Real time imaging of single extracellular vesicle pH regulation in a microfluidic cross-flow filtration platform

Abstract

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. We 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. We 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.

Label-free microfluidic cell sorting and detection for rapid blood analysis

“…Microfluidic filtration separate cells based on size and can be broadly divided into three categories: membrane filtration, 63,64 pillar filtration, 65 and tangential flow filtration. 66 While readers are highly encouraged to learn more about the various filtration mechanisms from other excellent review articles, 45,67,68 this section focuses on their applications to isolate CTCs, [63][64][65]69 leukocytes, 70 platelets, 66,71 bacteria, 72 and EVs [73][74][75] from whole blood. In general, key advantages of filtration methods include scalable throughput, low-cost and simple operation.…”

Microfluidic Devices: A Tool for Nanoparticle Synthesis and Performance Evaluation

“…This enables building specific designs that resemble physical and chemical microenvironments to answer specific challenges (e.g., the blood-brain barrier, vascular circuits, extravasation, or tumor permeation). Indeed, microfluidic platforms can be designed for several specific applications, such as single-cell studies, , cell trapping, , cell filtration, cell rolling, , cell migration, drug screening and discovery, biomarkers detection, , organ-on-a-chip, and body-on-a-chip. , Moreover, microfluidic devices operating in a continuous-perfusion mode allow for enhancement and optimize the microenvironment for cell functions. Indeed, as previously mentioned, this dynamic condition allows for the efficient delivery of nutrients and oxygen to the cells while metabolic wastes are removed.…”

From Conventional to Microfluidic: Progress in Extracellular Vesicle Separation and Individual Characterization

“…Microfluidic device [ 257] Single EV proteins EVs in plasma N/A Flow rate (10 μL min −1 ) First mechanistic description of single EV function 𝜆-DNA mediated viscoelastic microfluidics [ 258] Single EV proteins and nucleic acids…”

“…This is the first mechanistic description of EV functions at the level of single vesicles. [257] There is no doubt that the characterization of single exosomal proteins has made considerable progress in recent years, owing to the improvement of exosome isolation methods and the development of advanced instrumentations. Due to the heterogeneity of EVs, it is believed that proteomics research at the singleparticle level will become more intense.…”