Table 1.

Overview of the Described EV Isolation Methods and Their Main Advantages and Disadvantages

Method	Principle	Advantage	Disadvantage	Scalabilitya	Costa	Refs
UC	Isolation by differential centrifugation	Low protein contamination	Low throughput, isolates similarly sized particles, potential damage to EVs	+	$$$$	[60., 61., 62., 63.]
Density gradient	Separates EVs by density after initial isolation by UC or alternative techniques	Increased purity	Low throughput, lower yield	++	$$$	[58,59]
SEC	Separates by hydrodynamic volume	Reduced contamination with high-abundance proteins, gentle	Low resolution and dilution of EV isolates	++++	$$	[44,69,70]
Filtration	Uses membranes with specific pore sizes	Simple, time efficient, and relatively gentle	Low sample recovery, extrusion effects, possible irreproducibility	++++	$	[72., 73., 74., 75.]
Immunoaffinity-based isolation strategies	Capture EVs using antibodies	Increased purification efficiency, target specific population	Costly, nonspecific binding	++	$$$$	[76., 77., 78.]
Commercial reagents	Precipitate EVs using polymers	High yield, simple workflows	High protein contamination, various degrees of compatibility with profiling techniques	++	$$$	[87., 88., 89.]
Microfluidics	Based on physical, mechanical, and/or surface chemistry properties	Low sample volumes, low cost, low consumption, high throughput, high size selectivity	Prone to clogging, possible irreproducibility	++	$$$	[91., 92., 93., 94., 95., 96.]
AF4	Laminar flow	Gentle, isolation of EV subpopulations	Low resolution, possible irreproducibility	++	$$$$	[7,8,98,99]
Nano-FCM	Flow-cytometry based	High fidelity sorting	Swarm detection, simultaneous detection of multiple EVs, inadequate size assessment	++	$$$$	[100,101]

^aPotential for scalability and cost rankings shown in arbitrary units using a range of 0–4 units.