Intestinal Organoid Hypoxia-Reoxygenation Assay

The mice intestinal organoids were extracted and cultured as described in our previous publication [25 (link)]. In this experiment, the proximal small intestinal crypts were obtained from mice aged 4–8 weeks old, and were cultured in matrigel drops (356231, Coning). IntestiCult medium (06005, STEMCELL Technologies) was used to promote cell confluence and differentiation. For hypoxia-reoxygenation (H/R) experiments, organoids were precultured in ILA (0.5 mM), CH-223191 (10 μM, T2448, TargetMol) or VP (5 μM) for 24 h. It was then placed in a 95% N₂-5% CO₂ humidity incubator for 12 h and further moved into a 95% O₂-5% CO₂ incubator for 4 h. The organoids were randomized into five groups before being subjected to H/R experiments: (1) Control + DMSO; (2) H/R + DMSO; (3) H/R + ILA; (4) H/R + ILA + VP; (5) H/R + VP. The negative control (NC) group was treated with DMSO without H/R injury while the H/R group was treated with DMSO and subjected to H/R injury.