Table 1.

Overview of various small extracellular vesicles (EVs) isolation techniques.

Isolation method	Principle	Processing time	Advantages	Disadvantages	Purity degree	Reference
Ultracentrifugation	Sedimentation rates (depending on size and shape)	3–16 h	Easy to use, high yields	High equipment cost, time-consuming, low portability, not scalable, high-speed centrifugation may induce vesicle disruption	Low: contamination by cell debris, large vesicles, vesicle aggregates, and protein complexes	(12–16)
Density gradient separation	Density	20–24 h	Adequate grade of purity	Laborious and time-consuming, low portability, not scalable	Medium: contamination by high-density lipoprotein and other vesicles	(17–20)
Ultrafiltration	Size	1–6 h	Fast, good portability, useful for urine concentration	High-speed centrifugation may induce vesicle disruption, membrane filter clogging, small EVs loss, not scalable	Low: protein co-isolation	(12, 21)
Size-exclusion chromatography	Size	6–12 h	Gravity flow preserves small EVs integrity, high reproducibility	Not scalable, time-consuming	High	(22)
Small EVs precipitation	Polymer mixtures that alter the solubility of small EVs	30 min–2 h	Fast, easy to use, no specialized equipment required, large and scalable sample capacity	Low purity, not recommended if coupled with mass spectrometry	Low: contamination by protein aggregates and other vesicles	(23–25)
Immunoaffinity capture	Presence of small EVs’ surface antigens	16–20 h	Unique method for the isolation of specific small EVs	High reagent cost, low capacity, low yields, time-consuming	Very high	(26–29)