A CAR/CD19 Jurkat cell model for bioprocess screening and expansion: potential use for CAR-T cells production.

Introduction: Cell expansion is one of the most critical steps in the development of CAR-T cell manufacturing processes because it directly influences cell yield and process performance. The systematic optimization of expansion parameters using primary human T cells is often limited by inter-donor variability, cost, and experimental complexity. So, it is necessary to study more homogeneous populations that allow to stablish standardized conditions in a controlled environment that were able to be used as standard. So, in this study, we developed a Jurkat CAR-CD19 cell-based platform as a reproducible model for optimizing the cell expansion bioprocess under controlled culture conditions. Methods: CAR-CD19-expressing Jurkat cells were pre-generated, molecularly characterized and subsequently used to evaluate key bioprocess variables, including glucose concentration, inoculum density, culture medium composition (RPMI + 10% FBS versus Nutri-T without serum), shaking speed, and different culture operating modes (batch, fed-batch, and semi-perfusion). Cell growth kinetics, metabolite consumption, metabolite production, and CAR expression were evaluated throughout the optimization process. Results: An intermediate glucose concentration supported cell expansion without additional benefit from higher glucose supplementation. Serum-free Nutri-T medium promoted higher viable cell densities than RPMI (used as control media) under the evaluated conditions. Among the operating modes evaluated, semi-perfusion achieved the highest viable cell density, maintaining high cell viability and a homogeneous CAR-positive cell population at the end of culture in Nutri-T media. Conclusion: These findings demonstrate that the Jurkat CAR-CD19 model provides a robust and reproducible experimental platform for optimizing cell expansion bioprocesses. While further validation in primary human CAR-T cells is required, this model allows for the systematic evaluation of culture variables, minimizing experimental variability and facilitating early-stage process development for adoptive cell therapies.