Development of multi-lineage differentiated liver ductal organoids in suspension culture that generates therapeutic extracellular vesicles for sclerosing cholangitis treatment

Organoid-based therapy, as an approach in regenerative medicine, offers new options for previously untreatable diseases. However, the limitations of organoid transplantation, such as immunogenicity, tumorigenic potential, and ethical issues, restrict its clinical translation. For the first time, a novel three-dimension matrigel-free suspension culture system is developed to generate liver ductal organoid-derived extracellular vesicles (3D OEVs) for primary sclerosing cholangitis (PSC) treatment. The results show that the developed suspension culture provides a more favorable mechanical microenvironment, which significantly enhances the functional maturation of liver ductal organoids and their extracellular vesicles. This improvement is facilitated by a multidimensional regulatory network that encompasses the PI3K-AKT and RAP1 pathways. Moreover, 3D OEVs significantly attenuate hepatic inflammation and fibrosis because they can remodel the immune microenvironment, polarizing more macrophages into the anti-inflammatory M2 phenotypic macrophages. Furthermore, miR-1299 enriched in 3D OEVs is identified as the core effector molecule for macrophage reprogramming, which was confirmed by both in vivo and in vitro experiments to effectively repair biliary damage through suppressing the EGR1/FOS/RAS signaling axis. As a result, a scalable platform for the production of therapeutic OEVs was successfully developed in a 50 mL bioreactor utilizing a matrigel-free methodology, thus offering a bioactive material for liver regeneration.