Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo. Their complex cargo requires pH stability in EVs while traversing diverse body fluids. Researchers at the University of Chicago used a filtration-based platform to capture and stabilize EVs based on their size and studied their pH regulation at the single EV level. Dead-end filtration facilitated EV capture in the pores of an ultrathin (100 nm thick) and nanoporous silicon nitride (NPN) membrane within a custom microfluidic device. Immobilized EVs were rapidly exposed to test solution changes driven across the backside of the membrane using tangential flow without exposing the EVs to fluid shear forces. The epithelial sodium-hydrogen exchanger, NHE1, is a ubiquitous plasma membrane protein tasked with the maintenance of cytoplasmic pH at neutrality. The researchers show that NHE1 identified on the membrane of EVs is functional in the maintenance of pH neutrality within single vesicles. This is the first mechanistic description of EV function on the single vesicle level.
NPN membrane-based platform for investigation of EV properties in real time
a Schematic cross-sectional diagram of the microfluidic device used to study functional responses of individual, membrane-stabilized EVs in response to changes in ionic composition of external solution. b Stack of PDMS layers comprising the device, including the housing layer patterned for insertion of the NPN chip. c Photograph of the device assembled on the stage of laser scanning confocal microscope. Inlet and outlet tubing allowed for the flow of solutions over the NPN membrane-immobilized vesicles, at a rate of 5–10 µl/min, maintained by a pressure-driven microfluidic flow system (ElveFlow®). d Image of fluorescent beads (100 nm in diameter), trapped and retained on the NPN membrane under conditions of tangential flow (scale bar 300 nm). e Schematic depiction of how the ultrathin (100 nm thick) nanoporous NPN membrane (gray) separates perfusion flows: top compartment with high shear force provides fast solution exchange (red) which diffuses rapidly through nanopores to the bottom compartment where trapped vesicles (yellow) are exposed to low shear force (green).