Advanced dmem f12 medium

Gastric glands from the corpus region of the stomach were isolated and cultured into gastroids as described previously [26 (link), 27 (link)]. In brief, the corpus gastric mucosa was separated from muscle layers, cut into small pieces and incubated in chelation buffer containing 10 mM EDTA (3 h, shaking, 4 °C). EDTA removal was followed by mechanical dissociation by carefully pipetting the tissue to release glands. Isolated glands were mixed with Matrigel (BD Biosciences, Franklin Lanes, NJ, USA), distributed in culture plates and grown in Advanced DMEM/F12 medium (Invitrogen, Carlsbad, CA, USA), 50% Wnt3a conditioned medium, 10% R-Spondin1 and Noggin conditioned medium supplemented with 10 mM HEPES, 1X N-2, 1X B27, 1X GlutaMAX (Invitrogen), 2.5 mM N-Acetylcysteine (Sigma-Aldrich, St. Louis, MO, USA), 50 ng/ml EGF, 100 ng/ml FGF10 (Peprotech, Rocky Hill, NJ, USA) and 10 nM gastrin (Sigma-Aldrich). For the first 3 days, 10 μM ROCK inhibitor (Y-27632, Sigma-Aldrich) was added. Three days after the first passage, gastroids were treated with IFN-γ (Peprotech) and all wells were scanned microscopically every 24 h using a Cytation 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). Montages formed of stitched images were used to count the number of dead gastroids. Several figure panels show stitching junctions and for clarity these are not marked.

Gastric organoids (gastroids) collected from the gastric glands in the corpus of the stomach were isolated from Wildtype, Ifrd1^−/−, Trp53^−/−and Ddit4^−/− mice. After dissecting the stomach, flush stomachs with PBS and cut the forestomach and the antrum off. Wash the corpus with cold chelating solution, remove the muscle layer and cut the mucosa into small pieces. Immerse the small pieces in chelating solution + 10 mM EDTA for 3hours with gently shaking as described previously(Barker et al., 2010 (link); Osaki et al., 2019 (link)). Approximately 100 gastric glands were mixed with 50 μL of Matrigel (Corning), plated in 24-well plates and cultured in Advanced DMEM/F12 medium (Invitrogen), 50% Wnt3a conditioned medium, 10% R-Spondin1 and Noggin conditioned medium(Osaki et al., 2019 (link)) supplemented with 10mM HEPES, 1X N-2, 1X B27, 1X glutamax (Invitrogen), 1.25 mM N-Acetylcysteine (Sigma-Aldrich), 50 ng/mL EGF, 100 ng/mL FGF10 (Peprotech), 10 nM gastrin (Sigma-Aldrich) and 0.1% Primocin (Invivogen). 10 μM ROCK inhibitor (Y-27632, Sigma-Aldrich) was provided for the first generation gastroids to prevent anoikis. Conditioned medium was changed every 3 days.

Derivation and maintenance of HIOs followed published protocols^{1 (link), 25 (link)}. Briefly, HIOs were embedded in Matrigel (BD Biosciences) and overlaid with Advanced DMEM-F12 medium (Invitrogen, Carlsbad, CA) containing 1X B27 supplement (Invitrogen), 1X GlutaMAX (Life Technologies, Carlsbad, CA), 10 µM Hepes, 10% pen/strep, 100 ng/mL rhNoggin (R&D Systems), 100 ng/mL epidermal growth factor (R&D Systems), and approximately 500 ng/mL R-Spondin1 (RSPO1). RSPO1 was obtained from conditioned media collected from a HEK293 cell line that was stably transfected and zeocin-selected for the RSPO1 expression vector. Media was changed every two to four days, and HIOs were transferred to fresh Matrigel once a week until they reached approximately 2 to 3 mm in diameter for experiments. This size was reached on average 48 days after initial spheroid formation.