Acoustophoretic rapid media exchange and continuous-flow electrotransfection of primary human T cells for applications in automated cellular therapy manufacturing

Abstract

Autologous cellular therapies based on modifying T cells to express chimeric antigen receptor genes have been highly successful in treating hematological cancers. Deployment of these therapies is limited by the complexity and costs associated with their manufacturing. Transitioning these processes from virus-based methods for gene delivery to a non-viral method, such as electroporation, has the potential to greatly reduce cost and manufacturing time while increasing safety and efficacy. Major challenges with electroporation are the negative impacts on cell health associated with exposure to high-magnitude electric fields, and that most commercial bulk electroporators are low-precision instruments designed for manually-operated, lower-throughput batch processing of cells. Negative effects on cell health can be mitigated by use of specialized electroporation medias, but this adds processing steps, and long-term exposure to these medias can reduce transfection efficiency and post-transfection viability. To enable automated, clinical-scale production of cellular therapies using electrotransfection in specialized medias, we developed a high-precision microfluidic platform that automatically and continuously transfers cells from culture media into electroporation media using acoustophoresis, and then immediately applies electric fields from integrated electrodes. This limits cell residence time in electroporation media to seconds, and enables high transfection efficiency with minimum impact on cell viability. We tested our system by transferring primary human T cells from a standard cell media to electroporation media, and then transfecting them with mRNA encoding an mCherry fluorescent protein. We achieved a media exchange efficiency of 86% and transfection efficiency of up to 60%, with less than a 5% reduction in viability.