Gastric organoids generation

The protocol for generating gastric organoids (GOs) was previously described (^{Bartfeld et al., 2015}). The mice were sacrificed, and the mouse stomach was collected, and the forestomach was removed. Then, the reserved stomach tissue was cut through the lesser curvature, and the stomach was rinsed with ice-cold PBS with 1% penicillin/streptomycin to remove blood. The tissue samples were carefully immersed in chelating buffer (sterile distilled water with 5.6 mmol/L Na₂HPO₄, 8.0 mmol/L KH₂PO₄, 96.2 mmol/L NaCl, 1.6 mmol/L KCl, 43.4 mmol/L sucrose, 54.9 mmol/L D-sorbitol, 0.5 mmol/L DL-dithiothreitol, pH 7) in a 10 cm dish, then the tissue was transferred to a dry dish. The epithelial layer was peeled and minced into pieces using forceps. Minced epithelial pieces were placed into 10 mL cold chelating buffer, followed by robust pipetting up and down to rinse the tissue until the supernatant was clear. A 20 mL chelating buffer was prepared with 10 mM EDTA under room temperature, and the tissue was incubated in there for 10 min. The tissue was tenderly pipetted gently once up and down, and the pieces were allowed to settle. The tissue was then moved to the clean bench. Most of the water was removed, and the tissue pieces were carefully placed in the middle of a sterile 10 cm dish. A glass microscopy slide was put on top of the tissue and pressure was added upon the slide until the tissue pieces seemed cloudy. The cloudy tissue pieces were then flushed from the slides in 30 mL of cold Advanced DMEM/F12. The large tissue fragments were allowed to sediment by gravity. The cloudy supernatant was transferred to two 15 ml tubes. The tubes were then centrifuged for 5 min at 200 g and 4°C. The supernatant was carefully removed and resuspended with Matrigel-medium mixture (12 μL Matrigel mix with 8 μL GOs culture medium/well). Approximately 40 glands per 20 μL Matrigel-medium mixture per well of a 48-well plate were seeded. The plate was steadily transferred to the incubator to let it solidify for 10 minutes. Then, 500 μL of GOs culture medium was added to cover the dome, and the plate was incubated at 37 °C with 5% CO2. The medium was changed every 2 days.