Abstract 443: Cushioned-Density Gradient Ultracentrifugation (C-DGUC) Improves the Isolation Efficiency of Exosomes for their use in Atherosclerosis Research

Background and Purpose: Ultracentrifugation (UC) is recognized as a robust approach for the isolation of exosomes especially when combined with a second step that involves density gradient ultracentrifugation (DGUC). However, recent studies have highlighted limitations associated with the use of UC including low recovery efficiencies and possible aggregation of exosomes. Such effects could subsequently impact on downstream assessments of exosome function in biological systems. Methods: We tested the benefit of using a liquid cushion of iodixanol during the first UC step to improve the yield of exosomes that are concentrated from the conditioned media (CM) of J774.1 murine macrophages in a method we recently termed Cushioned(C)-DGUC. We also compared the yield and purity of exosomes isolated by C-DGUC with those isolated by first subjecting CM to two other forms of concentration that included: ultrafiltration (UF) and polyethylene glycol (PEG) sedimentation prior to DGUC. Results: Our data show that the concentration step largely determines the yield and purity of exosomes isolated following the second DGUC step. The use of a high-density iodixanol cushion in cushioned-UC (C-UC) led to a threefold improvement in exosome yield over conventional UC. Although subjecting the CM to UF resulted in a similar exosome recovery efficiency, it retained eight-fold more soluble proteins than C-UC method. Strikingly, PEG precipitation of the CM generated a substantial number of non-exosomal nanoparticles, which could not be efficiently eliminated by the DGUC step. Western blot analysis reproducibly detected exosome markers CD-81, TSG101 and Alix in fractions 6 and 7. Finally, an in vitro assay of exosome-mediated microRNA delivery confirmed that C-DGUC provided the highest yield of functional exosomes. Conclusions: Collectively, our data demonstrate that the use of a high-density liquid cushion of iodixanol during the concentration step of C-DGUC substantially improves the yield and purity of exosomes derived from cell culture media. This approach can therefore facilitate functional studies of cell-derived exosomes in atherosclerosis.