells were negative,which met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition). All the cells were adherent and fibriform,which were human cells type B. The cell banks were free from contamination with bacteria,fungi,viruses or mycoplasma,and no tumorigenicity was observed. The cells from established cell banks formed monolayer on three kinds of culture dishes within the specified time. The viable cell concentration in unit area was(2. 01 ~ 2. 59)× 105 cells / cm2,while th e titer of harvested virus was 5. 0 ~ 5. 25 lg PFU / ml,which met the internal requirements for live attenuated varicella vaccine in Chengdu Institute of Biological Products Co.,Ltd. Conclusion The MCB and WCB of 2BS cells were established,which provided a cell source and a theoretical basis for application of 2BS cells to development and production of live attenuated varicella vaccine.
Objective To establish the Master Cell Bank(MCB) and Working Cell Bank(WCB) of human diploid cell(2BS strain). Methods Human diploid cells(2BS strain)were cultured in a large scale and stored lyophilized to establish the master and working cell banks. The cells in established cell banks were counted by trypan blue staining, calculated for cell activity,and tested for chromosome,adventitious viral agents,identity,microorgan contamination and tumorigenicity. The cells from working cell bank qualified in overall control tests were inoculated to disposable T75 culture flask,3 L glass flask and 10-layer cell factory,then observed for morphology and counted,onto which varicella virus was inoculated at MOIs of0. 010 5,0. 012 8 and 0. 018 8. The bulk was harvested and determined for virus titer. Results The cell activities of MCB and WCB were 94. 1% and 94. 4% respectively. The result of karyotyping of 1 000 cells at mitotic phase was within the specified range of requirements for chromosome analysis of human diploid cells in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition). Both the determination results of human adventitious agents in supernatant and lysate of cells were negative,which met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition). All the cells were adherent and fibriform,which were human cells type B. The cell banks were free from contamination with bacteria,fungi,viruses or mycoplasma,and no tumorigenicity was observed. The cells from established cell banks formed monolayer on three kinds of culture dishes within the specified time. The viable cell concentration in unit area was(2. 01 ~ 2. 59)× 105 cells / cm2,while th e titer of harvested virus was 5. 0 ~ 5. 25 lg PFU / ml,which met the internal requirements for live attenuated varicella vaccine in Chengdu Institute of Biological Products Co.,Ltd. Conclusion The MCB and WCB of 2BS cells were established,which provided a cell source and a theoretical basis for application of 2BS cells to development and production of live attenuated varicella vaccine.
引文
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[2]Oxman MN.Zoster vaccine:current status and future prospects[J].Clin Infect Dis,2010,51(2):197-213.
[3]Baxter R,Ray P,Tran TN,et al.Long-term effectiveness of varicella vaccine:a 14-year,prospective cohort study[J].Pediatrics,2013,131(5):e1389-e1396.
[4]Nordgren J,Bucardo F,Svensson L,et al.Novel light-uponextension real-time PCR assay for simultaneous detection,quantification,and genogrouping of group A rotavirus[J].J Clin Microbiol,2010,48(5):1859-1865.
[5]Liang HY,Zou Y.Development status and trend of freeze dried live attenuated varicella vaccine[J].Chin J N Drug,2012,21(10):1076-1079,1161.(in Chinese)梁慧颖,邹勇.冻干水痘减毒活疫苗发展现状及趋势[J].中国新药杂志,2012,21(10):1076-1079,1161.
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