Characterization of Urinary Exosomes Purified with Size Exclusion Chromatography and Ultracentrifugation

Abstract

Exosomes, a subtype of extracellular vesicles secreted by mammalian cells with a typical size range of 30–150 nm, have been implicated in many biological processes as intercellular communication carriers. The isolation of exosomes is an essential and challenging step before subsequent analysis and functional studies, due to the complexity of body fluids, as well as the small size and low density of exosomes. Ultracentrifugation (UC) and size exclusion chromatography (SEC) are two methods that have been extensively used for exosomes isolation in biological studies in recent years. In this work, we compared the characteristics of urinary exosomes extracted with SEC and UC methods in detail. Results showed that the SEC isolation method was superior to UC in the recovery of exosomal particles and proteins. The results of proteomics analysis showed that more purified exosomes were extracted with the SEC method. We also observed that parts of exosomes were ruptured and precipitated insufficiently during UC isolations. It not only led to a low recovery of exosome proteins but also resulted in a considerable loss of exosomal particles. Moreover, the exosomal rupture and particle loss in UC could not be avoided by resuspension of the exosomal particles. Our results also showed that exosomes from SEC purifications possessed a high internalization capability from 4 to 6 h when incubated with EA.hy926 and HCV29 cell lines.

A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?

“…The recovery and purity of SEC-based exosomal isolation is especially recognizable when compared to other isolation methods, as shown in comparative studies with PEG-based isolation and dUC and UF [ 93 , 97 , 98 ]. dUC has been reported to potentially damage exosomes and can alter the exosome proteome, lipome, and/or genome [ 50 ]; UF can lead to the deformation and break-up of larger vesicles due to the pressure and contact with filter membranes [ 19 ]; PEG can co-precipitate non-EV components and alter exosomal protein signatures [ 93 ].…”

Characterization of exosome-like vesicles derived from Taenia pisiformis cysticercus and their immunoregulatory role on macrophages

“…Database search parameters were set as follows: trypsin as enzyme; peptide mass tolerance of 20 ppm and fragment mass tolerance of 0.05 Da; + 1, + 2, + 3 as peptide charge; a maximum of one missed cleavage; carbamidomethyl (C), iTRAQ8plex (N-term), iTRAQ8plex (K) as fixed modifications and oxidation (M), Gln-> pyro-Glu (N-term Q), deamidated (NQ) as variable modifications. False discovery rate (FDR) lower than 0.01 was used as a screening condition [34,35].…”

Analysis of RNA yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation

“…Some authors have proposed that, given the great centrifugal force (100 000 g) required in order to obtain EV, DU is associated with increased damage to the integrity of EV structure when compared with other isolation methods [45,46]. This has been linked to a lower EV recovery [34,47].…”