Characterization of extracellular vesicles isolated by size exclusion chromatography

Abstract

Introduction: In a search to decipher the role extracellular vesicles (EVs) play on pathological processes, novel methods for its detection and isolation are of paramount importance. Recently, at the ISEV meeting in Budapest, Rienk Nieuwland inferred that size exclusion chromatography (SEC) holds promise as a simple and efficient method for the isolation of plasma vesicles. Therefore, the aim of our study is to adopt a multifactorial platform to validate the potential of SEC to isolate pure plasma EVs in clinical samples. Methods: Platelet-free plasma (PFP) from 5 healthy donors were applied to a Sepharose CL-2B column and separated by size exclusion chromography. The eluated fractions (F) were then analysed by: (a) nanoparticle tracking analysis (NTA) for total vesicle concentration, (b) quantitative mass spectroscopy followed by functional annotation of the identified proteins, (c) extracellular vesicle (EV) array for capturing and phenotyping, (d) transmission electron microscopy (TEM) for morphology and (e) silver staining of SDS-PAGE gels, lipoprotein levels, protein and RNA to estimate purity. Results: We report that for fractions 710, a high particle count with a concomitant drop in protein and lipoprotein concentration is noted. Conversely, for fractions 1725, we observe a 50% reduction in particle count by means of NTA with an increased total protein concentration. In addition, SEC-purified EVs retained RNA concentration and morphology. Furthermore, TEM demonstrated that SEC holds the potential of removing lipoprotein particles from plasma. EV array indicated that pooled fractions 1725 exhibited higher EV content than fractions 710, although phenotypical details remained unchanged compared to control plasma. Mass spectroscopy revealed elevated apolipoprotein levels in the fractions 710 and augumented levels of EVs in fractions 1720. Summary/conclusion: Our results accentuate that this method holds potential in the research field of EVs. Interestingly, our EV array and MS data suggests that a prerequisite for preserving the authenticity of NTA data is SEC purification of plasma samples, as it allows for removal of lipoproteins.