Minimal dmem

Primary cultured colonic MSCs were seeded at 4 × 10⁴ cells per well in XF24 cell culture microplates (Agilent Technologies) and cultured in MSC culture medium overnight. One hour before measurement, culture medium was replaced with OCR assay medium (minimal DMEM [Sigma-Aldrich] and supplemented with 20 mmol/L glucose [Junsei Chemical, Tokyo, Japan], 2 mmol/L GlutaMax [Gibco], and 5 mmol/L pyruvate [Sigma-Aldrich]) or ECAR assay medium (minimal DMEM and supplemented with 2 mmol/L GlutaMax) in the 37°C non-CO₂ incubator. The Seahorse Bioscience XF24 analyzer (Agilent Technologies) was used to measure OCR and ECAR. For OCR measurements, 1 μmol/L oligomycin (Sigma-Aldrich), 1 μmol/L FCCP (Sigma-Aldrich), and 1 μmol/L rotenone and antimycin (Sigma-Aldrich) were injected.. For ECAR measurements, 10 mmol/L glucose, 2 μmol/L oligomycin, and 50 mmol/L 2-deoxy-D-glucose (Sigma-Aldrich) were injected. The parameters of glycolytic function, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification were calculated using Seahorse Wave software V2.6.

Oxidative respiration and glycolytic acidification were measured with Seahorse XF-96 Analyzer (Agilent). MTs were collected into a 15 mL Falcon tube and washed three times in assay medium + 0.2% BSA. 4h before measurement, four MTs (or five for the CMEC MT group) per well were plated on 96-well Matrigel-coated (0.167 mg/ml) plates. Measurements were made in minimal DMEM (Sigma) supplemented with 15 mM glucose (Sigma-Aldrich), 2 mM pyruvate (Thermo Fisher Scientific) and 1 mM L-glutamine (Thermo Fisher Scientific) (assay medium). For Mitochondrial stress test, the following drugs were diluted in assay medium: 3.5 μM Oligomycin, 4 μM FCCP, 2 μM Antimycin A and 2 μM Rotenone (all from Sigma-Aldrich). For the glycolytic stress test, MTs were plated in medium without glucose and the injections contained 15 mM glucose, 3.5 μM Oligomycin, and 100 mM 2-DG (Sigma-Aldrich). Normalization was performed by lysing the MTs with a chloride-based lysis buffer (10 mM Tris, 1 mM EDTA, 50 mM KCl, 2 mM MgCl₂) supplemented with 200 μg/ml Prot K, for 2 h at 60°C. DNA content was measured using a Picogreen assay (Thermo Fisher Scientific). Analysis was performed using R and R-studio.

Organoids were dissociated with TrypLE Express for 10 min at 37 °C. Dissociated single cells were seeded at 5 × 10³ cells per well with Matrigel in XF24 cell culture microplates (Agilent Technologies) and cultured in EN medium for 5–6 days. One hour before measurement, culture medium was replaced with OCR assay media [minimal DMEM (Sigma Aldrich) supplemented with GlutaMAX (2 mM, Thermo Fisher), pyruvate (5 mM, Thermo Fisher), glucose (20 mM, Junsei Chemical, Tokyo, Japan)] in the 37 °C non-CO₂ incubator for 1 h. We used a Seahorse Bioscience XF24 analyzer (Agilent Technologies) to measure OCR. Oligomycin (1 μM), FCCP (1 μM), and rotenone and antimycin (1 μM) were injected for OCR measurements. All reagents were purchased from Sigma Aldrich. After the measurements, cells were lysed with RIPA buffer (Thermo Fisher) and isolated proteins were quantified with Pierce BCA Protein Assay Kit (Thermo Fisher) for normalization.