"In-bone" utricle cultures--a simplified, atraumatic technique for in situ cultures of the adult mouse (Mus musculus) utricle.

The "in-bone" method of culturing utricles described here is a reliable and atraumatic technique for culturing mature mouse hair cells and studying hair cell death and protection. The current in vitro technique for studying hair cells of the mature mouse utricle involves removal from the temporal bone and free floating culture in media. This technique can be problematic because of variability in the preservation of the sensory epithelium and a steep learning curve that results in injury of the sensory epithelium in less experienced hands. We present a new atraumatic technique of culturing the utricle in situ within the temporal bone. Leaving the temporal bone largely intact, a window is opened in the bony vestibule overlying the mouse utricle. The entire temporal bone is then placed into culture media. Utricles were cultured in situ for several days with minimal damage to the epithelium. The utricles are then fixed in situ, removed from the temporal bone, and processed. A standardized aminoglycoside-induced hair cell damage protocol was developed. Mature mouse utricles maintained hair cell numbers for 3 days in culture. Exposure to neomycin resulted in significant dose-dependent hair cell toxicity (p < 0.0001, 1-way analysis of variance). Exposure to the protective drug tacrine resulted in significant protection against neomycin (p < 0.05, 3-way analysis of variance). The "in-bone" technique is a reliable and atraumatic method for culturing mature mouse utricles and studying hair cell death and protection. It is easily mastered and can make in vitro study of hair cells accessible to more research groups.