乙型脑炎减毒活疫苗病毒滴定用BHK21细胞密度和代次的确认  

Verification of BHK21 cell densities and passages used in virus titration for Japanese encephalitis vaccine

作　　者：易维维 彭维[1] 刘晓华[1] 江虹霞 廖海棠[1] 马玉晓[1] 吕钢 杨文杰[1] 刘菊[1] 卓金蓉[1] Yi Weiwei;Peng Wei;Liu Xiaohua;Jiang Hongxia;Liao Haitang;Ma Yuxiao;Lyu Gang;Yang Wenjie;Liu Ju;Zhuo Jinrong(Quality Control Department,Chengdu Institute of Biological Products Co.,Ltd.,Chengdu 610023 ,China)

机构地区：[1]成都生物制品研究所有限责任公司质量检定室,610023

出　　处：《国际生物制品学杂志》2018年第6期270-274,共5页International Journal of Biologicals

摘　　要：目的确定乙型脑炎(乙脑)减毒活疫苗病毒滴定使用的BHK21细胞的密度和代次范围。方法抽取20批不同代次BHK21细胞在6孔细胞板内培养成单层,进行细胞计数,计算细胞密度,滴定同批次乙脑病毒滴度国家参考品。将BHK21细胞从P95传代至P105,观察细胞形态及生长情况,与已用于检定的BHK21细胞(<P95)对比。用P95—P105 BHK21细胞重复6次滴定同批参考品,对滴度结果进行双因素方差分析,并与<P95 BHK21细胞的病毒滴度检测结果进行趋势分析比较。结果20批6孔板单层BHK21细胞密度均值1.55×10^6个/ml,差异较大(95%置信区间:7.89×10^4~3.02×10^6个/ml)。病毒滴度结果均在规定范围内(6.48~7.12 lg蚀斑形成单位/ml,相对标准偏差<5%),单层细胞密度对滴度结果无显著影响。P95—P105 BHK21细胞的生长情况及细胞形态与<P95基本一致,病毒滴定结果均在规定范围内。不同试验次数(F=0.13,P=0.721 4)、不同细胞代次(F=1.17,P=0.337 6)及两者的交互作用(F=0.30,P=0.978 0)对检测结果的影响无统计学意义,试验结果与较低代次细胞(<P95)检测的结果趋势一致,均整体稳定。结论BHK21细胞可连续传代至P105用于乙脑减毒活疫苗病毒滴定,且单层BHK21细胞密度对滴度检测结果无显著影响。Objective To verify the BHK21 cell densities and passages used in virus titer test for Japanese encephalitis vaccine (JEV).Methods Twenty batches of BHK21 cells at different passages were cultured in 6-well cell culture plates till forming monolayer. Cells were counted to calculate density before used to measure same national titration reference. BHK21 cells were continuously passed from P95 to P105, and cell morphology and growth situation were observed to compare with early passages (<P95). The same national titration reference was titrated 6 times with P95-P105 and <P95 BHK21 cells. Titration results were compared using two-factor variance analysis and trend analysis.Results The average density of 20 batch BHK21 cell was 1.55×10^6 cell/ml, with great batch-to-batch variance (95% confidence interval: 7.89×10^4-3.02×10^6 cell/ml). Titration results were within specified limits (6.48-7.12 lg plaque-forming unit/ml, relative standard deviation <5%), and the density of monolayer cells had no significant effect on titration results. Cell morphology and growth situation of P95-P105 and <P95 BHK21 cells were similar, and the titration results were both within specified limits. There was no statistical significance in effects on test results coming from different numbers of test (F=0.13, P=0.721 4), different passages (F=1.17, P=0.337 6) and interaction between the two factors (F=0.30, P=0.978 0). The titration trends were consistent between P95-P105 and <P95 BHK21 cells, and overall stable.Conclusion BHK21 cells can be used for JEV virus titration till passage P105, and the density of monolayer cells has no significant effect on titration results.

关 键 词：日本脑炎疫苗 BHK21细胞 细胞密度 细胞代次 病毒滴定 

分 类 号：R392[医药卫生—免疫学]