High‐Throughput Formation of Pre‐Vascularized hiPSC‐Derived Hepatobiliary Organoids on a Chip via Nonparenchymal Cell Grafting

Abstract Liver organoids have been increasingly adopted as a critical in vitro model to study liver development and diseases. However, the pre‐vascularization of liver organoids without affecting liver parenchymal specification remains a long‐lasting challenge, which is essential for their application in regenerative medicine. Here, the large‐scale formation of pre‐vascularized human hepatobiliary organoids (vhHBOs) is presented without affecting liver epithelial specification via a novel strategy, namely nonparenchymal cell grafting (NCG). Endothelial and mesenchymal cells are grafted to human hepatobiliary organoids (hHBOs) at the different liver epithelial differentiation stages without supplementing with nonparenchymal culture medium and growth factors. Endothelial grafting at the stage of hepatic maturation offers an optimal integration efficiency compared to the stage of hepatic specification. Additionally, grafting with mesenchymal proves crucial in endothelial invading and sprouting into the liver epithelial cells during the establishment of vhHBOs. Ectopic liver implants into mice further displayed integration of vhHBOs into mice vascular networks. Notably, transplanted vhHBOs self‐organized into native liver tissue like hepatic zone and bile ducts, indicating their potential to regenerate damaged hepatic and bile duct tissues. It is believed that nonparenchymal cell grafting will offer a novel technical route to form a high‐fidelity complex in vitro model for tissue engineering and regenerative medicine.