1.1. Classification

EVs comprise submicron particles heterogeneous in size, delimited by a lipid bilayer that cannot replicate. Traditionally, they have been classified according to their size and biogenesis, distinguishing: small particles or exosomes of endosomal origin with diameters ranging from 30 to 150 nm; ectosomes or microvesicles directly shed from the plasma membrane and polydisperse in size (100–1000 nm); and apoptotic bodies generated as a consequence of programmed cell death (1000–5000 nm) ^[1][2]. However, in recent years it has become apparent that the picture is more complex than expected. Assigning an EV to a particular biogenesis pathway still remains extraordinarily difficult given the overlap in size-distribution and protein-expression patterns among different EV types, especially when referring to exosomes and microvesicles, challenging the attempts to define a more precise nomenclature for EV classes ^[3]. Consequently, the latest recommendations of the ISEV encourage authors to define EV subtypes considering their physical characteristics—attending to: (a) size (small, medium/large) or density (low, medium, high) according to a defined range, (b) biochemical composition relaying on specific markers (e.g., CD63, CD81, annexin V, etc.), (c) isolation conditions (e.g., hypoxia, serum conditioning), and/or d) cellular origin (platelet, endothelial, cardiomyocytes, etc.)—rather than by the use of the traditional terms exosomes or microvesicles ^[1]. A summary of EVs’ physical and biochemical properties as well as parental cell conditions is provided in 

Table 1

EVs are released into the extracellular space by most cell types and can be found in a wide range of body fluids as reservoirs of lipids, proteins, nucleic acids, and carbohydrates of their parental cells. Current knowledge supports the view that each cell type tunes EVs’ biogenesis, depending on its activation status. Moreover, their cargo is particular to the stimulus or biological condition triggering their formation and release, suggesting the existence of intracellular selective cargo-sorting mechanisms. Subsequently, EVs’ composition will directly affect their fate and function ^[2][8][9]. There is a consensus about two major EV biogenesis pathways, giving rise to the most widely studied subpopulations, exosomes and microvesicles. Exosomes, generated within the endosomal system, are intraluminal vesicles formed by the inward budding of the endosomal membrane during maturation of multivesicular endosomes (MVEs) and are released to the extracellular space by fusion of MVEs with the cell membrane ^[10]. As for microvesicles, they originate by outward budding of the plasma membrane ^[11]. Microvesicle biogenesis determines the expression of surface-specific antigens from the cell origin, as well as the externalization of phosphatidylserine on the outer membrane leaflet ^[12], although the latter is not a prerequisite in all microvesicles ^[13]. Despite the aforementioned differences in biogenesis, the overlap in size, density, or composition, together with the lack of appropriate technology, hampers the possibility of distinguishing EV subpopulations once released to the extracellular medium, favoring the use of the generic term EVs, instead of a more specific nomenclature ^[1].

1.3. Mechanism of Action

The lipid bilayer protects EVs’ content from degradation by nucleases and proteinases present in biofluids, enabling the transfer of proteins, lipids, or nucleic acids from parental cells to recipient cells ^[14]. As such, EVs contribute to normal homeostasis, but also to the progression of several pathologies, including CVDs ^[15][16]. The mechanisms by which EVs mediate intercellular communication are not completely understood but are supposed to involve specific interactions between proteins or lipids enriched at the EVs surface (e.g., tetraspanins, integrins, lectins, phosphatidylserine) and receptors at the plasma membrane of recipient cells (e.g., intercellular adhesion molecules (ICAMs), annexin V, galectin 5) ^{[2][17][18][19][20]}. After docking at the cell membrane, EVs can remain at the binding site eliciting functional responses in recipient cells by activating downstream molecular pathways, or by direct interaction with extracellular matrix components ^[21] (

Figure 1). They can also be internalized by endocytosis or by fusion with the plasma membrane undergoing different fates. For instance, endocytosed EVs can reach the MVEs and be targeted for degradation by lysosomes, they can escape digestion by back fusion with the MVEs’ membrane, or they can be re-secreted to the extracellular space via the early endocytic recycling pathway ^{[21][22][23][24][25]}. Either by direct fusion with the plasma membrane or after escaping lysosomal degradation, EVs can release their content into the cytoplasm of recipient cells and regulate cellular processes ^[26] (

Figure 1

Figure 1.

 Mechanism of action of EVs. After released from donor cells, EVs may induce a response in recipient cells by different mechanisms. First, EVs may remain at the binding site on the cell membrane eliciting functional responses by activating downstream molecular pathways. Alternatively, EVs may be internalized by endocytosis or fusion with the cell membrane undergoing different intracellular fates. They can be targeted for degradation by lysosomes, they can escape degradation and modulate cell behavior, or they can be re-secreted to the extracellular space.

1.4. Separation and Characterization of Extracellular Vesicles

EVs can be separated from the cell culture medium and most body fluids (liquid biopsy), blood being the most frequently studied source of EVs. Before EV separation, some preanalytical parameters should be considered ^[27][28], such as the use of serum-free media or EV-depleted serum for EV separation from conditioned medium ^[1][29]. Moreover, differences in the physicochemical and biochemical properties of the selected separation methods can impact the enriched EV subpopulations ^{[30][31][32][33][34]} and do not enable an absolute purification of EVs from other contaminants ^[35].

Ultracentrifugation (UC) is the most commonly used EV separation and enrichment technique based on particle density, involving multiple centrifugation and ultracentrifugation steps ^[30]. Speeds of 10,000–20,000 g enable the separation of medium/large vesicles, while small-sized vesicles are recovered at higher speeds (100,000 g).

Size exclusion techniques include ultrafiltration and chromatography. Ultrafiltration is usually based on cellulose filters defined by molecular mass and size exclusion range ^[31], while size exclusion chromatography (SEC), separates fractions by elution with phosphate buffered saline (PBS) and has been proven to be reliable and scalable for various applications ^[36].

Immune affinity isolation is based on the immunolabeling of proteins on the surface of EVs, enabling the separation of specific particle subpopulations from other EV classes, contaminant protein aggregates or lipoproteins. Usually, specific antibodies are conjugated to magnetic beads and EVs are separated using magnets ^[37][38].

A range of commercial kits are also available, some based on polymer precipitation- methods ^[31][32][33] and others on non-precipitation alternatives, for instance, those selective for phosphatidylserine positive vesicles ^[29]. Alternative or complementary techniques to classical procedures are also emerging, including microfluidics, asymmetric flow field-flow fractionation, or high-resolution flow cytometry ^[35].

After separation, EVs’ purity should be tested by the use of multiple complementary methods: (i) western blotting to analyze EVs markers (e.g., CD63, Alix, etc.) and co-isolated contaminants ^[1]; (ii) nanoparticle tracking analysis (NTA), which can determine particle size and concentration ^[39]; (iii) conventional transmission electron microscopy (TEM) and the more strongly recommended cryo-TEM ^[40], or (iv) nanoflow cytometry, that enables the determination of cell surface antigens, the quantification of EV subpopulations based on parental cell markers ^[41][42], and the lipid nature of the studied particles with cell-permeant, non-fluorescent pro-dyes . Also, current advances in EV-adapted proteomic, lipidomic, and genomic technologies will greatly help to delimit the molecular signature of the EV subpopulations under research.