Three-dimensional reconstruction of a biliary system in a bioengineered liver using decellularized scaffold

Bioengineered livers are potential alternatives for liver transplantation in patients with end-stage liver disease. Liver scaffolds engineered through decellularization techniques have been developed for clinical applications; however, reconstruction of a functional biliary system remains a challenge. This study aimed to establish a three-dimensional biliary system in bioengineered livers through recellularization of rat primary hepatocytes (PHs) and liver ductal organoids (LDOs). Decellularized rat liver scaffolds were recellularized using rat PHs and green fluorescent protein (GFP)-expressing LDOs. Dissociated LDOs were injected via the bile duct and cultured for 5 days using a perfusion device, followed by PH injection and 2 days of culture. During co-culture, biliary drainage fluid and culture medium were collected to compare total bile acid concentrations using enzyme-linked immunosorbent assay. Histological and immunofluorescence analyses were performed after 7 days of perfusion culture. Histological analyses confirmed the engraftment of GFP-expressing LDOs into bile ducts and PHs into the parenchymal space. Engrafted PHs expressed ZO-1 and MRP2, forming bile canaliculi. In specific regions, MRP2-positive PHs and KRT19-positive LDO-repopulated cells adhered to each other, resembling the native liver structure. In the samples exhibiting such structures, total bile acid concentrations in the biliary drainage fluid tended to be higher than in the culture medium.This study suggested that a three-dimensional functional biliary system could be reconstructed in bioengineered livers. These findings represent a significant step toward the realization of bioengineered livers using decellularization and recellularization techniques.