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      Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single‐particle analysis platforms


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          We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single‐particle interferometric reflectance imaging sensing (SP‐IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP‐IRIS events could not be used to estimate particle concentrations. For sizing, SP‐IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP‐IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP‐IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single‐particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

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          Most cited references48

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          Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

          ABSTRACT 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.
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            Biological properties of extracellular vesicles and their physiological functions

            In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.
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              EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research

              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.

                Author and article information

                J Extracell Vesicles
                J Extracell Vesicles
                Journal of Extracellular Vesicles
                John Wiley and Sons Inc. (Hoboken )
                06 April 2021
                April 2021
                : 10
                : 6 ( doiID: 10.1002/jev2.v10.6 )
                [ 1 ] Department of Molecular and Comparative Pathobiology Johns Hopkins University School of Medicine Baltimore Maryland USA
                [ 2 ] Department of Urology Johns Hopkins University School of Medicine Baltimore Maryland USA
                [ 3 ] Department of Cell Biology Johns Hopkins University School of Medicine Baltimore Maryland USA
                [ 4 ] Department of Neurology Johns Hopkins University School of Medicine Baltimore Maryland USA
                [ 5 ] Johns Hopkins Drug Discovery Johns Hopkins University School of Medicine Baltimore Maryland USA
                [ 6 ] Department of Epidemiology Johns Hopkins University Bloomberg School of Public Health Baltimore Maryland USA
                [ 7 ] Faculty of Science Universidad de la República Montevideo Uruguay
                [ 8 ] Functional Genomics Unit Institut Pasteur de Montevideo Montevideo Uruguay
                [ 9 ] Department of Nutrition University of California Davis Davis California USA
                [ 10 ] Bioprocess Measurements Group National Institute of Standards and Technology Gaithersburg Maryland USA
                [ 11 ] Center for Nanomedicine at the Wilmer Eye Institute Johns Hopkins University School of Medicine Baltimore Maryland USA
                [ 12 ] The Richman Family Precision Medicine Center of Excellence in Alzheimer's Disease Johns Hopkins University School of Medicine Johns Hopkins Medicine and Johns Hopkins Bayview Medical Center Baltimore Maryland USA
                Author notes
                [*] [* ] Correspondence

                Kenneth W. Witwer, 733 North Broadway, Miller Research Building, Room 827, Baltimore, MD 21205.

                Email: kwitwer1@ 123456jhmi.edu

                © 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 6, Tables: 2, Pages: 20, Words: 11766
                Funded by: National Institute on Drug Abuse , open-funder-registry 10.13039/100000026;
                Award ID: R01DA040385
                Award ID: R01DA047807
                Funded by: National Institute of Mental Health , open-funder-registry 10.13039/100000025;
                Award ID: R21/R33MH118164
                Funded by: National Institute of Allergy and Infectious Diseases , open-funder-registry 10.13039/100000060;
                Award ID: R01AI144997
                Funded by: National Cancer Institute , open-funder-registry 10.13039/100000054;
                Award ID: UG3CA241694
                Funded by: Michael J. Fox Foundation , open-funder-registry 10.13039/100000864;
                Award ID: 00900821
                Funded by: Richman Family Precision Medicine Center of Excellence in Alzheimer's Disease at Johns Hopkins Medicine
                Research Article
                Research Articles
                Custom metadata
                April 2021
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.0.1 mode:remove_FC converted:06.04.2021

                ectosomes,exosomes,extracellular vesicles,microvesicles,nanoflow cytometry,nanoparticle tracking analysis,resistive pulse sensing,single particle interferometric reflectance imaging sensing


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