Ion exchange chromatography for small extracellular vesicles isolation v1

In the last few years, extracellular vesicles have become of great interest due to its potential as biomarkers, drug delivery systems and, in particular, as therapeutic agents. However, there is no consensus on which is the best way to isolate these vesicles. The choice of the isolation method depends on the starting material (i.e conditioned culture media, urine, serum, ecc) and their downstream applications. Even though there are numerous methods to isolate sEV, few of them are compatible with clinical applications, as they are not scalable. In the present work, we set up a protocol to isolate sEV from conditioned culture media by ion exchange chromatography, which is a simple, fast and scalable method, suitable for clinical production of sEV. We performed the isolation using an anion exchange resin (Q sepharose) and eluted the sEV using 500mM NaCl. We characterized the elution profile by measuring protein and lipid concentration and CD63 by ELISA. Moreover, we immunophenotyped all the eluted fractions, evaluated the presence of TSG101, calnexin and cytochrome C by western blot and analysed nanoparticle size and distribution by NTA and tRPS and morphology by TEM. Finally, we evaluated the immunomodulatory activity in vitro. We found that most sEV are eluted and concentrated in fraction 4, with a mean size <150nm, which are positive for CD9, CD63, CD81 and TSG101, while most proteins are eluted in fraction 5. Moreover, sEV in fraction 4 exerted an anti-inflammatory activity on LPS-stimulated macrophages. In summary, we set up a scalable and clinically compatible method to chromatographically isolate small extracellular vesicles, from conditioned culture media, that retain their biological activity.