Tabriz University of Medical Sciences Advanced Pharmaceutical Bulletin 2228-5881 15 1 2025 04 15 An Optimized Method for Single Cell Cloning of Human CAR-T Cells Based on FBS-Coated Plates 133 142 10.34172/apb.43798 EN Mahdie Jafari https://orcid.org/0000-0002-0991-1818 Shahriyar Abdoli Masoud Moghaddam Pour Mohammad Ali Shokrgozar https://orcid.org/0000-0001-5123-4155 Zahra Sharifzadeh https://orcid.org/0000-0002-4419-0315 Journal Article 10.34172/apb.43798 2024 09 19 2024 12 21 Purpose: 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 fetal bovine serum (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 green fluorescent protein (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. Furthermore, this method improves T cell adherence, proliferation, and functional effectiveness, offering a potential foundation for advancing CAR-T cell therapies aimed at solid tumors. Future research should concentrate on optimizing culture conditions and testing this method in preclinical animal models to ensure its clinical applicability and efficacy.