Mesenchymal stem cell exosome characterisation and high-throughput quantification by fluorescence polarisation spectroscopy

Background \u0026 Aim Mesenchymal Stem Cells (MSC) are a heterogenous subpopulation of adherent cells that are isolated from adult connective tissue, multipotent and capable of self-renewal. They are an attractive tool in cell therapy for multiple diseases and recent studies have demonstrated a therapeutic effect using MSC-derived exosomes instead of the cells themselves. Exosomes play an important role in cell-to-cell communication and have diverse biological roles; however, the production of EVs as a medicinal product presents serious challenges in terms of isolation, quantification and release. Current methods for quantification and testing rely on either the expression of specific membrane-associated proteins, such as CD81 and CD63, or on the measurement of physical properties such as size or density. The aim of this work is to develop a novel and high-throughput EV quantification platform for MSCs-derived exosomes using fluorescent polarisation (FP). FP is an accessible spectroscopic method that relies on the interaction between a small labeled probe and a target protein, taking advantage of differences in rotational energy of the free vs. bound probe. The change in polarisation between the unbound and bound probe can be used the devise a quantitative test of the concentration of EVs. Methods, Results \u0026 Conclusion EV-binding probes should exhibit strong and specific binding to the target and be highly stable in solution. Based on these characteristics, different probes have been identified in the current study. As prominent exosomes surface markers, the CD81 and CD63 tetraspanins have been targeted and candidate probes have been chosen based on their interaction with these markers. An enzyme-linked immunosorbent assay (ELISA) system has been established to assess the ability of the candidate probes to detect surface markers and capture exosomes. Following probe validation, candidate probes will be labeled with a suitable fluorophore and their utility in an FP assay format assessed in comparison with gold standard techniques for exosome quantification. As part of this study, MSCs-derived exosomes have been harvested from various culture conditions using ultracentrifugation and characterised through standard methods including western blot, Dynamic Light Scattering and Transmission Electron Microscopy. This assay will provide a rapid and high-throughput solution for exosome quantification directly in cell culture conditioned media, in a cost effective and timely manner.