High throughput intracellular delivery by viscoelastic mechanoporation

Abstract

Brief pulses of electric field (electroporation) and/or tensile stress (mechanoporation) have been used to reversibly permeabilize the plasma membrane of mammalian cells and deliver materials to the cytosol. However, electroporation can be harmful to cells, while efficient mechanoporation strategies have not been scalable due to the use of narrow constrictions or needles which are susceptible to clogging. Here we report a high throughput approach to mechanoporation in which the plasma membrane is stretched and reversibly permeabilized by viscoelastic fluid forces within a microfluidic chip without surface contact. Biomolecules are delivered directly to the cytosol within seconds at a throughput exceeding 250 million cells per minute. Viscoelastic mechanoporation is compatible with a variety of biomolecules including proteins, RNA, and CRISPR-Cas9 ribonucleoprotein complexes, as well as a range of cell types including HEK293T cells and primary T cells. Altogether, viscoelastic mechanoporation appears feasible for contact-free permeabilization and delivery of biomolecules to mammalian cells ex vivo.

High throughput cell mechanotyping of cell response to cytoskeletal modulations using a microfluidic cell deformation system

“…This efficiency highlights time intensive semi-manual analysis methods as major bottlenecks to the full potential of the MCDS. Future work should be done to implement a machine learning algorithm, such as U-net segmentation, to expedite the analysis process [19]; however, in the current work, we intentionally chose to use an ImageJ-based analysis method that is readily understood and simple to implement into almost any lab. Given that manual analysis across several individuals yielded qualitative comparable results that exhibited with detectable variation, (Supplemental Figure 3) all data collected and presented was analyzed by a single individual.…”

“…Furthermore, cells were often pushed up or down against a wall of the deformation device, likely due to the unstable flow. A previous study noted unstable flow in the constricting region of a similar microfluidic device following at high flow rates, resulting in non-streamline cell movement [19]. Therefore, in addition to having flow rates that induce viable and effective cell deformation, our MCDS needed to meet the engineering requirement of achieving stable flow rates that center cells in the channel.…”