Cell engineering with microfluidic squeezing preserves functionality of primary immune cells in vivo

Abstract

SignificanceEx vivo manipulation of primary cells is critical to the success of this emerging generation of cell-based therapies, such as chimeric antigen receptor T cells for the treatment of cancer and CRISPR for the correction of developmental diseases. However, the limitations of existing delivery approaches may dramatically restrict the impact of genetic engineering to study and treat disease. In this paper, we compared electroporation to a microfluidic membrane deformation technique termed “squeezing” and found that squeezed cells had dramatically fewer side effects than electroporation and gene expression profiles similar to those of unmanipulated cells. The significant differences in outcomes from the two techniques underscores the importance of understanding the impact of intracellular delivery methods on cell function for research and clinical applications.

Intracellular delivery of mRNA to human primary T cells with microfluidic vortex shedding

“…Electroporation and viral-based transduction of human T cells has been shown to induce significant and dramatic changes in gene expression profiles, including genes related to T cell activation and survival 11,13,39 . Therefore, we evaluated if processing cells via μVS would alter the expression levels of canonical T cell activation markers compared to non-processed cells.…”

“…These markers included CCR7, a chemokine receptor involved in T cell homing following antigen stimulation, CD69, an early activation marker necessary for Th17 differentiation, and the leukocyte adhesion molecule, CD44. Interestingly, changes to these markers, as well as genes involved in DNA damage, cell proliferation, and growth factor production, were detected in genetically-modified T cells as a result of electroporation and dramatically reduced their capacity to suppress tumor growth in vivo 11,13 . Collectively, our results indicate the μVS -based delivery does not perturb the native state of transfected cells, highlighting its potential to maintain the functionality and desired therapeutic effects of engineered cells upon administration.…”

“…Unlike viral-based methods, electroporation can be used to deliver a broader range of bioactive constructs into a variety of cell types, while bypassing the extensive safety and regulatory requirements for GMCT manufacturing using viruses 8,9 . However, the significant reductions in cell numbers and viabilities, accompanied by changes in gene expression profiles that negatively impact cell function, make physical transfection methods like electroporation less than ideal for GMCT applications 2,3,9,[11][12][13] . Therefore, the ideal intracellular delivery method to generate GMCTs would permit transfection of various constructs to multiple cell types while having minimal effects on cell viability and cell recovery, and minimal perturbation to normal and/or desired (i.e.…”

Genome Editing with mRNA Encoding ZFN, TALEN, and Cas9

“…113 Considering its ability to deliver diverse loads into various types of cells with relatively low cellular toxicity, development of a suitable squeeze protocol for genome editing is valuable. 115 Hydrodynamic injection causes transient deformation of the cell membrane to facilitate entry of nucleic acids into hepatocytes in vivo. 116 Hydrodynamic injection of a plasmid DNA expressing CRISPR-Cas9 and a single-stranded DNA donor resulted in the correction of a point mutation of fumarylacetoacetate hydrolase (Fah) in hepatocytes in a mouse model of hereditary tyrosinemia.…”

Efficient and Innocuous Live‐Cell Delivery: Making Membrane Barriers Disappear to Enable Cellular Biochemistry

“…A notable difference, however, is observable in how cells respond to these treatments, microfluidic squeezing being markedly less disruptive to cells. In particular, genome‐wide profiling shows that 34% of genes are misexpressed 6 h after electroporation of T cells (8141 transcripts out of 23 786 probed) while only 9% are affected 6 h after squeezing (2211/23 766) …”