Universitätsklinikum Würzburg, Würzburg, Germany.
Curr Protoc Immunol. 2020 Mar;128(1):e93. doi: 10.1002/cpim.93.
One promising approach to treat hematologic malignancies is the usage of patient-derived CAR T cells. There are continuous efforts to improve the function of these cells, to optimize their receptor, and to use them for the treatment of additional types of cancer and especially solid tumors. In this protocol, an easy and reliable approach for CAR T cell generation is described. T cells are first isolated from peripheral blood (here: leukoreduction system chambers) and afterwards activated for one day with anti-CD3/CD28 Dynabeads. The gene transfer is performed by lentiviral transduction and gene transfer rate can be verified by flowcytometric analysis. Six days after transduction, the stimulatory Dynabeads are removed. T cells are cultured in interleukin-2 conditioned medium for several days for expansion. There is an option to expand CAR T cells further by co-incubation with irradiated, antigen-expressing feeder cell lines. The CAR T cells are ready to use after 10 (without feeder cell expansion) to 24 days (with feeder cell expansion). © 2020 The Authors. Basic Protocol: Generation of CAR T cells by lentiviral transduction.
一种有前途的治疗血液系统恶性肿瘤的方法是使用患者来源的 CAR T 细胞。人们一直在努力提高这些细胞的功能，优化其受体，并将其用于治疗其他类型的癌症，特别是实体瘤。在本方案中，描述了一种简单可靠的 CAR T 细胞生成方法。首先从外周血（此处：白细胞减少系统室）中分离 T 细胞，然后用抗 CD3/CD28 Dynabeads 激活一天。通过慢病毒转导进行基因转移，并且可以通过流式细胞术分析验证基因转移率。转导后 6 天，去除有刺激作用的 Dynabeads。将 T 细胞在白细胞介素 2 条件培养基中培养数天以扩增。有一个选择是通过与辐照的、表达抗原的饲养细胞系共孵育进一步扩增 CAR T 细胞。CAR T 细胞在转导后 10 天（无饲养细胞扩增）至 24 天（有饲养细胞扩增）后即可使用。© 2020 作者。基本方案：通过慢病毒转导生成 CAR T 细胞。
