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

Chimeric antigen receptor (CAR) -T cell therapy became a revolutionary treatment for hematologic malignancies, but faced numerous challenges in solid tumors. The isolation and expansion of single T cells is a crucial step in the development of T cell-based therapies including CAR-T cells. Cell yield and quality, particularly viability and purity, necessitate cost-effective and scalable culture systems. Materials and Methods: This study tests the effectiveness of a two-dimensional (۲D) culture system for single cell isolation of CAR-T cells targeting prostate-specific membrane antigen (PSMA), where the matrices are pre-coated with ۰.۲% glutaraldehyde and then coated with fetal bovine serum (FBS). Jurkat cells were transduced with a lentiviral vector encoding the anti-PSMA CAR construct. Single-cell isolation and clonal expansion were done on FBS-coated and Matrigel-coated (a commercialized matrix) matrices. Activation and proliferation experiments were conducted on the CAR-T cells to confirm their functionality after single-cell cloning and expansion processes. Results: High transfection efficiency was achieved with ۸۸.۴% of Lenti-X ۲۹۳T cells expressing GFP. ۵۵.۸% of Jurkat cells showed GFP expression post-transduction, of which ۳۴.۱% showed surface expression of anti-PSMA CAR. Clonal expansion of CAR T cells onto the FBS-coated matrix was efficient, with ۹۲.۱% of isolated cells being GFP-positive. Functional assays showed that the CAR T cells co-cultured with LNCaP cells had significantly higher proliferation and activation (CD۶۹ expression) compared to those co-cultured with Du۱۴۵ cells. Conclusion: The optimized ۲D culture system, using FBS-coated matrix, provides a simple, cost-effective, and scalable technique for isolating single homogenic and clonal expansion T-cells. This approach improved the cell adhesion, proliferation, and functional efficacy of CAR-T cells and provided a promising strategy for the further advancement of CAR-T cell therapies, in particular for solid tumors. Future studies should concentrate on developing better culture conditions and on the validation of this system in preclinical models to assure clinical applicability and efficacy.

کلیدواژه ها:

CAR-T cells ، FBS coating ، Single cell isolation ، Clonal expansion ، Cancer immunotherapy