Highly efficient engineering of human immune cells and hematopoietic stem and progenitor cells using microfluidic transfection 2651

Abstract Description We developed a novel mechanoporation-based system that enables efficient intracellular cargo delivery with minimal cellular perturbations. Using this CellPore™ Transfection System, we optimized workflows to genetically engineer unactivated pan T cells, T cell subsets, NK cells, and hematopoietic stem and progenitor cells (HSPCs). We achieved high knockout efficiency of surface MHC-I (74%) or TCRαβ (94%) by delivering CRISPR/Cas9 ribonucleoproteins (RNP) to unactivated T cells using our workflow. T cells transfected by CellPore™ remained unactivated, while electroporation caused T cell activation and secretion of pro-inflammatory cytokines. Similarly, we optimized a workflow to deliver mRNA constructs and RNPs to freshly isolated NK cells. Transfection with RNPs resulted in significant knockout of TIGIT (85%) or NKp46 (87%) surface markers. Importantly, NK cells transfected with CellPore™ maintained their cytotoxic and expansion capacity. Using CellPore™ to deliver RNPs to CD34+ HSPCs, we achieved high knockout efficiencies of either MHC-I (92%) or CD45 (84%) with a shortened process timeline. The bulk HSPC and primitive hematopoietic stem cell populations were preserved, with minimal impact on their differentiation, expansion, and clonogenic potential. In summary, the CellPore™ Transfection System enables robust and efficient cargo delivery to mature immune cells and HSPCs without impacting cell function, in a simple workflow that can be easily integrated into research protocols. Funding Sources N/A Topic Categories Technological Innovations in Immunology (TECH)