嵌合抗原受体T(CAR-T)细胞体外培养体系及慢病毒侵染条件的优化

Optimization of CAR-T cell culture system and lentivirus transduction conditions

目的优化嵌合抗原受体T(CAR-T)细胞的体外培养体系及慢病毒侵染条件。方法CD3磁珠分离纯化健康人外周血单个核细胞(PBMC)及健康孕妇脐带血单个核细胞(UCBMC),于8种不同培养体系进行扩增培养,培养体系包括以下成分的不同组合:重组人白细胞介素2(rhIL-2)、rhIL-12、rhIL-18、rhIL-7、rhIL-21、TWS119。分别于第0、3、5、7、10、18天进行细胞计数检测细胞增殖情况,流式细胞术检测细胞程序性死亡蛋白1(PD-1)的表达,ELISA检测γ干扰素(IFN-γ)释放,优化T细胞体外培养体系。采用(0、20、50)μg/mL重组人纤连蛋白(RetroNectin^■),(250、500、1000)ng/mL抗人CD3/CD28抗体包被培养板,以感染复数(MOI)=3、5的阴性对照慢病毒侵染T细胞72 h,于荧光显微镜下观察绿色荧光蛋白(GFP)表达情况,初步判断病毒侵染效率;流式细胞术检测CD3/GFP阳性率,以获得慢病毒侵染条件。包装CD19 CAR慢病毒,实时荧光定量PCR、Western blot法检测是否成功构建CD19 CAR慢病毒载体,采用以上优化的病毒侵染条件及T细胞培养体系,包括建立rhIL-2、rhIL-12联合rhIL-18培养体系,使用1μg/mL抗人CD3/CD28,20μg/mLRetroNectin^■包被培养板,制备CD19 CAR-T细胞。结果细胞增殖能力较佳体系为rhIL-2联合rhIL-18,IFN-γ释放最强体系为rhIL-2、rhIL-12联合rhIL-18。抗CD3/CD28抗体剂量为1μg/mL,RetroNectin^■20μg/mL,MOI为3时病毒侵染效率最优。该优化条件下CAR-T细胞阳性率达34%。结论获得优化的CD19 CAR-T细胞体外培养体系及慢病毒侵染原代T细胞条件。

Objective To optimize the culture system of chimeric antigen receptor T(CAR-T)cells in vitro and lentivirus infection conditions.Methods Peripheral blood mononuclear cells(PBMCs)of healthy people and umbilical cord blood mononuclear cells(UCBMCs)of healthy pregnant women were isolated and purified by CD3 magnetic beads,and then they were cultured in different cell culture systems.There were eight cell culture systems containg different combinations of the following components:recombinant human interleukin 2(rhIL-2),rhIL-12,rhIL-18,rhIL-7,rhIL-21,TWS119.Cell proliferation was detected by counting the cells at 0,3,5,7,10,18 days after the cells were seeded into cell plates.Flow cytometry was used to detect the expression of programmed death 1(PD-1),and ELISA was used to detect the expression of interferon-γ(IFN-γ).Cell culturing plates were coated with serial concentrations of recombinant human fibronectin fragment(RetroNectin^■)(0,20,50μg/mL),and antibodies against human CD3/CD28(250,500,1000 ng/mL).Then T cells cultured in the above plates were infected with negative control lentivirus at different multiplicity of infection(MOI=3,5);72 hours later,expression of green fluorescent protein(GFP)was observed under a fluorescence microscope to preliminarily determine virus infection efficiency.Flow cytometry was used to detect CD3/GFP positive rate to obtain lentivirus infection conditions.CD19 CAR lentivirus was packaged.Real-time quantitative PCR and Western blotting were performed to detect whether the CD19 CAR vector was successfully constructed.Finally,T cells were cultured in 1μg/mL anti-human CD3/CD28 and 20μg/mL RetroNectin^■-coated culture plates,and rhIL-2,rhIL-12,rhIL-18 were added in the culture medium,then the cells were infected with CD19 CAR lentivirus at the optimized virus infection conditions.Results The cell culture system with the best proliferation ability was rhIL-2 combining with rhIL-18;the cell culture system with the strongest release of IFN-γwas rhIL-2 and rhIL-12 combined with rhIL-18.When the dose of antibodies against CD3/CD28 was 1μg/mL,RetroNectin^■ was 20μg/mL,and MOI was 3,the virus infection efficiency was optimal.The positive rate of CAR-T cells was 34%under the optimal condition.Conclusion The study achieved the optimal cell culture system of CD19 CAR-T cells in vitro and the conditions of lentivirus infection on primary T cells.