Characterisation of extracellular vesicles surface markers and co-expression studies with single particle interferometric imaging platform

Background & Aim Extracellular vesicle (EVs)-based therapeutics have been anticipated as an alternative to cell therapies based on the premise that EVs shed by stem cells exert similar therapeutic effects. A major challenge in EV research workflow is the lack of a platform that offers characterisation at the single EV level. In light of this, we tested and validated the single particle interferometric imaging using conditioned media in comparison against purified EVs obtained using ultracentrifugation and size exclusion chromatography. We performed multiplexed measurement of typical EV surface markers (e.g. tetraspanins) and simultaneously measured the size of antigen positive EVs. Methods, Results & Conclusion Human amniotic epithelial cells (hAECs) were cultured in serum free media for 96 hours. We prepared both unpurified (conditioned media, concentrated conditioned media) and purified EV samples (ultracentrifugation and size exclusion chromatography). Single particle interferometric imaging measurement was performed using the ExoView platform (Nanoview Biosciences). CD9, CD63 and CD81-positive EVs were immunocaptured on a tetraspanin microarray chip and imaged as single particles. Co-expression of tetraspanin proteins were then assessed by labeling the captured EVs with a cocktail of fluorescence antibodies conjugated to CD81-Alexa555, CD63-Alexa647 and CD9-Alexa488. The chips were then imaged with ExoView R100 reader and analysed using ExoViewer software with sizing thresholds set to 50-200nm diameter. The label free measurement used in this study captured particles based on their expression of classical EV markers (CD9/CD63/CD81) and provided specific EV particle characterisation down to a resolution of 50nm. Further labelling the captured EVs with fluorescence antibodies proved to increase sensitivity and include particles <50nm which were previously undetected by light scatter. This approach also provides co-expression data (Fig 1 and Fig 2). As hAECs are currently being used as cell therapy products in clinical trials and we are interested in the therapeutic potential of hAEC-derived EVs, a robust characterization method is needed to support EV research workflow. The ability of single particle interferometric imaging to probe distinctive hAEC-derived EV characteristics from the unpurified and purified EVs could streamline EV production. Extracellular vesicle (EVs)-based therapeutics have been anticipated as an alternative to cell therapies based on the premise that EVs shed by stem cells exert similar therapeutic effects. A major challenge in EV research workflow is the lack of a platform that offers characterisation at the single EV level. In light of this, we tested and validated the single particle interferometric imaging using conditioned media in comparison against purified EVs obtained using ultracentrifugation and size exclusion chromatography. We performed multiplexed measurement of typical EV surface markers (e.g. tetraspanins) and simultaneously measured the size of antigen positive EVs. Human amniotic epithelial cells (hAECs) were cultured in serum free media for 96 hours. We prepared both unpurified (conditioned media, concentrated conditioned media) and purified EV samples (ultracentrifugation and size exclusion chromatography). Single particle interferometric imaging measurement was performed using the ExoView platform (Nanoview Biosciences). CD9, CD63 and CD81-positive EVs were immunocaptured on a tetraspanin microarray chip and imaged as single particles. Co-expression of tetraspanin proteins were then assessed by labeling the captured EVs with a cocktail of fluorescence antibodies conjugated to CD81-Alexa555, CD63-Alexa647 and CD9-Alexa488. The chips were then imaged with ExoView R100 reader and analysed using ExoViewer software with sizing thresholds set to 50-200nm diameter.