Postnatal Mouse Cochlear Explant Culture

The organ culture procedures were modified from published reports (Zheng and Gao, 1996 (link), Ding et al., 2002 (link)). In brief, mice of postnatal day 3 (p3) were euthanized after antisepsis with 70% ethanol, and the inner ears were removed. The cochleae were immersed in cold Hank’s Balanced Salt Solution and the lateral wall tissues (stria vascularis and spiral ligament) and the auditory nerve bundle were dissected away leaving the organ of Corti and spiral ganglion neurons. The explants were placed onto a previously prepared culture dish: a 15-μL drop of rat tail collagen solution was allowed to polymerize for approximately 15 min on a 35 mm dish, then 1 mL of serum-free medium, including Basal Medium Eagle (BME), 1% serum-free supplement (Gibco catalogue number 51500-056; Invitrogen, Carlsbad, CA), 1% bovine serum albumin, 5 mg/mL glucose and 10 U/mL penicillin G were added. The cochlear explants were incubated (37°C, 5% CO₂) for 4 h, then an additional 1 mL of culture medium was added to submerse the explants.