Isolation, Cultivation, and Transient Transfection of Primary Human T Cells to Generate Chimeric Antigen Receptor (CAR) T Cells.

To assess the efficacy and potency of chimeric antigen receptor (CAR) T cells, it is essential to isolate, activate, expand, characterize, and genetically engineer primary human T cells. Compared to common T cell lines, primary cells can provide a more realistic model system, since they are highly variable, capable of T cell-mediated killing, and able to differentiate. Here, we provide a step-by-step protocol for all essential steps for the cultivation of primary T cells, including their isolation from blood products, activation, in vitro expansion, and transient transfection. We describe differences in expansion kinetics and differentiation of primary T cells upon activation with two different reagents and provide an antibody panel for flow cytometric analysis of T cell phenotype and activation state. We offer detailed instructions for electroporation of human T cells with mRNA encoding a CAR and assessing CAR expression kinetics over time. This protocol provides recommendations on ideal timepoints for downstream potency and efficacy assays of CAR T cells and the influence of different amounts of mRNA on signal intensity. Given the transient nature of this method, it provides a rapid and easy-to-implement possibility to test and compare many CAR constructs in parallel.