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

Vladimir Riazanski,James L Mcgrath,Samuel Walker,Gerardo Mauleon,Adriana M Zimnicka,Deborah J Nelson,Kilean Lucas

doi:10.1038/s42003-021-02965-7

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.

Highlights

Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo
After selectively capturing secreted EVs in the pores of ultrathin nanoporous silicon nitride (NPN) membranes based on their size, we use real-time fluorescence imaging to measure the kinetics of pH changes in single vesicles brought about by the activity of the Na/H antiporter
Vesicles in pre-cleared isolation solution from either cell culture media or murine bronchoalveolar lavage (BAL) following high-speed centrifugation steps were loaded onto the NPN membranes by dead-end filtration using SepCon® spin columns specially designed to accommodate the “membrane chip” (Fig. 1a)

Summary

Introduction

Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo. EVs and their cargo have been identified diversely as biomarkers for disease as well as effective vehicles for molecular target therapies Independent of their identified function, either as genetic signaling elements or genetic delivery vehicles, maintenance of a stable pH in the lumen of vesicles, both microvesicles derived from a plasma membrane scission and exosomes secreted from endocytically derived multivesicular bodies (MVBs), is essential. Vesicle-associated transporter function is generally studied in preparations that are amenable to bulk analysis, e.g. neuronal synaptic vesicles[9,10,11], chromaffin cell catecholamine containing granules[12], or hormone-containing secretory granules[13] This is not the case with EVs. EVs isolated from either cultured cells or primary bodily fluid samples yield heterogeneous preparations with potentially important subclasses that may go undetected in a bulk analysis of transport. After selectively capturing secreted EVs in the pores of ultrathin nanoporous silicon nitride (NPN) membranes based on their size, we use real-time fluorescence imaging to measure the kinetics of pH changes in single vesicles brought about by the activity of the Na/H antiporter

Methods

Results

Conclusion