A hybridized mechano-electroporation technique for efficient immune cell engineering

Immune cell engineering, which involves genetic modification of T cells, natural killer cells, and macrophages, is shifting the paradigm in immunotherapy for treating hematologic malignancies. These modified cells can be viewed as living drugs and offer advantages, including dynamic functionality, active local trafficking, and boosting the immune system while recognizing and eliminating malignant cells. Among the current technologies employed for the modification of immune cell functions, electroporation stands as a predominant approach, but it suffers from heterogeneity arising from the treatment of a bulk population of immune cells during the manufacturing procedures. To address this challenge of the field, here we present a hybrid approach to induce consecutive gentle mechanical and electric shocks. This approach enhances the treatment homogeneity and improves outcomes in difficult-to-load immune cells. The hybrid approach aims to enhance the treatment homogeneity by passing individual immune cells through a microengineered filter membrane with micropores smaller than the cell diameter. This facilitates the creation of transient pores in the cell membrane, followed by efficient delivery of biomolecules through the complementary use of a gentle electric shock. Using this hybrid mechano-electroporation (HMEP) system, we could successfully deliver fluorescein isothiocyanate (FITC) dextran molecules from the smallest (4 kDa) to the largest (2000 kDa) size and GFP expressing plasmid DNA into different immune cell types. We also provide insight into the delivery performance of the HMEP system in comparison with the benchtop electroporation since both methods hinge on membrane disruption as their permeabilization mechanism. Immune cells treated with the HMEP protocol demonstrated higher delivery efficiencies while maintaining cell viability compared to those experiencing conventional electroporation. Therefore, membrane-based mechanoporation can be a cost-effective and efficient approach to pre-treat the hard-to-deliver immune cells before electroporation, elevating the treatment homogeneity and delivery of exogenous cargoes to a higher level.