DNA-origami-engineered NAC-linkers enable the construction of humanized liver organoids for modeling ischemia-reperfusion injury

Hepatic ischemia-reperfusion injury (IRI) is a major cause of liver dysfunction and failure following surgical procedures such as liver resection and transplantation. Although numerous studies have reported various underlying mechanisms involving both parenchymal and nonparenchymal cells, there is an urgent need to develop human-relevant models that faithfully recapitulate clinical IRI. To address this, we established a novel three-dimensional liver organoid model using NAC linker technology to simulate hepatic IRI in both murine and human contexts. By coculturing primary hepatocytes with nonparenchymal cells (NPCs) and immune components such as peripheral blood mononuclear cells (PBMCs) or THP-1 monocytes, we developed multicellular organoids that recapitulate key structural and functional features of the liver. We demonstrated that oxidative stress induced by H 2 O 2 triggers inflammatory and apoptotic responses consistent with IRI, which are exacerbated by immune cell involvement. Furthermore, we identified the YAP signaling pathway as a critical protective mechanism: its activation attenuated cellular damage and inflammation, whereas its inhibition worsened injury. These findings highlight the utility of NAC-liver organoids as a robust platform for studying IRI mechanisms and screening therapeutic agents, with YAP agonists emerging as promising candidates for mitigating IRI-related damage. • Development of a novel NAC-linker-based 3D organoid platform co-culturing hepatocytes, non-parenchymal cells, and immune cells to accurately model hepatic IRI in both murine and human systems. • Demonstration that immune cells significantly exacerbate oxidative stress-induced damage in organoids. • Identification of YAP activation as a protective mechanism against IRI in humanized liver organoids, highlighting its potential as a therapeutic target.