Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations

Abstract

Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples.

Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy

“…The diversity of the mechanisms that lead to the release of small particles from cells contributes to the heterogeneity of the isolated material, which often contains populations of submicron particles of variable compositions and morphologies. Nowadays, it is well known that the field of EV studies suffers from poor reproducibility and inadequacy in terms of the separation of (and appropriate assignation of specific effects to) vesicles, lipoproteins, large protein complexes/aggregates, and other assemblies of biomolecules with similar/overlapping size and physico-chemical properties [ 26 ]. The system-interfering analytics and indirectness of the evidence also limit the relevance of the studies from the perspective of pursuing natural pathways (in vivo occurring processes).…”

“…Due to the small size of EVs and their heterogeneity in terms of size and composition, the quantification of EVs is still a challenge [ 23 , 24 ]. In addition, the properties of the isolated material strongly depend on the method of isolation [ 25 , 26 , 27 , 28 ]. Even when the same type of equipment and protocols are used, the reproducibility of isolation tends to be poor [ 22 , 29 ].…”

Exosomal Components and Modulators in Colorectal Cancer: Novel Diagnosis and Prognosis Biomarkers

“…As Allelein et al confirmed, heterogenous populations vary greatly. This phenomenon must exist in clinical samples [144]. In order to overcome these bottlenecks, we suggest that more studies need to sort out detailed information, including source cell type, extraction method, purity, modified vesicle type, etc.…”

The Important Role of Endothelium and Extracellular Vesicles in the Cellular Mechanism of Aortic Aneurysm Formation

“…The vesicles size is varied and ranges from 30 nm to 5 μm. Although EVs have a similar structure, there are major differences regarding the mechanism of their formation and physicochemical properties [ 80 , 81 , 82 , 83 ]. Apart from the density, isolation methods, or the presence of specific markers, the size and diameter of the vesicles are the most common EVs classification criteria.…”

“…The mechanism of initiation and the course of migration depends on many complex processes, among which migration is the most important. It plays a key role in embryonic development but is also responsible for the movement of immune cells, wound healing, and tissue regeneration [ 80 , 81 , 82 , 83 , 84 , 85 , 86 ]. Cell movement is also a pivotal factor during the formation of tumor metastases and in the course of inflammation.…”