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      Small extracellular vesicles isolation and separation: Current techniques, pending questions and clinical applications

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          Abstract

          Extracellular vesicles, especially small extracellular vesicles (sEVs) are now accepted as important messengers in cell-to-cell communication and as a promising drug delivery platform. They are involved in nearly all physiological and pathological processes and are involved in disease diagnosis and therapy. However, their heterogeneity of physicochemical properties and functions is not fully understood, which hinders further clinical applications. To obtain highly bioactive sEVs with both high yield and purity, will certainly facilitate their future study and application. This review informs up-to-date research on frequently-used and cutting-edge technologies of sEVs isolation and makes a deep comparison and analysis of different methods, including their advantages, limitations and applications. Pending questions about the inherent property of these small vesicles as well as isolation strategies are discussed. Additionally, an overview of their applications in disease diagnosis and treatment, including some of the on-going clinical trials, are also reviewed.

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          Most cited references198

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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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            The biology, function, and biomedical applications of exosomes

            The study of extracellular vesicles (EVs) has the potential to identify unknown cellular and molecular mechanisms in intercellular communication and in organ homeostasis and disease. Exosomes, with an average diameter of ~100 nanometers, are a subset of EVs. The biogenesis of exosomes involves their origin in endosomes, and subsequent interactions with other intracellular vesicles and organelles generate the final content of the exosomes. Their diverse constituents include nucleic acids, proteins, lipids, amino acids, and metabolites, which can reflect their cell of origin. In various diseases, exosomes offer a window into altered cellular or tissue states, and their detection in biological fluids potentially offers a multicomponent diagnostic readout. The efficient exchange of cellular components through exosomes can inform their applied use in designing exosome-based therapeutics.
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              Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.

              Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called "exosomal shuttle RNA" (esRNA).
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                Author and article information

                Journal
                Theranostics
                Theranostics
                thno
                Theranostics
                Ivyspring International Publisher (Sydney )
                1838-7640
                2022
                6 September 2022
                : 12
                : 15
                : 6548-6575
                Affiliations
                [1 ]Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, Institute of Stem Cells, School of Medicine, Jiangsu University, Zhenjiang, China.
                [2 ]Department of Clinical Laboratory, The Affiliated Yixing Hospital of Jiangsu University, Yixing, China.
                [3 ]Aoyang Institute of Cancer, Affiliated Aoyang Hospital of Jiangsu University, 279 Jingang Road, Suzhou, Jiangsu, China.
                Author notes
                ✉ Corresponding authors: Shi Hui, E-mail: shihui@ 123456ujs.edu.cn ; Sun Yaoxiang, E-mail: staff2037@ 123456yxph.com ; Qian Hui, E-mail: lstmmmlst@ 123456163.com .

                #These authors contributed equally to this work.

                Competing Interests: The authors have declared that no competing interest exists.

                Article
                thnov12p6548
                10.7150/thno.74305
                9516236
                36185597
                0571295a-5554-4969-bf1e-f386283f118e
                © The author(s)

                This is an open access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.

                History
                : 22 April 2022
                : 25 August 2022
                Categories
                Review

                Molecular medicine
                small extracellular vesicles,exosomes,isolation techniques,clinical applications

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