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.

The immunosuppressive tumor microenvironment (TME) plays a critical role in the exacerbation of chemotherapy-induced multidrug resistance (MDR). To overcome MDR, a natural nanovesicle-based biomimetic nanosystem was created using low-frequency vibrational magnetic fields (VMF) and an 808 nm laser to help overcome drug resistance in tumors. This advanced platform integrates M1 macrophage-derived exosomes (M1-EXO) with magnetic nanoparticles, Ce6 labeled antisense oligonucleotides of heat shock protein 70 (HSP-70) and doxorubicin. This approach enables a triple-modality synergistic therapy that integrates immunotherapy, gene therapy, and enhanced photothermal therapy (PTT). Specifically, M1-EXO effectively reprogram tumor-associated M2 macrophages (TAMs) toward the antitumor M1 phenotype, while Ce6-mediated photodynamic therapy (PDT) amplifies reactive oxygen species (ROS)-dependent M1 repolarization. Further, the nanovesicles target HSP70 to suppress heat shock protein expression, thereby overcoming the thermal resistance of tumor cells and enhancing PTT. Concurrently, nanovesicles' favorable magnetic responsiveness enables direct destruction of cancer cells under VMF exposure, which greatly contributed to immunogenic cell death (ICD) activation. Consequently, this synergistic strategy initiates antitumor immunity and activates cytotoxic T lymphocytes. Both in vitro and in vivo studies demonstrate that this biomimetic nanovesicle reduces the half-maximal inhibitory concentration (IC₅₀) of doxorubicin-resistant breast cancer cells by 96-fold. In conclusion, this nanoplatform successfully tackles drug resistance by actively targeting pathways that activate the immune system, and promotes exosomes from human peripheral blood mononuclear cells for transplantation immunotherapy in the future.