甲基丙烯酰化明胶3D培养的CAR-T细胞靶向杀伤脑胶质瘤细胞的研究

目的研究在甲基丙烯酰化明胶中培养的CAR-T细胞功能及活性的变化，以及用GelMA为载体，负载缓释CAR-T细胞的可行性。方法基因编辑靶向表皮生长因子受体（EGFR）的第二代CAR-T细胞，制备负载CAR-T的甲基丙烯酸酯明胶，通过水凝胶成胶过程图像、储存及损耗模量验证水凝胶成功制备；使用荧光显微镜荧光成像、流式细胞分析验证CAR-T的成功转染；应用CCK-8实验检测水凝胶的生物相容性；构建共培养模型，利用 Elisa试剂盒、细胞毒性LDH试剂盒检测上清液中释放的细胞因子、LDH，检测CAR-T是否成功活化，评估 CAR-T细胞杀伤效应。结果荧光显微镜下观察转染第4天的CAR-T细胞，见CAR-T细胞聚集成团并散发绿色荧光；第9天流式细胞术示CD3+T细胞的慢病毒转染率为42.2％，CD8+T细胞：CD4+T细胞约为1∶1，CAR-T细胞成功制备。CCK8实验示水凝胶无毒性。Elisa试剂盒示上清液中细胞活性因子IFN-γ释放量显著增多，提示CAR-T细胞识别EGFR抗体后，可成功活化。当效靶比为1∶1时，U87细胞毒性为71.75%，而U87 EGFRvⅢ细胞毒性为18.13%。结论甲基丙烯酸酯化明胶负载培养的CAR-T细胞活性良好，功能正常，可设计使用该水凝胶负载递送CAR-T细胞，实现CAR-T细胞的精确靶向治疗。

Objective To study the changes of function and activity of CAR-T cells cultured in methacryloylated gelatin and to explore the feasibility of loading sustained-release CAR-T cells with GelMA as carrier. Methods CAR-T loaded methacrylate gelatin was prepared by gene editing targeting the second generation CAR-T cells of epidermal growth factor receptor（EGFR）. The successful preparation of hydrogel was verified by hydrogel formation process image, storage and loss modulus. The successful transfection of CAR-T was verified by fluorescence microscope fluorescence imaging and flow cytometry analysis. CCK-8 test was used to detect the biocompatibility of hydrogel. The co-culture model was constructed, and the cytokines and LDH released from the supernatant were detected by Elisa kit and cytotoxic LDH kit, to detect whether CAR-T was activated successfully and evaluate the killing effect of CAR-T cells. Results The CAR-T cells transfected on the 4th day were observed under the fluorescence microscope, and the CAR-T cells gathered into clusters and emitted green fluorescence. On the 9th day, flow cytometry showed that the lentivirus transfection rate of CD3+T cells was 42.2%. CD8+T cells: CD4+T cells were about 1∶1. CCK8 test showed that the hydrogel was non-toxic. Elisa kit showed a significant increase in the release of cytoactive factor IFN- γ in the supernatant, suggesting that CAR-T cells could be activated successfully after recognizing EGFR antibody.With a target ratio of 1∶1, U87 cytotoxicity was 71.75%, while U87 EGFRvIII cytotoxicity was 18.13%. Conclusions The CAR-T cells loaded with methacrylate gelatin have good activity and normal function. The hydrogel can be designed to deliver CAR-T cells to achieve accurate targeted therapy of CAR-T cells.