Table 1.

Evolution of extracellular vesicles (EV) dichotomous classification according to the accumulation of recent knowledge.

Historical Criteria	Early Knowledge	Current Knowledge
Size	MVs range between 100 and 1000 nm, while exosomes have dimeters smaller than 100 nm [23,27]	Classification no longer in use, MVs can be smaller than 100 nm, exosomes have an upper limit based on endosomal size (up to 150 nm or larger); “small EVs” and “medium/large EVs” nomenclature is preferred [25]
Protein content	Different marker profiles due to biogenesis: GTP-binding proteins (ARF6), vesicle-associated membrane protein 3 (VAMP3), proteasomes, mitochondria-related proteins for MVs, transduction or scaffolding proteins (Syntenin 1), extracellular matrix, cell adhesion, receptor binding proteins and endosome-binding proteins (TSG101) for exosomes [24,28,29]	No molecular markers that could characterize specifically each EV subtype, yet validation with three markers from three different classes is required in order to evaluate tissue specificity, lipid, or membrane-binding ability and purity [25]
Lipid content	Enriched contents according to the EV subtype: ceramides and sphingomyelins in MVs, cardiolipins in exosomes [29]	Lipid ratios in EVs are not yet established [25]; more studies are needed in order to compare the lipid profiles of EVs with co-isolated lipoproteins and validate characteristic EV lipid contents such as lysoglycerophospholipids [30]
Nucleic acid content	DNA, mRNA, ncRNA, and especially miRNA in both MVs and exosomes; origin-specific miRNA profiles for exosomes [8,31]	Confirmed specific incorporation of RNAs into subtypes of EVs [25]
Isolation and purification methods	Differential centrifugation or ultracentrifugation (10,000–20,000× g for MVs, 100,000–125,000× g for exosomes), size exclusion chromatography, immunoaffinity capture [32,33,34,35,36]	No “golden standard” method to isolate and/or purify EVs, the choice is to be made based on the downstream applications, recovery, and specificity rates [24,25]