Table 1.

Requirements for extracellular vesicles’ separation and classification based on their physical and biochemical properties as well as donor cell conditions.

Physical properties	Size [10]	Small EVs	<100 nm
		Small/medium EVs	<200 nm
		Medium/large EVs	>200 nm
	Density (in sucrose) [23]	Low	1.13–1.19 g/mL
		Medium	1.16–1.28 g/mL
		High	>1.28 g/mL
Biochemical composition	Surface antigens [10]	Tetraspanins MHC class I Integrins Transferrin receptor LAMP1/2 Heparan sulfate	Proteoglycans EMMPRIN ADAM10 GPI-anchored 5ʹnucleotidase CD73 Complement-binding proteins CD55 and CD59 Sonic hedgehog
	Lipids [10,24]	Phosphatidylserine Phosphatidylinositol Phosphatidylethanolamine Phosphatidylcholine	Cholesterol Ceramide Diacylglycerol Glycosphingolipids
	Internal cargo [10,25,26]	Proteins	TSG101 ALIX VPS4A/B ARRDC1 Flotillins-1 and 2	Caveolins Annexins Heat shock proteins HSC70 and HSP84 Syntenin
		Cardiac-related miRNAs	let-7 miR-16 miR-17-92 miR-19b miR-20a/b miR-21a miR-24 miR-26a miR-34 miR-93 miR-94a miR-107a miR-125b miR-126	miR-130a/b miR-132 miR-143 miR-145 miR-146a miR-181b miR-182 miR-208a miR-210 miR-214 miR-294 miR-302a miR-451
Conditions at EVs harvest	Cell culture conditions [10]	Normoxia Hypoxia Surface coating	Treatment Grade of confluency Passage number
	Donor status [10]	Age Biological sex Circadian variation Body mass index	Pathological/healthy condition Exercise level Diet Medication

ADAM10: ADAM metallopeptidase domain 10; ALIX: ALG-2-interacting protein X; ARRDC1: arrestin domain-containing protein 1; EMMPRIN: extracellular matrix metalloproteinase inducer; EVs: extracellular vesicles; GPI: glycosylphosphatidylinositol; LAMP: lysosomal-associated membrane protein; MHC: major histocompatibility complex; miRNA: microRNA; TSG101: tumor susceptibility gene 101; VPS4A/B: Vacuolar Protein Sorting 4 Homolog A/B.