Abstract
Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma.
Highlights
Extracellular vesicles (EVs) are small, membrane-coated particles released by cells with heterogeneous morphological and functional properties
Based on their different release mechanisms and size, EVs can be categorized into small vesicles that arise from a fusion of multivesicular endosomes with the plasma membrane and larger vesicles that are released through direct budding of the plasma membrane or via yet unknown pathways involving multivesicular bodies [1]
By comparing the major separation approaches used, namely ultracentrifugation (UC), size exclusion chromatography (SEC), and immunoaffinity isolation (IP), we found that (i) all methods are suitable to isolate EVs; (ii) the yield of EVs differs considerably between the different methods; (iii) unexpectedly, the methods showed little differences in miRNA profile; (iv) UC-EVs were best suited for flow cytometric analysis; and (v) EVs isolated by IP are unsuitable for further protein analysis
Summary
Extracellular vesicles (EVs) are small, membrane-coated particles released by cells with heterogeneous morphological and functional properties. Based on their different release mechanisms and size, EVs can be categorized into small vesicles (small EVs) that arise from a fusion of multivesicular endosomes with the plasma membrane (sometimes termed “exosomes”) and larger vesicles that are released through direct budding of the plasma membrane or via yet unknown pathways involving multivesicular bodies (sometimes termed “microvesicles”) [1]. Despite attempts to set rigorous cut-off values for the size of these two types of EVs, some overlap exists. In oncology, EVs play a major role in the interaction between the tumor and the microenvironment and are involved in the regulation of cell proliferation, migration, immunosuppression, and angiogenesis [5–9]
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