Non-viral, high throughput genetic engineering of primary immune cells using nanostraw-mediated transfection

Transfection of proteins, mRNA, and chimeric antigen receptor (CAR) transgenes into immune cells remains a critical bottleneck in cell manufacturing. Current methods, such as viruses and bulk electroporation, are hampered by low transfection efficiency, unintended transgene integration, and significant cell perturbation. The Nanostraw Electro-actuated Transfection (NExT) technology offers a solution by using high aspect-ratio nanostraws and localized electric fields to precisely deliver biomolecules into cells with minimal disruption. We demonstrate that NExT can deliver proteins, polysaccharides, and mRNA into primary human CD8⁺ and CD4⁺ T cells, and achieve CRISPR/Cas9 gene knockout of CXCR4 and TRAC in CD8⁺ T cells. We showcase NExT's versatility across a range of primary human immune cells, including CD4⁺ T cells, γδ-T cells, dendritic cells, NK cells, T_reg cells, macrophages, and neutrophils. Finally, we developed a scalable, high-throughput multiwell NExT system capable of transfecting over 14 million cells and delivering diverse cargoes into multiple cell types from various donors simultaneously. This technology holds promise for streamlining high-throughput screening of allogeneic donors and reducing optimization costs for large-scale CAR-immune cell transfection.