Isolation of Extracellular Vesicles: General Methodologies and Latest Trends

Table 1

Main advantages and disadvantages of the currently available methods for EV isolation.

Method	Time	Advantages	Disadvantages	References
(1.1) Ultracentrifugation, differential centrifugation: 300 ×g, 10000 ×g, 100000–200000 ×g (1.5 h)	140–600 min	Cost (in the case of ultracentrifugation), isolation from large volumes, absence of additional chemicals	Equipment, complexity, nonexosomal impurities, low reproducibility, low RNA yield, damage of exosomes; efficiency is affected by the type of rotor, force , sample viscosity; only six samples can be concurrently processed in one ultracentrifuge	[35, 49–54]
(1.2) Density gradient ultracentrifugation, sucrose or iodixanol density gradient, differential centrifugation	250 min–2 days	Pure preparations; no contamination with viral particles after iodixanol centrifugation; absence of additional chemicals	Complexity, loss of sample, ultracentrifugation; fails to separate large vesicles with similar sedimentation rates; contamination with viral particles after sucrose density gradient procedure	[36–39, 55, 56]
(2.1) Ultrafiltration, nanomembrane or filters with a pore diameter of 0.8–0.1 µm	130 min	Simple procedure allowing for concurrent processing of many samples; pure preparations; additional chemicals; no limitations on sample volume	Filter plugging, loss of sample, contamination (proteins); deformation of vesicles; small quantity of exosomal proteins	[37, 38, 57, 58]
(2.2) Hydrostatic dialysis, membrane separation at concentration gradient	30 min 1 h per 75 ml	Appropriate for analysis of highly diluted samples (urine); cost; no additional chemicals; standardizes sample concentration, volume, electrolyte composition	Need in additional urine sample purification from bacteria	[59–61]
(2.3) Size-exclusive chromatography (SEC), columns filled with polymers with heterogeneous pores	1 ml/min + column washing	Reproducibility and purity; preserves vesicle integrity; use of the buffers with a high ionic strength enhances elimination of nonspecific impurities; high sensitivity, no losses, scalability, large amount of exosomal proteins; prevents EV aggregation; insensitive to high viscosity of samples; no additional chemicals	Limitations on sample volume and number of separated peaks (necessary difference of the components in molecular weight, ≥10%); specialized equipment; complexity; coisolation of large protein aggregates and lipoproteins; processing no more than one sample in each procedure; cost	[37–40, 42, 53, 57, 62–65]
(3.1.1) Precipitation with polymers, polyethylene glycol caused EV precipitation	65 min	Cost and simplicity of procedure; preservation of EV integrity; no need in additional equipment; pH close to physiological range; high ion concentrations	Contamination and retention of the polymer	[42, 66]
(3.1.2) Commercial kits for polymer precipitation (ExoQuick, TEI, and Norgen), polymer precipitates EVs	45–65 min (sometimes overnight)	Simple procedure; preservation of EV integrity; no need in additional equipment; pH close to physiological range; high ion concentrations	Cost (especially for diluted samples, such as urine); poor reproducibility; impurities and retention of polymer; low content of exosomal proteins	[37, 39, 40, 55, 57, 66–72]
(3.2) Precipitation with protamine	55 min + incubation (overnight)	Cost; simple procedure; preservation of EV integrity and biological activity; purity; efficiency	Need in purification of the isolated fraction from protamine and lipoproteins (heparin + gel filtration); long duration	[43]
(3.3) Precipitation with sodium acetate, , 0.1 M acetate	130 min	Cost; simple procedure; and the possibility of processing samples of large volume	Contamination with non-EV proteins	[44]
(3.4) Precipitation of proteins with organic solvent PROSPR, cold acetone	105 min	Cost and simplicity	Aggregation in multivesicles	[42, 73]
(4) Two-phase isolation, incubation in PEG-dextran mixture	75–195 min	Cost; simple procedure; no EV deformation; purity; efficiency	Repeated replacement of PEG phase and presence of polymer	[74, 75]
(5.1) Use of antibodies to EV receptors, in particular, tetraspanins (CD9, CD63, CD81), TSG101, EpCAM	about 240 min	Purity and high selectivity	High selectivity, cost, availability of antibodies; difficulties with detachment of molecules and analysis of intact vesicles (eluting buffers can damage EV functional activity); nonspecific binding	[36–38, 40, 47, 56, 69, 76]
(5.2) Use of phosphatidylserine-binding proteins (annexin and Tim4)	12 h incubation	Readily reversible binding and simplicity	Cost	[77, 78]
(5.3) Use of heparin-modified sorbents	24 h incubation	Cost and preservation of EV functional integrity	Need in the initial purification and concentration (ultracentrifugation)	[79]
(5.4) Binding of heat shock proteins	<1 h	Preservation of EV functional integrity	Cost	[80, 81]
(5.5) Use of lectins	12 h incubation	Cost, simplicity, purity	Need in the initial purification and concentration (ultracentrifugation of centrifugation at 20000 ×g)	[82]
(6) Microfluidic technologies	1–14 µl/min	Rapidness, purity, efficiency	Complexity of devices and need in additional equipment; cost	[45, 46, 48, 76, 83–87]
(7) KeepEX, protocol for urine dilution	<2 h	Higher EV yield as compared with ultracentrifugation	Equipment, laborious procedure, limitation on the number of concurrently processed samples (to six sample)	[88]