Name: Enzymatic Disaggregation of the Auditory Epithelium to Obtain Auditory Hair Cells
Uploaded: 2023-09-15T12:40:00
Description: Watch this Scientific Journal Video about Enzymatic Disaggregation of the Auditory Epithelium to Obtain Auditory Hair Cells at JoVE.com

enzymatic disaggregation of the auditory epithelium to obtain auditory hair cells

Cultivo in vitro eficiente de células ciliadas cocleares de camundongos

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 sound. Inner hair cells are responsible for detecting sounds. After graded depolarization, acoustic information is transmitted to the brain through the auditory nerve fibers2.
Hearing loss may be caused by genetic defects, aging, noise trauma, or the excessive use of ototoxic drugs, which constitute a major health concern worldwide3,4. Hearing loss mainly results from irreversible damage to hair cells5. Regarding noise-induced hearing loss, although researchers have reached a consensus on several details of its etiology, a comprehensive understanding of the numerous underlying mechanisms is lacking. Outer hair cells are particularly vulnerable to acoustic overexposure6. Mechanosensitive cochlear hair cells are involved in age-related hearing loss; however, the molecular and cellular mechanisms underlying hair cell degeneration remain unknown. Several changes in the molecular processes lead to hair cell aging, oxidative stress, DNA damage response, autophagy, and dysregulation of the expression and transcription of genes related to hair cell specialization7.
As the inner ear is encased in the temporal bone, deep in the hardest bone of the body, it is experimentally inaccessible, posing a challenge to investigations into the mechanisms of hair cell repair and regeneration. Hence, establishing in vitro cultures for investigating the function of hair cells has become an ideal method for research on the regeneration and injury mechanisms of the inner ear. The procedures for preparing cochlear organotypic cultures have been described in earlier studies8,9,10. Investigators worldwide have employed various cochlear microdissection and surface preparation techniques. Despite the persistent challenges, various primary hair cell culture systems have been successfully established in vitro. Cochlear organ cultures contain various cell types, including hair cells, Deiters cells, Hensen&#39;s cells, pillar cells, and auditory nerve fibers. An in-depth understanding of the changes in hair cells at the cellular and molecular levels after injury will enable the development of more powerful research tools. This study aimed to demonstrate the steps for isolating cochlear organs from neonatal mice and enzymatically detaching the abundant hair cells for in vitro studies. The nature of the cultured cells was confirmed using immunofluorescence staining.

Autor em destaque: Avanços no cultivo de células ciliadas de camundongos para pesquisa auditiva 

Isolamento e Cultura de Células Ciliadas Cocleares Primárias de Camundongos Neonatais

The inner ear is encased in the temporal bone, deep in the hardest bone of the body, posing a challenging to investigations. The cultivation of primary hair cells is indispensable for investigating cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells.We demonstrate the steps for isolating cochlear organs from your natural mice and asymmetrically detaching the abundant hair cells for in-vitro studies. The nature of the cultured cells, was confirmed using immunofluorescence staining. There are three primary culture method established by isolating living cells from cochlear organs, and immediately coaching them, pair to be a valuable tool for extensive research, are all three system.This method can enhance our understanding of the biological characteristics of in-vivo cultured hair cells, and demonstrate the efficiency of cochlear hair cell cultures, establishing a solid methodological foundation for further auditory research.

Watch this Scientific Journal Video about Enzymatic Disaggregation of the Auditory Epithelium to Obtain Auditory Hair Cells at JoVE.com

Enzymatic Disaggregation of the Auditory Epithelium to Obtain Auditory Hair Cells  

Disagregação enzimática do epitélio auditivo para obtenção das células ciliadas auditivas

Para começar, isole o epitélio auditivo do osso temporal de camundongos recém-nascidos e transfira-o para 10 mililitros de DMEM fresco contendo 0,25% de tripsina, incube a mistura a 37 graus Celsius por 12 minutos. Usando uma ponteira de pipeta de 200 microlitros, separe suavemente as células ciliadas da lâmina basal e outras células usando um microscópio cirúrgico. Em seguida, adicione mais 10 mililitros de meio de cultura para inibir a desagregação.Filtre as células suspensas através de um filtro de 70 micrômetros. Recolher o filtrado num tubo limpo de 50 mililitros e centrifugar-o a 300G durante cinco minutos. Usando uma ponteira de pipeta de 1000 microlitros, ressuspenda as células ciliadas em cinco mililitros de meio de cultura por pipetagem suave.Agora coloque uma tampa no fundo de uma placa de seis poços com antecedência. Conte as células e cultive-as a 10 a sexta células por mililitro de densidade na placa de seis poços, cultive as células de aderência em dois mililitros de DMEM a 37 graus Celsius e 5% de dióxido de carbono. Após um dia de cultivo, as células ciliadas aderiram firmemente ao fundo do prato.No terceiro dia, o número de células dobrou.

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Biology

Enzymatic Disaggregation of the Auditory Epithelium to Obtain Auditory Hair Cells

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice

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 ...