Table 2.
Methodologies used for uEV isolation.
| Technique | Isolation method | Advantages | Disadvantages |
|---|---|---|---|
| Ultracentrifugation | Progressive ultracentrifugation (14) | Reproducible results; high yield of intact proteins and nucleic acids | 5-7 h to process single sample; contamination by highly abundant proteins; expensive equipment. |
| Double-cushion ultracentrifugation (93) | Less contamination of highly abundant proteins; reproducible results | Long processing time; tedious separation techniques; expensive equipment | |
| Sucrose gradient ultracentrifugation (94) | |||
| Ultracentrifugation-size exclusion chromatography (95) | |||
| Filtration | Nanomembrane filtration (96) | Shorter processing time (0.5-2 h); many samples can be processed at one time; relatively inexpensive; Can be used in a clinical setting |
Possible clogging of membrane; sample loss; contamination by highly abundant proteins |
| Micromembrane filtration (97) | |||
| Precipitation | Precipitation by ExoQuick-TC (15) | Shorter processing time (0.5-2 h); Yields intact RNA; Relatively inexpensive; can be used in a clinical setting. |
Low purity of protein; Modified protocol. |
| Hydrostatic dialysis | Hydrostatic filtration dialysis (73) | Low cost, simple system, efficient pre-processing and concentration for biobanking purposes; suitable to any downstream analyses; patients can be the end-users | Protein purity is not as good as ultracentrifugation, but acceptable; contain THP contamination; Comparing to ultracentrifugation, large vesicle fraction (>500 nm) was underrepresented, low proportion of small EVs (60-140 nm) and more very small size EV-like participles (<40nm) (72). |
| Acoustic trapping | Polystyrene beads model (98) | Rapid, automated, low-volume compatible, robust; no impact on the integrity or miRNA content of trapped vesicles | Amplifier used to drive the piezo may limit the possibilities for device parallelization (99) |
| Immunocapture | Antibody-based affinity capture on magnetic beads (100) | Less expensive equipment, less expertise, purer uEV fractions | Capture proteins displayed on the outer surface of uEVs |