HL-60/IL-6体外热原检测方法学研究及对细胞治疗产品的适用性考查  

The study on HL-60/IL-6 in vitro pyrogen detection method and its applicability to cell therapy products

作　　者：王冲 徐琳 华晓东 Wang Chong;Xu Lin;Hua Xiaodong(Tianjin Institute for Drug Control,Tianjin 300070)

机构地区：[1]天津市药品检验研究院,天津300070

出　　处：《天津药学》2020年第2期6-10,共5页Tianjin Pharmacy

摘　　要：目的:建立用于体外热原检测的HL-60/IL-6法,并对细胞治疗产品进行适用性考查。方法:以细菌内毒素为工具药物,对可能影响HL-60/IL-6法试验结果的不同细胞代次、孵育时间、细胞数量、显色时间及血清含量5种因素进行考查,优化最佳试验条件。选用人源间充质干细胞(MSC)、树突细胞(DC)、T淋巴细胞、嵌合抗原T细胞(CAR-T)治疗产品进行方法适用性研究。应用CD3-APC抗体及凋亡试剂盒检测T及CAR-T细胞对HL-60细胞的凋亡作用,更为直观地验证其干扰作用。结果:经优化的HL-60/IL-6法的试验条件为:10~25代细胞、共同孵育24 h、细胞数量为2×10^5个/孔,血清含量为2%。采用本法对细胞治疗产品进行检测,干扰试验的回收率均在50%~200%之间,热原物质检测结果均为未检出或阴性,与细菌内毒素动态显色方法结果均一致。流式细胞检测仪结果表明,与T及CAR-T细胞共同孵育24 h,对HL-60细胞无凋亡作用,不会对试验系统产生干扰。结论:HL-60/IL-6法检测敏感性高,结果重复性好,可应用于细胞治疗产品热原物质的检查。Objective:To establish a HL-60/IL-6 method for in vitro pyrogen detection and to investigate the applicability for cell therapy products by the established method. Methods:By using bacterial endotoxin as a tool drug, five factors that might affect HL-60/IL-6 test results including cell generation, incubation time, cell number, chromogenic time and serum content, were evaluated to optimize the best test conditions. Human mesenchymal stem cells(MSC),dendritic cells(DC),T lymphocytes,and chimeric antigen receptor T-cells(CAR-T) were selected for method applicability research. CD3-APC antibody and apoptosis detection kit were used to detect the apoptosis induction effect of T cells and CAR-T cells on HL-60 cells.Results: The optimized HL-60/IL-6 test conditions were as follows:the cells generations were 10 to 25 passages;the incubated period was 24 h;the number of cells was 2 × 10^5/well,and the serum content was 2%. The results showed that the recovery of interference test was between 50% and 200%,and the results of pyrogen test were negative or undetected, which were consistent with the results of the bacterial endotoxin dynamic coloration method. Flow cytometry results showed that co-incubation with T cells and CAR-T cells for 24 h had no apoptotic effect on HL-60 cells and did not interfere with the test system. Conclusion:The HL-60/IL-6 method has high sensitivity and good repeatability,and can be applied to the detection of pyrogen in cell therapy products.

关 键 词：HL-60 IL-6 热原检测 细胞治疗产品 

分 类 号：R927.1[医药卫生—药学]