Ductular Network Formation by Rat Biliary Epithelial Cells in the Dynamical Culture with Collagen Gel and Dimethylsulfoxide Stimulation

Abstract

Formation of bile ducts in culture is important for reconstructing hepatic organoids with bile drainage systems. However, morphogenic factors of biliary epithelial cells (BECs) have been poorly understood because of the lack of experimental models. Here, we demonstrated that rat BECs formed bile ductular networks in dynamic culture, when culture conditions were sequentially controlled. BEC morphogenesis was achieved through two-dimensional culture on collagen gel, collagen gel sandwich configuration, and 1% dimethylsulfoxide stimulation. In this culture system, BECs developed into large bile duct structures (LBDs) that formed interconnected networks of continuous lumens. LBD luminal surfaces possessed well developed microvilli, consisted of 7 to 10 BECs, and their inner diameters measured 20 to 50 microm. Quantitative PCR analysis revealed that the cells in LBDs expressed apical and basal domain markers of BECs. Immunofluorescent staining identified apical domain markers such as Cl(-)/HCO(3)(-) anion exchanger 2 and cystic fibrosis transmembrane regulator on the luminal surface of LBDs, responding to secretin stimulation as well as laminin protein surrounding LBDs. Furthermore, the cells in LBDs transported metabolized fluorescein from the basal side to the luminal space, further demonstrating that the reconstructed LBDs were functionally and morphologically similar to the bile ducts in vivo. The culture model described here will be useful in reconstructing hepatic tissues as well as in understanding the mechanism of bile duct development and its disruption in disease.