Advancing Allogeneic NK Cell Immunotherapy through Microfluidic Gene Delivery

Abstract 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 6 cells min −1 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.