Date Published: April 11, 2019
Publisher: Public Library of Science
Author(s): Phat Duong, Allen Chung, Laura Bouchareychas, Robert L. Raffai, Ronald Hancock.
Ultracentrifugation (UC) is recognized as a robust approach for the isolation of extracellular vesicles (EVs). However, recent studies have highlighted limitations of UC including low recovery efficiencies and aggregation of EVs that could impact downstream functional analyses. We tested the benefit of using a liquid cushion of iodixanol during UC to address such shortcomings. In this study, we compared the yield and purity of EVs isolated from J774A.1 macrophage conditioned media by conventional UC and cushioned-UC (C-UC). We extended our study to include two other common EV isolation approaches: ultrafiltration (UF) and polyethylene glycol (PEG) sedimentation. After concentrating EVs using these four methods, the concentrates underwent further purification by using OptiPrep density gradient ultracentrifugation (DGUC). Our data show that C-DGUC provides a two-fold improvement in EV recovery over conventional UC-DGUC. We also found that UF-DGUC retained ten-fold more protein while PEG-DGUC achieved similar performance in nanoparticle and protein recovery compared to C-DGUC. Regarding purity as assessed by nanoparticle to protein ratio, our data show that EVs isolated by UC-DGUC achieved the highest purity while C-DGUC and PEG-DGUC led to similarly pure preparations. Collectively, we demonstrate that the use of a high-density iodixanol cushion during the initial concentration step improves the yield of EVs derived from cell culture media compared to conventional UC. This enhanced yield without substantial retention of protein contaminants and without exposure to forces causing aggregation offers new opportunities for the isolation of EVs that can subsequently be used for functional studies.
Extracellular vesicles (EVs), including those referred to as exosomes, are membrane-enclosed microparticles abundantly present in body fluids and are thought to be secreted by all cell types . Recent observations of RNA  and metabolite  exchange via EVs have led to a boom into their research [4, 5]. Although the exact nature of their biogenesis and function remains incompletely understood, EVs are recognized as intercellular messengers in health and disease . Moreover, EVs are potentially an ideal source of diagnostic biomarkers and delivery vehicles for therapeutic applications [6, 7]. Although there are growing interests in EV biology, progress in this field is hampered by variability and inconsistencies in reports of their function . The source of such biological noise has been proposed to include from their mode of isolation [8, 9]. There is thus a need for the development of new methodologies that can generate highly pure, intact EVs to improve rigor and reproducibility of experiments amongst different laboratories [5, 10–12].
Ever since their early report, including that by Rose Johnstone and colleagues , investigators have increasingly sought to understand the biological properties of EVs. Seminal findings by Valadi et al.  demonstrating the intercellular transfer of RNA by EVs, specifically exosomes, opened a new avenue of research centered on these tiny cellular particles in diverse physiological systems [4, 5]. However, the small size of many EVs including exosomes, ranging from 50 to 150 nm has imposed numerous technical hurdles for their isolation and study. The most popular method remains the one set up by Thery et al.  that makes use of ultracentrifugation to concentrate EVs and other microparticles in a pellet that is then resuspended for downstream applications. Because UC is limited in scale, cumbersome, and requires costly equipment numerous other traditional protein and virus concentration methods have been adopted for concentrating these vesicles. Ultrafiltration is a well-established method making use of membrane separation based on molecular size. When using this method, EVs and molecules more massive than the membrane pores are retained and concentrated . The use of polyethylene glycol, which long served as a fractional precipitating agent for virus  and lipoprotein sedimentation from plasma , has also gained popularity due to its rapid and simple-to-use nature. Because polyethylene glycol displays high solubility in water, EVs and macromolecules are sterically excluded from the solvent and can be concentrated by low-speed centrifugation .