Extracellular vesicle isolation and characterization: toward clinical application

Abstract

Two broad categories of extracellular vesicles (EVs), exosomes and shed microvesicles (sMVs), which differ in size distribution as well as protein and RNA profiles, have been described. EVs are known to play key roles in cell-cell communication, acting proximally as well as systemically. This Review discusses the nature of EV subtypes, strategies for isolating EVs from both cell-culture media and body fluids, and procedures for quantifying EVs. We also discuss proteins selectively enriched in exosomes and sMVs that have the potential for use as markers to discriminate between EV subtypes, as well as various applications of EVs in clinical diagnosis.

Figure 1. Characterization of distinct EV subtypes by cryoelectron microscopy.

Electron micrographs of EV subtypes released into human colon cancer cell line LIM1863 cell–culture media. EVs were harvested from cell-culture media by sequential centrifugal ultracentrifugation, as described (37). EVs range in diameter from small (50–120 nm) (exosomes and sMVs) to intermediate (~200–300 nm) (sMVs) and large (>500 nm) vesicles (sMVs).

Figure 2. Proposed molecular machineries involved in EV (exosome/sMVs) secretion and recipient cell uptake.

Components of donor cells are incorporated into EVs (exosomes, sMVs) that contain diverse cargoes, such as signaling proteins, transcriptional regulators, various RNA species, DNA, and lipids. Multiple machineries are thought to be involved in exosome biogenesis of ILVs and MVBs. ESCRT components are involved in an ESCRT-dependent intracellular pathway that traffics MVBs and their ILV contents (exosomes) out of the cell, while other ESCRT-independent pathways (lipid dependent) have also been described. Members of the Rab GTPase family have been shown to modulate exosome secretion and are thought to act on different MVBs along ESCRT-dependent and -independent endocytic pathways. It is not known whether each of these pathways acts on different MVBs or on the same MVB concomitantly or how exosome/ILV-loaded MVBs differ from those destined for lysosomes. Even less is known about the molecular machinery regulating outward budding of the plasma membrane and sMV release, apart from a requirement of ARF6, acid sphingomyelinase activity, and some ESCRT components (ESCRT-I). EVs may interact with recipient cells by direct signaling through ligand/receptor molecules on their respective surfaces or by their uptake via direct fusion of EV and recipient cell plasma membrane (110, 111) through lipid raft-, clathrin-, and calveolae-dependent endocytosis, macropinocytosis, and phagocytosis (–117). EVs can be involved in antigen presentation and in the transfer of both MHC molecules and antigens, thereby participating in immune regulation (–120). APC, antigen-presenting cell.