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

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

Biological properties of extracellular vesicles and their physiological functions

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

Biological properties of extracellular vesicles and their physiological functions

María Yáñez-Mó#, Pia R.-M. Siljander#, Zoraida Andreu, Apolonija Bedina Zavec, Francesc E. Borràs, Edit I. Buzas, Krisztina Buzas, Enriqueta Casal, Francesco Cappello, Joana Carvalho, Eva Colás, Anabela Cordeiro-da Silva, Stefano Fais, Juan M. Falcon-Perez, Irene M. Ghobrial, Bernd Giebel, Mario Gimona, Michael Graner, Ihsan Gursel, Mayda Gursel, Niels H. H. Heegaard, An Hendrix30, Peter Kierulf, Katsutoshi Kokubun, Maja Kosanovic, Veronika Kralj-Iglic, Eva-Maria Krämer-Albers, Saara Laitinen, Cecilia Lässer, Thomas Lener, Erzsébet Ligeti, Aija Linē, Georg Lipps, Alicia Llorente, Jan Lötvall, Mateja Manček-Keber, Antonio Marcilla, Maria Mittelbrunn, Irina Nazarenko, Esther N.M. Nolte-‘t Hoen, Tuula A. Nyman, Lorraine O'Driscoll, Mireia Olivan, Carla Oliveira, Éva Pállinger, Hernando A. del Portillo, Jaume Reventós, Marina Rigau, Eva Rohde, Marei Sammar, Francisco Sánchez-Madrid, N. Santarém1, Katharina Schallmoser, Marie Stampe Ostenfeld, Willem Stoorvogel, Roman Stukelj, Susanne G. Van der Grein, M. Helena Vasconcelos, Marca H. M. Wauben and Olivier De WeverIn 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.Peer reviewe

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

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

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Erratum: Intestinal cell barrier function in vitro is severely compromised by keratin 8 and 18 mutations identified in patients with inflammatory bowel disease (PLoS ONE (2014) 9, 6 (e99398) DOI: 10.1371/journal.pone.0099398)

10.1371/journal.pone.0104122PLoS ONE97e10412

Intestinal cell barrier function in vitro is severely compromised by keratin 8 and 18 mutations identified in patients with inflammatory bowel disease

10.1371/journal.pone.0099398PLoS ONE96e9939

Characterization of Nanohybridosomes from Lipids and Spruce Homogenate Containing Extracellular Vesicles

Vesna Spasovski,1,2,* Anna Romolo,1,3,* Urška Zagorc,4 Vesna Arrigler,4 Matic Kisovec,5 Apolonija Bedina Zavec,5 Matevž Arko,1 Adrienn Molnár,6,7 Gitta Schlosser,7 Aleš Iglič,3,8 Ksenija Kogej,4 Veronika Kralj-Iglič1 1University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia; 2Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia; 3University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Physics, Ljubljana, Slovenia; 4University of Ljubljana, Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia; 5National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia; 6Hevesy György PhD School of Chemistry, ELTE Eötvös Loránd University, Budapest, Hungary; 7MTA-ELTE Lendület Ion Mobility Mass Spectrometry Research Group, Faculty of Science, Institute of Chemistry, ELTE Eötvös Loránd University, Budapest, Hungary; 8University of Ljubljana, Faculty of Medicine, Laboratory of Clinical Biophysics, Ljubljana, Slovenia*These authors contributed equally to this workCorrespondence: Veronika Kralj-Iglič, Email [email protected]: Lipid nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called nano-sized hybridosomes or nanohybridosomes, NSHs) were considered.Methods: We formed NSHs by mixing appropriate amounts of lecithin, glycerol and supernatant of isolation of extracellular vesicles from spruce needle homogenate. We visualized NSHs by light microscopy and cryogenic transmission electron microscopy and assessed them by flow cytometry, dynamic light scattering, ultraviolet–visual spectroscopy, interferometric light microscopy and liquid chromatography–mass spectrometry.Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry and interferometric light microscopy measurements showed that the majority of the particles were nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well on the average hydrodynamic radius of the particles Rh (between 140 and 180 nm), while the concentrations of the particles were in the range between 1013 and 1014/mL indicating that NSHs present a considerable (more than 25%) of the sample which is much more than the yield of natural extracellular vesicles (EVs) from spruce needle homogenate (estimated less than 1%). Spruce specific lipids and proteins were found in hybridosomes.Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient formation procedure suggest that large-scale production of NSHs from lipids and spruce needle homogenate is feasible.Plain Language Summary: Cells shed into their exterior nanoparticles (here referred to as extracellular vesicles – EVs) that are free to move, reach distant cells and are taken up by them. As they carry bioactive constituents, EVs may have important impact on the recipient cells. The mechanisms of EV formation and mediation can be employed in designing therapeutic, prophylactic and diagnostic methods for various medical issues. EVs can be harvested from biological samples; however, their yield is small,12 and there are potential side effects. Artificial vesicles – liposomes – have high yield; however, in vivo, they can be degraded before reaching the target and their reproducibility is yet insufficient. In order to combine advantages of both types of nanoparticles, we have composed nanohybridosomes (NSHs) from soya lecithin, water and supernatant of isolation of EVs from spruce needle homogenate, visualized them by cryogenic electron microscopy and characterized them with respect to their size, concentration and protein/nucleic acid content. We have applied a recently developed interferometric light microscopy to determine the hydrodynamic radius and the concentration of EVs. We found that the majority of composed particles are nano-sized and that they enclose more than 25% of the incoming volume of liquid, which is considerably more than about 1% that can be harvested by isolation of EVs from spruce needle homogenate by (ultra)centrifugation.Keywords: hybridosomes, liposomes, nanovesicles, extracellular particles, small cellular particles, drug deliver

Nanoparticles isolated from blood: a reflection of vesiculability of blood cells during the isolation process

Vid Šuštar1, Apolonija Bedina-Zavec1,2, Roman Štukelj1, Mojca Frank3, Goran Bobojevic1, Rado Janša4, Eva Ogorevc5, Peter Kruljc6, Keriya Mam7, Boštjan Šimunic8, Mateja Mancek-Keber9, Roman Jerala9, Blaž Rozman3, Peter Veranic10, Henry Hägerstrand11, Veronika Kralj-Iglic11Laboratory of Clinical Biophysics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; 2Laboratory of Biosynthesis and Biotransformation, National Institute of Chemistry, Ljubljana, Slovenia; 3Departments of Rheumatology; 4Gastroenterology, Ljubljana University Medical Centre, Ljubljana, Slovenia; 5Laboratory of Biophysics, Faculty of Electrical Engineering; 6Clinics for Reproduction and Horses, Faculty of Veterinary Medicine, University of Ljubljana, Ljubljana, Slovenia; 7FEI Quanta, Eindhoven, The Netherlands; 8Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana, Slovenia; 9University of Primorska, Science and Research Centre of Koper, Koper, Slovenia; 10Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; 11Department of Biosciences, Biocity, Åbo Akademi University, Åbo/Turku, FinlandBackground: Shedding of nanoparticles from the cell membrane is a common process in all cells. These nanoparticles are present in body fluids and can be harvested by isolation. To collect circulating nanoparticles from blood, a standard procedure consisting of repeated centrifugation and washing is applied to the blood samples. Nanoparticles can also be shed from blood cells during the isolation process, so it is unclear whether nanoparticles found in the isolated material are present in blood at sampling or if are they created from the blood cells during the isolation process. We addressed this question by determination of the morphology and identity of nanoparticles harvested from blood.Methods: The isolates were visualized by scanning electron microscopy, analyzed by flow cytometry, and nanoparticle shapes were determined theoretically.Results: The average size of nanoparticles was about 300 nm, and numerous residual blood cells were found in the isolates. The shapes of nanoparticles corresponded to the theoretical shapes obtained by minimization of the membrane free energy, indicating that these nanoparticles can be identified as vesicles. The concentration and size of nanoparticles in blood isolates was sensitive to the temperature during isolation. We demonstrated that at lower temperatures, the nanoparticle concentration was higher, while the nanoparticles were on average smaller.Conclusion: These results indicate that a large pool of nanoparticles is produced after blood sampling. The shapes of deformed blood cells found in the isolates indicate how fragmentation of blood cells may take place. The results show that the contents of isolates reflect the properties of blood cells and their interaction with the surrounding solution (rather than representing only nanoparticles present in blood at sampling) which differ in different diseases and may therefore present a relevant clinical parameter.Keywords: nanoparticles, nanovesicles, microparticles, microvesicles, cell–cell communicatio