Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9
Justin A. Jarrell, Brandon J. Sytsma, Leah H. Wilson, Fong L. Pan, Katherine H. W. J. Lau, Giles T. S. Kirby, Adrian A. Lievano, Ryan S. Pawell
Abstract
Abstract Microfluidic vortex shedding ( µVS ) can rapidly deliver mRNA to T cells with high yield and minimal perturbation of the cell state. The mechanistic underpinning of µVS intracellular delivery remains undefined and µVS -Cas9 genome editing requires further studies. Herein, we evaluated a series of µVS devices containing splitter plates to attenuate vortex shedding and understand the contribution of computed force and frequency on efficiency and viability. We then selected a µVS design to knockout the expression of the endogenous T cell receptor in primary human T cells via delivery of Cas9 ribonucleoprotein (RNP) with and without brief exposure to an electric field ( eµVS ). µVS alone resulted in an equivalent yield of genome-edited T cells relative to electroporation with improved cell quality. A 1.8-fold increase in editing efficiency was demonstrated with eµVS with negligible impact on cell viability. Herein, we demonstrate efficient processing of 5 × 10 6 cells suspend in 100 µl of cGMP OptiMEM in under 5 s, with the capacity of a single device to process between 10 6 to 10 8 in 1 to 30 s. Cumulatively, these results demonstrate the rapid and robust utility of µVS and eµVS for genome editing human primary T cells with Cas9 RNPs.