Updating the MISEV minimal requirements for extracellular vesicle studies: building bridges to reproducibility

Abstract

On separation of EVs (also referred to as isolation, purification or concentration), MISEV2014 acknowledged that there is no consensus on an “optimal” method, such as ultracentrifugation, size exclusion or immunoaffinity. Instead, selection of a method should be guided by the relevant scientific question and downstream applications, and all details of the method should be provided to ensure reproducibility.\nMISEV2014 recommended that EVcharacterization be performed at both the population and single vesicle levels. For the former, both positive and negative protein marker characterization was endorsed, following four categories of proteins: (1) transmembrane or lipid-bound EV protein, (2) EV cytosolic protein, (3) intracellular but not associated with plasma membrane or endosomes (i.e. relatively less abundant in exosomal EVs than in cells) and (4) extracellular but not typically EV-associated proteins. Several examples of proteins in each category were provided.\nFor characterization of single vesicles, as a means to assess population heterogeneity, MISEV2014 called for the use of at least two different but complementary technologies. For example, electron or atomic force microscopy could be paired with one of the single particle tracking methods. Furthermore, close-up images should be accompanied by wide-field views to allow assessment of heterogeneity.\nFor EV functional studies, MISEV2014 stated that quantitation of EVs and their activity should include dose–response studies. Process controls should be included, since contaminants might contribute activity. Use of density gradients was encouraged to show that any observed activity is intrinsic to EVs and not to closely associated, commonly co-purifying particles; alternatively, this activity should be depletable, e.g. by immunodepletion of EVs. Labelling studies were also recommended.

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Affiliated Institutions

• University of Gothenburg SE
• Pohang University of Science and Technology KR
• Instituto de Ciências Farmacêuticas BR
• Johns Hopkins Medicine US
• University College London GB
• Southern Medical University CN
• La Trobe University AU
• Hiroshima University JP
• University of Colorado Boulder US
• Icahn School of Medicine at Mount Sinai US
• Cedars-Sinai Medical Center US
• University of Amsterdam NL
• Nanfang Hospital CN
• Ikerbasque ES
• Rhode Island Hospital US
• Sysmex (Japan) JP
• Utrecht University NL
• Inserm FR
• CIC bioGUNE ES
• Institut Curie FR
• QIMR Berghofer Medical Research Institute AU
• Universidade Federal de São Paulo BR
• Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas ES
• University of California, San Diego US
• Vanderbilt University US
• Tsinghua University CN
• Providence College US
• Semmelweis University HU
• Johns Hopkins University US
• Scintillon Institute US
• Université Paris Sciences et Lettres FR

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