Scientific Reports (Jun 2021)

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

DOI
https://doi.org/10.1038/s41598-021-91307-y
Journal volume & issue
Vol. 11, no. 1
pp. 1 – 13

Abstract

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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.