Publication protocol
Hepatocytes were isolated by a two-step collagenase perfusion technique with modifications. Briefly, the inferior vena cava (IVC) was cannulated with a 24-gauge 3/4 inch angiocatheter (BD) and the portal vein was cut. The liver was perfused via the inferior vena cava with 100 mL of Liver Perfusion Medium (LP, Invitrogen) at 37 °C, followed by perfusion with 100 mL of collagenase type IV (Wellington) in HBSS containing Ca2+ and Mg2+ (GIBCO). After the liver was digested, it was dissected out and cut into small pieces and passed through a 100 μm strainer (Falcon). Hepatocytes were separated from non-parenchymal cells (NPCs) by low-speed centrifugation (50 g × 5 mins × 3, brake = 2), and further purified by Percoll gradient centrifugation (50 % v/v, Sigma). For 3D culture, hepatocytes were embedded in growth-factor reduced (GFR) matrigel (BD), at approximately 1000 cells per 20 μL of matrigel droplets. William E media containing 1% (v/v) Glutamax, 1% (v/v) Non-Essential Amino Acids, 1% (v/v) penicillin/streptomycin (all from Gibco), 0.2% (v/v) normocin (Invivogen), 2% (v/v) B27 (Gibco), 1% (v/v) N2 supplement (Gibco), 100 mM nicotinamide (Sigma-Aldrich), 1.25 mM N-acetylcysteine (Sigma-Aldrich), 10 μM Y27632 (Peprotech), 1 μM A83-01 (Tocris) was used as basal media. Expansion media contained 3 μM CHIR99021 (Peprotech), 25 ng/mL EGF (Peprotech), 50 ng/mL HGF (Peprotech) and 100 ng/mL TNFα (Peprotech). Media was replaced every 2 – 3 days. After approximately 2 weeks in culture, matrigel was digested with dispase (Stem Cell Technologies), and released intact organoids were embedded in new matrigel droplets and cultured for another one to two weeks. Of note, hepatocyte organoids were not dissociated by dispase. From the second passage onwards, after dispase digestion of matrigel, released organoids were further incubated with TrypLE Express (Gibco) for 5 minutes at 37 °C. Then, 3D colonies were broken up into multiple-cell aggregates by gentle pipetting. To ensure high viability of hepatocytes, prolonged enzyme incubation or mechanical dissociation that resulted in single-cell suspension was avoided. Dissociated cell aggregates were embedded in matrigel at appropriate densities (usually at 500 – 2000 cells per 20 μL drops). Remaining cells were frozen in Bambanker (Wako) at −80 °C and could be thawed following standard procedures for subsequent cultures. For long term culture, media was supplemented with 50 ng/mL noggin (Peprotech) for the first 4 – 7 days of culture from the second passage onwards. Cultures were typically passaged every 14 – 20 days, when the size of organoids was > 200 – 300 μm, or when the matrigel droplets were overcrowded. For culture without TNFα, the cytokine was omitted from the media, but otherwise contained all other factors present in the standard expansion media. For culture with IL-6, 50 ng/ml IL-6 was used in the expansion media without TNFα.
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