Biliary epithelial cells play an essential role in the reconstruction of hepatic tissue with a functional bile ductular network.

One of the most challenging issues in liver tissue engineering is the incorporation of a functional bile duct (BD) network in the hepatic tissue. In this study, we investigated the feasibility of a three-dimensional (3D) coculture of adult rat primary biliary epithelial cells (ABECs) and rat fetal liver cells (FLCs) to form functional BD structures. 3D hetero-spheroids containing various ratios of the two cell types were successfully obtained in low-adherence, round-bottom, 96-well plates. Histological analyses and functional analysis using fluorescein diacetate demonstrated that the ABECs critically contributed to the reconstruction of continuous ductular networks. Metabolized fluorescein was transported toward cystic structures in a time- and ABEC ratio-dependent manner. In particular, the duct-like structures containing fluorescein accumulations interlinked the large cystic accumulations. Furthermore, transplantation experiments demonstrated that ABECs, but not FLCs, enabled the establishment of BD networks in vivo. This study provided important insights into the development of transplantable liver tissues with bile excretion functionality.