Methods for Separation and Characterization of Extracellular Vesicles: Results of a Worldwide Survey Performed by the ISEV Rigor and Standardization Subcommittee

Abstract

Research on extracellular vesicles (EVs) is growing exponentially due to an increasing appreciation of EVs as disease biomarkers and therapeutics, an expanding number of EV-containing materials under study, and application of new preparation, detection, and cargo analysis methods. Diversity of both sources and methodologies imposes challenges on the comparison of measurement results between studies and laboratories. While reference guidelines and minimal requirements for EV research have achieved the important objective of assembling community consensus, it is also essential to understand which methodologies and quality controls are currently being applied, and how usage trends are evolving. As an initial response to this need, the International Society for Extracellular Vesicles (ISEV) performed a worldwide survey in 2015 on “Techniques used for the isolation and characterization of extracellular vesicles” and published the results from this survey in 2016. In 2019, a new survey was performed to assess the changing state of the field. The questionnaire received more than 600 full or partial responses, and the present manuscript summarizes the results of this second worldwide survey. The results emphasize that separation methods such as ultracentrifugation and density gradients are still the most commonly used methods, the use of size exclusion chromatography has increased, and techniques based on tangential flow and microfluidics are now being used by more than 10% of respondents. The survey also reveals that most EV researchers still do not perform sample quality controls before or after isolation of EVs. Finally, the majority of EV researchers emphasize that separation and characterization of EVs should receive more attention.

Rigor and standardization of extracellular vesicle research: Paving the road towards robustness

“…In 2015, ISEV members were surveyed on methods used for isolation and characterization of EVs, and the results were published in 2016 (Gardiner et al., 2016). A second 'pulse‐taking' of the community was implemented in 2019 and published by Rigor and Standardization (Royo et al., 2020). Although neither the questions nor the respondents were identical, and thus a perfect comparison cannot be made for most questions and answers, the results of the two surveys can nevertheless provide some insights into trends in EV research methodologies and goals.…”

“…While differential ultracentrifugation remains the workhorse of the field, its use appears to have declined slightly (Gardiner et al., 2016, Royo et al., 2020). In contrast, use of size exclusion chromatography has more than doubled, and gradient ultracentrifugation and affinity methods have also increased (Gardiner et al., 2016, Royo et al., 2020). Polymer‐based precipitation is used by more than 20% of respondents, although it is perhaps the 'dirtiest' method available unless combined with other approaches.…”

“…EV biomolecules of interest . The 2015 survey asked about 'downstream applications,' while the 2019 survey focused on molecules of interest (Gardiner et al., 2016, Royo et al., 2020). Nevertheless, we can make some comparisons.…”

“…EV characterization methods . Major techniques for EV characterization, such as Western blotting, single particle tracking, electron microscopy, and flow cytometry, appeared to maintain their relative ranks, roughly, from 2015 to 2019 (Gardiner et al., 2016, Royo et al., 2020). Importantly, though, respondents in 2019 reported using more–and more diverse–characterization methods.…”

“…Polymer-based precipitation is used by more than 20% of respondents, although it is perhaps the ' dirtiest' method available unless combined with other approaches. In 2019, the survey also provided options for microfluidics, field-flow, tangential flow for the first time (Royo et al, 2020).…”