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.