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Table 4 Common isolation protocols used for MSC-derived EVs

From: Heterogeneity of mesenchymal stem cell-derived extracellular vesicles is highly impacted by the tissue/cell source and culture conditions

Method   References
Differential centrifugation (dUC) Prior to the ultracentrifugation (100,000–200,000 × g, 1-2 h, 4 °C) several low to intermediate-speed centrifugation steps are required to remove cells, cell debris, apoptotic bodies, and aggregates:
300–400 × g for 10 min sediment cells
1500–2000 × g for 15–20 min. at 4 °C remove cell debris
10,000 × g 15–30 min at 4 °C removal of other structures with a higher buoyant density that MSC-EVs
Density gradient isolation Hereby, a continuous density gradient is formed by layering different concentrations of iodixanol. The MSC-EV-rich conditioned medium (CM) is overlaid on top and subjected to high-speed centrifugation (100,000 × g, 18 h, 4 °C), resulting in gradient fractions containing EV-like vesicles of different concentrations. Subsequently, these fractions are further processed in another high-speed centrifugation step (100,000 × g, 1-2 h, 4 °C) to separate MSC-EVs from other proteins and nucleoproteins [111]
Size-exclusion chromatography (SEC) CM is concentrated using a 100 kDa molecular weight cut-off filter to reduce total volume prior to the loading onto the column. The most common stationary phase used for EV isolation using SEC is Sepharose CL-2B, which is extensively washed and then packed into a column or syringe. The CM is loaded on top and EV-rich fractions are collected immediately and pooled after elution and again concentrated for further analytical procedures [109]
Precipitation/Phase separation The majority of protocols use polyethylene glycol (PEG)-based volume exclusion which precipitates EVs to a pellet. Hereby, CM is centrifuged at intermediate speed (6,000–10,000 × g, 45 min, 4 °C), filtered (0.22 µm), added to PEG solution to a final concentration of 10% (or 75 mM), and incubated for 8–16 h at 4 °C. Subsequently, the suspension is centrifuged and the EV-rich pellet is washed a few times with 0.9% NaCl. Lastly, the suspension is ultracentrifuged (100,000 × g, 130 min, 4 °C) and the resulting pellet is dissolved in buffer [112]