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      Ultrafiltration and size exclusion chromatography combined with asymmetrical-flow field-flow fractionation for the isolation and characterisation of extracellular vesicles from urine

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          ABSTRACT

          Extracellular vesicles (EVs) have a great potential in clinical applications. However, their isolation from different bodily fluids and their characterisation are currently not optimal or standardised. Here, we report the results of examining the performance of ultrafiltration combined with size exclusion chromatography (UF-SEC) to isolate EVs from urine. The results reveal that UF-SEC is an efficient method and provides high purity. Furthermore, we introduce asymmetrical-flow field-flow fractionation coupled with a UV detector and multi-angle light-scattering detector (AF4/UV-MALS) as a characterisation method and compare it with current methods. We demonstrate that AF4/UV-MALS is a straightforward and reproducible method for determining size, amount and purity of isolated urinary EVs.

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          Most cited references18

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          Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics.

          Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the "gold-standard" Folch or Bligh and Dyer recipes.
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            Characterization and comprehensive proteome profiling of exosomes secreted by hepatocytes.

            Exosomes represent a discrete population of vesicles that are secreted from various cell types to the extracellular media. Their protein and lipid composition are a consequence of sorting events at the level of the multivesicular body, a central organelle which integrates endocytic and secretory pathways. Characterization of exosomes from different biological samples has shown the presence of common as well as cell-type specific proteins. Remarkably, the protein content of the exosomes is modified upon pathological or stress conditions. Hepatocytes play a central role in the body response to stress metabolizing potentially harmful endogenous substances as well as xenobiotics. In the present study, we described and characterized for the first time exosome secretion in nontumoral hepatocytes, and with the use of a systematic proteomic approach, we establish the first extensive proteome of a hepatocyte-derived exosome population which should be useful in furthering our understanding of the hepatic function and in the identification of components that may serve as biomarkers for hepatic alterations. Our analysis identifies a significant number of proteins previously described among exosomes derived from others cell types as well as proteins involved in metabolizing lipoproteins, endogenous compounds and xenobiotics, not previously described in exosomes. Furthermore, we demonstrated that exosomal membrane proteins can constitute an interesting tool to express nonexosomal proteins into exosomes with therapeutic purposes.
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              Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles.

              The research field of extracellular vesicles (EVs), such as microparticles and exosomes, is growing exponentially. The goal of this review is to provide an overview of recent developments relevant to the readers of the Journal of Thrombosis and Haemostasis. We will discuss nomenclature, the presence of EVs in fluids, methods of isolation and detection, and emerging clinical implications. Although research on EVs has been performed within the ISTH for over a decade, most of the recent research on EVs has been brought together by the International Society on Extracellular Vesicles (ISEV). To achieve an overview of recent developments, the information provided in this review comes not only from publications, but also from latest meetings of the ISEV (April 2015, Washington, DC, USA), the International Society on Advancement of Cytometry (June 2015, Glasgow, UK), and the ISTH (June 2015, Toronto, Canada).
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                Author and article information

                Journal
                J Extracell Vesicles
                J Extracell Vesicles
                ZJEV
                zjev20
                Journal of Extracellular Vesicles
                Taylor & Francis
                2001-3078
                2018
                03 July 2018
                : 7
                : 1
                : 1490143
                Affiliations
                [a ]Sustainable Health Department, Flemish Institute for Technological Research (VITO), Mol , Belgium
                [b ]Centre for Proteomics (CFP), University of Antwerp , Antwerp, Belgium
                [c ]Pharmafluidics , Gent, Belgium
                [d ]Phase I – Early Clinical Trials Unit, Oncology Department, Antwerp University Hospital (UZA) & Edegem & Center for Oncological Research, University of Antwerp , Antwerp, Belgium
                [e ]Pathological Anatomy Department, Antwerp University Hospital (UZA) , Edegem, Belgium
                [f ]Department of Clinical Oncology, Queen Elizabeth Hospital , Kowloon, Hong Kong
                Author notes
                CONTACT Inge Mertens inge.mertens@ 123456vito.be
                Author information
                http://orcid.org/0000-0003-3227-7250
                http://orcid.org/0000-0003-1870-6389
                http://orcid.org/0000-0002-0661-931X
                http://orcid.org/0000-0002-6252-7065
                http://orcid.org/0000-0002-5109-0267
                http://orcid.org/0000-0002-8553-1921
                http://orcid.org/0000-0002-7178-0185
                http://orcid.org/0000-0003-1524-9389
                http://orcid.org/0000-0003-4174-4586
                http://orcid.org/0000-0002-4888-3485
                Article
                1490143
                10.1080/20013078.2018.1490143
                6032024
                29988836
                9e6ffc34-8983-45ea-8be1-14af4b7f7388
                © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 10 October 2017
                : 13 June 2018
                Page count
                Figures: 8, Tables: 2, References: 43, Pages: 13
                Funding
                Funded by: Vlaamse Instelling voor Technologisch Onderzoek 10.13039/501100007610
                This work was supported by the Flemish Institute for Technological Research (VITO), Belgium.
                Categories
                Research Article

                extracellular vesicles,small extracellular vesicles,exosomes,urine,isolation,size exclusion chromatography,asymmetrical-flow field-flow fractionation

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