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.

Breast milk is a complex liquid with immune-competent cells and soluble proteins that provide immunity to the infant and affect the maturation of the infant’s immune system. Exosomes are nanovesicles (30–100 nm) with an endosome-derived limiting membrane secreted by a diverse range of cell types. Because exosomes carry immunorelevant structures, they are suggested to participate in directing the immune response. We hypothesized that human breast milk contain exosomes, which may be important for the development of the infant’s immune system. We isolated vesicles from the human colostrum and mature breast milk by ultracentrifugations and/or immuno-isolation on paramagnetic beads. We found that the vesicles displayed a typical exosome-like size and morphology as analyzed by electron microscopy. Furthermore, they floated at a density between 1.10 and 1.18 g/ml in a sucrose gradient, corresponding to the known density of exosomes. In addition, MHC classes I and II, CD63, CD81, and CD86 were detected on the vesicles by flow cytometry. Western blot and mass spectrometry further confirmed the presence of several exosome-associated molecules. Functional analysis revealed that the vesicle preparation inhibited anti-CD3-induced IL-2 and IFN-γ production from allogeneic and autologous PBMC. In addition, an increased number of Foxp3⁺CD4⁺CD25⁺ T regulatory cells were observed in PBMC incubated with milk vesicle preparations. We conclude that human breast milk contains exosomes with the capacity to influence immune responses.

Background: Exosomes are 30-100 nm membrane vesicles of endocytic origin produced by numerous cells. They can mediate diverse biological functions, including antigen presentation. Exosomes have recently been shown to contain functional RNA, which can be delivered to other cells. Exosomes may thus mediate biological functions either by surface-to-surface interactions with cells, or by the delivery of functional RNA to cells. Our aim was therefore to determine the presence of RNA in exosomes from human saliva, plasma and breast milk and whether these exosomes can be taken up by macrophages. Method: Exosomes were purified from human saliva, plasma and breast milk using ultracentrifugation and filtration steps. Exosomes were detected by electron microscopy and examined by flow cytometry. Flow cytometry was performed by capturing the exosomes on anti-MHC class II coated beads, and further stain with anti-CD9, anti-CD63 or anti-CD81. Breast milk exosomes were further analysed for the presence of Hsc70, CD81 and calnexin by Western blot. Total RNA was detected with a Bioanalyzer and mRNA was identified by the synthesis of cDNA using an oligo (dT) primer and analysed with a Bioanalyzer. The uptake of PKH67-labelled saliva and breast milk exosomes by macrophages was examined by measuring fluorescence using flow cytometry and fluorescence microscopy. Results: RNA was detected in exosomes from all three body fluids. A portion of the detected RNA in plasma exosomes was characterised as mRNA. Our result extends the characterisation of exosomes in healthy humans and confirms the presence of RNA in human saliva and plasma exosomes and reports for the first time the presence of RNA in breast milk exosomes. Our results also show that the saliva and breast milk exosomes can be taken up by human macrophages. Conclusions: Exosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA. Furthermore, exosomes are readily taken up by macrophages, supporting the notion that exosomal RNA can be shuttled between cells.

Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs. Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery.

Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co‐isolated entities, a combination of methods is needed. However, this is time‐consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 μl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size‐exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC‐MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non‐EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non‐EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.

Exosomes are 30–100 nm diameter vesicles formed by inward budding of endosomal compartments and are produced by several cell types, including T‐cells, B‐cells and dendritic cells (DC)s. Exosomes from DCs express major histocompatibility complexes (MHC) class I and II, and co-stimulatory molecules on their surface, and can induce antigen-specific activation of T‐cells.The aims of the present study were to investigate for the presence of exosomes in bronchoalveolar lavage fluid (BALF) from healthy individuals, and to establish if these exosomes bear MHC and co-stimulatory molecules.The authors analysed BALF taken from seven healthy volunteers and used exosomes from monocyte-derived DC (MDDC) cultures as a reference. After ultracentrifugation, exosomes were bound to anti‐MHC class II coated magnetic beads and analysed by flow cytometry and electron microscopy.The authors report for the first time that exosomes are present in BALF. These exosomes are similar to MDDC derived exosomes as they express MHC class I and II, CD54, CD63 and the co-stimulatory molecule CD86.The results demonstrate that exosomes are present in the lung, and since they contain both major histocompatibility complex and co-stimulatory molecules it is likely that they are derived from antigen presenting cells and might have a regulatory role in local immune defence.