Microfluidic-based rapid generation of chimeric antigen receptor T cells improves low-multiplicity-of-infection...

Conventional chimeric antigen receptor T-cell (CAR-T) manufacturing requires prolonged ex vivo processing and substantial viral input. We investigated whether brief T-cell activation combined with closed-loop microfluidic recirculation could improve low-multiplicity-of-infection (MOI) lentiviral transduction while generating functional CD19 CAR-T cells within a 24-h core process.

Primary human T cells were activated with CD3/CD28 beads for 4 h and transduced with CD19 CAR/green fluorescent protein reporter (CAR/GFP) lentivirus at MOI 0.5 or 1.0 in donor-matched microfluidic-chip and static-plate comparisons. Unless otherwise stated, microfluidic rapid-manufactured CAR-T (MF-rmCAR-T) products were generated at MOI 1.0 by 20 h closed-loop recirculation. Conventionally manufactured CAR-T (cmCAR-T) products served as the product-level comparator. Day 7 CAR/GFP positivity, bulk-product vector copy number (VCN), viability, expansion, and phenotype were assessed, followed by in vitro functional testing and exploratory evaluation in a systemic Raji-Luc xenograft model. Donor-matched data were analyzed using paired t-tests or two-way repeated-measures ANOVA with Šídák correction, as appropriate.

Microfluidic processing increased Day 7 CAR/GFP positivity versus matched static transduction at MOI 0.5 (14.6% ± 1.7% vs. 7.3% ± 1.2%; p < 0.001) and MOI 1.0 (22.1% ± 1.5% vs. 13.9% ± 1.7%; p < 0.001) without reducing viability. MF-rmCAR-T and cmCAR-T products showed comparable CAR/GFP positivity, viability, viable-cell recovery, and Day 7 expansion, whereas MF-rmCAR-T products had lower bulk-product VCN (1.2 ± 0.2 vs. 2.8 ± 0.4 copies/cell; p < 0.01), greater central-memory representation, and lower TIM-3 and LAG-3 expression. MF-rmCAR-T cells showed numerically higher bulk Raji-Luc killing and greater lysis after normalization to equivalent CAR/GFP-positive effector numbers, together with higher IFN-γ release and lower post-co-culture PD-1 expression. Both CAR-T products reduced tumor burden and prolonged survival versus untransduced T-cell controls, with no significant difference between the CAR-T groups.

Closed-loop microfluidic recirculation enabled a 24-h CD19 CAR-T manufacturing workflow with improved low-MOI transduction, lower bulk-product VCN, and preserved antitumor activity. These findings support evaluation in larger preclinical studies and scalable closed manufacturing systems.