Novel Vascularized Human Liver Organoids for Modeling Alcohol‐Induced Liver Injury and Developing Hepatoprotective Therapy

Abstract Liver organoids have emerged as transformative tools for disease modeling and therapy development. However, their inability to form functional multivascular networks significantly limits their capacity to replicate the multifaceted functions of the native human liver with high fidelity. Here, novel vascularized liver organoids (3HLOs) are constructed by co‐culturing robust human reprogrammed hepatocyte‐like cells (hrHLs) with endothelial cells (HUVECs) and mesenchymal stem cells (HUMSCs) in a 3D system. After optimization, 3HLOs form vascular architecture featuring CD31 + endothelial networks and CK19 + biliary ducts, while demonstrating enhanced hepatic function, including upregulated glycogen storage, elevated albumin secretion, accelerated indocyanine green uptake, and improved rhodamine‐123 excretion. To improve physiological relevance, 3HLOs‐on‐chip models are developed for modeling alcohol‐induced liver injury (ALI) pathogenesis and testing hepatoprotective agents. The in vitro findings closely match in vivo observations, confirming the high physiological relevance of 3HLOs‐on‐chip ALI models. Subcutaneous implantation of 3HLOs significantly attenuated liver injury and promoted hepatic regeneration in end‐stage ALI mice, primarily through establishing functional anastomoses with host microvasculature. Multi‐omics analysis revealed that 3HLOs secreted human hepatoprotective proteins into the host circulating system, thereby modulating inflammatory responses and lipid metabolism pathways. Collectively, this work offers a robust and reliable platform for ALI modeling, drug testing, and liver regeneration.