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      Lab-on-Chip for Exosomes and Microvesicles Detection and Characterization

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          Abstract

          Interest in extracellular vesicles and in particular microvesicles and exosomes, which are constitutively produced by cells, is on the rise for their huge potential as biomarkers in a high number of disorders and pathologies as they are considered as carriers of information among cells, as well as being responsible for the spreading of diseases. Current methods of analysis of microvesicles and exosomes do not fulfill the requirements for their in-depth investigation and the complete exploitation of their diagnostic and prognostic value. Lab-on-chip methods have the potential and capabilities to bridge this gap and the technology is mature enough to provide all the necessary steps for a completely automated analysis of extracellular vesicles in body fluids. In this paper we provide an overview of the biological role of extracellular vesicles, standard biochemical methods of analysis and their limits, and a survey of lab-on-chip methods that are able to meet the needs of a deeper exploitation of these biological entities to drive their use in common clinical practice.

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          Most cited references147

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          ExoCarta: A Web-Based Compendium of Exosomal Cargo.

          Exosomes are membranous vesicles that are released by a variety of cells into the extracellular microenvironment and are implicated in intercellular communication. As exosomes contain RNA, proteins and lipids, there is a significant interest in characterizing the molecular cargo of exosomes. Here, we describe ExoCarta (http://www.exocarta.org), a manually curated Web-based compendium of exosomal proteins, RNAs and lipids. Since its inception, the database has been highly accessed (>54,000 visitors from 135 countries). The current version of ExoCarta hosts 41,860 proteins, >7540 RNA and 1116 lipid molecules from more than 286 exosomal studies annotated with International Society for Extracellular Vesicles minimal experimental requirements for definition of extracellular vesicles. Besides, ExoCarta features dynamic protein-protein interaction networks and biological pathways of exosomal proteins. Users can download most often identified exosomal proteins based on the number of studies. The downloaded files can further be imported directly into FunRich (http://www.funrich.org) tool for additional functional enrichment and interaction network analysis.
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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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              Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer.

              Exosomes are small vesicles (50-150 nm) of endocytic origin that are released by many different cell types. Exosomes in the tumor microenvironment may play a key role in facilitating cell-cell communication. Exosomes are reported to predominantly contain RNA and proteins. In this study, we investigated whether exosomes from pancreatic cancer cells and serum from patients with pancreatic ductal adenocarcinoma contain genomic DNA. Our results provide evidence that exosomes contain >10-kb fragments of double-stranded genomic DNA. Mutations in KRAS and p53 can be detected using genomic DNA from exosomes derived from pancreatic cancer cell lines and serum from patients with pancreatic cancer. In addition, using whole genome sequencing, we demonstrate that serum exosomes from patients with pancreatic cancer contain genomic DNA spanning all chromosomes. These results indicate that serum-derived exosomes can be used to determine genomic DNA mutations for cancer prediction, treatment, and therapy resistance.
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                Author and article information

                Journal
                Sensors (Basel)
                Sensors (Basel)
                sensors
                Sensors (Basel, Switzerland)
                MDPI
                1424-8220
                20 September 2018
                October 2018
                : 18
                : 10
                : 3175
                Affiliations
                [1 ]CNR NANOTEC Institute of Nanotechnology, via Monteroni, 73100 Lecce, Italy; monica.bianco@ 123456nanotec.cnr.it (M.B.); elisabetta.primiceri@ 123456nanotec.cnr.it (E.P.); francesco.ferrara@ 123456st.com (F.F.); valentina.arima@ 123456nanotec.cnr.it (V.A.); giuseppe.maruccio@ 123456unisalento.it (G.M.)
                [2 ]Institute of Experimental Neurology, Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy; nigro.annamaria@ 123456hsr.it (A.N.); romano.alessandro@ 123456hsr.it (A.R.); quattrini.angelo@ 123456hsr.it (A.Q.); furlan.roberto@ 123456hsr.it (R.F.)
                [3 ]STMicroelectronics, Via Monteroni, I-73100 Lecce, Italy
                [4 ]Department of Mathematics and Physics, University of Salento, via Monteroni, 73100 Lecce, Italy
                Author notes
                Author information
                https://orcid.org/0000-0002-7195-267X
                https://orcid.org/0000-0002-1791-7232
                https://orcid.org/0000-0002-7290-9542
                https://orcid.org/0000-0002-8226-8320
                https://orcid.org/0000-0001-7376-9425
                https://orcid.org/0000-0002-3429-8365
                https://orcid.org/0000-0001-7669-0253
                Article
                sensors-18-03175
                10.3390/s18103175
                6210978
                30241303
                4036e44a-264c-4c21-8fb2-83a94471df3d
                © 2018 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 08 August 2018
                : 16 September 2018
                Categories
                Review

                Biomedical engineering
                lab-on-chip,microfabrication,microfluidics,biosensors,extracellular vesicles,exosomes

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