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

Sci Rep. 2020 Oct 22;10(1):18045. doi: 10.1038/s41598-020-73755-0.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Cell- and Tissue-Based Therapy / methods*
  • Cells, Cultured
  • Electroporation / methods*
  • Gene Editing / methods*
  • Gene Transfer Techniques*
  • Humans
  • Microfluidics / methods*
  • RNA, Messenger / genetics*
  • T-Lymphocytes*
  • Transfection / methods*

Substances

  • RNA, Messenger