Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis
TL;DR: The purpose of this review is to not only introduce the different types of extracellular vesicles but also to summarize their differences and similarities, and discuss different methods of exosome isolation and analysis currently used.
Abstract: The use of extracellular vesicles, specifically exosomes, as carriers of biomarkers in extracellular spaces has been well demonstrated Despite their promising potential, the use of exosomes in the clinical setting is restricted due to the lack of standardization in exosome isolation and analysis methods The purpose of this review is to not only introduce the different types of extracellular vesicles but also to summarize their differences and similarities, and discuss different methods of exosome isolation and analysis currently used A thorough understanding of the isolation and analysis methods currently being used could lead to some standardization in the field of exosomal research, allowing the use of exosomes in the clinical setting to become a reality
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TL;DR: In this article, the authors summarized the most up-to-date know-kedge about the complex biological journey of exosomes from biogenesis and secretion, transport and uptake to their intracellular signalling.
Abstract: The use of exosomes in clinical settings is progressively becoming a reality, as clinical trials testing exosomes for diagnostic and therapeutic applications are generating remarkable interest from the scientific community and investors. Exosomes are small extracellular vesicles secreted by all cell types playing intercellular communication roles in health and disease by transferring cellular cargoes such as functional proteins, metabolites and nucleic acids to recipient cells. An in-depth understanding of exosome biology is therefore essential to ensure clinical development of exosome based investigational therapeutic products. Here we summarise the most up-to-date knowkedge about the complex biological journey of exosomes from biogenesis and secretion, transport and uptake to their intracellular signalling. We delineate the major pathways and molecular players that influence each step of exosome physiology, highlighting the routes of interest, which will be of benefit to exosome manipulation and engineering. We highlight the main controversies in the field of exosome research: their adequate definition, characterisation and biogenesis at plasma membrane. We also delineate the most common identified pitfalls affecting exosome research and development. Unravelling exosome physiology is key to their ultimate progression towards clinical applications. Video Abstract.
388 citations
TL;DR: A panoramic view of current exosome isolation techniques is provided, providing perspectives toward the development of novel approaches for high-efficient exosomes isolation from various types of biological matrices.
Abstract: Exosomes are small extracellular vesicles with diameters of 30-150 nm. In both physiological and pathological conditions, nearly all types of cells can release exosomes, which play important roles in cell communication and epigenetic regulation by transporting crucial protein and genetic materials such as miRNA, mRNA, and DNA. Consequently, exosome-based disease diagnosis and therapeutic methods have been intensively investigated. However, as in any natural science field, the in-depth investigation of exosomes relies heavily on technological advances. Historically, the two main technical hindrances that have restricted the basic and applied researches of exosomes include, first, how to simplify the extraction and improve the yield of exosomes and, second, how to effectively distinguish exosomes from other extracellular vesicles, especially functional microvesicles. Over the past few decades, although a standardized exosome isolation method has still not become available, a number of techniques have been established through exploration of the biochemical and physicochemical features of exosomes. In this work, by comprehensively analyzing the progresses in exosome separation strategies, we provide a panoramic view of current exosome isolation techniques, providing perspectives toward the development of novel approaches for high-efficient exosome isolation from various types of biological matrices. In addition, from the perspective of exosome-based diagnosis and therapeutics, we emphasize the issue of quantitative exosome and microvesicle separation.
386 citations
TL;DR: This book discusses exosome function in the tumor microenvironment and cancer progression, and the role of exosomes in this environment and its role in cancer progression.
304 citations
TL;DR: The status of the field is presented, the interest of the research community in these molecules is increasing as their role in extracellular vesicles is starting to be acknowledged, and what is needed is described.
242 citations
TL;DR: In comparison with their donor cells, MSC-derived exosomes offer more stable entities and diminished safety risks regarding the administration of live cells, e.g. microvasculature occlusion risk.
Abstract: Mesenchymal stem cells (MSCs) have captured great attention in regenerative medicine for over a few decades by virtue of their differentiation capacity, potent immunomodulatory properties, and their ability to be favorably cultured and manipulated. Recent investigations implied that the pleiotropic effects of MSCs is not associated to their ability of differentiation, but rather is mediated by the secretion of soluble paracrine factors. Exosomes, nanoscale extracellular vesicles, are one of these paracrine mediators. Exosomes transfer functional cargos like miRNA and mRNA molecules, peptides, proteins, cytokines and lipids from MSCs to the recipient cells. Exosomes participate in intercellular communication events and contribute to the healing of injured or diseased tissues and organs. Studies reported that exosomes alone are responsible for the therapeutic effects of MSCs in numerous experimental models. Therefore, MSC-derived exosomes can be manipulated and applied to establish a novel cell-free therapeutic approach for treatment of a variety of diseases including heart, kidney, liver, immune and neurological diseases, and cutaneous wound healing. In comparison with their donor cells, MSC-derived exosomes offer more stable entities and diminished safety risks regarding the administration of live cells, e.g. microvasculature occlusion risk. This review discusses the exosome isolation methods invented and utilized in the clinical setting thus far and presents a summary of current information on MSC exosomes in translational medicine.
170 citations
Cites background or methods from "Overview of Extracellular Vesicles,..."
...The concentrated exosome aliquot is then subjected to a short UC at ∼100,000 g and resuspended in PBS for further analysis [41]....
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...Finally, exosomes are retrievd by a long (60–120 min) ultracentrifugation (UC) step at 100,000–200,000 g and subsequent washing of the pellet in PBS; b rate-zonal ultracentrifugation (RZUC): RZUC is a type of density gradient UC (DGUC) where sample is placed at the surface of a gradient density medium such as sucrose, and following a step of UC at 100,000 g, sample components migrate through the gradient density and separate according to their size and shape; c isopycnic ultracentrifugation (IPUC): IPUC is another type of DGUC that separates particles based on their density....
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...Finally, exosomes are retrieved by UC at approximately 100,000–120,000 g for 60–120 min and subsequent washing in a proper medium like phosphate buffered saline (PBS) [28]....
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...Further purification is then performed by 0.22 microfiltration and elimination of apoptotic bodies through centrifugation at 10,000 g. Finally, exosomes are retrieved by UC at approximately 100,000–120,000 g for 60–120 min and subsequent washing in a proper medium like phosphate buffered saline (PBS) [28]....
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...However, the gradient construction in this strategy was extremely time-consuming and further precaution was required to inhibit the gradient damage during acceleration and deceleration step [28]....
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References
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TL;DR: Apoptosis seems to be involved in cell turnover in many healthy adult tissues and is responsible for focal elimination of cells during normal embryonic development, and participates in at least some types of therapeutically induced tumour regression.
Abstract: The term apoptosis is proposed for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations. Its morphological features suggest that it is an active, inherently programmed phenomenon, and it has been shown that it can be initiated or inhibited by a variety of environmental stimuli, both physiological and pathological.The structural changes take place in two discrete stages. The first comprises nuclear and cytoplasmic condensation and breaking up of the cell into a number of membrane-bound, ultrastructurally well-preserved fragments. In the second stage these apoptotic bodies are shed from epithelial-lined surfaces or are taken up by other cells, where they undergo a series of changes resembling in vitro autolysis within phagosomes, and are rapidly degraded by lysosomal enzymes derived from the ingesting cells.Apoptosis seems to be involved in cell turnover in many healthy adult tissues and is responsible for focal elimination of cells during normal embryonic development. It occurs spontaneously in untreated malignant neoplasms, and participates in at least some types of therapeutically induced tumour regression. It is implicated in both physiological involution and atrophy of various tissues and organs. It can also be triggered by noxious agents, both in the embryo and adult animal.
15,416 citations
"Overview of Extracellular Vesicles,..." refers background in this paper
...Unlike exosomes and MVs, apoptotic bodies contain intact organelles, chromatin, and small amounts of glycosylated proteins [3,48,60,99]....
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TL;DR: This review focuses on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function.
Abstract: Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and RNA. Deficiencies in our knowledge of the molecular mechanisms for EV formation and lack of methods to interfere with the packaging of cargo or with vesicle release, however, still hamper identification of their physiological relevance in vivo. In this review, we focus on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function.
6,141 citations
"Overview of Extracellular Vesicles,..." refers background in this paper
...NTA is capable of determining particle size between 10 and 1000 nm in diameter, which is within the size of exosomes which are known to be between 50–150 nm [5,137]....
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...The factors that determine the fate of a specific MVB are not well understood [5]....
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...While there is a broad range of potential applications and uses of exosomes in the clinical setting, more standardized methods for exosome isolation and analysis are needed in order to meet the regulatory requirements of the FDA and other regulatory agencies to use exosomes as biomarkers, vaccines, drug delivery devices, and therapeutic tools [5]....
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...Thus, the challenge remains to develop isolation techniques that can differentiate the different types of EVs in the extracellular matrix and do so rapidly, efficiently, reproducibly, and in a clinically friendly manner [5]....
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...The primary focus of this review will be on the protein content of EVs, however, the nucleic acid and lipid composition of EVs is well described in [1,2,5] and [6–8], respectively....
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Clotilde Théry1, Kenneth W. Witwer2, Elena Aikawa3, María José Alcaraz4 +414 more•Institutions (209)
TL;DR: The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities, and a checklist is provided with summaries of key points.
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.
5,988 citations
University of Helsinki1, Semmelweis University2, University of Szeged3, Hungarian Academy of Sciences4, University of Palermo5, University of Porto6, Institute of Molecular Pathology and Immunology of the University of Porto7, Autonomous University of Barcelona8, Instituto de Biologia Molecular e Celular9, Ikerbasque10, Harvard University11, University of Duisburg-Essen12, Paracelsus Private Medical University of Salzburg13, Salk Institute for Biological Studies14, University of Colorado Denver15, Bilkent University16, Middle East Technical University17, University of Southern Denmark18, Statens Serum Institut19, Ghent University Hospital20, Oslo University Hospital21, University of Belgrade22, University of Ljubljana23, University of Mainz24, Finnish Red Cross25, University of Gothenburg26, Latvian Biomedical Research and Study centre27, University of Applied Sciences and Arts Northwestern Switzerland FHNW28, University of Valencia29, Centro Nacional de Investigaciones Cardiovasculares30, University of Freiburg31, Utrecht University32, Trinity College, Dublin33, Catalan Institution for Research and Advanced Studies34, University of Barcelona35, International University Of Catalonia36, Aarhus University Hospital37
TL;DR: A comprehensive overview of the current understanding of the physiological roles of EVs is provided, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia.
Abstract: In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.
3,690 citations
"Overview of Extracellular Vesicles,..." refers background or methods in this paper
...Further, it has been demonstrated that the proteomic profiles of EVs from the same source are dependent on their isolation method [2]....
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...Extracellular vesicles (EVs) are lipid bound vesicles secreted by cells into the extracellular space [1,2]....
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...However, substantial overlap of protein profiles is often observed, due in part to the lack of standardized isolation and analysis methods of EVs [2,12]....
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...The focus of this review will remain on the proteome of MVs, however, the glycome of MVs is thoroughly discussed in [2]....
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...Originally, it was thought that, like exosomes, MVs were a cellular dumping or maintenance mechanism, by which the cell would get rid of unwanted material [2]....
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TL;DR: It is shown that exosomes—endogenous nano-vesicles that transport RNAs and proteins—can deliver short interfering (si)RNA to the brain in mice, and the therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA and protein knockdown of BACE1, a therapeutic target in Alzheimer's disease, in wild-type mice.
Abstract: To realize the therapeutic potential of RNA drugs, efficient, tissue-specific and nonimmunogenic delivery technologies must be developed. Here we show that exosomes-endogenous nano-vesicles that transport RNAs and proteins-can deliver short interfering (si)RNA to the brain in mice. To reduce immunogenicity, we used self-derived dendritic cells for exosome production. Targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide. Purified exosomes were loaded with exogenous siRNA by electroporation. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and non-specific uptake in other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease, in wild-type mice.
3,442 citations
"Overview of Extracellular Vesicles,..." refers background in this paper
...Also, because of these inherent advantages of exosomes, they are also ideal for the development of drug delivery systems [79]....
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