Table 1.
Methods commonly used to isolate extracellular vesicles.
| Noncommercial Methods | |||
|---|---|---|---|
| Methods | Procedures | Time | Reference |
| UC | Clearance: CM is collected and centrifuged at 2700× g for 5 min and afterwards filtered using a 0.2 μm filter. Isolation: Supernatant is centrifuged at 100,000× g overnight at 4 °C |
>16 h | Klymiuk et al. [49] |
| UC | Clearance: CM is collected and filtered using 0.2 μm filter. Filtrate is subjected to two centrifugation steps: (1) 2000× g for 10 min and (2) 10,000× g for 70 min. Isolation: Supernatant is centrifuged at 100,000× g for 70 min and afterwards, washed in PBS and centrifuged at 100,000× g for 70 min |
>3 h | Klymiuk et al. [49] |
| UC | Clearance: CM is collected and subjected to two centrifugation steps: (1) 300× g for 10 min and (2) 10,000× g for 30 min. Next, the supernatant is filtered using a 0.2 μm filter. Isolation: Filtrate is centrifuged at 100,000× g for 90 min, discarding the supernatant. Exosome pellet is washed in PBS and then centrifuged 100,000× g for 90 min |
≈3 h | Haraszti et al. [67] |
| UC | Clearance: CM is collected and subjected to two centrifugation steps: (1) 300× g and (2) 1200× g, both for 10 min. Supernatant is filtered using 0.2 μm filter. Isolation: Filtrate is centrifuged at 100,000× g for 14 h at 4 °C. |
>14 h | Antounians et al. [89] |
| UC | Clearance: CM is collected and subjected to two centrifugation steps: (1) 400× g for 10 min and (2) 2000× g for 20 min. Supernatant is filtrated using a 0.2 μm filter. Isolation: Filtrate is centrifuged at 100,000 × g for 3 h |
≈4 h | Duong et al. [134] |
| UC | Clearance: CM is collected and subjected to two centrifugation steps: (1) 300× g and (2) 1200× g, both for 10 min. Isolation: Filtrate is centrifuged at 100,000× g for 14 h |
≈15 h | Antounias et al. [89] |
| UC | Clearance: CM is collected and centrifuged at 2700× g for 5 min and afterwards filtered using a 0.2 μm filter. Filtrate is subjected to two centrifugation steps: (1) 2000× g for 10 min and (2) 10,000× g for 30 min. Isolation: Supernatant is centrifuged at 100,000× g for 70 min, discarding the supernatant. Exosome pellet is washed in PBS and then centrifuged 100,000× g for 70 min. |
≈4 h | Klymiuk et al. [49] |
| UC | Clearance: CM is collected and subjected to three centrifugation steps: (1) 300× g for 10 min, (2) 2000× g for 30 min, and (3) 20,000 for 30 min. Isolation: Supernatant is centrifuged at 100,000× g for 70 min, discarding the supernatant. Exosome pellet is washed in PBS and then centrifuged 100,000× g for 70 min. |
≈4 h | Narbute et al. [135] |
| CUC | Clearance: CM is collected and subjected to two centrifugation steps: (1) 400× g for 10 min and (2) 2000× g for 20 min. Supernatant iscollected Isolation: Filtrate is loaded on 60% iodixanol cushion. Sample is centrifuges at 100,000× g for 3 h. |
≈4 h | Duong et al. [134] |
| CUC | Clearance: CM is collected and subjected to two centrifugation steps: (1) 300× g and (2) 6000× g, both for 30 min. Supernatant is filtrated using a using a 0.2 μm filter. Isolation: Filtrate is loaded on 30% sucrose/D2O cushion. Samples is centrifuged at 120,000× g for 90 min, discarding the supernatant. Exosome pellet is washed in PBS and next, centrifuged at 120,000× g for 90 min. |
≈6 h | Li et al. [136] |
| DGUC | Clearance: CM is collected and subjected to two centrifugation steps: (1) 400× g for 10 min and (2) 2000× g for 20 min. Supernatant is filtrated using a 0.2 μm filter. Isolation: Exosomes, previously isolated using UC method, is loaded on 5, 10 or 20% iodixanol gradient solution with 0.25 mm sucrose, 1 mM Tris-HCl pH 7.4 and centrifuged at 100,000× g for 18 h |
≈20 h | Duong et al. [134] |
| PEG-B | Clearance: not applied Isolation: CM is collected and transferred to centrifugation tubes containing 1:4 (v/v) of 50% PEG 6000 with 375 mm NaCl—CM. Sample is incubated for 18 h at 4 °C and next, centrifuged at 1500× g for 30 min. |
≈19 h | Duong et al. [134] |
| PEG-B | Clearance: CM is collected and centrifuged at 3000× g for 20 min. Supernatant is filtrated using a 0.2 μm filter. Isolation: Filtrate is incubated with 50% PEG 35.000 and 2% protaminesulfate for 1 h at 4 °C and, next, centrifuged at 12,000× g for 10 min. |
≈2 h | Klymiuk et al. [49] |
| UF | Clearance: CM is collected and centrifuged at 3000× g for 20 min. Supernatant is filtrated using a 0.2 μm filter. Isolation: Filtrate obtained is filtrated using 3K Ultra Filter and then, centrifuged at 2700× g for 10 min. Pellet is washed with PBS and, next, centrifuged at 2700× g for 10 min. |
≈0.5 h | Klymiuk et al. [49] |
| TFF | Isolation: CM is subjected to ultrafiltration in a tangential flow filtration (TFF) system, using a 500 kDa cutoff TFF cartridge with a flow rate of 120 mL/min and a transmembrane pressure < 3.5 psi. Then, the filtrate is concentrated 9-fold and exchange with 6× volume of PBS. Finally, the exosomes are filtrated using a 0.2 μm filter. | ≈2 h | Haraszti et al. [67] |
| Commercial Methods | |||
| Methods | Procedures | Time | Reference |
| Total Exosome Isolation (TEI) | Procedure: CM is collected and centrifuged at 2000× g for 30 min at 4 °C. Supernatant is incubated overnight at 4 °C with the Total Exosome Isolation reagent. Then, the sample is centrifuged at 10,000× g for 60 min at 4 °C. | >20 h | Thermo Fischer Scientific |
| ExoQuick Preciptation Solution | Procedure: CM is collected and centrifuged at 3000× g for 15 min at 4 °C. Supernatant is filtered using a 0.2 μm filter. Filtrated is incubated overnight at 4 °C with the ExoQuick reagent. Then, the sample is centrifuged at 1500× g for 5 min at 4 °C. | >20 h | System Biosciences |
| ExoMAX Opti Enhancer Reagent | Procedure: CM is collected and centrifuged at 3000× g for 30 min (clearance). Supernatant is incubated overnight with ExoMAX Opti Enhancer Reagent. Then, the sample is centrifuged at 1500× g for 30 min. Pellet is washed and centrifuged at 75,000× g for 70 min. | >20 h | BioCat |
| qEV Exosome Isolation | Procedure: CM is collected and centrifuged at 16,000× g for 30 min at 4 °C (clearance). Supernatant is incubated overnight at 4 °C with the Exo-Spin reagent. Then, the sample is centrifuged at 16,000× g for 60 min at 4 °C. | >20 h | Cell Guidance Systems |
| MagCaptureTM Exosome Isolation Kit | Procedure: CM is collected and loaded on a stationary phase consisting of porous resin particles, in which particles between 35–350 nm enter into the pores. | ≈2 h | IZON |
| Exo-SpinTM Exosome Purification | Procedure: CM is collected and centrifuged at 300× g for 10 min. Next, the supernatant is filtered using a 0.2 μm filter (clearance). Filtrate is incubated overnight with Tim4 protein solidified magnetic beads able to bind to phosphatidylserine on the surface of extracellular vesicles. Finally, exosomes are captured using a magnetic rack. | >20 h | Wako |
UC—ultracentrifugation; CUC—cushion ultracentrifugation; DGUC—density-gradient ultracentrifugation, PEG-B—polyethylene glycol-based method, UF—ultrafiltration, TFF—tangential flow filtration.