In vitro culture of mammalian inner ear hair cells

Abstract

Auditory function in vertebrates depends on the transduction of sound vibrations into electrical signals by inner ear hair cells. In general, hearing loss resulting from hair cell damage is irreversible because the human ear has been considered to be incapable of regenerating or repairing these sensory elements following severe injury. Therefore, regeneration and protection of inner ear hair cells have become an exciting, rapidly evolving field of research during the last decade. However, mammalian auditory hair cells are few in number, experimentally inaccessible, and barely proliferate postnatally in vitro. Various in vitro primary culture systems of inner ear hair cells have been established by different groups, although many challenges remain unresolved. Here, we briefly explain the structure of the inner ear, summarize the published methods of in vitro hair cell cultures, and propose a feasible protocol for culturing these cells, which gave satisfactory results in our study. A better understanding of in vitro hair cell cultures will substantially facilitate research involving auditory functions, drug development, and the isolation of critical molecules involved in hair cell biology.

概 要

由于内耳的血脑屏障作用, 药物渗入到内耳比较困难。 新生小鼠内耳毛细胞的体外培养体系的建立, 为体外进行支持细胞转分化机制的研究和进 行体外药物损伤毛细胞实验等提供实验技术的前提。

为了避免毛细胞体外培养过程中污染杂菌, 解剖内耳耳蜗的整个过程十分重要。 处死小鼠后, 将其浸泡在 75%酒精中 1~3 分钟, 防止鼠毛污染培养基。 打开内耳耳蜗之前, 选用添加了青霉素的磷酸盐缓冲液 (1× PBS) ; 培养过程中使用的是仅仅添加青霉素的培养基来减少对毛细胞的损伤。 在基底膜培养的第一步, 选用 DMEM (包含 5%马血清体积比和 5%胎牛血清) 作为组织粘附培养的培养基, 保证足够的营养, 同时更好地维持整个基底膜培养状态下的形态。 在之后的培养中, 选用 DMEM (添加了 10%胎牛血清、 1%N2 和 1%B27) 作为长期的培养基。 使用含有表皮生长因子的 N2 和 B27 的培养基进行基底膜以及椭圆囊之后的培养, 有助于维持毛细胞的体外生长时间。 选用鼠尾胶包被盖玻片后培养,可以增加基底膜和椭圆囊的粘附作用, 保证毛细胞静纤毛的向上生长。

该文章展现了哺乳动物毛细胞的体外培养的具体方法, 能够较好地维持耳蜗基底膜在体外培养的形态, 并增加毛细胞体外培养的存活时间。