Intracellular delivery of mRNA to human primary T cells with microfluidic vortex shedding

Abstract

Intracellular delivery of functional macromolecules, such as DNA and RNA, across the cell membrane and into the cytosol, is a critical process in both biology and medicine. Herein, we develop and use microfluidic chips containing post arrays to induce microfluidic vortex shedding, or VS, for cell membrane poration that permits delivery of mRNA into primary human T lymphocytes. We demonstrate transfection with VS by delivery of a 996-nucleotide mRNA construct encoding enhanced green fluorescent protein (EGFP) and assessed transfection efficiencies by quantifying levels of EGFP protein expression. We achieved high transfection efficiency (63.6 {+/-} 3.44% EGFP+ viable cells) with high cell viability (77.3 {+/-} 0.58%) and recovery (88.7 {+/-} 3.21%) in CD3+ T cells 19 hrs after VS processing. Importantly, we show that processing cells via VS does not negatively affect cell growth rates or alter cell states. We also demonstrate processing speeds of greater than 2.0 x 106 cells s-1 at volumes ranging from 0.1 to 1.5 milliliters. Altogether, these results highlight the use of VS as a rapid and gentle delivery method with promising potential to engineer primary human cells for research and clinical applications.