High-throughput continuous-flow microfluidic electroporation of mRNA into primary human T cells for applications in cellular therapy manufacturing
Autor: | Daniel K. Freeman, Vienna L. Mott, Nerses J. Haroutunian, Jenna L. Balestrini, Vishal Tandon, Ernest S. Kim, Andrew Czarnecki, Charles A Lissandrello, Aaron G Stoddard, Michaela Welch, Peter Hsi, Jonathan R. Coppeta, Jordan P. Chesin, Jose A. Santos, Deborah A Flusberg |
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Rok vydání: | 2020 |
Předmět: |
0301 basic medicine
Computer science T-Lymphocytes Microfluidics Cell T cells Cell- and Tissue-Based Therapy lcsh:Medicine Cancer immunotherapy Transfection Genome Article Viral vector Cell therapy 03 medical and health sciences Gene therapy 0302 clinical medicine Genome editing medicine Humans CRISPR Gene delivery RNA Messenger Viability assay lcsh:Science Cells Cultured Gene Editing Multidisciplinary Lab-on-a-chip Cas9 Electroporation lcsh:R Gene Transfer Techniques Cell biology 030104 developmental biology medicine.anatomical_structure 030220 oncology & carcinogenesis lcsh:Q CRISPR-Cas Systems Million Cells Biomedical engineering |
Zdroj: | Scientific Reports, Vol 10, Iss 1, Pp 1-16 (2020) Scientific Reports |
ISSN: | 2045-2322 |
DOI: | 10.1038/s41598-020-73755-0 |
Popis: | 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$$^8$$ 8 –10$$^9$$ 9 cells, and that it is well-suited to scale for production of trillions of cells to support emerging allogeneic therapies. |
Databáze: | OpenAIRE |
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