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

Accompanying the growing awareness that extracellular vesicles (EVs) are useful for diagnostics and therapeutics is the realization that EV applications must be established with rigor, reproducibility, and standardization. The small size and heterogeneity of most EVs are well-known barriers to rigorous studies. However, progress is also hampered by the largely unknown influence of a host of pre-analytical variables and a lack of quality controls, differences in EV separation and characterization techniques (van Deun et al., 2017), a general lack of dedicated reference materials and standards (Welsh et al., 2020), and analyses that are poorly defined and thus irreproducible. The goal of this Editorial is to summarize the past, present, and future contributions of the International Society for Extracellular Vesicles (ISEV) to improving rigor and standardization in EV research. We highlight in particular the Rigor and Standardization Subcommittee and the recently published results of a survey on the methods used for separation and characterization of EVs (Royo, Théry, Falcón-Pérez, Nieuwland, & Witwer, 2020). Major challenges remain. Even so, we predict that within a few years, the expansion of an ISEV infrastructure dedicated to rigor and standardization will enable us to turn the ugly duckling of current challenges into a beautiful swan of comparable and reproducible EVmeasurement results. Because we are working in a new and developing field of research, ISEV has the unique opportunity to set high standards for rigor, and we may even lead the way for other research fields and societies. Rigor and standardizationwith community input have been part of the ISEVmission since its founding. Impetus for founding a society arosewith a 2011meeting in Paris that was organized byClotilde Théry andGraçaRaposo. The incipient community voted to emphasize ’extracellular vesicles’ in the name of the society rather than limiting focus to specific subtypes of EVs. Following the first annual meeting of ISEV in 2012, the first ISEV Workshop was organized and held in October, 2012 in New York City. This Workshop gave rise to the first two ISEV position papers, on standardization of sample collection and processing (Witwer et al., 2013) and RNA analysis (Hill et al., 2013). Subsequent workshops and co-sponsored events have given birth to additional position papers and other products, most of them published in the Journal of Extracellular Vesicles (JEV) (Lötvall et al., 2012; Théry, Gho, & Quesenberry, 2019), that provide guidance on topics ranging from non-mammalian EVs (Soares et al., 2017) to EV RNA (Mateescu et al., 2017) and membranes (Russell et al., 2019) to biomarker considerations (Clayton et al., 2018) and therapeutic uses of EVs (Lener et al., 2015; Reiner et al., 2017). In 2014, the first position statement was published on the ’Minimal experimental requirements for definition of extracellular vesicles’ (MISEV) (Lötvall et al., 26913). Following a community guidance survey (Witwer et al., 2017) and a series of intensive information-gathering surveys and exchanges with the ISEV board, MISEV was updated as the 382-author ’MISEV2018’ (Théry et al., 2018). The goal of these manuscripts has not changed over the years, because it was and still is a challenge to purify and detect EVs, and to attribute specific biological cargo and functions to EVs. Working towards rigor and standardization, ISEV is not alone. Over the years, formal and informal collaborations with other societies and groups have coalesced. An early development was the founding of the Extracellular Vesicle Flow CytometryWorking Group (www.evflowcytometry.org), which first met at the ISEV2015 annual meeting. Members of ISEV, ISTH (International Society on Thrombosis andHaemostasis) and ISAC (International Society for Advancement of Cytometry) have prepared guidelines to improve the reproducibility of EV detection by flow cytometry, culminating in the 2020 position paper ’MiFlowCyt-EV: a framework for standardized reporting of extracellular vesicles flow cytometry experiments’ (Welsh et al., 2020). Additional papers are planned, including an educationalmanuscript about ’do’s and don’t’s’ of EV flow cytometry. A guidelines publication was produced by several ISEV Board of Directors (BoD) members with members of ISTH and the American Heart Association (AHA) (Coumans et al., 2017). ISEVhasworkedwith groups such as SOCRATES and the International Society for Cell andGeneTherapy (ISCT) on issues of clinical translation of EVs (Börger et al., 2020; Witwer et al., 2019). Importantly, the EV-TRACK consortium and knowledgebase, led by the Hendrix group, was launched to improve transparent reporting of EV research and is endorsed by ISEV/JEV (van Deun et al., 2017).