Scalable intracellular delivery via microfluidic vortex shedding enhances the function of chimeric antigen receptor T-cells.

Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies, as well being developed for treatment of solid tumors, autoimmune disorders, heart disease and aging. Despite unprecedented clinical outcomes, CAR-T and other engineered cell therapies face a variety of manufacturing and safety challenges. Traditional methods, like lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies, such as CAR-T. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7 to 2x higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis and increased pro-inflammatory and regulatory cytokine secretion in addition to improved CAR-T yield by day 5 post-transfection. We demonstrated scaled-up hydroporation can process 5 × 10 8 cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T cell manufacturing with the potential for improved therapeutic outcomes.

Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9.

Microfluidic vortex shedding (µVS) can rapidly deliver mRNA to T cells with high yield and minimal perturbation of the cell state. The mechanistic underpinning of µVS intracellular delivery remains undefined and µVS-Cas9 genome editing requires further studies. Herein, we evaluated a series of µVS devices containing splitter plates to attenuate vortex shedding and understand the contribution of computed force and frequency on efficiency and viability. We then selected a µVS design to knockout the expression of the endogenous T cell receptor in primary human T cells via delivery of Cas9 ribonucleoprotein (RNP) with and without brief exposure to an electric field (eµVS). µVS alone resulted in an equivalent yield of genome-edited T cells relative to electroporation with improved cell quality. A 1.8-fold increase in editing efficiency was demonstrated with eµVS with negligible impact on cell viability. Herein, we demonstrate efficient processing of 5 × 106 cells suspend in 100 µl of cGMP OptiMEM in under 5 s, with the capacity of a single device to process between 106 to 108 in 1 to 30 s. Cumulatively, these results demonstrate the rapid and robust utility of µVS and eµVS for genome editing human primary T cells with Cas9 RNPs.