Mouse intesticult

Murine enteroids were derived from C57BL/6 mice and maintained as previously described [24 (link)]. Briefly, established enteroids were maintained by embedding them in 50 μl Matrigel (Corning) and covering the domes with Mouse IntestiCult (Stemcell Technologies) 1×Pen-Strep (Corning), incubated at 37°C and 5% CO₂. Medium was replaced every 2 to 4 days and enteroids were subcultured every 5 to 7 days by mechanical sheering in Gentle Cell Dissociation Reagent (Stemcell Technologies), washing in DMEM/F-12 15 mM HEPES (Stemcell Technologies) and re-embedding in Matrigel at a 1:3 to 1:4 splitting ratio.

Stomach corpus tissues were removed from C57BL/6 wild-type mice or Mist1-Kras mice, cut along the greater curvature of the stomach and washed in ice cold PBS. The antrum tissue area was removed with a razor blade and stomach corpus mucosa was separated from serosa along the muscle layer using cell scrapers. The corpus mucosa was incubated in the pre-warmed digestion buffer (Advanced DMEM/F12 + 5% FBS + 1 mg/mL collagenase type Ia + 1/100 DNAse I) at 37 °C with vigorous shaking at 220 rpm for 30 min and added stopping buffer (Advanced DMEM/F12 + Y-27632 and 1 mM DTT), then strained through 100 μm cell strainer to remove remaining tissue clumps. The dissociated glands were centrifuged at 300×g for 5 min to pellet glands, removed supernatant and repeated twice. The supernatant was removed and the pellets were resuspended with ice cold Matrigel (ECM, Sigma). Thirty microliter of gland/Matrigel mixture containing about 100–200 glands was plated in wells of 48 well plates and left the plates at 37 °C incubator for 30 min. Three hundred microliter of Mouse Intesticult (StemCell Technology) medium with a ROCK inhibitor, Y-27632, was added in each well and medium was replaced every 3 days.