Extracellular vesicles are a heterogeneous and dynamic group of lipid bilayer membrane nanoparticles that can be classified into three different groups depending on their cellular origin: exosomes, microvesicles, and apoptotic bodies. They are produced by different cell types and can be isolated from almost all body fluids. EVs contain a variety of proteins, lipids, nucleic acids, and metabolites which regulate a number of biological and pathological scenarios both locally and systemically. Different techniques have been described in order to determine EV isolation, release, uptake, and cargo. Although standard techniques such as immunoblotting, fluorescent microscopy, and electron microscopy are still being used to characterize and visualize EVs, in the last years, more fine-tuned techniques are emerging. For example, EV uptake can be specifically determined at a single cell level using the Cre reporter methodology and bioluminescence based-methods reports have been employed to determine both EV release and uptake. In addition, techniques for cargo identification have also enormously evolved during these years. Classical mass spectrometry and next generation sequencing have been used in the past, but nowadays, advances in these tools have facilitated a more in depth characterization of the EV content. Researchers from Queen Mary University of London assess the standard and latest technical advances for studying EV biology in different biological systems.
Schematic representation of established and new evolving techniques used for the study of EV biology organized across the biogenesis and uptake of EVs
(A) Techniques to isolate EVs. (B) Methods to visualize and characterize purified EVs. (C) Techniques to allow for the EVs labeling and uptake visualization. (D) Identification of EV cargo. GA, Golgi Apparatus; RER, Rough Endoplasmic Reticulum; SEC, Size Exclusion Chromatography; EM, Electron Microscopy; SEM, Scanning Electron Microscopy; TEM, Transmission Electron Microscopy; Cryo-EM, Cryo-Electron Microscopy; NTA, Nanoparticle Tracking Analysis; TRSP, Tunable resistive pulse-sensing; AFM, Atomic Force Microscopy.