Advancing Allogeneic NK Cell Immunotherapy through Microfluidic Gene Delivery.

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized cancer treatment, yet challenges such as manufacturing complexity, high costs, and safety concerns have spurred the development of alternatives like CAR-natural killer (NK) cell immunotherapies. CAR-NK cell therapies provide innate cytotoxicity with antigen-independent targeting, reducing safety risks while improving therapeutic efficacy. However, efficient genomic engineering and large-scale production of allogeneic NK cells remain significant obstacles. To address these challenges, a novel microfluidic gene delivery platform is developed, the Y-hydroporator, designed for allogeneic NK cell immunotherapy. This platform features a Y-shaped microchannel where NK cells experience rapid hydrodynamic stretching near the stagnation point, creating transient membrane discontinuities that facilitate the uptake of exogenous cargo. The Y-hydroporator achieves high delivery and transfection efficiency, processing ≈2 × 10 cells min while maintaining long-term cell viability (>89%) and functionality. Using this platform, human primary CAR-NK cells and NKG2A-knockout NK cells are successfully generated by delivering anti-CD19 CAR mRNA and CRISPR/Cas9 ribonucleoproteins, respectively. These engineered NK cells demonstrated enhanced cytotoxicity, underscoring the potential of the Y-hydroporator as a transformative tool for advancing allogeneic NK cell-based immunotherapies.