Isolation and Culture of Intrahepatic Cholangiocyte Organoids

As previously described,¹⁵ (link)^,16 (link) ICOs (n = 5) were initiated and cultured using an established protocol. In brief, donor liver tissue was minced, rinsed twice with Earle's balanced salt solution (EBSS, Thermo-Fisher, Waltham, USA), and digested in collagenase solution (2.5 mg/mL collagenase Type A, Sigma–Aldrich, St. Louis, USA) in Advanced DMEM/F-12 medium (Invitrogen, Carlsbad, USA) for 15 min at 37 °C. Cold Advanced DMEM/F-12 was added to stop the digestion. The single-cell suspension was filtered using a 70 μm Nylon cell strainer and centrifuged at 1500 rpm, 5 min, at 4 °C. After that, the cell pellets were collected at the bottom and subsequently mixed with BME (Basement Membrane Extract, Type 2, R&D Systems, Minneapolis, USA). Cell droplets were then seeded in multi-well plates. Initiating medium was added after the solidification of BME and the cells were cultured at 37 °C in a humidified atmosphere with 5% CO². After three days of culture in the initiating medium, the cells were refreshed with an expansion medium in which noggin, Wnt, Y27632, and hES cell cloning recovery supplement were deprived. Components supplemented in Advanced DMEM/F12 medium for initiating and expansion medium were listed in Table S2.