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      Microfluidics-based on-a-chip systems for isolating and analysing extracellular vesicles

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          ABSTRACT

          Extracellular vesicles (EVs), which can be found in almost all body fluids, consist of a lipid bilayer enclosing proteins and nucleic acids from their cells of origin. EVs can transport their cargo to target cells and have therefore emerged as key players in intercellular communication. Their potential as either diagnostic and prognostic biomarkers or therapeutic drug delivery systems (DDSs) has generated considerable interest in recent years. However, conventional methods used to study EVs still have significant limitations including the time-consuming and low throughput techniques required, while at the same time the demand for better research tools is getting stronger and stronger. In the past few years, microfluidics-based technologies have gradually emerged and have come to play an essential role in the isolation, detection and analysis of EVs. Such technologies have several advantages, including low cost, low sample volumes, high throughput and precision. This review summarizes recent advances in microfluidics-based technologies, compares conventional and microfluidics-based technologies, and includes a brief survey of recent progress towards integrated “on-a-chip” systems. In addition, this review also discusses the potential clinical applications of “on-a-chip” systems, including both “liquid biopsies” for personalized medicine and DDS devices for precision medicine, and then anticipates the possible future participation of cloud-based portable disease diagnosis and monitoring systems, possibly with the participation of artificial intelligence (AI).

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          Most cited references65

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          Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication.

          Normal and malignant cells shed from their surface membranes as well as secrete from the endosomal membrane compartment circular membrane fragments called microvesicles (MV). MV that are released from viable cells are usually smaller in size compared to the apoptotic bodies derived from damaged cells and unlike them do not contain fragmented DNA. Growing experimental evidence indicates that MV are an underappreciated component of the cell environment and play an important pleiotropic role in many biological processes. Generally, MV are enriched in various bioactive molecules and may (i) directly stimulate cells as a kind of 'signaling complex', (ii) transfer membrane receptors, proteins, mRNA and organelles (e.g., mitochondria) between cells and finally (iii) deliver infectious agents into cells (e.g., human immuno deficiency virus, prions). In this review, we discuss the pleiotropic effects of MV that are important for communication between cells, as well as the role of MV in carcinogenesis, coagulation, immune responses and modulation of susceptibility/infectability of cells to retroviruses or prions.
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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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              Continuous particle separation through deterministic lateral displacement.

              We report on a microfluidic particle-separation device that makes use of the asymmetric bifurcation of laminar flow around obstacles. A particle chooses its path deterministically on the basis of its size. All particles of a given size follow equivalent migration paths, leading to high resolution. The microspheres of 0.8, 0.9, and 1.0 micrometers that were used to characterize the device were sorted in 40 seconds with a resolution of approximately 10 nanometers, which was better than the time and resolution of conventional flow techniques. Bacterial artificial chromosomes could be separated in 10 minutes with a resolution of approximately 12%.
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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
                20 August 2018
                : 7
                : 1
                : 1508271
                Affiliations
                [a ]Institute of Microsurgery on Extremities, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital , Shanghai, China
                [b ]Department of Orthopaedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital , Shanghai, China
                [c ]Faculty of Health and Life Science, Oxford Brookes University , Oxford, UK
                Author notes
                CONTACT Shi-Cong Tao jerrytao1990@ 123456outlook.com Department of Orthopaedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital , Shanghai200233, China
                Author information
                http://orcid.org/0000-0001-6285-6750
                http://orcid.org/0000-0002-8706-6138
                Article
                1508271
                10.1080/20013078.2018.1508271
                6104604
                30151077
                ac065953-addb-4ddc-9a66-9529d94a6873
                © 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
                : 02 April 2018
                : 25 July 2018
                : 13 June 2018
                Page count
                Figures: 4, References: 127, Pages: 16
                Funding
                Funded by: National Health and Family Planning Commission of the People’s Republic of China 10.13039/501100004572
                Award ID: 201402016
                Funded by: National Natural Science Foundation of China 10.13039/501100001809
                Award ID: 81301589
                Funded by: Joint Project Funding for Major Diseases in Shanghai
                Award ID: 2014ZYJB0301
                This work was supported by the National Natural Science Foundation of China [81802226, 81871834, 81301589 and 81472066].
                Categories
                Review Article

                extracellular vesicle,exosomes,biomarker,cell-free therapy,drug delivery system,dds,microfluidics,on-a-chip,protein,rna

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