Four well culture dishes

The P1 mice were euthanized and decapitated, and their heads were placed in 75% ethanol and quickly transferred to pre‐cooled HBSS. The temporal bones were dissected out, the cochlea was isolated from the temporal bones using sterile procedures in ice‐cold HBSS, and the basilar membranes were removed with tweezers. Explants of the organ of Corti were placed intact on polyphenol‐protein‐coated cover glasses (Cell‐Tak Cell and Tissue Adhesive, Corning, USA) and maintained in four‐well culture dishes (Corning, USA) in culture medium composed of DMEM/F‐12 supplemented with N2, B27, and Ampicillin. The explant tissues were incubated at 37°C in an atmosphere at 5% CO₂. After 12 h of incubation, AAV was added to each well and incubated for 48 h, and then the culture medium was replaced with fresh culture medium.

hMSCs were cultured in four-well culture dishes (1 × 10⁴ cells per well; Corning Incorporated). The cells were treated with 500 μM H₂O₂ for 6 hours with or without prior let-7b transfection. After 6 hours of H₂O₂ treatment, the cells were washed with ice-cold phosphate-buffered saline (PBS; Life Technologies Corporation) for 5 minutes and fixed with 4 % formalin (Sigma) for 10 minutes. After blocking with 500 μl Annexin-binding buffer, the cells were stained with Annexin V-fluorescein isothiocyanate (FITC) at room temperature in the dark for 15 minutes. The dishes were washed with ice-cold PBS, stained with propidium iodide Annexin V/PI staining (PI), and diluted with Annexin-binding buffer at room temperature in the dark for 5 minutes. All images of Annexin V/PI-positive cells were detected by laser scanning confocal microscopy (LSM 700; Carl Zeiss, Thornwood, NY USA), and the images were transferred to a computer equipped with ZEN Lite (Carl Zeiss).