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      Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

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          Abstract

          The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments.

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          Most cited references200

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            Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.

            Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, in this study we performed a comprehensive evaluation of current methods used for exosome isolation including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40-100nm vesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and Western blotting. We employed a proteomic approach to profile the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, we found IAC-Exos to be the most effective method to isolate exosomes. For example, Alix, TSG101, CD9 and CD81 were significantly higher (at least 2-fold) in IAC-Exos, compared to UG-Exos and DG-Exos. Application of immunoaffinity capture has enabled the identification of proteins including the ESCRT-III component VPS32C/CHMP4C, and the SNARE synaptobrevin 2 (VAMP2) in exosomes for the first time. Additionally, several cancer-related proteins were identified in IAC-Exos including various ephrins (EFNB1, EFNB2) and Eph receptors (EPHA2-8, EPHB1-4), and components involved in Wnt (CTNNB1, TNIK) and Ras (CRK, GRB2) signalling. Copyright © 2012 Elsevier Inc. All rights reserved.
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              Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates

              Purpose To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates. Methods Standard polystyrene beads of sizes ranging from 60 to 1,000 nm and physical mixtures thereof were analyzed with NTA and DLS. The influence of different ratios of particle populations was tested. Drug delivery nanoparticles and protein aggregates were analyzed by NTA and DLS. Live monitoring of heat-induced protein aggregation was performed with NTA. Results NTA was shown to accurately analyze the size distribution of monodisperse and polydisperse samples. Sample visualization and individual particle tracking are features that enable a thorough size distribution analysis. The presence of small amounts of large (1,000 nm) particles generally does not compromise the accuracy of NTA measurements, and a broad range of population ratios can easily be detected and accurately sized. NTA proved to be suitable to characterize drug delivery nanoparticles and protein aggregates, complementing DLS. Live monitoring of heat-induced protein aggregation provides information about aggregation kinetics and size of submicron aggregates. Conclusion NTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates.
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                Author and article information

                Journal
                J Extracell Vesicles
                J Extracell Vesicles
                JEV
                Journal of Extracellular Vesicles
                Co-Action Publishing
                2001-3078
                27 May 2013
                2013
                : 2
                : 10.3402/jev.v2i0.20360
                Affiliations
                [1 ]Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, MD, USA
                [2 ]Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary
                [3 ]Department of Biomedical Sciences, University of Minnesota Medical School Duluth, Duluth, MN, USA
                [4 ]Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA
                [5 ]Krefting Research Centre, Department of Internal Medicine and Clinical Nutrition, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
                [6 ]Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
                [7 ]Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH, USA
                [8 ]Massachusetts General Hospital Cancer Center, Boston, MA, USA
                [9 ]Exosome Diagnostics, Inc., New York, USA
                [10 ]INSERM, Cedex, Paris, France
                [11 ]Institut Curie, Paris, France
                Author notes
                [* ] Kenneth W. Witwer, 733 N. Broadway, Miller Research Building Rm 829, Baltimore, MD 21205, USA. Tel: +(011) 1-410-955-9770, Fax: +(011) 1-410-955-9823. Email: kwitwer1@ 123456jhmi.edu

                Except for KWW, EIB and FH, who initiated writing of this article, with KWW compiling the diverse contributions and finalizing the manuscript, all authors are listed in alphabetical order.

                Article
                20360
                10.3402/jev.v2i0.20360
                3760646
                24009894
                9e0b8e24-ebf7-41e6-bc56-e30cc4dc77e1
                © 2013 Kenneth W. Witwer et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 23 February 2013
                : 05 April 2013
                : 11 April 2013
                Categories
                An ISEV Position Paper

                extracellular vesicle,exosome,microvesicle,standardization,isolation

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