Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9

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...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	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
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/600c810d0b0d45d6a04aaa482fec0696
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:600c810d0b0d45d6a04aaa482fec0696
record_format	dspace
spelling	oai:doaj.org-article:600c810d0b0d45d6a04aaa482fec06962021-12-02T18:25:04ZNumerical optimization of microfluidic vortex shedding for genome editing T cells with Cas910.1038/s41598-021-91307-y2045-2322https://doaj.org/article/600c810d0b0d45d6a04aaa482fec06962021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91307-yhttps://doaj.org/toc/2045-2322Abstract 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.Justin A. JarrellBrandon J. SytsmaLeah H. WilsonFong L. PanKatherine H. W. J. LauGiles T. S. KirbyAdrian A. LievanoRyan S. PawellNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q 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 Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9
description	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.
format	article
author	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
author_facet	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
author_sort	Justin A. Jarrell
title	Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9
title_short	Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9
title_full	Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9
title_fullStr	Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9
title_full_unstemmed	Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9
title_sort	numerical optimization of microfluidic vortex shedding for genome editing t cells with cas9
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/600c810d0b0d45d6a04aaa482fec0696
work_keys_str_mv	AT justinajarrell numericaloptimizationofmicrofluidicvortexsheddingforgenomeeditingtcellswithcas9 AT brandonjsytsma numericaloptimizationofmicrofluidicvortexsheddingforgenomeeditingtcellswithcas9 AT leahhwilson numericaloptimizationofmicrofluidicvortexsheddingforgenomeeditingtcellswithcas9 AT fonglpan numericaloptimizationofmicrofluidicvortexsheddingforgenomeeditingtcellswithcas9 AT katherinehwjlau numericaloptimizationofmicrofluidicvortexsheddingforgenomeeditingtcellswithcas9 AT gilestskirby numericaloptimizationofmicrofluidicvortexsheddingforgenomeeditingtcellswithcas9 AT adrianalievano numericaloptimizationofmicrofluidicvortexsheddingforgenomeeditingtcellswithcas9 AT ryanspawell numericaloptimizationofmicrofluidicvortexsheddingforgenomeeditingtcellswithcas9
_version_	1718378041754779648

Numerical optimization of microfluidic vortex shedding for genome editing T cells with Cas9

Ejemplares similares