Different isolation techniques for isolation of extracellular vesicles

Despite reports on several techniques there is currently no consensus for the optimal extracellular vesicle (EV) isolation method with respect to yield, quality, purity, and small patient plasma volumes. We compared four different techniques (precipitation (PP), size-exclusion (SEC), membrane affinity (MA), and ultracentrifugation (UCF)) for EV isolation from plasma. EV characterization included electron microscopy and nanoparticle tracking analysis. EV preparation quality and purity was assessed using protein quantification methods, flow cytometry, and Western Blotting. Purification was performed via ultrafiltration and Albumin out kit. Each tested method resulted in approximately the same particle size (115–147nm) with a similar amount of time. PP and MA showed the highest protein purity. SEC showed higher purity on electron microscopy and the highest values of exosome markers CD9 (99%), CD63 (82%), and CD81 (97%). Purification techniques did not further improve purity levels. In conclusion, SEC seems to be the most promising method for EV isolation. Nevertheless, certain applications require higher purity, so efforts should be made to improve isolation purity.

Publication History

Article published online:
17 May 2022