Scalable and ultrafast CAR-T cell production using microfluidics.

Chimeric antigen receptor T cell (CAR-T) therapy has recently gained recognition as a transformative treatment of cancer, particularly of hematological malignancies. However, CAR-T manufacturing remains a major bottleneck of this treatment modality; in standard cases, it takes up to two weeks, resulting in a phenotypic shift toward terminally differentiated T-cells and a significant depletion of T-cells with naive-like phenotype (Tnlp), crucial for sustained clinical efficacy. Leveraging the current progress in microfluidic technologies, we develop and optimize a microfluidic device (MFD) for CAR-T cell production via an ultrafast protocol that integrates T-cell activation and lentiviral transduction in a single step within 24 hours. The MFD geometry allowed reaching a transduction rate of 27% (for MOI 3) compared to 17% and 8% transduction (MOI 3) in 48- and 6-well plates, respectively, used as controls. Notably, in the ultrafast protocol in our MFD, the amount of CD3+ Tnlp is approximately six times higher than that remaining after the standard 9 day protocol (18.07 ± 6.03% vs. 3.97 ± 2.37%). A similar pattern is noted for CD4+ and CD8+ Tnlp, with percentages of 11.07 ± 6.08% vs. 3.56 ± 3.52% and 29.2 ± 7.11% vs. 4.18 ± 1.69%, respectively, in the final CAR-T product. Our results highlight MFDs as a scalable platform to streamline CAR-T manufacturing, with the potential to improve clinical accessibility and outcomes by reducing the production time while preserving essential T-cell phenotypes.