Table 3.
EV isolation methods.
| Isolation Method | Principle | EV Type | Sample | Advantage | Limitations | [Ref.] |
|---|---|---|---|---|---|---|
| Centrifugation | ||||||
| Ultracentrifugation | Density | Exosomes and MVs | CM (conditioned mesdium)/ urine |
Isolation of large volumes, cost, simple procedure | Time-consuming, operator-sensitive, damage of EVs, low efficiency, impurity and co-isolation of aggregates | [117,118] |
| Density gradient ultracentrifugation | Density | Possible subtype isolation | CM/ urine |
Purity, better removal of contaminating protein aggregates | Complex procedure, loss of sample | [119,120] |
| Precipitation | ||||||
| Precipitation | Solubility | Exosomes and MVs | CM/ urine |
Cost, EVs integrity, high yield | May present contamination of polymers, co-isolation of proteins and aggregates | [121,122] |
| Filtration | ||||||
| Ultrafiltration | Size | Possible subtype isolation | CM/ urine |
Fast and simple procedure, isolation of large volumes, scalable | Filter plugging, low puricity (protein contamination), damage of EVs | [123,124] |
| TFF (Tangential Flow Filtration) |
Size with tangential flow | Possible subtype isolation | CM/ urine |
The tangential flow reduces clog of the pore membrane, high yield, large scale, EVs integrity | Contamination of proteins and lipid impurities | [125,126] |
| Hydrostatic filtration | Size | Exosomes and MVs | Urine | Does not require centrifugation, cost, isolation of large volumes | Combination of other techniques to obtain EVs subpopulations | [127,128] |
| Size exclusive chromatography (SEC) | ||||||
| SEC | Hydrodynamic volume or molecular size | Possible subtype isolation | CM/urine | Scalability, EVs integrity, efficiency and purity | Specialized equipment, cost, coisolation of aggregates and proteins, further concentration steps needed | [129,130] |
| Two-dimensional SEC | Size | Possible subtype isolation | CM/urine | Improve exosome isolation, higher efficiency and purity than SEC | Specialized equipment, sample volume is limited | [129,131] |
| Filed-flow fraction | ||||||
| Asymmetrical filed-flow fraction (AsFFF) | Diffusion coefficient | EV subtype isolation | Urine | Less time consuming, possible to isolate EVs from plasma contaminants | Specialized equipment | [132,133] |
| Affinity | ||||||
| Immunoaffinity | Antibodies binding | EV subtype isolation, specific exosomes | CM/ urine |
Simple and fast procedure, specificity and purity | Non-specific binding, availability of antibodies, costs | [129,134] |
| Aptamers affinity | Aptamers binding | EV subtype isolation, specific exosomes | CM/ urine |
Higher affinity and specificity than immunoaffinity methods | Costs, low yield, prior knowledge of EVs characteristics | [135,136] |
| Microfluidics | ||||||
| Multistage filtration | Size | EV subtype isolation | Urine | Efficient, high purity | Low sample capacity | [137,138] |
| Deterministic lateral displacement (DLD) | Size | EV subtype isolation | CM/urine | Less time consuming | Specialized equipment, scalability | [139,140] |
| Combination with affinity method | Binding and size | EV subtype isolation, specific exosomes | CM/urine | Allows quantification and characterization of EVs | Specialized equipment, costs | [141,142] |
| Viscoelasticity-based | Viscoelasticity/size | EV subtype isolation | CM | High purity and faster than DLD method | Specialized equipment | [143,144] |
| Acoustophoresis | Size | EV subtype isolation | CM | High purity and yield | Need of high-frequency power supply | [145,146] |