3.5. Precipitation-Based Isolation (Sodium Acetate, PEG, Protamine)

Precipitation-based isolation has the following two mechanisms: polymeric precipitation and neutralizing charges [47]. In polymeric precipitation, a soluble polymer, usually polyethylene glycol (PEG), is mixed with EV samples and the mixture is incubated overnight, and EVs are sedimented by low-speed centrifugation at 1500 g. PEG precipitation enables a simple process for a large number of samples. Commercial kits, such as ExoQuick (System Biosciences), total exosome isolation reagent (Invitrogen), EXO-Prep (HansaBioMed), exosome purification kit (Norgen Biotek), and miRCURY exosome isolation kit (Exiqon), are based on this principle. For the other precipitation method, all EVs possess negative charges, so positively charged molecules (i.e., sodium acetate and protamine) are chosen for the precipitation. This method is popular due to its straightforward protocol; however, these precipitation methods lead to low sEV purity due to co-precipitation of the components from CM or biofluids, such as protein, DNA and RNA, and hence further purification is required.