基于AO/PI荧光染色原理的杀伤细胞数量及活率检测方法学验证和细胞培养、冻存、复苏稳定性研究  

Validation of analytical methods for CIK cells quantity and viability analysis based on AO/PI fluorescence staining principle and stability study of cell culture,cryopreservation and resuscitation

作　　者：储王龙 朱丽萍 张芬 钟振忠 柯晖 梁晓 曾秀萍 刘沐芸 CHU Wanglong;ZHU Liping;ZHANG Fen;ZHONG Zhenzhong;KE Hui;LIANG Xiao;ZENG Xiuping;LIU Muyun(Shenzhen Beike Biotechnology Co.,Ltd.,Shenzhen 518000,China;Shenzhen Kenuo Medical Laboratory,Shenzhen 518000,China;National Engineering Research Center of Foundational Technologies for CGT Industry,Shenzhen 518000,China)

机构地区：[1]深圳市北科生物科技有限公司,广东深圳518000 [2]深圳科诺医学检验实验室,广东深圳518000 [3]细胞产业关键共性技术国家工程研究中心,广东深圳518000

出　　处：《药物评价研究》2023年第2期377-383,共7页Drug Evaluation Research

基　　金：科诺医学细胞质量检测技术公共服务平台(S-2020-M74-500513)。

摘　　要：目的验证基于吖啶橙(AO)/碘化丙啶(PI)荧光染色原理的自动细胞分析仪检测细胞因子诱导的杀伤细胞(CIK)数量及活率的可行性,并对CIK细胞培养全过程及冻存复苏后至使用前过程进行检测,建立相应的数量及活率标准。方法采集健康供者的外周血分离、培养CIK细胞,取培养过程中的细胞使用AO/PI荧光染色法、应用自动细胞分析仪检测细胞数量及活率,对结果的专属性、准确度、精密度、数量线性及范围、活率线性及范围进行验证。应用建立的方法对外周血分离后的外周血单个核细胞(PBMC)、培养过程中、冻存前及复苏后的CIK细胞数量及活率进行全过程检测。结果专属性、准确度、精密度、数量线性及范围、活率线性及范围验证均符合预期要求。CIK细胞培养全过程活率均不低于80%,样本平均倍增时间为(42.26±1.17)h。冻存前的CIK细胞在加入含5%DMSO的冷冻保护剂后90 min内活率稳定,复苏后未经稀释或洗涤处理120 min内活率稳定,复苏后经洗涤去除DMSO处理后12 h内活率稳定。结论基于AO/PI荧光染色原理的自动细胞分析仪可以用于检测CIK细胞的数量及活率,其结果可以用于细胞放行评价及稳定性研究。Objective To verify the feasibility of the automatic cell analyzer based on the fluorescent staining principle of acridine orange(AO)/propyl iodide(PI)to detect the number and viability of cytokine induced killer cells(CIK),and to detect the whole process of CIK cell culture and the process from freezing resuscitation to pre-use,and establish the corresponding number and viability standards.Methods Peripheral blood samples collected from the healthy donors were used to induce CIK cells.The quantity and viability of the cells during culturing were evaluated using AO/PI fluorescence staining method,and the specificity,accuracy,precision,quantity linearity,viability linearity and range were validated.Furthermore,the quantity and viability of PBMC separated from peripheral blood,CIK cells during the cultivation,before freezing and after thawing were evaluated to provide a basis for the monitoring and release of CIK cells.Results The specificity,accuracy,precision,quantity linearity,viability linearity and range all met with the expected criterions.The viability of the entire process of the CIK cell culturing was greater than 80%,and the average population doubling time was(42.26±1.17)h.Before cryopreservation,the viability of CIK cells was stable within 90 min after adding cryoprotectant containing 5%DMSO.After thawing,the viability was stable within 120 min without dilution or washing treatment,and it was stable within 12 hours after washing and removing DMSO.Conclusion Automatic cell analyzer based on AO/PI fluorescence staining can be used to measure the number and viability of CIK cells,and the results can be used for cell release evaluation and stability study.

关 键 词：细胞因子诱导的杀伤细胞(CIK) AO/PI荧光 自动细胞分析仪 细胞数量 活率 验证 稳定性 

分 类 号：R965.2[医药卫生—药理学]