48 well suspension culture plates

Intrahepatic cholangiocyte organoids (ICO) (n = 5) were initiated from donor liver biopsies (0.5 cm³–1 cm³) following a previously published protocol [5 (link),22 (link)]. The use of liver biopsies for research purposes was approved by the medical ethics committee of the Erasmus University Medical Center (MEC-2014-060). Cholangiocarcinoma (CCA) tumor tissue biopsies (n = 3) were obtained through liver resections performed at the Erasmus MC (MEC-2013-143) and CCAO were initiated as described previously [9 (link)]. All patients gave written informed consent to use their tissue for research purposes. In short, the biopsies were digested in 2.5 mg/mL collagenase type A (Sigma-Aldrich, St. Louis, MO, US) for 20–120 min at 37 °C. Next, the suspension was strained (70 µm cell strainer), washed with cold ADV+ (Table S1), resuspended in basement membrane extract (BME, Cultrex, R&D systems, Minneapolis, MN, United States), and plated in 25 µL droplets in 48-well suspension culture plates (Greiner Bio One, Alphen aan den Rijn, The Netherlands). The BME was allowed to solidify for 45–60 min at 37 °C before 250 µL startup expansion medium (SEM, Table S2) was added. After 72 h, SEM was replaced with Expansion Medium (EM, Table S2) and refreshed every 3 to 4 days. Organoids were passaged by mechanical dissociation every 7 days in 1:3 to 1:6 split ratios.

For infection with VSVΔ51, organoids were seeded onto the BME layer and infected with 10⁶ PFU. For that, 48-well suspension culture plates (Greiner Bio-One) were moistened using culture medium and each well was evenly covered with 35 µL of undiluted BME and left overnight at room temperature to let it solidify. Confluent organoids were collected, pelleted, washed from old BME, and each confluent dome was resuspended in 500 µL of the respective culture medium but without N-acetyl-cysteine. This 500 µL of organoid suspension was carefully added to the center of the BME-covered well. Organoids were grown for 24 h before the addition of 4-OI (125 µM) and incubated for another 24 h before VSVΔ51 infection. On the day of infection, the media was carefully replaced with fresh media containing either DMSO (control), 4-OI alone (125 µM), VSVΔ51 (10⁶ PFU) plus DMSO, or VSVΔ51 (10⁶ PFU) plus 4-OI at the same concentration.