Liver Tumor Organoid Cultivation

Tumor organoid lines were generated from liver biopsy or resection tissue according to published protocols^{8 (link),9 (link)}. Briefly, tumor tissues were dissociated to small-cell clusters and seeded in domes of basement membrane extract type 2 (BME2, R&Dsystems, Cat. No. 3533-005-02). Polymerized BME2 domes were overlaid with expansion medium (EM): advanced DMEM/F-12 (Gibco, Cat. No. 12634010) supplemented with 1× B-27 (Gibco, Cat. No. 17504001), 1× N-2 (Gibco, Cat. No. 17502001), 10 mM Nicotinamide (Sigma, Cat. No. N0636), 1.25 mM N-Acetyl-L-cysteine (Sigma, Cat. No. A9165), 10 nM [Leu¹⁵]-Gastrin (Sigma, Cat. No. G9145), 10 μM Forskolin (Tocris, Cat. No. 1099), 5 μM A83-01 (Tocris, Cat. No. 2939), 50 ng/ml EGF (Peprotech, Cat. No. AF-100-15), 100 ng/ml FGF10 (Peprotech, Cat. No. 100-26), 25 ng/ml HGF (Peprotech, Cat. No. 100-39), 10% RSpo1-conditioned medium (v/v, homemade). Cultures were kept at 37 °C in a humidified 5% CO₂ incubator. Organoids were passaged weekly at 1:4–1:6 split ratios using 0.25% Trypsin-EDTA (Gibco, Cat. No. 25200056). Frozen stocks were prepared at regular intervals. All organoid cultures were regularly tested for Mycoplasma contamination using the MycoAlert™ Mycoplasma detection kit (Lonza, Cat. No. LT07-118).