dissection and removal of the temporal bone to collect auditory epithelia from newborn mice

小鼠耳蜗毛细胞的高效体外培养

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.

作者聚焦：培养小鼠毛细胞用于听觉研究的进展 

新生小鼠原代耳蜗毛细胞的分离和培养

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 Dissection and Removal of the Temporal Bone to Collect Auditory Epithelia from Newborn Mice at JoVE.com

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

解剖和切除颞骨以收集新生小鼠的听觉上皮

首先，将安乐死的新生小鼠的头部牢固地固定到位。使用微操作剪刀，沿着矢状缝合线打开头皮。然后，用手指将头皮分开并固定在双侧。用剪刀切断头盖骨，然后将其向前翻转以进入头骨。使用小型骨膜升降机提取大脑，然后用剪刀尖轻轻刮擦大脑，露出颅底。现在，检查颅骨底部的双侧颞骨。用剪刀沿中线切开颅骨底部。然后，刮掉皮肤并消除任何不必要的骨头。保存颞骨并将其转移到装有新鲜HBSS的35毫米无菌培养皿中。接下来，使用一对 5 个数字尖镊子从颞骨的岩部消除 bola 和周围组织。用一只手握住镊子，以稳定颞骨的半圆形部分。用另一只手将镊子的下端插入圆形窗位，将耳蜗的外侧骨与前庭的鳞片分开。小心地去除颞骨的岩部，不要接触皮质上皮器官。然后，从基底到顶端，小心翼翼地分离并提取耳蜗的骨迷路。使用5个数字尖镊子，小心地将Corti器官的感觉上皮与modiolus进行微分离。接下来，使用微操作镊子固定螺旋韧带，并轻轻地将其与血管纹分开。使用200微升移液管将干净的听觉上皮转移到含有HBSS的3毫米无菌培养皿中。将分离的听觉上皮细胞放入装有HBSS的100毫米无菌培养皿中，用于后续制备步骤。

dissection-removal-temporal-bone-to-collect-auditory-epithelia-from

Research

JoVE Journal

Biology

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

185 次观看.  The Second Affiliated Hospital of Xi’an Jiaotong University. 首先，将安乐死的新生小鼠的头部牢固地固定到位。使用微操作剪刀，沿着矢状缝合线打开头皮。然后，用手指将头皮分开并固定在双侧。用剪刀切断头盖骨，然后将其向前翻转以进入头骨。使用小型骨膜升降机提取大脑，然后用剪刀尖轻轻刮擦大脑，露出颅底。现在，检查颅骨底部的双侧颞骨。用剪刀沿中线切开颅骨底部。然后，刮掉皮肤并消除任何不必要的骨...

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

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 ...

科研

生物学

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.

Enzymatic Disaggregation of the Auditory Epithelium to Obtain Auditory Hair Cells

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.