Background: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited.Methods: We examined the effect of applying differential centrifugation g‐forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin.Results: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g‐force which also removed most contaminating microsomes. Optimal vesicle‐to‐protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics.Conclusions: Specific g‐force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines.

Background: Exosomes have been implicated in tumour progression and metastatic spread. Little is known of the effect of mechanical and innate immune interactions of malignant cell‐derived exosomes on endothelial integrity, which may relate to increased extravasation of circulating tumour cells and, therefore, increased metastatic spread.Methods: Exosomes isolated from non‐malignant immortalized HCV‐29 and isogenic malignant non‐metastatic T24 and malignant metastatic FL3 bladder cells were characterized by nanoparticle tracking analysis and quantitative nanomechanical mapping atomic force microscopy (QNM AFM) to determine size and nanomechanical properties. Effect of HCV‐29, T24 and FL3 exosomes on human umbilical vein endothelial cell (HUVEC) monolayer integrity was determined by transendothelial electrical resistance (TEER) measurements and transport was determined by flow cytometry. Complement activation studies in human serum of malignant and non‐malignant cell‐derived exosomes were performed.Results: FL3, T24 and HCV‐29 cells produced exosomes at similar concentration per cell (6.64, 6.61 and 6.46×10⁴ exosomes per cell for FL3, T24 and HCV‐29 cells, respectively) and of similar size (120.2 nm for FL3, 127.6 nm for T24 and 117.9 nm for HCV‐29, respectively). T24 and FL3 cell‐derived exosomes exhibited a markedly reduced stiffness, 95 MPa and 280 MPa, respectively, compared with 1,527 MPa with non‐malignant HCV‐29 cell‐derived exosomes determined by QNM AFM. FL3 and T24 exosomes induced endothelial disruption as measured by a decrease in TEER in HUVEC monolayers, whereas no effect was observed for HCV‐29 derived exosomes. FL3 and T24 exosomes traffic more readily (11.6 and 21.4% of applied exosomes, respectively) across HUVEC monolayers than HCV‐29 derived exosomes (7.2% of applied exosomes). Malignant cell‐derived exosomes activated complement through calcium‐sensitive pathways in a concentration‐dependent manner.Conclusions: Malignant (metastatic and non‐metastatic) cell line exosomes display a markedly reduced stiffness and adhesion but an increased complement activation compared to non‐malignant cell line exosomes, which may explain the observed increased endothelial monolayer disruption and transendothelial transport of these vesicles.

In the last two decades, extracellular vesicles (EVs) from the three domains of life, Archaea, Bacteria and Eukaryotes, have gained increasing scientific attention. As such, the role of EVs in host-pathogen communication and immune modulation are being intensely investigated. Pivotal to EV research is the determination of how and where EVs are taken up by recipient cells and organs in vivo, which requires suitable tracking strategies including labelling. Labelling of EVs is often performed post-isolation which increases risks of non-specific labelling and the introduction of labelling artefacts. Here we exploited the inability of helminths to de novo synthesise fatty acids to enable labelling of EVs by whole organism uptake of fluorescent lipid analogues and the subsequent incorporation in EVs. We showed uptake of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-Rho) in Anisakis spp. and Trichuris suis larvae. EVs isolated from the supernatant of Anisakis spp. labelled with DOPE-Rho were characterised to assess the effects of labelling on size, structure and fluorescence of EVs. Fluorescent EVs were successfully taken up by the human macrophage cell line THP-1. This study, therefore, presents a novel staining method that can be utilized by the EV field in parasitology and potentially across multiple species.