enzymatic disaggregation of the auditory epithelium to obtain auditory hair cells

쥐 달팽이관 유모 세포의 효율적인 In vitro 배양

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.

저자 스포트라이트: 청각 연구를 위한 쥐 유모 세포 배양의 발전 

신생아 마우스의 일차 달팽이관 유모 세포의 분리 및 배양

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  

청각 유모 세포를 얻기 위한 청각 상피의 효소 분해

시작하려면 갓 태어난 쥐의 측두골에서 청각 상피를 분리하고 0.25 % 트립신을 함유 한 신선한 DMEM 10 밀리리터로 옮기고 혼합물을 섭씨 37도에서 12 분 동안 배양합니다. 200 마이크로리터 피펫 팁을 사용하여 작동 현미경을 사용하여 기저층 및 다른 세포에서 유모 세포를 부드럽게 분리합니다. 그런 다음 응집을 억제하기 위해 10ml의 배양 배지를 더 추가합니다.70마이크로미터 필터를 통해 부유 세포를 여과합니다. 깨끗한 50mL 튜브에 여과액을 모으고 300G에서 5분 동안 원심분리합니다. 1000 마이크로리터 피펫 팁을 사용하여 부드러운 피펫팅으로 5 밀리리터의 배양 배지에 유모 세포를 다시 현탁시킵니다.이제 미리 6웰 플레이트의 바닥에 커버 슬립을 놓습니다. 세포를 계수하고 6 웰 플레이트에서 밀리리터 밀도 당 10-6 개의 세포로 배양하고 섭씨 37 °C에서 2 밀리리터의 DMEM과 5 % 이산화탄소로 부착 세포를 성장시킵니다. 배양 하루 후 유모 세포가 접시 바닥에 단단히 부착되었습니다.셋째 날이 되자 세포의 수가 두 배로 늘어났습니다.

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

Research

JoVE Journal

Biology

Enzymatic Disaggregation of the Auditory Epithelium to Obtain Auditory Hair Cells

102 조회수.  The Second Affiliated Hospital of Xi’an Jiaotong University. 시작하려면 갓 태어난 쥐의 측두골에서 청각 상피를 분리하고 0.25 % 트립신을 함유 한 신선한 DMEM 10 밀리리터로 옮기고 혼합물을 섭씨 37도에서 12 분 동안 배양합니다. 200 마이크로리터 피펫 팁을 사용하여 작동 현미경을 사용하여 기저층 및 다른 세포에서 유모 세포를 부드럽게 분리합니다. 그런 다음 응집을 억제하기 위해 10ml의 배양 배지를 더 추가합니다.70마이크로?...

비디오: 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 ...

연구

생물학

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.

신생아 쥐의 청각 상피를 수집하기 위한 측두골의 해부 및 제거

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.