Liver biopsies (0.5–1 cm3) were obtained from donor and explant livers during liver transplantation performed at the Erasmus MC, Rotterdam. The Medical Ethical Council of the Erasmus Medical Center approved the use of this material for research purposes, and informed consent was provided from all patients. For EpCAM sorting experiments and hepatocyte isolation, primary human liver tissue was obtained with informed consent and approval by the Regional Ethics Board, from the CLINTEC division of Karolinska institute (Dnr: 2010/678-31/3) (Jorns et al., 2014 (link)). Liver cells were isolated from human liver biopsies (0.5–1 cm3) by collagenase-accutase digestion, as described in the Extended Experimental Procedures . The different fractions were mixed and washed with cold Advanced DMEM/F12 and spun at 300–400 × g for 5 min. The cell pellet was mixed with Matrigel (BD Biosciences) or reduced growth factor BME 2 (Basement Membrane Extract, Type 2, Pathclear), and 3,000–10,000 cells were seeded per well in a 48-well/plate. Non-attaching plates were used (Greiner). After Matrigel or BME had solidified, culture medium was added. Culture media was based on AdDMEM/F12 (Invitrogen) supplemented with 1% N2 and 1% B27 (both from GIBCO), 1.25 mM N-Acetylcysteine (Sigma), 10 nM gastrin (Sigma), and the growth factors: 50 ng/ml EGF (Peprotech), 10% RSPO1 conditioned media (homemade), 100 ng/ml FGF10 (Peprotech), 25 ng/ml HGF (Peprotech), 10 mM Nicotinamide (Sigma), 5 uM A83.01 (Tocris), and 10 uM FSK (Tocris). For the establishment of the culture, the first 3 days after isolation, the medium was supplemented with 25 ng/ml Noggin (Peprotech), 30% Wnt CM (homemade prepared as described in Barker et al. [2010] (link)), and 10 uM (Y27632, Sigma Aldrich) or hES cell cloning recovery solution (Stemgent). Then, the medium was changed into a medium without Noggin, Wnt, Y27632, hES cell cloning recovery solution. After 10–14 days, organoids were removed from the Matrigel or BME, mechanically dissociated into small fragments, and transferred to fresh matrix. Passage was performed in a 1:4–1:8 split ratio once every 7–10 days for at least 6 months. To prepare frozen stocks, organoid cultures were dissociated and mixed with recovery cell culture freezing medium (GIBCO) and frozen following standard procedures. When required, the cultures were thawed using standard thawing procedures and cultured as described above. For the first 3 days after thawing, the culture medium was supplemented with Y-27632 (10 μM).
Growth curves and expansion ratios were performed and calculated as described in theExtended Experimental Procedures .
Growth curves and expansion ratios were performed and calculated as described in the
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