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.

Trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas Disease, shed extracellular vesicles (EVs) enriched with glycoproteins of the gp85/trans‐sialidase (TS) superfamily and other α‐galactosyl (α‐Gal)‐containing glycoconjugates, such as mucins. Here, purified vesicles from T. cruzi strains (Y, Colombiana, CL‐14 and YuYu) were quantified according to size, intensity and concentration. Qualitative analysis revealed differences in their protein and α‐galactosyl contents. Later, those polymorphisms were evaluated in the modulation of immune responses (innate and in the chronic phase) in C57BL/6 mice. EVs isolated from YuYu and CL‐14 strains induced in macrophages higher levels of proinflammatory cytokines (TNF‐α and IL‐6) and nitric oxide via TLR2. In general, no differences were observed in MAPKs activation (p38, JNK and ERK 1/2) after EVs stimulation. In splenic cells derived from chronically infected mice, a different modulation pattern was observed, where Colombiana (followed by Y strain) EVs were more proinflammatory. This modulation was independent of the T. cruzi strain used in the mice infection. To test the functional importance of this modulation, the expression of intracellular cytokines after in vitro exposure was evaluated using EVs from YuYu and Colombiana strains. Both EVs induced cytokine production with the appearance of IL‐10 in the chronically infected mice. A high frequency of IL‐10 in CD4+ and CD8+ T lymphocytes was observed. A mixed profile of cytokine induction was observed in B cells with the production of TNF‐α and IL‐10. Finally, dendritic cells produced TNF‐α after stimulation with EVs. Polymorphisms in the vesicles surface may be determinant in the immunopathologic events not only in the early steps of infection but also in the chronic phase.

Cell culture‐conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM‐derived EVs (CCM‐EV). The CCM‐EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell‐specific recommendations may need to be established for non‐mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM‐EV research.

Trypanosoma cruzi, the aetiologic agent of Chagas disease, releases vesicles containing a wide range of surface molecules known to affect the host immunological responses and the cellular infectivity. Here, we compared the secretome of two distinct strains (Y and YuYu) of T. cruzi, which were previously shown to differentially modulate host innate and acquired immune responses. Tissue culture‐derived trypomastigotes of both strains secreted extracellular vesicles (EVs), as demonstrated by electron scanning microscopy. EVs were purified by exclusion chromatography or ultracentrifugation and quantitated using nanoparticle tracking analysis. Trypomastigotes from YuYu strain released higher number of EVs than those from Y strain, enriched with virulence factors trans‐sialidase (TS) and cruzipain. Proteomic analysis confirmed the increased abundance of proteins coded by the TS gene family, mucin‐like glycoproteins, and some typical exosomal proteins in the YuYu strain, which also showed considerable differences between purified EVs and vesicle‐free fraction as compared to the Y strain. To evaluate whether such differences were related to parasite infectivity, J774 macrophages and LLC‐MK2 kidney cells were preincubated with purified EVs from both strains and then infected with Y strain trypomastigotes. EVs released by YuYu strain caused a lower infection but higher intracellular proliferation in J774 macrophages than EVs from Y strain. In contrast, YuYu strain‐derived EVs caused higher infection of LLC‐MK2 cells than Y strain‐derived EVs. In conclusion, quantitative and qualitative differences in EVs and secreted proteins from different T. cruzi strains may correlate with infectivity/virulence during the host–parasite interaction.

Trypanosoma cruzi, Trypanosoma bruceiandLeishmania(Trypanosomatidae: Kinetoplastida) are parasitic protozoan causing Chagas disease, African Trypanosomiasis and Leishmaniases worldwide. They are vector borne diseases transmitted by triatomine bugs, Tsetse fly, and sand flies, respectively. Those diseases cause enormous economic losses and morbidity affecting not only rural and poverty areas but are also spreading to urban areas. During the parasite-host interaction, those organisms release extracellular vesicles (EVs) that are crucial for the immunomodulatory events triggered by the parasites. EVs are involved in cell-cell communication and can act as important pro-inflammatory mediators. Therefore, interface between EVs and host immune responses are crucial for the immunopathological events that those diseases exhibit. Additionally, EVs from these organisms have a role in the invertebrate hosts digestive tracts prior to parasite transmission. This review summarizes the available data on how EVs from those medically important trypanosomatids affect their interaction with vertebrate and invertebrate hosts.