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 timeconsuming 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 (Exo-Quick 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.
Extracellular vesicles (EV) are candidates for cancer immunotherapy because of their capacity to stimulate tumor-specific activity in vivo. However, clinical trials using peptide-loaded autologous EVs have so far only showed moderate T cell responses, suggesting a need for optimization of EV-induced immunity in humans. We previously demonstrated that induction of Ag-specific CD8 + T cells and antitumor responses to whole Ag were independent of MHC class I on EVs and hypothesized that multiple injections of allogeneic EVs could potentiate Ag-specific responses. In this study, we show that the allogeneic EV from mouse bone marrowderived dendritic cells enhances Ag-specific CD8 + T cell, follicular helper T cell, and Ag-specific Ab responses. EV-injected mice demonstrated Ag-specific memory after 4 mo, with the highest Ab avidity in mice receiving double allogeneic EV injections. Reduced B16mOVA melanoma tumor growth was shown in all EV-injected groups. Our findings support the application of allogeneic EVs for therapeutic use in clinical studies in which an adaptive immune response is desired.
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