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  • 标题:Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9
  • 本地全文:下载
  • 作者:Justin A. Jarrell ; Brandon J. Sytsma ; Leah H. Wilson
  • 期刊名称:Scientific Reports
  • 电子版ISSN:2045-2322
  • 出版年度:2021
  • 卷号:11
  • DOI:10.1038/s41598-021-91307-y
  • 语种:English
  • 出版社:Springer Nature
  • 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.
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