Fig. 1

The methods applied in the process of chimeric antigen receptor T cells culture influence cell differentiation and exhaustion state of the final product. In vitro culture process of CAR T cells mainly includes T cell collection, purification, activation, gene transduction and cell expansion. Enrichment of T cells by magnetic adsorption of CD3/CD28 antibody coated beads is commonly used but the purity cannot be guaranteed. Different methods for T cell activation leading to different activation strength or activity of pathways, and the viral or non-viral gene editing resulting in random or site-specific gene integration, ultimately influence the state of cell differentiation and exhaustion. Reducing the duration of ex vivo culture yields less differentiated CAR T cells, and addition of cytokines such as IL-7, IL-15 and IL-21 increases the frequencies of memory stem CAR T cells