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 × 106 cells suspend in 100 µl of cGMP OptiMEM in under 5 s, with the capacity of a single device to process between 106 to 108 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.
Highlights
Microfluidic vortex shedding can rapidly deliver mRNA to T cells with high yield and minimal perturbation of the cell state
Using our computational fluid dynamics (CFD) model, we demonstrated that reductions in splitter plate lengths correlated with hydrodynamic conditions to enhance vortex shedding
Our investigation of microfluidic vortex shedding via simulations and experimentation provides a better understanding of the fluid dynamics contributing to μVS as a method for intracellular delivery
Summary
Microfluidic vortex shedding (μVS) can rapidly deliver mRNA to T cells with high yield and minimal perturbation of the cell state. The development of μVS as an effective platform for cellular modification requires an improved understanding of vortex shedding to apply μVS to the intracellular delivery of additional constructs, cell types and indications To this end, we developed a series of three-dimensional, transient, single-phase computational fluid dynamics models of μVS. Using our computational fluid dynamics (CFD) model, we demonstrated that reductions in splitter plate lengths correlated with hydrodynamic conditions to enhance vortex shedding These effects were subsequently verified through the fabrication of splitter plate devices and delivery of eGFP mRNA to activated primary human T cells via μVS (see “Methods”). This demonstrates μVS and eμVS are rapid and robust alternatives to electroporation for genome editing activated human primary T cells
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