摘要：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.
