Rat Enteric Nervous System Primary Culture

Primary culture of rat ENS were generated using pregnant Sprague–Dawley rats (Janvier Laboratories SA, Le Genest-St-Isle, France) as previously described [11 (link)]. All housing and experimental procedures were carried out in compliance with the local ethical review panel of INSERM (agreement E. 44,011; INSERM, Nantes, France). Pregnant rats were killed by an overdose of CO₂ followed by severing the carotid arteries. The small intestines of rat embryos were removed, diced in Hank’s Buffered Salt Solution (Sigma, Saint-Quentin Fallavier, France) and collected in 5 mL of Dulbecco’s modified Eagle’s medium (DMEM)-F12 (Gibco®, Life Technologies, Villebon sur Yvette, France) (1:1) for digestion at 37 °C for 15 min in 0.1% (v/v) trypsin (Sigma). The trypsin reaction was stopped by adding medium containing 10% fetal calf serum and then treatment with DNase I 0.01% (v/v) (Sigma) for 10 min at 37 °C. After triturating with a 10 mL pipette, cells were centrifuged at 750 rpm for 10 min. Cells were counted and then seeded at a density of 2.4 × 10⁵ cells/cm² on 24-well plates previously coated with a solution of 0.5% (v/v) gelatin in sterile phosphate buffered saline. After 24 h, the medium was replaced with a serum-free medium DMEM-F12 (1:1) containing 1% (v/v) of N-2 supplement (Life Technologies). Cultures were maintained for 14 days.