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Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9.
- Source :
- Scientific Reports; 6/3/2021, Vol. 11 Issue 1, p1-13, 13p
- Publication Year :
- 2021
-
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 × 10<superscript>6</superscript> cells suspend in 100 µl of cGMP OptiMEM in under 5 s, with the capacity of a single device to process between 10<superscript>6</superscript> to 10<superscript>8</superscript> 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. [ABSTRACT FROM AUTHOR]
- Subjects :
- VORTEX shedding
T cells
GENOME editing
MESSENGER RNA
NUCLEOPROTEINS
Subjects
Details
- Language :
- English
- ISSN :
- 20452322
- Volume :
- 11
- Issue :
- 1
- Database :
- Complementary Index
- Journal :
- Scientific Reports
- Publication Type :
- Academic Journal
- Accession number :
- 150669683
- Full Text :
- https://doi.org/10.1038/s41598-021-91307-y