Acoustofluidic sonoporation for gene delivery to human hematopoietic stem and progenitor cells

Significance Commercial strategies to deliver biomolecular cargo ex vivo (e.g., electroporation, lipofection) to clinically relevant cell lines are limited by toxicity, cost, and throughput. These technical limitations have inhibited development of these technologies into streamlined clinical platforms for manufacturing gene-modified stem cells and cancer immunotherapies. Here, we demonstrate an acoustofluidic platform capable of delivering plasmids with high throughput to human T lymphocytes, peripheral blood mononuclear cells, and CD34+ hematopoietic stem and progenitor cells. Acoustofluidic-treated cells showed evidence of cytosolic DNA delivery, endocytic DNA aggregation, and nuclear membrane rupture. Collectively, these observations demonstrate the utility of this method as a research tool for gene editing applications and mechanistic studies of plasma membrane and nuclear membrane repair. Advances in gene editing are leading to new medical interventions where patients’ own cells are used for stem cell therapies and immunotherapies. One of the key limitations to translating these treatments to the clinic is the need for scalable technologies for engineering cells efficiently and safely. Toward this goal, microfluidic strategies to induce membrane pores and permeability have emerged as promising techniques to deliver biomolecular cargo into cells. As these technologies continue to mature, there is a need to achieve efficient, safe, nontoxic, fast, and economical processing of clinically relevant cell types. We demonstrate an acoustofluidic sonoporation method to deliver plasmids to immortalized and primary human cell types, based on pore formation and permeabilization of cell membranes with acoustic waves. This acoustofluidic-mediated approach achieves fast and efficient intracellular delivery of an enhanced green fluorescent protein-expressing plasmid to cells at a scalable throughput of 200,000 cells/min in a single channel. Analyses of intracellular delivery and nuclear membrane rupture revealed mechanisms underlying acoustofluidic delivery and successful gene expression. Our studies show that acoustofluidic technologies are promising platforms for gene delivery and a useful tool for investigating membrane repair.