Cell culture

Before seeding cholangiocytes or endothelial cells, a bottom channel of the PDMS device was pre-coated with fibronectin (1 µg/mL, Sigma-Aldrich). Cholangiocytes (HuCCT1 cells, JCRB0425, JCRB Cell Bank) or endothelial cells (Human Umbilical Vein Endothelial Cells: HUVECs, Lonza) were suspended at 5 × 106 cells/mL in RPMI-1640 medium containing 1×GlutaMAX (Thermo Fisher Scientific) and 10% fetal bovine serum (FBS) or EGM-2 Endothelial Cell Growth Medium-2 BulletKit (Lonza), respectively. Ten µL suspension medium was injected into the fibronectin-coated bottom channel. Then, the device was turned upside down and incubated for 1 h. After the incubation, the device was turned over, and the medium was injected into the bottom channel.

After 4 days, human hepatocytes (HUCPI, Lonza) were seeded into the top channel. Before seeding hepatocytes, the PDMS devices were pre-coated with Collagen I solution (30 µg/mL, Corning). The vials of human hepatocytes were rapidly thawed in a shaking water bath at 37°C. The contents of each vial were emptied into pre-warmed Cryopreserved Hepatocyte Recovery Medium (Thermo Fisher Scientific), and the suspension was centrifuged at 1,200 rpm for 5 min at room temperature. Hepatocytes were suspended at 5 × 106 cells/mL in Hepatocyte Culture Medium BulletKit (HCM, Lonza) containing 10% FBS. Ten µL suspension medium was injected into a Collagen Type I (Corning)-coated PDMS device. After 1 h, the medium was added into the top and bottom channels.