The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications.