Hydrogel nanovial technology for single-cell sorting based on extracellular vesicle production

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SUMMARY Secreted extracellular vesicles (EVs) are an emerging therapeutic category with significant potential in the treatment of disease. However, critical challenges remain with the study and production of these cell-derived therapeutics. Heterogeneous populations of cells, like mesenchymal stem cells (MSCs) that produce different amounts and types of EVs along with unknown mechanisms of their biogenesis hamper the scaling of uniform EV products for clinical translation. Sorting based on EV-secretion rate can enable the study of genetic underpinnings that lead to high EV biogenesis. Producer cells that are transfected to secrete biologics, such as antibody-based therapeutics, are traditionally sorted based on their secretion rate to isolate clones that produce large quantities of antibody, and similar approaches to sort and select highly-secreting populations will be advantageous for obtaining high quality EV-secreting cells. However, methods to rapidly sort out single cells secreting high levels of EVs are not currently available and cell-to-cell variability cannot be assessed in bulk, such that the separation of cells that produce EVs at higher rates or with more uniform contents is not feasible. We propose to develop a lab on a particle platform to sort MSCs based on EV secretion at the single-cell level using standard fluorescence activated cell sorters (FACS). Our approach leverages cavity-containing hydrogel microparticles, or nanovials, that allow seeding and adhesion of MSCs followed by the uniform formation of droplets around the nanovials with simple pipetting steps to confine secreted EVs for capture on the particles. Captured EVs can be stained and the corresponding secreting cells sorted based on the level of EVs secreted using commercial FACS machines. We propose to develop the on-nanovial immunoassay to detect secreted EVs and evaluate it in sorting of human MSCs that secrete the highest levels of EVs. We will identify the timeframe over which MSCs selected for this high secretion phenotype is stable. Our technology can aid in clinical studies for EV-based therapeutics, defining populations of cells that produce uniform EV therapeutics at high rates, and will further expand the ability to define the cellular underpinnings of EV-based products. Improved EV production can enable MSC derived EV-based (MSC-EV) treatments which have shown initial efficacy in one clinical trial for chronic kidney disease, and there are currently 10 on-going trials including two for COVID-19.