Recombinant murine gdnf

DRG excised from adult BALB/c mice (5–10-week-old) were collected in a dish containing cold F12 (Nutrient Mixture F12 Ham) medium (Sigma–Aldrich Inc., Italy). After accurate de-sheathing, DRG were transferred into a sterile 35 mm dish containing collagenase from Clostridium hystoliticum 0.125% (Sigma–Aldrich Inc., Italy) and DNase (Sigma–Aldrich Inc., Italy) in F12 (Nutrient Mixture F12 Ham) medium and incubated at 37 °C. After 1 h incubation, DRG were triturated using a 1000 µL tip. Myelin and nerve debris were eliminated by centrifugation through bovine serum albumin (BSA) cushion. Cell pellets were re-suspended in Bottenstein and Sato medium (BS): 30% F12 (Nutrient Mixture F12 Ham medium), 40% DMEM (Dulbecco’s Modified Eagle’s medium, Sigma–Aldrich Inc., Italy), 30% Neurobasal A medium (Life Technologies, Italy), 100X N2 supplement (Life Technologies, Italy), penicillin 10 U/mL and streptomycin 100 mg/mL (Sigma–Aldrich Inc., Italy), supplemented with Recombinant Human β-NGF, Recombinant Murine GDNF and Recombinant Human NT3 (Peprotech, Rocky Hill, NJ, USA) and plated onto 24 mm glass coverslips pre-coated with laminin (Sigma–Aldrich Inc., Italy). The administration of OHP and amiloride was done 48 h after the isolation of DRG neurons, and all the experiments were performed from 54 to 96 h of culture.

The small intestines and colons of male C57BL/6 mice were isolated and cut into 2‐4 mm sections after making a longitudinal incision along the entire length of the intestine, then incubated in 2 mmol/L ethylenediaminetetraacetic acid (EDTA) on a rocking platform at 100 rpm for 30 minutes (small intestine) or 60 minutes (colon) at 4°C. The tissue was then re‐suspended in clean PBS and pipetted several times. The supernatant was collected and filtered through a 70 μm filter, then centrifuged at 300 × g for 5 minutes at 4°C. Isolated crypts were suspended in Matrigel Matrix (Corning, NY, USA). The suspension was carefully pipetted into the centre of each well in a pre‐warmed 24‐well plate (Corning). After the Matrigel solidified, IntestiCult^™ Organoid Growth Medium (STEMCELL Technologies, Vancouver, Canada) supplemented with penicillin‐streptomycin (100 units/100 μg per mL) was added and refreshed every 3‐4 days. Organoids were kept in a humidified atmosphere containing 5% CO₂ at 37°C.
Organoid treatment: organoids cultured for 2 days were incubated with recombinant murine GDNF (PeproTech, London, UK) at different concentrations (50, 100 and 200 nmol/L) or RET kinase inhibitor GSK3179106 (MedChemExpress, La Jolla, CA) at 100 nmol/L for 96 hours. After stimulation, supernatants were used for the following analysis, and organoids were lysed for RNA isolation.