A new benchtop system for simple and versatile introduction of macromolecules into human lymphocytes and CD34₊ cells by microfluidic squeezing

Abstract Intracellular delivery of molecules is a key step in biological research that enables the development of new methods of cell and gene-based therapies. Traditional methods for intracellular delivery are limited in a) delivering a wide variety of cargos, b) efficient delivery to a broad range of cell types, and c) preventing unwanted genetic dysregulation. To address these challenges, we have developed the CellPore™ system, a benchtop device for gentle, yet efficient delivery of cargos to a range of primary cell types. The CellPore™ system is a microfluidic platform that creates transient disruptions in the plasma membrane via mechanical deformation by applying pressure, thereby allowing cargo entry to the cytosol. We developed a simple and straightforward workflow to determine optimal delivery conditions. This workflow consists of cell isolation, cargo preparation for delivery across a range of system pressures, followed by viability and delivery efficiency measurements. Optimized parameters were subsequently applied to deliver eGFP and mCherry mRNA as well as B2M-targeting Cas9 ribonucleoproteins to human unactivated T and CD34+ cells. Importantly, edited cells were functionally active after manipulation by the CellPore™ system, and subsequent studies in T cells revealed minimal transcriptional perturbations compared to electroporated samples. These results demonstrate the importance of having a comprehensive understanding of the impact a delivery modality can have in a biological system. The CellPore™ system enables efficient delivery of cargos to target cells, while safeguarding key functional attributes. This simple workflow allows the CellPore™ system to be easily integrated into cell therapy research protocols.