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High-throughput continuous-flow microfluidic electroporation of mRNA into primary human T cells for applications in cellular therapy manufacturing.


ABSTRACT: Implementation of gene editing technologies such as CRISPR/Cas9 in the manufacture of novel cell-based therapeutics has the potential to enable highly-targeted, stable, and persistent genome modifications without the use of viral vectors. Electroporation has emerged as a preferred method for delivering gene-editing machinery to target cells, but a major challenge remaining is that most commercial electroporation machines are built for research and process development rather than for large-scale, automated cellular therapy manufacturing. Here we present a microfluidic continuous-flow electrotransfection device designed for precise, consistent, and high-throughput genetic modification of target cells in cellular therapy manufacturing applications. We optimized our device for delivery of mRNA into primary human T cells and demonstrated up to 95% transfection efficiency with minimum impact on cell viability and expansion potential. We additionally demonstrated processing of samples comprising up to 500 million T cells at a rate of 20 million cells/min. We anticipate that our device will help to streamline the production of autologous therapies requiring on the order of 10[Formula: see text]-10[Formula: see text] cells, and that it is well-suited to scale for production of trillions of cells to support emerging allogeneic therapies.

SUBMITTER: Lissandrello CA 

PROVIDER: S-EPMC7582186 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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High-throughput continuous-flow microfluidic electroporation of mRNA into primary human T cells for applications in cellular therapy manufacturing.

Lissandrello Charles A CA   Santos Jose A JA   Hsi Peter P   Welch Michaela M   Mott Vienna L VL   Kim Ernest S ES   Chesin Jordan J   Haroutunian Nerses J NJ   Stoddard Aaron G AG   Czarnecki Andrew A   Coppeta Jonathan R JR   Freeman Daniel K DK   Flusberg Deborah A DA   Balestrini Jenna L JL   Tandon Vishal V  

Scientific reports 20201022 1


Implementation of gene editing technologies such as CRISPR/Cas9 in the manufacture of novel cell-based therapeutics has the potential to enable highly-targeted, stable, and persistent genome modifications without the use of viral vectors. Electroporation has emerged as a preferred method for delivering gene-editing machinery to target cells, but a major challenge remaining is that most commercial electroporation machines are built for research and process development rather than for large-scale,  ...[more]

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