DMEM/F-12

Protocol tips

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Defrost the Cultrex growth factor reduced BME type 2 in advance, on ice or in fridge

Upstream tips
Defrost the Cultrex growth factor reduced BME type 2 in advance, on ice or in fridge

Publication protocol

Organoid Culture: Tumor biopsy fragments designated for organoid generation typically measured ∼1 mm × 5–10 mm corresponding to a volume of ∼3.9–7.9 mm3. They were transported to the laboratory on ice and further processed for organoid generation within 20 min after collection. For tumor organoid generation, biopsies underwent a limited digestion to small-cell clusters. We avoided complete digestion into single cells because it has been reported that preservation of cell-cell contacts enhances derivation efficiency (Kondo et al., 2011). Tumor tissue was minced and shortly (maximum [max.] 2–4 min) digested with 2.5 mg/mL collagenase IV (Sigma), 0.1 mg/mL DNase (Sigma) at 37°C. The yield of the procedure varied because of differences in the size of the tumor biopsy available for the generation of organoids and the variable content of viable tumor cells in the biopsies. Cell clusters were then seeded into reduced growth factor BME2 (Basement Membrane Extract, Type 2; Amsbio). After polymerization of BME2, expansion medium (Huch et al., 2015) was added to the cells. The composition is advanced 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 μM forskolin (Tocris), 5 μM A83-01 (Tocris), 50 ng/mL EGF (PeproTech), 100 ng/mL FGF10 (PeproTech), 25 ng/ml HGF (PeproTech), 10% RSpo1-conditioned medium, (homemade), and 30% Wnt3a-conditioned medium (homemade). In the few cases for which enough biopsy material was available, we tried an adapted version of the culture medium in comparison with the normal one. The adapted medium lacked some of the original components reported to have a negative effect on HCC cell proliferation (forskolin, N-acetyl-L-cysteine, nicotinamide, HGF) and contained FGF19 because of the frequent amplification of the FGF19 gene detected in HCCs and its positive effect on proliferation of HCC cells. However, these attempts did not result in the establishment of additional HCC organoid lines. Organoid cultures from non-tumor liver biopsies were generated as previously described (Huch et al., 2015). Tumor organoids were passaged after dissociation with 0.25% Trypsin-EDTA (GIBCO). Non-tumor liver organoids were passaged by mechanical dissociation through a fire-polished Pasteur-pipette or incubation in 0.25% Trypsin-EDTA (GIBCO) for 2 min. Cryovials were prepared at regular intervals by dissociating organoids and resuspending in Recovery Cell Culture Freezing Medium (GIBCO) prior to freezing. We could prepare frozen stocks of early (≤P4) passages from all the samples that yielded tumor organoids. All organoid lines could be kept in long-term cultures with regular splitting for at least 1 year. All organoid cultures were regularly tested for Mycoplasma contamination with the MycoAlert Mycoplasma Detection Kit (Lonza) according to the manufacturer’s instructions.

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