Isolation and Culture of Small Intestinal Organoids

Small intestinal organoids were isolated and cultured as previously described^{55 (link)}. In brief, the small intestine was opened longitudinally and then washed in cold PBS with vigorous shaking, and the intestine was then cut into 2 mm pieces. The intestine samples were continuously washed in cold PBS with a pipette until the supernatant was clear. The samples were then incubated in 30 mM EDTA for 15 min at 4 °C and vortexed for 15 s to separate the crypts. Then the samples were filtered through a 70 μm cell strainer, followed by 200 × g centrifugation for 5 min. After being washed in cold PBS, the crypts were collected in a 50 ml centrifuge tube. For ISC isolation, the crypts were digested in 0.1% type I collagenase (Invitrogen) and incubated in 1× TrypLE Express (Life Technologies) supplemented with 1 kU mL⁻¹ DNase I (Worthington, USA) for single-cell preparation. Lgr5-GFP + ISC were classified using fluorescence-activated cell sorting (FACS) and incubated with IntestiCult medium (STEMCELL Technologies, Vancouver, Canada) for growing into organoids. To induce the organoid H/R model, organoids were placed in an anaerobic environment containing 5% CO₂, 2% O₂, and 93% N₂ in a 37 °C incubator for 4 h. Flow cytometry was performed using FACS Calibur instruments (BD Biosciences, San Jose, CA, USA), and data were analyzed using FlowJo (Tree Star Inc.).