An Optimized Method for Single Cell Cloning of Human CAR-T Cells based on FBS-coated Plates

Abstract

T cell-based immunotherapy, especially chimeric antigen receptor (CAR)-T cells, has emerged as an appropriate approach for treating hematologic malignancies and is currently under investigation in clinical trials for solid tumors. Despite significant improvements in CAR-T cell production processes, the isolation and expansion of CAR-engineered T cells continue to pose significant challenges. The aim of this research is to provide a simple and cost-effective method for the isolation and expansion of human CAR-T cells. This novel concept applies coated FBS culture plates and focuses on enhancing viability and functionality to improve the adherence of suspended T cells. Methods: This study evaluated a two-dimensional (2D) culture technique for isolating the CAR-T cells that target prostate-specific membrane antigen (PSMA) utilizing matrices pre-coated with 0.2% glutaraldehyde and FBS. Jurkat cells were transduced with a lentiviral vector encoding the anti-PSMA CAR construct. FBS-coated and commercialized Matrigel-coated matrices were used for single-cell isolation and clonal expansion. Functional tests were conducted to assess the activation and proliferation of CAR-T cells and the IFN-γ release assay subsequent to cloning and expansion. Results: Transfection efficiency markedly improved, with 88.4% of Lenti-X 293T cells demonstrating GFP expression. Among the Jurkat cells, 57.1% showed GFP expression post-transduction, of which 34.1% showed surface expression of anti-PSMA CAR. Clonal expansion on the FBS-coated matrix proved effective, yielding 92.1% GFP-positive isolated cells. Functional assays demonstrated that CAR-T cells co-cultured with LNCaP cells exhibited significantly enhanced proliferation, activation (as indicated by CD69 and CD25 expression), and cytokine release assay (IFN-γ) compared with those co-cultured with DU 145 and mock cells. Conclusion: This new approach is efficient, economical, and scalable for isolating specific homogenous T cells and promoting their clonal proliferation and expansion. Future research should concentrate on optimizing culture conditions and testing this method in preclinical animal models to ensure its clinical applicability and efficacy.

Keywords: Cancer immunotherapy, CAR-T cells, Clonal expansion, 2D culture system, FBS coating, Single cell isolation