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 μVS , for cell membrane poration that permits delivery of mRNA into primary human T lymphocytes. We demonstrate transfection with μVS 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 μVS processing. Importantly, we show that processing cells via μVS does not negatively affect cell growth rates or alter cell states. We also demonstrate processing speeds of greater than 2.0 × 10 6 cells s −1 at volumes ranging from 0.1 to 1.5 milliliters. Altogether, these results highlight the use of μVS as a rapid and gentle delivery method with promising potential to engineer primary human cells for research and clinical applications.

Deterministic lateral displacement (DLD) is a microfluidic size-based particle separation or filter technology with applications in cell separation and enrichment. Currently, there are no cost-effective manufacturing methods for this promising microfluidic technology. In this fabrication paper, however, we develop a simple, yet robust protocol for thermoplastic DLD devices using regulatory-approved materials and biocompatible methods. The final standalone device allowed for volumetric flow rates of 660 ll min À1 while reducing the manufacturing time to <1 h. Optical profilometry and image analysis were employed to assess manufacturing accuracy and precision; the average replicated post height was 0.48% less than the average post height on the master mold and the average replicated array pitch was 1.1% less than the original design with replicated posts heights of 62.1 6 5.1 lm (mean 6 6 standard deviations) and replicated array pitches of 35.6 6 0.31 lm.

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.

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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 μVS, for cell membrane poration that permits delivery of mRNA into primary human T lymphocytes. We demonstrate transfection with μVS 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 μVS processing.Importantly, we show that processing cells via μVS does not negatively affect cell growth rates or alter cell states. We also demonstrate processing speeds of greater than 2.0 x 10 6 cells s -1 at volumes ranging from 0.1 to 1.5 milliliters.Altogether, these results highlight the use of μVS as a rapid and gentle delivery method with promising potential to engineer primary human cells for research and clinical applications.