这项工作得到了中国国家自然科学基金（NFSC 82101224 to YG）的支持

所有动物实验均获得习交通大学动物使用与护理委员会批准（编号：2021-847）。
1.灭菌和材料准备
<ol><li>使用高温高压蒸汽消毒对解剖工具进行灭菌，并在50°C培养箱中干燥过夜。</li>
	<li>制备100mL含有10%胎牛血清（FBS）和10mg / mL青霉素/链霉素的培养基（将10mL FBS和10μL青霉素储备溶液加入90mL的Dulbecco改良Eagle培养基（EMEM）中）并储存在4°C。</li>
</ol>2.颞骨的解剖和切除以收集听觉上皮
<ol><li>通过在冰上斩首对总共10只新生小鼠（年龄在P3和P5之间）实施安乐死。如果需要，使用其他合乎道德的方法。</li>
	<li>将头部固定到位，使用微操作剪刀沿矢状缝合线打开头皮，并用手指将头皮分开并双侧固定，如图 1A所示。</li>
	<li>使用小型骨膜升降机取出大脑，并将基底颅骨一分为二。用剪刀剪开头盖骨，向前翻转以打开头骨;用剪刀尖刮开大脑，露出头骨底部。</li>
	<li>观察颅骨底部的双侧颞骨（图1C）。用剪刀沿中线切开颅骨底部，刮掉皮肤，去除任何不必要的骨头（图1D，D'）。</li>
	<li>将颞骨保留并转移到含有新鲜Hank平衡盐溶液（HBSS）的35mm无菌培养皿中（图1E）。</li>
	<li>使用两个#5尖镊子从颞骨的岩部去除大疱和周围组织（图1E）。 注意：在这个阶段，小鼠颞骨中的骨迷路不会完全钙化，并且很容易使用镊子解剖。</li>
	<li>一只手握住镊子固定颞骨的半圆形部分，另一只手将镊子的下脚插入圆窗龛，将耳蜗的外侧骨与鳞状前庭分开。小心地去除颞骨的岩部，不要接触OC上皮。随后，小心地分离并移除耳蜗的骨迷宫，从基底端到顶端（图1F）。</li>
	<li>使用#5尖镊子小心地将Corti感觉上皮器官与modiolus进行微分离（图1G）。</li>
	<li>握住螺旋韧带，用微操作镊子小心地将其与血管纹分离，并使用200μL移液管将干净的听觉上皮转移到含有HBSS的3mm无菌培养皿中（图1H）。</li>
	<li>从每只动物中收集20个标本，并将它们快速转移到含有HBSS的100mm无菌培养皿中，用于下一步制备步骤（图1）。</li>
</ol>3. 酶解聚获得听觉毛细胞
<ol><li>将听觉上皮转移到含有0.25%胰蛋白酶的10mL新鲜DMEM中，并在37°C孵育12分钟。</li>
	<li>使用200μL移液器吸头，在手术显微镜下轻轻地将毛细胞与基底层和其他细胞分开。</li>
	<li>再加入 10 mL 培养基以抑制分解。</li>
	<li>通过70μm过滤器过滤培养基中的悬浮细胞，将滤液收集在干净的50mL管中，并以300× g 离心5分钟。</li>
	<li>使用 1,000 μL 移液器吸头轻轻上下移液，将毛细胞重悬于至少 5 mL 培养基中。避免引入气泡。</li>
	<li>提前将盖玻片放在六孔板的底部。计数细胞，并在六孔板中以 106 个细胞/mL 的密度培养它们。</li>
	<li>在37°C和5%CO2下，在2mL DMEM（含有10%FBS，100单位/ mL青霉素和100μg/ mL链霉素）中培养贴壁细胞。每天更换培养基。 注意：应该提到的是，使用此协议不会导致获得纯毛细胞。基于这种原代细胞培养方法，我们建议使用 d2 或 d3 细胞进行进一步研究，因为毛细胞可能约占培养细胞的 70% 并且处于良好状态。</li>
</ol>4.免疫荧光染色
<ol><li>从d1到d6收获培养的细胞（每天一个孔）。吸出培养基并用磷酸盐缓冲盐水 （PBS） 冲洗细胞 2 次。</li>
	<li>在室温（RT）下用4%多聚甲醛固定细胞15分钟。</li>
	<li>取出固定剂并用PBS冲洗细胞3 x 3分钟。</li>
	<li>在室温下用含有0.2%Triton X-100的PBS透化细胞10分钟。</li>
	<li>在室温下，将透化的细胞与含有10%FBS的PBS封闭溶液孵育20分钟。</li>
	<li>用抗肌球蛋白单克隆抗体（在PBS中以1：200稀释）在4°C下染色细胞过夜。</li>
	<li>用无菌 PBS 洗涤细胞 3 次，并用二抗（Alexa Fluor 594 山羊抗兔 MYO7A，在 PBS 中以 1：500 稀释）和荧光素标记的鬼笔环肽（Alexa Fluor 488，以鉴定细胞结构）在室温下孵育 2 小时。</li>
	<li>用 PBS 冲洗细胞 3 次以去除二抗。</li>
	<li>在载玻片上加入1-2滴含有DAPI的封固剂，安装盖玻片，并将它们置于激光扫描共聚焦显微镜下以捕获细胞的照片。</li>
</ol>5. 统计分析
<ol><li>进行双向方差分析 （ANOVA），然后进行 Tukey 事后检验，以分析肌球蛋白-VIIa 和鬼笔环肽灰度值随时间的变化。使用字母标记方法标记统计差异。</li>
	<li>执行额外的单因素方差分析，然后进行 Tukey 的事后检验，以比较第 1 天至第 6 天细胞样本之间的鬼笔环肽阳性细胞比率和同一天的肌球蛋白-VII 阳性细胞比率。</li>
</ol>

小鼠耳蜗毛细胞的高效体外培养

<ol>
	<li>Tani, T., Koike-Tani, M., Tran, M. T., Shribak, M., Levic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+structural+development+of+mammalian+Basilar+Membrane+provides+anatomical+basis+for+the+maturation+of+tonotopic+maps+and+frequency+tuning.">Postnatal structural development of mammalian Basilar Membrane provides anatomical basis for the maturation of tonotopic maps and frequency tuning.</a> Sci Rep. 11 (1), 7581 (2021).</li><li>Goutman, J. D., Elgoyhen, A. B., Gomez-Casati, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+hair+cells:+The+sound-sensing+machines.">Cochlear hair cells: The sound-sensing machines.</a> FEBS Lett. 589 (22), 3354-3361 (2015).</li><li>Joo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Contribution+of+Ototoxic+Medications+to+Hearing+Loss+Among+Older+Adults.">The Contribution of Ototoxic Medications to Hearing Loss Among Older Adults.</a> J Gerontol A Biol Sci Med Sci. 75 (3), 561-566 (2020).</li><li>Nieman, C. L., Oh, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hearing+Loss.">Hearing Loss.</a> Ann Intern Med. 173 (11), ITC81-ITC96 (2020).</li><li>Mao, H., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise-Induced+Hearing+Loss:+Updates+on+Molecular+Targets+and+Potential+Interventions.">Noise-Induced Hearing Loss: Updates on Molecular Targets and Potential Interventions.</a> Neural Plast. 2021, 4784385 (2021).</li><li>Morioka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hearing+vulnerability+after+noise+exposure+in+a+mouse+model+of+reactive+oxygen+species+overproduction.">Hearing vulnerability after noise exposure in a mouse model of reactive oxygen species overproduction.</a> J Neurochem. 146 (4), 459-473 (2018).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cytological+profiling+of+biological+aging+of+mouse+cochlear+inner+and+outer+hair+cells.">Molecular and cytological profiling of biological aging of mouse cochlear inner and outer hair cells.</a> Cell Rep. 39 (2), 110665 (2022).</li><li>Ogier, J. M., Burt, R. A., Drury, H. R., Lim, R., Nayagam, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+Culture+of+Neonatal+Murine+Inner+Ear+Explants.">Organotypic Culture of Neonatal Murine Inner Ear Explants.</a> Front Cell Neurosci. 13, 170 (2019).</li><li>Ding, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin+ototoxicity+in+rat+cochlear+organotypic+cultures.">Cisplatin ototoxicity in rat cochlear organotypic cultures.</a> Hear Res. 282 (1-2), 196-203 (2011).</li><li>Abitbol, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin-induced+ototoxicity+in+organotypic+cochlear+cultures+occurs+independent+of+gap+junctional+intercellular+communication.">Cisplatin-induced ototoxicity in organotypic cochlear cultures occurs independent of gap junctional intercellular communication.</a> Cell Death Dis. 11 (5), 342 (2020).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin-VIIa+is+expressed+in+multiple+isoforms+and+essential+for+tensioning+the+hair+cell+mechanotransduction+complex.">Myosin-VIIa is expressed in multiple isoforms and essential for tensioning the hair cell mechanotransduction complex.</a> Nat Commun. 11 (1), 2066 (2020).</li><li>Kalinec, G. M., Park, C., Thein, P., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+with+auditory+HEI-OC1+cells.">Working with auditory HEI-OC1 cells.</a> J Vis Exp. (115), (2016).</li><li>Montgomery, S. C., Cox, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+dissection+and+immunofluorescence+of+the+adult+mouse+cochlea.">Whole mount dissection and immunofluorescence of the adult mouse cochlea.</a> J Vis Exp. (107), (2016).</li><li>Xu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mouse+cochlear+progenitors+that+develop+hair+and+supporting+cells+in+the+organ+of+Corti.">Identification of mouse cochlear progenitors that develop hair and supporting cells in the organ of Corti.</a> Nat Commun. 8, 15046 (2017).</li><li>Zheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prestin+is+the+motor+protein+of+cochlear+outer+hair+cells.">Prestin is the motor protein of cochlear outer hair cells.</a> Nature. 405 (6783), 149-155 (2000).</li><li>Ruel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+SLC17A8+encoding+vesicular+glutamate+transporter-3,+VGLUT3,+underlies+nonsyndromic+deafness+DFNA25+and+inner+hair+cell+dysfunction+in+null+mice.">Impairment of SLC17A8 encoding vesicular glutamate transporter-3, VGLUT3, underlies nonsyndromic deafness DFNA25 and inner hair cell dysfunction in null mice.</a> Am J Hum Genet. 83 (2), 278-292 (2008).</li><li>Seal, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorineural+deafness+and+seizures+in+mice+lacking+vesicular+glutamate+transporter+3.">Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3.</a> Neuron. 57 (2), 263-275 (2008).</li><li>Luo, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+distinct+Atoh1+enhancers+cooperate+for+sound+receptor+hair+cell+development.">Three distinct Atoh1 enhancers cooperate for sound receptor hair cell development.</a> Proc Natl Acad Sci U S A. 119 (32), e2119850119 (2022).</li><li>Kalinec, G., Thein, P., Park, C., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEI-OC1+cells+as+a+model+for+investigating+drug+cytotoxicity.">HEI-OC1 cells as a model for investigating drug cytotoxicity.</a> Hear Res. 335, 105-117 (2016).</li></ol>

作者没有要披露的利益冲突。

与HEI-OC1细胞系相比，毛细胞的原代培养更准确地复制了体内细胞的生理状态。因此，通过将活细胞从耳蜗器官中分离出来并立即培养它们而建立的听觉原代培养方法似乎是广泛研究听觉系统的宝贵工具。某些技术对于成功的文化至关重要。首先，最小化Corti器官与颞骨分离的持续时间可增强毛细胞持续活动的可能性。因此，研究人员必须尽量减少器官解剖和浸入PBS之间的时间间隔。根据我们的实?...

耳蜗毛细胞在声音检测和信号传递到听觉神经中起着重要作用。毛细胞是机械细胞，在脊椎动物中充当主要感觉受体并将声音振动转化为电信号。哺乳动物内耳的感觉上皮包括单排内毛细胞和三排外毛细胞。在不同的基本膜区域，毛细胞感知不同频率（20 至 2,000 Hz）的声音1。外毛细胞的功能是一个主动的机械放大过程，有助于微调哺乳动物的内耳，赋予对声音的高灵敏度。内毛细胞负责检测声音。分级去极化后，声学信息通过听觉神经纤维2传递到大脑。
听力损失可能是由遗传缺陷、衰老、噪音创伤或过度使用耳毒性药物引起的，这些都是世界范围内的主要健康问题 3,4。听力损失主要由毛细胞不可逆的损伤引起5.关于噪声引起的听力损失，尽管研究人员已就其病因的几个细节达成共识，但缺乏对众多潜在机制的全面了解。外毛细胞特别容易受到声学过度暴露的影响6.机械敏感的耳蜗毛细胞与年龄相关的听力损失有关;然而，毛细胞变性的分子和细胞机制仍然未知。分子过程的几种变化导致毛细胞老化、氧化应激、DNA 损伤反应、自噬以及与毛细胞特化相关的基因表达和转录失调7.
由于内耳被包裹在颞骨中，深入身体最坚硬的骨骼，因此在实验上无法进入，这对研究毛细胞修复和再生机制提出了挑战。因此，建立用于研究毛细胞功能的体外培养物已成为研究内耳再生和损伤机制的理想方法。制备耳蜗器官型培养物的程序已在早期研究中描述8,9,10。世界各地的研究人员采用了各种人工耳蜗显微切割和表面处理技术。尽管存在持续的挑战，但各种原代毛细胞培养系统已成功在体外建立。耳蜗器官培养物包含多种细胞类型，包括毛细胞、Deiters 细胞、Hensen 细胞、支柱细胞和听觉神经纤维。深入了解损伤后毛细胞在细胞和分子水平上的变化将有助于开发更强大的研究工具。本研究旨在证明从新生小鼠中分离耳蜗器官并酶促分离丰富的毛细胞用于体外研究的步骤。使用免疫荧光染色确认培养细胞的性质。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm BioLite cell culture dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>130182</td>
			<td>using for culture</td>
		</tr>
		<tr>
			<td>35 mm Nunc cell culture dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>150318</td>
			<td>using for culture</td>
		</tr>
		<tr>
			<td>6-well palate</td>
			<td>Thermo Fisher Scientific</td>
			<td>310109005</td>
			<td>using for culture</td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainers</td>
			<td>BD Company</td>
			<td>352350</td>
			<td>using for filter</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12379</td>
			<td>immunofluorescent staining</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG Alexa Fluor 488</td>
			<td>Thermo Fisher Scientifc</td>
			<td>A11008</td>
			<td>immunofluorescent staining</td>
		</tr>
		<tr>
			<td>day 3-5 neonatal murine&#160;</td>
			<td></td>
			<td></td>
			<td>provided by Xi&#39;an Jiaotong University</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td>using for culture</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>12483020</td>
			<td>using for culture</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td>5#</td>
			<td>using for dissection</td>
		</tr>
		<tr>
			<td>Leica anatomy microscope</td>
			<td>Germany</td>
			<td>S9i</td>
			<td>using for dissection</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td>using for culture</td>
		</tr>
		<tr>
			<td>Rabbit plyclonal to Myosin VIIa</td>
			<td>Abcam company</td>
			<td>ab92996</td>
			<td>immunofluorescent staining</td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>Belevor</td>
			<td>10cm/04.0524.10</td>
			<td>using for dissection</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich&#160;</td>
			<td>9036-19-5</td>
			<td>immunofluorescent staining</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200072</td>
			<td>using for culture</td>
		</tr>
	</tbody>
</table>

isolation and culture of primary cochlear hair cells from neonatal mice

耳蜗毛细胞是听觉系统的感觉受体。这些细胞位于内耳骨迷宫内负责听觉的感觉器官Corti器官中。耳蜗毛细胞由两种解剖学和功能上不同的类型组成：外毛细胞和内毛细胞。其中任何一个的损坏都会导致听力损失。值得注意的是，由于内部毛细胞无法再生，并且对它们的损害是永久性的。因此，原代毛细胞的 体外 培养对于研究耳蜗毛细胞的保护或再生作用是必不可少的。本研究旨在发现一种分离和培养小鼠毛细胞的方法。 
手动去除耳蜗侧壁后，在显微镜下从耳蜗中仔细解剖听觉上皮，在由0.25%胰蛋白酶-EDTA组成的混合物中在37°C下孵育10分钟，并使用200μL移液器吸头轻轻悬浮在培养基中。将细胞悬液通过细胞过滤器，离心滤液，并在 24 孔板中培养细胞。根据毛细胞表达参与运动张力的机械转导复合物肌球蛋白-VIIa的能力以及通过使用鬼笔环肽选择性标记 F-肌动蛋白来鉴定毛细胞。细胞在培养 4 天后达到 >90% 的汇合度。该方法可以增强我们对体外培养毛细胞生物学特性的理解，并证明耳蜗毛细胞培养的效率，为进一步的听觉研究奠定了坚实的方法学基础。

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.

按照该协议，我们接种了分离的细胞。如果原代耳蜗毛细胞种子没有漂浮在培养基中并在 24 小时内扩散，则认为这些细胞是成功的。我们确定了毛细胞粘附并扩散成培养皿底部的扁平聚集体后的数量。1天后，将活毛细胞紧紧粘附在培养皿底部，并用PBS冲洗除去非贴壁细胞。通常，培养3天后细胞数量增加一倍（图2）。
免疫荧光（IF）显示肌球蛋白-VIIa和...

Watch this Scientific Journal Video about Advancements in Cultivating Mouse Hair Cells for Auditory Research at JoVE.com

在此，我们提出了从小鼠中分离和培养原代耳蜗毛细胞的详细方案。最初，在显微镜下从新生儿（3-5天龄）鼠耳蜗中解剖Corti的器官。随后，将细胞酶消化成单细胞悬液，并在培养数天后使用免疫荧光进行鉴定。

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 ...

内耳被包裹在颞骨中，深深地扎在身体最坚硬的骨头中，对调查构成了挑战。原代毛细胞的培养对于研究耳蜗毛细胞是必不可少的。本研究旨在发现一种分离和培养小鼠毛细胞的方法。我们演示了从天然小鼠中分离耳蜗器官和不对称分离丰富的毛细胞以进行体外研究的步骤。使用免疫荧光染色确认培养细胞的性质。从耳蜗器官中分离活细胞，并立即指导它们，成为广泛研究的宝贵工具，主要培养方法有三种，这三种系统都是三种。该方法可以增强我们对体内培养毛细胞生物学特性的理解，并证明耳蜗毛细胞培养的效率，为进一步的听觉研究奠定了坚实的方法学基础。

isolation-culture-primary-cochlear-hair-cells-from-neonatal

Research

JoVE Journal

Biology

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice

1.6K 次观看.  The Second Affiliated Hospital of Xi’an Jiaotong University. 内耳被包裹在颞骨中，深深地扎在身体最坚硬的骨头中，对调查构成了挑战。原代毛细胞的培养对于研究耳蜗毛细胞是必不可少的。本研究旨在发现一种分离和培养小鼠毛细胞的方法。我们演示了从天然小鼠中分离耳蜗器官和不对称分离丰富的毛细胞以进行体外研究的步骤。使用免疫荧光染色确认培养细胞的性质。从耳蜗器官中分离活细胞，并立即指导它们，成为广泛?...

视频: 作者聚焦：培养小鼠毛细胞用于听觉研究的进展 

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.

科研

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.

解剖和切除颞骨以收集新生小鼠的听觉上皮

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.