Establishment of Hepatocellular Carcinoma Organoids

The stable HCC organoid lines used in this study have been reported previously^{18 (link)}. Briefly, ultrasound-guided tumor biopsies were obtained from patients undergoing diagnostic liver biopsy at the University Hospital Basel. Written informed consent was obtained from all patients and the study was approved by the ethics committee of the northwestern part of Switzerland (Protocol Number EKNZ 2014-099). Tumor needle biopsy tissue was rapidly transferred to the lab, minced, and digested with 2.5 mg/mL collagenase IV (Sigma), 0.1 mg/mL DNase (Sigma) at 37 °C for 5 min. Digested biopsy/cell clusters were then seeded into reduced growth factor BME2 (Basement Membrane Extract, Type 2; Amsbio). After polymerization of BME2, expansion medium was added to the BME2 droplets. The composition of the medium is: advanced Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (GIBCO) supplemented with 1:50 B-27 (GIBCO), 1:100 N-2 (GIBCO), 10 mM nicotinamide (Sigma), 1.25 mM N-acetyl-l-cysteine (Sigma), 10 nM [Leu15]-gastrin (Sigma), 10 mM forskolin (Tocris), 5 mM A83-01 (Tocris), 50 ng/mL EGF (PeproTech), 100 ng/mL FGF10 (PeproTech), 25 ng/mL HGF (PeproTech), and 10% RSpo1-conditioned medium (homemade). Tumor organoids were passaged by dissociation with 0.25% Trypsin-EDTA (GIBCO).