4.2. CRC Organoid Cultures

The Medical Research Council of Hungary (ETT-TUKEB, No 51323-4/2015/EKU) approved the experiments with human samples and informed consent was obtained from the patients. We used the CRC organoid lines established and characterized previously in our research group [22]. Organoids were cultured in CRC medium containing advanced DMEM/F12 (Gibco), 10 mM HEPES (Merck), penicillin/streptomycin (Gibco), glutamine (Gibco), B27 supplement (Gibco), 1 mM N-Acetyl-Cysteine (Merck), 10 mM Nicotinamide (Merck), 50 ng/mL EGF (Peprotech, London, UK), 10 uM SB202190-Monohydrochloride (Merck) and 500 nM A83-01 (Merck). The Rho kinase inhibitor Y27632 (Merck) was also added for 3 days after passaging to avoid anoikis. Organoids were removed from Matrigel every 5–6 days mechanically, centrifuged at 700× g for 5 min, washed with phosphate-buffered saline (PBS), and digested with TrypLE (Thermo Fisher, Waltham, MA, USA) until organoids were dissociated into cell clusters. Samples were then washed with advanced DMEM/F12 medium and embedded into Matrigel again in a 1:3 ratio. Organoids #1–4 have already been characterized and clinical data of the patients have been published [22,24].