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Biology

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice

Published: September 15th, 2023

DOI:

10.3791/65687

1Department of Core Research Laboratory, The Second Affiliated Hospital of Xi’an Jiaotong University, 2Department of Otolaryngology, The Second Affiliated Hospital of Xi’an Jiaotong University
* These authors contributed equally

Herein, we present a detailed protocol for isolating and culturing primary cochlear hair cells from mice. Initially, the organ of Corti was dissected from neonatal (aged 3-5 days) murine cochleae under a microscope. Subsequently, cells were enzymatically digested into a single-cell suspension and identified using immunofluorescence after several days in culture.

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for hearing, within the osseous labyrinth of the inner ear. Cochlear hair cells consist of two anatomically and functionally distinct types: outer and inner hair cells. Damage to either of them results in hearing loss. Notably, as inner hair cells cannot regenerate, and damage to them is permanent. Hence, in vitro cultivation of primary hair cells is indispensable for investigating the protective or regenerative effects of cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells.

After manual removal of the cochlear lateral wall, the auditory epithelium was meticulously dissected from the cochlear modiolus under a microscope, incubated in a mixture consisting of 0.25% trypsin-EDTA for 10 min at 37 °C, and gently suspended in culture medium using a 200 µL pipette tip. The cell suspension was passed through a cell filter, the filtrate was centrifuged, and cells were cultured in 24-well plates. Hair cells were identified based on their capacity to express a mechanotransduction complex, myosin-VIIa, which is involved in motor tensions, and via selective labeling of F-actin using phalloidin. Cells reached >90% confluence after 4 d in culture. This method can enhance our understanding of the biological characteristics of in vitro cultured hair cells and demonstrate the efficiency of cochlear hair cell cultures, establishing a solid methodological foundation for further auditory research.

Cochlear hair cells play important roles in sound detection and signal transmission to the auditory nerve. Hair cells are mechanistic cells that function as primary sensory receptors and convert sound vibrations into electrical signals in vertebrates. The sensory epithelium of the mammalian inner ear comprises a single row of inner hair cells and three rows of outer hair cells. In different basic membrane areas, hair cells perceive sounds at different frequencies (between 20 and 2,000 Hz)1. The function of outer hair cells is an active mechanical amplification process that helps fine-tune the mammalian inner ear, conferring high sensitivity to ....

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All animal experiments were approved (No. 2021-847) by the Xi'an Jiaotong University Committee on the Use and Care of Animals.

1. Sterilization and material preparation

  1. Sterilize the dissection tools using high-temperature and high-pressure steam disinfection and dry them in a 50 °C incubator overnight.
  2. Prepare 100 mL of the culture medium containing 10% fetal bovine serum (FBS) and 10 mg/mL penicillin/streptomycin (add 10 mL of FBS and 10 µ.......

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Following this protocol, we seeded the isolated cells. Primary cochlear hair cell seeds were considered successful if the cells did not float in the culture medium and spread within 24 h. We determined the number of hair cells after they adhered and spread into flat aggregates at the bottom of the dish. After 1 day, live hair cells were tightly adhered to the bottom of the culture dish and non-adherent cells were removed by rinsing with PBS. Typically, the number of cells doubled after 3 d of culture (

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Compared with the HEI-OC1 cell line, primary cultures of hair cells more accurately replicated the physiological state of cells in vivo. Therefore, the auditory primary culture method established by isolating living cells from cochlear organs and immediately culturing them appears to be a valuable tool for extensive research on auditory systems. Certain techniques are crucial for a successful culture. First, minimizing the duration of separation of the organ of Corti from the temporal bone enhances the likelihood of sust.......

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This work was supported by the National Natural Science Foundation of China (NFSC 82101224 to YG)

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Name Company Catalog Number Comments
100 mm BioLite cell culture dish Thermo Fisher Scientific 130182 using for culture
35 mm Nunc cell culture dish Thermo Fisher Scientific 150318 using for culture
6-well palate Thermo Fisher Scientific 310109005 using for culture
70 µm cell strainers BD Company 352350 using for filter
Alexa Fluor 488 Phalloidin Thermo Fisher Scientific A12379 immunofluorescent staining
Anti-rabbit IgG Alexa Fluor 488 Thermo Fisher Scientifc A11008 immunofluorescent staining
day 3-5 neonatal murine  provided by Xi'an Jiaotong University
Dulbecco’s Modified Eagle Medium Thermo Fisher Scientific 11965092 using for culture
Fetal Bovine Serum Thermo Fisher Scientific 12483020 using for culture
Forceps Dumont 5# using for dissection
Leica anatomy microscope Germany S9i using for dissection
Penicillin/streptomycin Thermo Fisher Scientific 15140-122 using for culture
Rabbit plyclonal to Myosin VIIa Abcam company ab92996 immunofluorescent staining
Scissor Belevor 10cm/04.0524.10 using for dissection
Triton X-100 Sigma Aldrich  9036-19-5 immunofluorescent staining
Trypsin Thermo Fisher Scientific 25200072 using for culture

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