Isolation and long-term culture of primary mouse cholangiocytes that retain biophysical properties and distinct...

Cultures of primary mouse bile duct epithelial cells are a valuable tool to study cholangiocyte secretion and bile formation. However, freshly isolated cells have a limited ability to expand in culture. Here we report a novel isolation and culture technique for normal mouse cholangiocytes (NMC) that enables long-term growth without compromising function. Mouse cholangiocytes were isolated and cultured in conditioned medium (CM) that was subsequently supplemented with ROCK inhibitor Y-27632. Expression of cholangiocyte markers was assessed by qPCR, immunofluorescence, and Western blotting. Patch clamp techniques were used to measure cAMP-activated Cl- current, Ca2+-activated Cl- current, and volume-stimulated Cl- current. We obtained NMC cultures that were polarized and maintained a cholangiocyte phenotype for over 50 passages. Functional studies show that ion channel activity is maintained in NMC regardless of the number of passages and despite removal of CM. NMC also perform other physiological functions such as ATP release and intracellular Ca2+ changes in response to stimulation with bile acids. Thus, our isolation procedure produces viable NMC that maintain biophysical properties in long-term culture. We also demonstrate the utility of NMC in studies investigating the cellular mechanisms responsible for cholangiocyte secretion and bile formation.