Massively-Parallelized, Deterministic Mechanoporation for Intracellular Delivery

Abstract

Microfluidic intracellular delivery approaches based on plasma membrane poration have shown promise for addressing the limitations of conventional cellular engineering techniques in a wide range of applications in biology and medicine. However, the inherent stochasticity of the poration process in many of these approaches often results in a trade-off between delivery efficiency and cellular viability, thus potentially limiting their utility. Herein, we present a novel microfluidic device concept that mitigates this trade-off by providing opportunity for deterministic mechanoporation (DMP) of cells en masse. This is achieved by the impingement of each cell upon a single needle-like penetrator during aspiration-based capture, followed by diffusive influx of exogenous cargo through the resulting membrane pore, once the cells are released by reversal of flow. Massive parallelization enables high throughput operation, while single-site poration allows for delivery of small and large-molecule cargos in difficult-to-transfect cells with efficiencies and viabilities that exceed both conventional and emerging transfection techniques. As such, DMP shows promise for advancing cellular engineering practice in general and engineered cell product manufacturing in particular.

Emerging Roles of 1D Vertical Nanostructures in Orchestrating Immune Cell Functions

“…VNS‐based platforms can be built in microfluidic devices to achieve massively parallelized, deterministic mechanoporation (DMP) for intracellular delivery into suspension cells. [ 149 ] SiNWs (or penetrators) were specially designed and fabricated in confined wells (Figure 8d,e), where only a single cell can be captured and porated by impingement upon the penetrator under a negative aspiration flow (Figure 8f); cells were released by reversal of flow after which intracellular delivery occurs via diffusive influx of exogenous cargos (pGFP) through the single transient plasma membrane pore. Moreover, high cell viability and transfection efficiency were detected (both >87%) for DMP‐transfected Jurkat cells; mean pGFP transfection yield by the DMP device (88%) was over four times that by conventional BEP methods (20%) (Figure 8g).…”

Intracellular Labeling with Extrinsic Probes: Delivery Strategies and Applications

“…Reproduced with permission. [ 150 ] Copyright 2019, American Chemical Society. f) Schematic illustration of the design and operating principles of the hydroporator device that permeabilizes cells by fluid shear forces at a cross‐sectional junction with opposing flows.…”

“…Following a similar concept, cell membrane disruption was also achieved by Dixit et al on a microfluidic platform that first aspirates cells onto a sharp tip, after which the flow direction is reversed to release the punctured cell (Figure 13e). [ 150 ] Although they did not apply this platform for intracellular label delivery, successful plasmid DNA delivery indicated that it could be used for that purpose as well, even with large‐sized labels. In another approach, cells were permeabilized by the fluid streams themselves, such as the hydroporator that was developed by Kizer et al (Figures 11c and 12f).…”

High-throughput and dosage-controlled intracellular delivery of large cargos by an acoustic-electric micro-vortices platform

“…To address the challenges facing viral and conventional non-viral techniques, microfluidics and nanotechnology have appeared as powerful tools that have shown tremendous potential for adoption in clinical settings and research labs ( 11 ). Notable examples include methods based on cell deformation ( 12, 13 ), nanostructures for localized electroporation ( 10, 14–16 ), mechanoporation ( 17, 18 ), acoustofluidics sonoporation ( 19 ), flow-through electroporation ( 20 ), droplet microfluidics ( 21 ), and inertial microfluidics ( 18, 22 ). For safe, efficient, and controllable intracellular delivery, these methods focus on precise control of cellular permeabilization and uptake, down to the single-cell level.…”