CAR-T细胞制备过程中磁珠法激活T细胞条件的优化  

Optimized Primary T Cells Activation for CAR-T Cells Preparation

作　　者：张章 邱顺芳 王潇霖 丁乾 王晶 于长明 徐俊杰 张晓鹏 ZHANG Zhang;QIU Shun-Fang;WANG Xiao-Lin;DING Qian;WANG Jing;YU Chang-Ming;XU Jun-Jie;ZHANG Xiao-Peng(Beijing Institute of Biotechnology,Beijing 100071;Institute of Health Sciences,Anhui University,Hefei 230000,China)

机构地区：[1]军事科学院军事医学研究院生物工程研究所,北京100071 [2]安徽大学健康科学学院,安徽合肥230000

出　　处：《生物技术通讯》2018年第6期789-795,共7页Letters in Biotechnology

基　　金：国家自然科学基金青年科学基金(81301965)

摘　　要：目的:研究CAR-T细胞制备过程中,磁珠法体外激活人类原代T细胞的最佳条件。方法:新鲜分离的人外周血单个核细胞(PBMCs)与anti-CD3/CD28抗体包被的Dynabeads磁珠按3种不同的比例,于22℃混合孵育30 min,以激活T细胞。随后,通过细胞自动计数,检测不同激活条件对细胞活力和直径的影响;利用流式细胞术检测CD3^+T细胞所占比例;连续监测并绘制T细胞生长曲线,分析激活条件对T细胞增殖速度的影响。结果:以3种激活条件处理的细胞,在激活早期,细胞活力随着磁珠数的增加而降低;流式细胞术检测结果表明,随着磁珠数的降低,CD3^+T细胞所占比例呈上升趋势。然而,只有当PBMCs数与磁珠数比例为1∶1时,T细胞的生长速度最快,在第15 d时可扩增50倍。结论:T细胞激活条件对细胞活力及增殖有重要影响,PBMCs数与磁珠数比例为1∶1有利于T细胞快速扩增,这对在较短时间内获得足够的T细胞,缩短整个CAR-T细胞制备周期具有较重要的意义。Objective:To investigate the effect of Dynabeads CD3/CD28CTS activation on human primary T cells for CAR-T cells preparation.Methods:Freshly isolated human peripheral blood mononuclear cells(PBMCs)were incubated with Dynabeads at different ratios(PBMCs∶beads=3∶1,1∶1or1∶3)for30min at22℃.Then the viability and mean diameter of cells were detected using Cellometer Auto2000,and the proportion of CD3^+T cells was examined by flow cytometry.Furthermore,the long-term growth of T cells was monitored to analyze the effects of different activation protocols.Results:The data showed that,at very early stage,both cells viability and CD3+T cells proportion were decreased with the increasing ratio of Dynabeads.However,T cells activated with equal number of Dynabeads exhibited the best proliferation rate,which expanded50-fold in15days.Conclusion:The activation protocol has significant influence on T cells viability and growth.T cells have higher proliferation rate when stimulating by equivalent Dynabeads CD3/CD28CTS,which is beneficial to yield more CAR-T cells or shorten the preparation procedure.

关 键 词：CAR-T细胞 CD3 CD28 细胞激活 细胞增殖 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]