A method for in vitro isolation and culture of inner ear hair cells

Abstract

The invention discloses a method for separating and culturing inner ear hair cells in vitro. The method is as follows: place the hair cells of the cochlear basement membrane on a four-hole cell culture plate coated with laminin in advance, and then add inner ear hair cells. Cell culture medium, replace the culture medium every 24 hours to obtain inner ear hair cells; the inner ear hair cell culture medium contains 2-15% horse serum at the final volume concentration, 2-15% fetal bovine serum at the final volume concentration, and 2-15% fetal bovine serum at the final volume concentration High glucose DMEM medium with 1‑2% N‑2 cytokine supplement. The method of the present invention can separate and obtain complete cochlear basement membrane more efficiently; Utilize the culture condition of the present invention, the morphology of basement membrane hair cells is closer to the in vivo state, and the arrangement of outer hair and inner hair is more complete; at the same time compared with the traditional method (10 % fetal calf serum high-sugar DMEM medium), the method of the present invention can prolong the survival time of basement membrane hair cells more than double.

Description

(1) Technical field [0001] The invention relates to a method for separating and in vitro culturing sensory hair cells of the inner ear. (2) Background technology [0002] Hair cells act as sensory cells in the inner ear that convert the vibrational signal of sound into an electrical signal. Damage and loss of hair cells can cause deafness. There are many reasons for hair cell damage, including drugs, environment, genetic factors and age. Inner ear hair cells are distributed in the inner ear cochlea, utricle, ampullary ridge, and balloon of mammals. The hair cells located in the utricle mainly sense linear velocity, while the hair cells located in the ampullary ridge sense rotational angular velocity. Only the hair cells located in the cochlea Cells convert sound vibration signals into the inner ear into electrical signals, which are eventually transmitted to the brain by the afferent nerves of the cochlea. The cochlea is embedded in the hard temporal bone, coupled with th...

AI Technical Summary

Problems solved by technology

However, at present, the isolation and culture of hair cells in vitro needs to be further improved. The current technology cannot cultivate hair cells in vitro for a long time and maintain a high survival rate (refer to Ding D et al.: Cisplatin ototoxicity in rat cochlear organotypic cultures.Hearing research 2011, 282( 1-2): 196-203. Ou HC et al.: "In-bone" utriclecultures--a simplified, atraumatic technique for in situ cultures of the adultmouse (Mus musculus) utricle. Otology & neurotology: official publication of the American Otological Society, American Neurotology Society&European Academy of Otology and Neurotology 2013,34(2):353-359.)

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