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 an integrated biliary system remains a challenge. This study aimed to structurally reconstruct a three-dimensional biliary architecture in bioengineered livers through recellularization of decellularized rat liver scaffolds with rat primary hepatocytes (PHs) and intrahepatic cholangiocyte organoids (ICOs), and to assess bile acid distribution in relation to the reconstructed hepatobiliary architecture. Decellularized rat liver scaffolds were recellularized using rat PHs and green fluorescent protein (GFP)-expressing ICOs. Dissociated ICOs 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 ICOs 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 ICO-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 provides evidence supporting the structural reconstruction of a three-dimensional biliary system in bioengineered livers. These findings represent a significant step toward the development of bioengineered livers using decellularization and recellularization techniques.
Horie et al. (Tue,) studied this question.