Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9
| Autor: | Brandon J. Sytsma, Ryan S. Pawell, Adrian A. Lievano, Leah H. Wilson, Justin A. Jarrell, Giles T. S. Kirby, Katherine H.W.J. Lau, Fong L. Pan |
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| Přispěvatelé: | Jarrell, Justin A, Sytsma, Brandon J, Wilson, Leah H, Pan, Fong L, Lau, Katherine HWJ, Kirby, Giles TS, Lievano, Adrian A, Pawell, Ryan S |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Cell Survival
Transgene Science mRNA T-Lymphocytes Cell Microfluidics Gene Expression Transfection Article Genome editing Genes Reporter CRISPR-Associated Protein 9 medicine Humans Viability assay Transgenes Ribonucleoprotein Gene Editing Multidisciplinary Chemistry Cas9 Electroporation Gene Transfer Techniques Models Theoretical Cell biology medicine.anatomical_structure Hydrodynamics Medicine Immunotherapy T cell receptor CRISPR-Cas Systems Intracellular Microfluidic vortex shedding (µVS) |
| Zdroj: | Scientific Reports Scientific Reports, Vol 11, Iss 1, Pp 1-13 (2021) |
| ISSN: | 2045-2322 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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