Impact of Isolation Techniques on the Content of Small Extracellular Vesicles

ABSTRACT An important topic of discussion amongst the extracellular vesicle (EV) research field is which genetic materials are considered true constituents of EV cargo. What were once regarded as non‐EV components have now evolved to be potentially essential to EV composition, serving as key mediators in communication. Researchers continue to pursue varying methods for the isolation of EVs with the desired content. However, the desired content depends on the source and the requirements of the intended downstream use of EVs. Different isolation methods can modify EV cargo, impacting functional EV‐induced effects and analysis of EV contents. Ensuring that any result produced is truly representative of EVs and not of co‐isolated materials is imperative. Here, we describe a side‐by‐side comparison of the outcomes of EV isolation from plasma of healthy individuals, using size exclusion chromatography and an ultrafast filtration system. Characterisation of EVs was performed by particle (nanoparticle tracking analysis), protein (Bradford assay) and lipid (sulfo‐phospho‐vanillin assay) quantification, morphology visualisation (transmission electron microscopy), microRNA expression (miRNA sequencing) and assessment of protein absence or presence (traditional and capillary‐based western blot analyses, ELISA and mass spectrometry). To our surprise, the isolation methods employed identified significant differences in the protein abundance and composition of the resulting EVs. SEC isolated plasma EVs with high abundance of EV transmembrane and cytosolic markers, as well as corona‐related complement, fibrinogen and extracellular matrix proteins. However, this was accompanied by high quantities of non‐EV immunoglobulins. In contrast, UFF isolated plasma EVs with high abundance of EV integrins and corona‐related complement proteins, albeit with high lipoprotein abundance. Each isolation approach produces EVs with different biomolecular properties, which might provide specific advantages and disadvantages, depending on the intended use of EVs. Therefore, isolation methods need to be tested intensively and selected carefully for downstream applications.