enzymatic disaggregation of the auditory epithelium to obtain auditory hair cells

マウス蝸牛有毛細胞の効率的なIn vitro培養

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.

著者スポットライト:聴覚研究のためのマウス有毛細胞の培養における進歩 

新生仔マウスの初代蝸牛有毛細胞の単離と培養

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  

聴覚上皮の酵素的解離による聴覚有毛細胞の採取

まず、新生児マウスの側頭骨から聴覚上皮を単離し、それを0.25%トリプシンを含む10ミリリットルの新鮮なDMEMに移し、混合物を摂氏37度で12分間インキュベートします。200マイクロリットルのピペットチップを使用して、手術用顕微鏡を使用して有毛細胞を基底層および他の細胞から静かに分離します。次に、さらに10ミリリットルの培地を加えて、解離を抑制します。懸濁細胞を70マイクロメートルのフィルターでろ過します。濾液を清潔な50ミリリットルのチューブに集め、300Gで5分間遠心分離します。1000マイクロリットルのピペットチップを使用して、5ミリリットルの培養液に有毛細胞を穏やかにピペッティングして再懸濁します。次に、事前に6ウェルプレートの下部にカバースリップを配置します。細胞をカウントし、6ウェルプレートで1ミリリットルあたり10〜6個の細胞密度で培養し、37°Cで2ミリリットルのDMEMと5%の二酸化炭素で接着細胞を増殖させます。培養の1日後、有毛細胞は皿の底にしっかりと付着しました。3日目までに、細胞数は2倍になりました。

enzymatic-disaggregation-auditory-epithelium-to-obtain-auditory-hair

Research

JoVE Journal

Biology

Enzymatic Disaggregation of the Auditory Epithelium to Obtain Auditory Hair Cells

102 ビュー.  The Second Affiliated Hospital of Xi’an Jiaotong University. まず、新生児マウスの側頭骨から聴覚上皮を単離し、それを0.25%トリプシンを含む10ミリリットルの新鮮なDMEMに移し、混合物を摂氏37度で12分間インキュベートします。200マイクロリットルのピペットチップを使用して、手術用顕微鏡を使用して有毛細胞を基底層および他の細胞から静かに分離します。次に、さらに10ミリリットルの培地を加えて、解離を抑制します。

ビデオ: 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 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 &#176;C, and gently suspended in culture medium using a 200 &#181;L 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 &gt;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.

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 ...

生物学

Dissection and Removal of the Temporal Bone to Collect Auditory Epithelia from Newborn Mice

To begin, securely hold the head of the euthanized newborn mice in place. Using micro operating scissors, open the scalp along the sagittal suture. Then, separate and immobilize the scalp bilaterally with the fingers.Use scissors to sever the cranium and flip it forward to access the skull. Employ a small periosteal elevator to extract the brain and then use the tip of the scissors to gently scrape the brain, revealing the base of the skull. Now, examine the bilateral temporal bones at the skull's base.Employ scissors to incise the base of the skull along the midline. Then, scrape off the skin and eliminate any unnecessary bone. Preserve and transfer the temporal bones to 35-millimeter sterile Petri dishes filled with fresh HBSS.Next, use a pair of 5 number pointed forceps to eliminate the bola and surrounding tissue from the petrous portion of the temporal bone. Hold the forceps with one hand to stabilize the semi-circular part of the temporal bone. Use the other hand to insert the lower tip of the forceps into the round window niche, separating the lateral bone of the cochlea from the scale of vestibule.Carefully remove the petrous portion of the temporal bone without contacting the Organ of Corti epithelium. Then, meticulously detach and extract the bony labyrinth of the cochlea, starting from the basal to the apical end. Using 5 number pointed forceps, carefully micro isolate the sensory epithelium of the Organ of Corti from the modiolus.Next, employ micro-operating forceps to hold the spiral ligament, and gently separate it from the stria vascularis. Transfer the clean auditory epithelium, using a 200 microliter pipette, to a three millimeter sterile culture dish containing HBSS. Place the isolated auditory epithelium in a 100 millimeter sterile Petri dish filled with HBSS for the subsequent preparation steps.

新生仔マウスの聴覚上皮を採取するための側頭骨の解剖と除去

To begin, isolate the auditory epithelium from the temporal bone of newborn mice and transfer it to 10 milliliters of fresh DMEM containing 0.25%Trypsin, incubate the mixture at 37 degrees Celsius for 12 minutes. Using a 200 microliter pipette tip, gently separate the hair cells from the basal lamina and other cells using an operating microscope. Then add another 10 milliliters of culture medium to inhibit the disaggregation.Filter the suspended cells through a 70 micrometer filter. Collect the filtrate in a clean 50 milliliter tube and centrifuge it at 300G for five minutes. Using a 1000 microliter pipette tip, re suspend the hair cells in five milliliters of culture medium by gentle pipetting.Now place a cover slip at the bottom of a six well plate in advance. Count the cells and culture them at 10 to the sixth cells per milliliter density in the six well plate, grow the adherence cells in two milliliters of DMEM at 37 degrees Celsius and 5%carbon dioxide. After one day of culturing the hair cells adhered firmly to the bottom of the dish.By the third day, the number of cells had doubled.