Ampicillin

Fresh tumor tissue was cut into pieces with a maximum diameter of 2 mm and implanted subcutaneously in anaesthetized NMRI-nu mice (RjOrl:NMRI-Foxn1^nu/Foxn1^nu, Janvier Labs SAS, Saint-Berthevin, France). After harvest from euthanized animals, tumor tissue was dissociated into a single cell suspension by the GentleMACS Dissociator (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) applying a standardized protocol. In brief, tissue was minced manually and subjected to repeated cycles of mechanical stirring and enzymatic digestion. The resulting suspension was filtered and centrifugalized. The pellet was gently resuspended in a medium (Airway Epithelial Cell Growth Medium with SupplementMix, PromoCell GmbH, Heidelberg, Germany) with the addition of 50 μg/ml ampicillin (Ratiopharm GmbH, Ulm, Germany). Cells were incubated at 37°C in 5% CO₂ for 4–7 days. Following that, the initial medium was gradually replaced by DMEM (Biowest, Nuaillé, France) with the addition of 10% fetal bovine serum, 14 μg/ml phosphoethanolamine, 11 μg/ml ethanolamine, 0.5 ng/ml EGF (all Biochrom AG, Berlin, Germany), 5.0 μg/ml hydrocortisone, 5.0 μg/ml insulin (Sigma-Aldrich, St. Louis, USA), 0.5 ng/ml FGF (R&D Systems Inc., Minneapolis, USA), and 50 μg/ml ampicillin.

For OP induction, the EBs were transferred into a plate coated with a poly-l-ornithine and laminin (both were from Sigma-Aldrich Chemical Co.)-coated plate and cultured for 3 days in an advanced Dulbecco’s modified Eagle’s medium (DMEM)/nutrient mixture F12 supplemented with 20% KSR, N2, B27 (ThermoFisher Scientific), human basic fibroblast growth factor (bFGF; 25 ng/mL; R&D Systems), human FGF19 (25 ng/mL; R&D Systems), human noggin (30 ng/mL; R&D Systems), human R-spondin1 (R&D Systems; 50 ng/mL), heparan sulphate (50 ng/mL; Sigma, USA) and ampicillin (50 µg/mL). However, the medium was replaced with the advanced DMEM/F12 supplemented with 15% KSR, N2, B27, bFGF (25 ng/mL), human FGF19 (25 ng/mL), human BMP4 (20 ng/mL; R&D Systems), heparan sulphate (50 ng/mL) and ampicillin (50 µg/mL) and cultured for 3 days^{5 (link),57} .