Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the “protein corona” of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery.

One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC‐3 and VCaP, in conventional cell culture dishes and in two‐chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition‐dependent differences by non‐targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor‐derived EVs (BR‐EVs) and the EVs obtained from cells grown in conventional cell cultures (C‐EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non‐polar metabolites when the BR‐EVs were compared to the C‐EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects.

Cell-secreted extracellular vesicles (EVs) have rapidly gained prominence as sources of biomarkers for non-invasive biopsies, owing to their ubiquity across human biofluids and physiological stability. There are many characterisation studies directed towards their protein, nucleic acid, lipid and glycan content, but more recently the metabolomic analysis of EV content has also gained traction. Several EV metabolite biomarker candidates have been identified across a range of diseases, including liver disease and cancers of the prostate and pancreas. Beyond clinical applications, metabolomics has also elucidated possible mechanisms of action underlying EV function, such as the arginase-mediated relaxation of pulmonary arteries or the delivery of nutrients to tumours by vesicles. However, whilst the value of EV metabolomics is clear, there are challenges inherent to working with these entities—particularly in relation to sample production and preparation. The biomolecular composition of EVs is known to change drastically depending on the isolation method used, and recent evidence has demonstrated that changes in cell culture systems impact upon the metabolome of the resulting EVs. This review aims to collect recent advances in the EV metabolomics field whilst also introducing researchers interested in this area to practical pitfalls in applying metabolomics to EV studies.

Background/Aim: Tumor-secreted extracellular vesicles (EVs) play an important role as mediators of intercellular communication. Hypoxia is a common feature of solid tumors frequently associated with an aggressive clinical behavior. This study aimed to gain a deeper understanding into the functions of EVs in intercellular communication between primary and metastatic cancer cells under hypoxic conditions. Materials and Methods: EVs were isolated from two isogenic colorectal cancer (CRC) cell lines SW480 and SW620 cultured under normoxic and hypoxic conditions. Their uptake and effects in SW480 and SW620 cells were studied using EV uptake, proliferation, spheroid-formation, wound healing and invasion assays. Results: Our data showed that hypoxia enhanced the release of EVs from CRC cells in a Hypoxia Induced Factor (HIF)-1-dependent manner. Hypoxic EVs were taken up by CRC cells more efficiently than normoxic EVs. Hypoxic EVs stimulated motility, invasiveness and stemness of primary tumour-derived SW480 cells, whereas they had a little effect on metastasis-derived SW620 cells. Conclusion: Hypoxic colorectal cancer-derived EVs confer aggressiveness and invasiveness to hypoxia-naïve cancer cells.

Renowned for their role in haemostasis and thrombosis, platelets are also increasingly recognized for their contribution in innate immunity, immunothrombosis and inflammatory diseases. Platelets express a wide range of receptors, which allows them to reach a variety of activation endpoints and grants them immunomodulatory functions. Activated platelets release extracellular vesicles (PEVs), whose formation and molecular cargo has been shown to depend on receptor‐mediated activation and environmental cues.This study compared the immunomodulatory profiles of PEVs generated via activation of platelets by different receptors, glycoprotein VI, C‐type lectin‐like receptor 2 and combining all thrombin‐collagen receptors. Functional assays in vivo in zebrafish and in vitro in human macrophages highlighted distinct homing and secretory responses triggered by the PEVs. In contrast, omics analyses of protein and miRNA cargo combined with physicochemical particle characterization found only subtle differences between the activated PEV types, which were insufficient to predict their different immunomodulatory functions. In contrast, constitutively released PEVs, formed in the absence of an exogenous activator, displayed a distinct immunomodulatory profile from the receptor‐induced PEVs.Our findings underscore that PEVs are tunable through receptor‐mediated activation. To truly comprehend their role(s) in mediating platelet functions among immune cells, conducting functional assays is imperative.