Mesenchymal stem/stromal cells (MSCs) provide therapeutic effects in many diseases. Contrary to initial hypotheses, they act in a paracrine rather than a cellular manner. To this end, extracellular vesicles (EVs) have been found to mediate the therapeutic effects, even when harvested from MSC-conditioned cell culture supernatants. Lacking self-replicating activity and being so small that MSC-EV preparations can be sterilized by filtration, EVs provide several advantages as therapeutic agents over cellular therapeutics. At present, methods allowing EV preparation from larger volumes are scarce and regularly require special equipment.
Researchers from the University Hospital Essen have developed a polyethylene glycol-based precipitation protocol allowing extraction of EVs from several liters of conditioned medium. MSC-EVs prepared with this method have been successfully applied to a human graft-versus-host disease patient and to several animal models. Although the method comes with its own limitations, it is extremely helpful for the initial evaluation of EV-based therapeutic approaches. Here, the researchers introduce the technique in detail and discuss all critical steps.
Harvested MSC‐CM is pooled and spun down for 45 min at 6800 × g, 4°C. The supernatant (SN) is filtered through a 0.22‐µm pore‐size filter, adjusted to concentrations of 10% PEG 6000 and 75 mM NaCl, and incubated for 8‐16 hr at 4°C. The suspension is centrifuged for 30 min at 1500 × g, 4°C. The pellet is resuspended and washed in NaCl. EVs are reprecipitated by ultracentrifugation for 130 min at 100,000 × g, 4°C. The obtained EV pellet is resuspended in buffer and stored at −80°C until use.