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

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 deliver...

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Bibliographic Details
Published in:	Scientific reports Vol. 10; no. 1; p. 18045
Main Authors:	Lissandrello, Charles A., Santos, Jose A., Hsi, Peter, Welch, Michaela, Mott, Vienna L., Kim, Ernest S., Chesin, Jordan, Haroutunian, Nerses J., Stoddard, Aaron G., Czarnecki, Andrew, Coppeta, Jonathan R., Freeman, Daniel K., Flusberg, Deborah A., Balestrini, Jenna L., Tandon, Vishal
Format:	Journal Article
Language:	English
Published:	London Nature Publishing Group UK 22-10-2020 Nature Publishing Group
Subjects:	631/1647/2300 631/1647/2300/1851 631/1647/277 631/250/1619/554 631/61/201 631/67/1059/2325 639/166/985 Cell therapy Cell viability Cell- and Tissue-Based Therapy - methods Cells, Cultured CRISPR CRISPR-Cas Systems Electroporation Electroporation - methods Gene Editing - methods Gene Transfer Techniques Genome editing Genomes Humanities and Social Sciences Humans Lymphocytes Lymphocytes T Manufacturing Microfluidics Microfluidics - methods mRNA multidisciplinary RNA, Messenger - genetics Science Science (multidisciplinary) T-Lymphocytes Transfection Transfection - methods
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Summary:	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 8 –10 9 cells, and that it is well-suited to scale for production of trillions of cells to support emerging allogeneic therapies.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	2045-2322 2045-2322
DOI:	10.1038/s41598-020-73755-0