Abstract
Lentiviral vector (LV) production for CAR-T therapy remains challenging due to the limited scalability of adherent HEK293 cell cultures. To address this, we adapted HEK293FT cells directly to serum-free FreeStyle 293 Expression Medium, generating a novel suspension cell line, HEK293FT-DS. We characterized growth kinetics, stability over 35 passages, and transient transfection parameters (PEI:DNA ratio, cell density). The suspension-adapted cells grew with >93% viability and a specific growth rate of 0.54 day−1, and maintained stable viable cell density (1.51 ± 0.03 × 106 cells/mL) over 35 passages. The optimal PEI:DNA ratio was 2.5:1, and increasing the transfection cell density to 16 × 106 cells/mL boosted functional titers to approximately 9 × 106 TU/mL in clarified supernatant. Scaled production (1 L culture supernatant per batch) yielded up to 5 × 109 TU. The resulting vectors efficiently transduced primary human T cells (57% CAR-positive cells) in a CliniMACS Prodigy-based process. The platform based on suspension-adapted HEK293FT-DS cells enables high-density transfection and provides a cost-effective, scalable alternative to commercial LV production systems, particularly suited for academic CAR-T cell manufacturing.