Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Abstract

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Sex Differences in Plasmacytoid Dendritic Cell Levels of IRF5 Drive Higher IFN-α Production in Women

“…Cells were resuspended in R-10 at 1,000 cells/μL and were mixed with Cascade Blue-labeled 3-kDa dextran molecules, for control of delivery, and with either 0.03–0.06 μg/μL of IRF5 recombinant protein (Abcam or Origene, Rockville, MD) or 0.05–0.1 μg/μL of the control TUBA1A recombinant protein (Abcam), and subsequently placed in the device’s inlet reservoir. Delivery was performed using a vector-free microfluidic platform as previously described (51, 52) and illustrated in Supplemental Fig. 1A.…”

“…In brief, cells were mechanically deformed while passing through the microfluidic device (SQZ Biotechnologies, USA) at a pressure of 80 or 120psi, resulting in the transient formation of holes in the cell membrane allowing content from the surrounding buffer to diffuse into the cytosol. Cells were incubated at room temperature in the delivery solution for 5 min after treatment to ensure closure of membrane holes before being subjected to any further treatment, as previously described (52). Delivery efficiency was assessed using FITC-labeled 70 kDa dextran probes and/or Cascade Blue-labeled 3 kDa dextran molecules mimicking protein and siRNA deliveries respectively.…”

Plasma membrane recovery kinetics of a microfluidic intracellular delivery platform

“…Delivery experiments were conducted as described previously 23 . Briefly, the suspension of cells and delivery material was passed through the device at the desired pressure.…”

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

“…In general, microfluidic methods have improved macromolecule delivery into cells by scaling microfluidic channel geometries with cell dimensions. Intracellular delivery methods utilizing microfluidics include electroporation [14][15][16] , microinjection 17 , cell constriction or squeezing [18][19][20][21][22][23] , fluid shear 24,25 and electrosonic jet ejection 26,27 . These methods offer appealing alternatives to conventional transfection systems, however, their production output (i.e.…”

“…microfluidic jet ejections) 26,27 or use arrays with spacing smaller than a cell's diameter (i.e. squeezing) [18][19][20][21][22][23] that dramatically reduce processing rates. Interestingly, the hydrodynamic conditions that facilitate µVS in a microfluidic post array with spacing greater than a cell's diameter suggests that our device can efficiently deliver material into cells while addressing the limitations of physical transfection methods.…”