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

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

Technical approaches to reduce interference of Fetal calf serum derived RNA in the analysis of extracellular vesicle RNA from cultured cells

Foetal calf serum (FCS) is a common supplement of cell culture medium and a known source of contaminating extracellular vesicles (EV) containing RNA. Because of a high degree of sequence similarity among homologous non-coding RNAs of mammalian species, residual FCS-RNA in culture medium may interfere in the analysis of EV-RNA released by cultured cells. Recently, doubts have been raised as to whether commonly used protocols for depletion of FCS-EV efficiently remove FCS-RNA. Moreover, technical details in FCS-EV depletion protocols are known to vary between labs, which may lead to inter-study differences in contaminating FCS-RNA levels. Here, we investigated how technical modifications of EV-depletion protocols affect the efficiency with which bovine RNAs are depleted from FCS, and determined the contribution of contaminating bovine RNA to EV-RNA purified from cell cultures. Our data show differences in depletion efficiency between and within various classes of small non-coding RNA. Importantly, we demonstrate that variations in FCS-EV depletion protocols affect both the quantity and type of residual FCS-RNAs in EV-depleted medium. By using optimised FCS-EV depletion protocols combined with methods for high-grade purification of EV the levels of contaminating bovine RNA in EV populations isolated from cell culture medium can be reduced. With illustrative datasets we also demonstrate that the abundance of a specific RNA in cell culture EV can only be determined if measured relative to background levels of this RNA in medium control samples. These data highlight the need for optimisation and validation of existing and novel FCS-EV depletion methods and urge for accurate descriptions of these methods in publications to increase experimental reproducibility