Microfluidic generation of transient cell volume exchange for convectively driven intracellular delivery of large macromolecules

Abstract

Efficient intracellular delivery of target macromolecules remains a major obstacle in cell engineering and other biomedical applications. We discovered a unique cell biophysical phenomenon of transient cell volume exchange by using microfluidics to rapidly and repeatedly compress cells. This behavior consists of brief, mechanically induced cell volume loss followed by rapid volume recovery. We harness this behavior for high-throughput, convective intracellular delivery of large polysaccharides (2000 kDa), particles (100 nm), and plasmids while maintaining high cell viability. Successful proof of concept experiments in transfection and intracellular labeling demonstrated potential to overcome the most prohibitive challenges in intracellular delivery for cell engineering.

Cell Mechanical and Physiological Behavior in the Regime of Rapid Mechanical Compressions that Lead to Cell Volume Change

“…Flow rate through the microchannel was varied from 3.5 to 275 mm s −1 with high speed video recording to observe cell responses during mechanical compression. A compression gap of 9 µm, previously characterized to cause volume change in K562 myelogenous leukemia cells, was used for this study . When calculating cell volume from 2D imaging, the uncertainty of the 3D shape of the compressed cell is a potential source of error.…”

“…This permeabilization can have lasting physiological effects, particularly in nerve cells . However, numerous human cell types, including epithelial cells, chondrocytes, and leukocytes, have demonstrated the ability to recover from mechanical compression without significant impact on viability and function …”

“…Therefore, to isolate the impact of compression rate on volume exchange, the spacing between ridges proportional to the volumetric flow rate to keep the cell recovery time between compressions constant was varied. Devices with 7 ridges were used to avoid saturation or maximization of delivery that would otherwise obscure the effects of compression rate …”

“…This effect was also shown to deliver extracellular liquid and target molecules into the cell on convective fluid currents. Target molecule delivery was found to be nonendosomal and distributed throughout the cell interior . Therefore the phenomenon resulting from brief, rapid compressions can cause a transient exchange of fluid and macromolecules between the cell interior and its surroundings for intracellular macromolecule delivery .…”

Intracellular Labeling with Extrinsic Probes: Delivery Strategies and Applications

“…[ 144 ] To further increase the efficiency of probe delivery, two sequential constriction sites have been used as well for double membrane permeabilization ( Figure b). [ 145 ] In this way, 80% of cells were successfully labeled with TRITC 2000 kDa. Along the same line, a design with two sequential deformation sites with different geometries was fabricated by Modaresi et al to create pores at different locations in the cell membrane (Figure 13c), thus increasing the number of positive cells.…”