Gibco™Advanced DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the BME type 2 in advance, on ice or in fridge	Seed 2.000-5.000 cells were seeded per well in a 24-multi-well plate Grow half of the sample in classical isolation medium and half in tumouroid specific isolation medium, to ensure growth

Upstream tips
Defrost the BME type 2 in advance, on ice or in fridge
Protocol tips
Seed 2.000-5.000 cells were seeded per well in a 24-multi-well plate Grow half of the sample in classical isolation medium and half in tumouroid specific isolation medium, to ensure growth

Publication protocol

Isolation and Culture of human liver healthy and tumoural organoids: Healthy liver-derived organoids were isolated and cultured using our previously described method24,25 while tumour-derived organoids (tumouroids) were isolated by adapting this method as follows. Briefly, ¼ of the patient-derived or healthy donor specimen (~0.25 to 1cm3) was minced and incubated at 37°C with the digestion solution. Incubation was performed for 30min-1h for healthy donor tissue (as described in ref 24) while for patient-derived tissue digestion was left for 2-5 hours to overnight (O/N) according to the degree of liver fibrosis, which was evaluated in a patient-specific basis by visual inspection under a stereomicroscope as well as according to the resistance of the tissue to be minced. For patient-derived tissue, after 2-5h digestion, the digestion preparation was visually inspected and either digestion was stopped or, if a significant part of the original tissue was still under-digested (>50% of starting material, depending on the fibrotic status of the tissue), the preparation was left o/n at 37°C in the digestion solution, in order to get a good yield of tumoural cells. This increase in the digestion times compared to healthy tissue (>2h-o/n) facilitated reducing the number of viable healthy contaminating duct cells. In all cases, the digestion was stopped once no pieces of tissue were left, and the suspension was then filtered through a 100µm nylon cell strainer and spun 5 min at 300-400G. The pellet was washed in cold Advanced DMEM/F12 (GIBCO) then mixed with BME (Basement Membrane Extract, Type 2, Pathclear). 2.000-5.000 cells were seeded per well in a 24-multi-well plate. After BME had solidified, half of the wells obtained for each sample were cultured in the classical human liver organoid isolation medium (Advanced DMEM/F12 supplemented with 1% Penicillin/Streptomycin, 1% Glutamax, 10 mM HEPES, 1:50 B27 supplement (without Vitamin A), 1:100 N2 supplement, 1.25mM n-Acetyl-L-cysteine, 10% (vol/vol) Rspo-1 conditioned medium, 30% (vol/vol) Wnt3a conditioned medium, 10mM nicotinamide, 10nM recombinant human [Leu15]-Gastrin I, 50ng/ml recombinant human EGF, 100ng/ml recombinant human FGF10, 25ng/ml recombinant human HGF, 10μM Forskolin, 5μM A8301, 25ng/ml Noggin and 10μM Y27632 as described in ref 24). The other half were cultured in a tumouroid specific isolation medium (classical human liver organoid isolation medium without Noggin, Rspo-1 and Wnt3a conditioned media but supplemented with 3nM Dexamethasone (Sigma Aldrich). Thus, the tumoroid isolation medium contained: Advanced DMEM/F12 supplemented with 1% Penicillin/Streptomycin, 1% Glutamax, 10 mM HEPES, 1:50 B27 supplement (without Vitamin A), 1:100 N2 supplement, 1.25mM n-Acetyl-L-cysteine, 10mM nicotinamide, 10nM recombinant human [Leu15]-Gastrin I, 50ng/ml recombinant human EGF, 100ng/ml recombinant human FGF10, 25ng/ml recombinant human HGF, 10μM Forskolin, 5μM A8301, 10μM Y27632 and 3nM Dexamethasone). It is important to always culture half of the sample in classical isolation medium and half in our tumouroid specific isolation medium, to ensure growth of the cultures. For instance, CC-1 patient material only grew in classical isolation medium because it requires Rspo-1 to grow. For this line, though, we enriched for the tumouroids by hand-picking out contaminating healthy organoids (as described in Supplementary Fig. 1). After isolation medium was changed twice a week. For healthy-donor derived organoids, isolation medium was changed to “human healthy liver-derived organoids expansion medium” after 1-week in culture (see composition below). For tumouroids, isolation medium (classical or tumouroid specific) was maintained until the first split. For tumouroid culture establishment, after 2-3 weeks in culture (depending on the sample) the growing structures were visually inspected and, if required, contaminating healthy organoids were hand-picked to prevent these from outgrowing the tumouroid structures. Upon attainment of dense culture (healthy liver-derived organoids (1-2 weeks after isolation) and tumour-derived organoids (2-3 weeks after isolation) were passaged by mechanical dissociation into small fragments via trituration with a glass Pasteur pipet, and transferred to fresh matrix in the previously defined “human healthy liver-derived organoids expansion medium”24,25 : Advanced DMEM/F12 supplemented with 1% Penicillin/Streptomycin, 1% Glutamax, 10 mM HEPES, 1:50 B27 supplement (without Vitamin A), 1:100 N2 supplement, 1.25mM n-Acetyl-L-cysteine, 10% (vol/vol) Rspo-1 conditioned medium, 10mM nicotinamide, 10nM recombinant human [Leu15]-Gastrin I, 50ng/ml recombinant human EGF, 100ng/ml recombinant human FGF10, 25ng/ml recombinant human HGF, 10μM Forskolin and 5μM A83-01)24. Expansion medium was changed twice a week and cultures were split upon attainment of dense culture. All cultures were tested every month for mycoplasma using the ‘PCR Mycoplasma Test kit I/C’ kit from Promega in accordance with the manufacturer’s instructions. 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 3-4 days (organoids) or first 2 weeks (tumouroids) after thawing, the culture medium was supplemented with Y-27632 (10μM). Organoid pictures were taken with either a Leica M80 stereoscope and Leica MC170 HD camera or with an inverted microscope Leica DMIL and Leica DFC 450C camera.

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