CELL INTELLIGENCE · 肿瘤细胞治疗研究

Efficient and reproducible lentiviral vector production and T-cell transduction remain important technical challenges in CAR-T (Chimeric Antigen Receptor T-cell) cell manufacturing. In this study, we optimized HEK293T transfection and primary T-cell transduction parameters for lentiviral CAR constructs targeting BCMA (B-cell maturation antigen) and GPRC5D (G-protein coupled receptor family C group 5 member D). Lipofectamine 3000 and TurboFectin 8. 0 were compared across different seeding densities and reagent-to-DNA ratios, with vector yields quantified by qPCR (Quantitative Polymerase Chain Reaction) and p24 ELISA (Enzyme-linked Immunosorbent Assay). Lipofectamine 3000 consistently generated higher viral titers and transduction efficiencies, as reflected by a greater proportion of GFP-positive (Green Fluorescent Protein) cells than TurboFectin 8. 0, reaching peak titers of 9.

65 10 8 copies/mL for the anti-GPRC5D and 5. 33 10 8 copies/mL for the anti-BCMA vectors. Under optimized conditions, transduction efficiencies reached 43. 8% GFP + cells for BCMA-CAR and approximately 13-14% GFP-positive transduced cells for the GPRC5D construct within the tested TU/mL range. Co-transduction experiments yielded approximately 62-66% GFP + cells with detectable BCMA-binding and presumptive GPRC5D-CAR-expressing subpopulations identified based on GFP reporter expression.

Immunophenotypic analysis demonstrated a relatively stable CD4/CD8 distribution (~65/35), enrichment of effector memory CD8 + cells, and expression of activation-associated markers. Collectively, these findings describe an optimized lentiviral transfection and transduction workflow that may support the further development of dual-targeting BCMA/GPRC5D CAR-T manufacturing strategies in research and early translational settings.