Stable two- and three-dimensional cholangiocyte culture systems from extrahepatic bile ducts of biliary atresia patients: use of structural and functional bile duct epithelium models for in vitro analyses.

We herein report two- (2D) and three-dimensional (3D) culture methods of cholangiocytes originating from extrahepatic bile ducts of biliary atresia (BA) patients. Cells were stabilized for in vitro analyses, and 3D culture by two different methods showed the structural and functional features of cholangiocytes in the gel scaffold. First, cells were obtained from gallbladder contents or resected tissues of patients at surgery and then cultured in our original conditioned medium with a cocktail of signaling inhibitors that maintains the immaturity and amplification of cells. Cells were immortalized by inducing SV40T and hTERT genes using lentivirus systems. Immunostaining with CK19 and Sox9 antibodies confirmed the cells as cholangiocytes. 3D organoids were formed in Matrigel in two different ways: by forming spheroids or via vertical growth from 2D cell sheets (2 + 1D culture). Organoids generated with both methods showed the uptake and excretion of rhodamine-123, and duct-like structures were also found. Our culture methods are simpler than previously reported methods and still show the structural and functional characteristics of cholangiocytes. Thus, this system is expected to be useful for the in vitro investigation of cholangiocyte damage or regeneration in BA patients.