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.

Release of membrane vesicles, a process conserved in both prokaryotes and eukaryotes, represents an evolutionary link, and suggests essential functions of a dynamic extracellular vesicular compartment (including exosomes, microparticles or microvesicles and apoptotic bodies). Compelling evidence supports the significance of this compartment in a broad range of physiological and pathological processes. However, classification of membrane vesicles, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Here, we give a comprehensive overview of extracellular vesicles. After discussing the technical pitfalls and potential artifacts of the rapidly emerging field, we compare results from meta-analyses of published proteomic studies on membrane vesicles. We also summarize clinical implications of membrane vesicles. Lessons from this compartment challenge current paradigms concerning the mechanisms of intercellular communication and immune regulation. Furthermore, its clinical implementation may open new perspectives in translational medicine both in diagnostics and therapy.

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.

Introduction: In recent years, there has been an exponential increase in the number of studies aiming to understand the biology of exosomes, as well as other extracellular vesicles. However, classification of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross‐contamination.Method: EVs released from three different kinds of cell lines: HMC‐1, TF‐1 and BV‐2 were isolated using two centrifugation‐based protocols. In protocol 1, apoptotic bodies were collected at 2,000×g, followed by filtering the supernatant through 0.8 µm pores and pelleting of microvesicles at 12,200×g. In protocol 2, apoptotic bodies and microvesicles were collected together at 16,500×g, followed by filtering of the supernatant through 0.2 µm pores and pelleting of exosomes at 120,000×g. Extracellular vesicles were analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a Bioanalyzer^®.Results: RNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no RNA except for microvesicles collected from TF‐1 cell cultures. The different vesicle pellets showed highly different distribution of size, shape and electron density with typical apoptotic body, microvesicle and exosome characteristics when analyzed by transmission electron microscopy. Flow cytometry revealed the presence of CD63 and CD81 in all vesicles investigated, as well as CD9 except in the TF‐1‐derived vesicles, as these cells do not express CD9.Conclusions: Our results demonstrate that centrifugation‐based protocols are simple and fast systems to distinguish subpopulations of extracellular vesicles. Different vesicles show different RNA profiles and morphological characteristics, but they are indistinguishable using CD63‐coated beads for flow cytometry analysis.