Name: three-dimensional organotypic cultures of vestibular and auditory sensory organs
Uploaded: 2018-06-01T14:00:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs at JoVE.com

我们感谢贾格布博士、j. Salvi 博士和 Petelski 为本议定书所依据的原始研究所作的贡献。我们还感谢美洲骆驼和 w Makmura 提供技术援助和畜牧业。我们承认 NIDCD 培训补助金 T32 DC009975, NIDCD 赠款 R01DC015530, 罗伯逊治疗发展基金, 和家庭基金会的资金。最后, 我们承认霍华德休斯医学研究所的支持, Hudspeth 博士是一名调查员。

这里描述的所有方法都得到了洛克菲勒大学和南加州大学动物保育和使用委员会的批准。
1. (可选) 用小鼠尾肌腱制备胶原 i 型溶液
注意:胶原蛋白 I 解决方案可在商业上使用。按照制造商的说明进行凝胶制剂。
<ol><li>弄死 5-10 个年轻成人 (3-5 周大) 的小鼠, 任何野生型菌株与二氧化碳按照有关机构动物护理和使用委员会批准的议定书16。收集尾巴和消毒他们通过淹没在70% 乙醇至少在室温下4小时。 注意:培养超过48小时应避免, 因为它导致过多的组织脱水和阻碍肌腱提取过程。</li>
	<li>从每条尾巴的皮肤, 通过引入一个切割刀刀片和缩回整个皮肤的镊子。将剥皮的尾巴转移到一个100毫米的培养皿中, 里面装满干净的70% 乙醇, 切成10毫米的段。 注意:丢弃尾部最薄的部分, 这是难以操作的。</li>
	<li>使用镊子, 确保尾部的每一段到培养皿的底部, 并使用第二对精钳从尾部一次提取肌腱。个别肌腱纤维应出现的阻力最小。 注意:陈旧的, 沉闷的 #5 钳为这一步的工作很好。</li>
	<li>在不育的分子级水中, 准备100毫升的醋酸0.1% 溶液 (按体积), 并在不育的100毫米培养皿中加入10毫升的溶液。将肌腱转移到培养皿中, 在室温下留1小时变性胶原蛋白 i。</li>
	<li>使用无菌手术刀或虹膜剪刀钝, 弯曲的提示, 以切碎肌腱成 1-2 毫米碎片。将切碎的肌腱转移到不育的50毫升管中, 加入0.1% 醋酸, 使体积增加到50毫升。 注意:使用四或五尾为每50毫升的酸, 以实现胶原 i 浓度的 2.0-2.5 毫克/毫升。</li>
	<li>将胶原蛋白 i 溶液冷藏4摄氏度, 最小为48小时, 以促进蛋白质的完全变性。涡旋与桌面涡设置为最大速度为1分钟两次一天。</li>
	<li>用二辛可宁酸法测定溶液的蛋白质浓度, 通过添加0.1% 醋酸溶液, 将胶原 i 浓度调整为 2.0-2.5 毫克/毫升, 并贮存在4摄氏度。 注意:胶原 i 溶液可以储存一到两年。</li>
	<li>选离心的胶原 i 溶液在 2000 x g 1 小时在4摄氏度, 并使用半透明的顶部分数 (约一半的体积), 以实现光学明确的胶原凝胶在4节。</li>
</ol>2. 前庭和听觉器官的解剖
<ol><li>根据有关机构动物保育和使用委员会批准的议定书, 弄死怀孕小鼠任何与二氧化碳有关的菌株16。提取并斩首胚胎。 注意:Lfng-CreERT2/tdTomato 鼠标可用于在暴露于 4-hydroxytamoxifen17时允许永久标记支持单元格。</li>
	<li>消毒所有的工作表面和解剖器械, 包括两对 #5 钳和一条发刀18, 通过清洁70% 乙醇。</li>
	<li>通过采用无菌手术刀刀片或使用两对 #5 钳, 将每个头分成两半。提取的颞骨, 其中包含内耳19, 并把它们放入15毫升的冰冷汉克的平衡盐溶液 (HBSS) 在60毫米的培养皿。把盘子放在冰上。 注意:从 E13.5 到 E18.5 的各个阶段的胚胎已经成功使用。</li>
	<li>选在50毫升的锥形管中轻轻摇动它们, 其中包含30毫升冰冷的 HBSS, 并更换 HBSS 三或四倍。把管子放在冰上。 注意:这一步骤限制了可能的组织培养的污染, 并迅速带来的温度为4摄氏度, 这可以防止组织退化和细胞死亡。</li>
	<li>使用两对精细 #5 钳, 分开的内耳从颞骨和转移的耳朵, 每次三或四, 成一个60毫米的培养皿充满冰冷的 HBSS。</li>
	<li>解剖前庭感官器官。<ol>
<li>将耳朵内侧侧向上定位, 并找到囊。用两对 #5 钳, 除去前庭器官周围的软骨。轻轻地切断前庭神经, 囊和囊之间的连接, 以及半圆形运河, 如图 1所示。轻轻拉囊和 ampullae 的上级和水平半圆运河从耳朵。 注意:该方法可用于 E16.5 E18.5 阶段的内耳。保存下面3节的软骨碎片。</li>
	</ol>
</li>
	<li>解剖耳蜗。<ol>
<li>用两对 #5 钳去除听觉器官周围的软骨组织。轻轻地切断耳蜗底座和囊之间的连接, 如图 1所示。 注意:对于分期 E13.5-E14.5, 软化软骨前解剖通过治疗内耳与0.25% 胶原酶 I 在磷酸盐缓冲盐水 (PBS) 溶液5分钟室温。保存下面3节的软骨碎片。</li>
	</ol>
</li>
	<li>使用 200-µL 吸管装有宽的不粘锅尖端, 转移胞果和耳蜗到一个30毫米培养皿填充生长培养基, 包括 DMEM/F12 补充33毫米 Dglucose, 19 毫米碳酸氢钠, 15 毫米 4 (2 羟乙基)-1piperazineethanesulfonic 酸 (HEPES), 1 毫米谷氨酰胺, 1 毫米烟酰胺, 20 毫克/升表皮生长因子, 20 毫克/升成纤维细胞生长因子, 10 毫克/升胰岛素, 5.5 毫克/升转铁蛋白, 5 µg/升亚硒钠。</li>
	<li>保持囊的准备为3小时, 在37摄氏度的组织培养孵化器与5% 二氧化碳, 以允许愈合的切口在解剖过程中引入的上皮。耳蜗的准备工作应转移到胶原凝胶后10分钟的解剖。 注意:因为解剖是在冰冷的 HBSS 中进行的, 所以没有必要将生长培养基预热到37摄氏度。</li>
</ol>3. (可选) 通过增加不同浓度的软骨细胞来调节胶原蛋白 i 凝胶的硬度
注意:软骨细胞隔离的方法从戈塞特et 。20
<ol><li>准备1% 溶液的胶原酶 I 在无菌 1x PBS 和存储 100-200 µL 整除数在-80 摄氏度。在冰上解冻时需要, 避免多个冻融循环。</li>
	<li>用细钳收集从前庭和听觉器官解剖遗留下来的软骨碎片, 从 10-12 耳。从软骨中分离结缔组织和内耳组织。将软骨转移到30毫米培养皿中, 加入300µL 的无菌生长培养基。</li>
	<li>使用无菌手术刀刀片或虹膜剪刀钝弯曲提示, 以粉碎组织, 以达到大约0.5 毫米的软骨片长度。</li>
	<li>添加胶原酶 I 到培养基与软骨, 以达到最终的酶浓度为0.25%。把盘子转移到一个组织培养孵化器在37°c 毒气与5% 二氧化碳。吸管积极使用1000µL 吸管每20分钟, 直到软骨部分离解, 并已不再区分在解决方案, 然后孵化额外20分钟。 注意:在囊准备的情况下, 可以在步骤2.9 中执行软骨细胞隔离;它需要大约2小时 (3-4 吹打回合)。</li>
	<li>收集细胞悬浮在一个15毫升圆锥管和调整体积为10毫升与无菌 1x PBS。离心机在 800 x g 5 分钟在4摄氏度。取出上清液, 并用重悬10毫升无菌 1x PBS 中的细胞。离心机在 800 x g 5 分钟在4°c 和去除上清。 注意:两次冲洗细胞是至关重要的;任何剩余的胶原酶, 我会消化胶原蛋白 i 凝胶。</li>
	<li>使用 200-µL 吸管, 添加100µL 培养基的细胞颗粒和吸管轻轻地并用重悬。使用 hemocytometer 计算单元格并在冰上维持它们。</li>
	<li>为了增加凝胶的硬度, 添加软骨细胞到中性胶原 i 凝胶溶液 (1 节), 并迅速混合在整个凝胶之前分配细胞凝固。 注意:胶原 i 型凝胶的弹性模量 (硬度的测量), 随着软骨细胞数量的增加而增大11。该关系由实验确定的线性函数E = 206·描述。Nc + 15, 其中E是帕斯卡中的弹性模数, Nc是数以百万计的软骨细胞数。有关凝胶刚度测量的详细协议, 请参阅原始文章11。</li>
</ol>4. 将前庭或听觉感觉器官放置在胶原蛋白 i 凝胶中
<ol><li>在不育的1.5 毫升管, 制备胶原 i 聚合溶液的混合160µL 10x PBS 与苯酚红色 pH 值, 133 µL 0.34 米氢氧化钠, 70 µL 的0.9 米碳酸氢钠, 40 µL 1 米 HEPES。把管子放在冰上。 注意:这个食谱提供了聚合解决方案, 以准备2毫升胶原蛋白 i 凝胶, 但可以根据需要缩放。</li>
	<li>混合100µL 的聚合溶液和400µL 的胶原 i 溶液在冰上轻轻吹打上下在一个冷冻1.5 毫升管。为了增加凝胶的硬度, 添加50µL 的生长培养基中含有软骨细胞, 并轻轻混合, 如第3节所述。</li>
	<li>将中性胶原 i 溶液的500µL 转移到一个冷冻的30毫米培养皿中, 用10毫米的玻璃底部插入物或一个四井板的井。 注意:保持冰上的所有试剂, 防止胶原蛋白的快速和不均匀聚合是至关重要的。</li>
	<li>快速将耳蜗或胞果转移到中性胶原 i 溶液中, 用不育的发刀18或一对精钳将器官调整到所需的位置. 注意:胶原 i 聚合在 1-2 分钟以后变得引人注目, 因为溶液变得混浊。</li>
	<li>在溶液周围的组织已聚合, 放置培养皿或四井板20分钟37°c 在一个组织培养孵化器与5% 二氧化碳毒气, 以确保完全聚合。</li>
	<li>添加3毫升的生长培养基辅以0.5% 胎牛血清 (血清) 每培养皿或500µL 的同一培养基每井四井板。保持37°c 的文化在一个组织培养孵化器与5% 二氧化碳毒气。如果需要, 补充生长培养基与10µM 5-乙炔-2´脱氧尿苷 (教育局) 标签增殖细胞。 注意:如果需要, 可以使用更高的血清浓度。</li>
</ol>5. 三维的前庭和听觉知觉器官的病毒注射
<ol><li>在冰上解冻所需的病毒, 并在0.5 毫升锥形管中与台盼蓝溶液混合, 达到最终染料浓度的0.05%。使用 10-20x 染料, 以避免大量稀释的病毒。保持冰块。 注意:腺病毒5型对感染囊19中的支持细胞最有效, 而腺相关病毒 Anc80 可以感染囊和耳蜗21中的毛发细胞和支持细胞。</li>
	<li>在一双双目解剖显微镜的最高放大倍数下, 用干净的细钳在微拉拔器上折断玻璃针的尖端。 注意:优化拔针器的设置非常重要。针与 9-12 毫米小腿和 20-30-µm 开口工作最好。</li>
	<li>从孵化器中移除感官器官的培养。将针贴在 microinjector 上, 用 2-3 µL 染料和病毒混合物填充。在双目解剖显微镜下观察, 将针头推进感官器官。</li>
	<li>通过三维 utricular 培养的屋顶的间质和上皮层轻轻地驱动针尖。注入病毒混合物, 直到囊和 ampullae 的空洞填充蓝色染料。 注意:因为它允许容易接触感官器官, 一个10毫米的玻璃底部培养皿是最佳的注射。</li>
	<li>在5% 二氧化碳的组织培养孵化器中孵育37摄氏度。 注意:在病毒结构中使用绿色或红色荧光蛋白时, 荧光在病毒传导后明显24小时。</li>
</ol>

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作者没有什么可透露的。

研究了在发育过程中调节内耳生长和分化的分子信号5,6,7,8,9,10。然而, 从 utricular 模型系统获得的证据表明, 通过细胞连接感知的机械线索和河马信号的活化, 在这些过程中也扮演着重要的角色2,11,<...

体外系统促进了哺乳动物器官发育的大多数方面的研究。现在有两种主要的方法用于前庭感觉器官的培养: 自由浮动的1和黏附的2准备。这两种方法都允许调查毛细胞漏洞3和再生1,4 在体外。此外, 凹槽5、6、Wnt7、8和表皮生长因子受体 (EGFR)9、10信号级联在内耳中的发展角色有在某种程度上是通过使用体外的感官上皮培养而建立的。然而, 细胞生长和分化的控制, 不仅通过格局来说信号, 而且还通过物理和机械的线索, 如细胞间接触, 僵硬的细胞外基质, 和机械伸展或收缩。这种机械刺激的作用在开发内耳体内的研究中具有挑战性。此外, 现有的游离漂浮和贴附培养方法不适用于这种研究在体外。在这里, 我们描述了一个三维脊髓培养的方法, 胶原蛋白 i 凝胶的变化刚度。此方法主要保留前庭和耳蜗感官器官的体内结构, 并允许对机械力对生长和分化的影响进行调查11。
因为已知的机械刺激可以激活下游分子事件, 如河马信号通路12,13,14,15, 这是很重要的, 能够结合机械刺激生化和基因操控。这里描述的文化方法允许病毒介导的基因交付, 因此可以用来研究在内耳发育过程中的机械和分子信号学11。

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three-dimensional organotypic cultures of vestibular and auditory sensory organs

内耳的感官器官由于无法进行实验操作和光学观察, 对哺乳动物的研究具有挑战性。此外, 虽然现有的文化技术允许生物化学的扰动, 这些方法并没有提供一个方法来研究机械力和组织刚度的影响, 在发展的内耳感官器官。在这里, 我们描述了一个三维脊髓文化的完整的小鼠囊和耳蜗克服这些限制的方法。在这里描述的三维矩阵刚度的调整技术允许操纵的弹性力反对组织生长。因此, 该方法可以用来研究机械力在内耳发育过程中的作用。此外, 这些文化还允许病毒介导的基因传递, 可用于增益和功能损失的实验。这种培养方法保留了固有的毛发细胞和支持细胞, 作为传统的前庭和听觉知觉器官的二维文化的潜在优越替代品。

从胚胎耳中培养的前庭和听觉知觉器官 40-Pa 胶原蛋白 i 型凝胶模拟低刚度胚胎条件11, 保留相对正常的三维结构 (图 1) 并保持毛发细胞和支持单元格 (图 2和图 3)。虽然支持的细胞密度降低了30% 以上 (学生的t测试: n = 4, p < 0.004) 和毛细胞密度下降 60% (学生的t测试: n = 5, ...

Watch this Scientific Journal Video about Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs at JoVE.com

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

三维脊髓培养的小鼠囊和耳蜗在光学透明胶原 i 凝胶保存固有的组织形态学, 允许机械刺激通过调整基质刚度, 并允许病毒介导的基因传递。

三维脊髓前庭和听觉感官器官的培养

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

three-dimensional-organotypic-cultures-vestibular-auditory-sensory

Research

JoVE Journal

Developmental Biology

8.1K 次观看.  Keck School of Medicine of the University of Southern California. 三维脊髓培养的小鼠囊和耳蜗在光学透明胶原 i 凝胶保存固有的组织形态学, 允许机械刺激通过调整基质刚度, 并允许病毒介导的基因传递。

视频: Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of this cellular mosaic often requires that the organ of Corti is cross-sectioned. In particular, the nonsensory pillar and Deiters' cells, whose nuclei are located basally with respect to the hair cells, cannot be visualized without cross-sectioning the organ of Corti. However, the delicate cytoarchitecture of the mammalian organ of Corti, including the fine cytoplasmic processes of the pillar and Deiters' cells, is difficult to preserve by routine histological procedures such as paraffin and cryo-sectioning, which are compatible with standard immunohistochemical staining techniques.
Here I describe a simple and robust procedure consisting of vibratome sectioning of the cochlea, immunohistochemical staining of these vibratome sections in whole mount, followed by confocal microscopy. This procedure has been used widely for immunhistochemical analysis of multiple organs, including the mouse limb bud, zebrafish gut, liver, pancreas, and heart (see 3-6 for selected examples). In addition, this procedure was sucessful for both imaging and quantitificaton of pillar cell number in mutant and control organs of Corti in both embryos and adult mice 7. This method, however, is currently not widely used to examine the mammalian organ of Corti. The potential for this procedure to both provide enhanced preservation of the fine cytoarchitecture of the adult organ of Corti and allow for quantification of various cell types is described.

A simple procedure of vibratome sectioning the organ of Corti, followed by immunohistochemistry and confocal microscopy is described. This procedure allows for improved preservation of the fine cytoarchitecture of the mammalian organ of Corti, and consequently allows for accurate quantification of cell types.

Vibratome切片增强保尔蒂哺乳动物器官的细胞构筑

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of ...

科研

神经科学

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

一个孤立的半完整的上下游鼠标前庭上皮细胞电生理和高分辨率的双光子显微镜

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

生物学

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

发育生物学

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

In this study, we present a real-time imaging method using confocal microscopy to observe cells moving toward damaged tissue by ex vivo incubation with the cochlear epithelium containing the organ of Corti.

体外延时活细胞成像探索细胞向科尔蒂器官迁移

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

医学

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

从新生儿啮齿动物中分离和培养前庭和螺旋神经节 Somata 以进行膜片钳记录

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

Whole Neonatal Cochlear Explants as an In vitro Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. 
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. 
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Whole Neonatal Cochlear Explants as an In vitro Model

The current protocol updates previous protocols and incorporates relatively straightforward approaches for culturing high-quality cochlear explants. This provides reliable data acquisition and high-resolution imaging in live and fixed cells. This protocol supports the ongoing trend of studying inner ear cells.

整个新生儿耳蜗外植体作为 体外 模型

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

Harvest of Vestibular End-Organs under Physiologic Conditions during Labyrinthectomy

The living human inner ear is challenging to study because it is encased within dense otic capsule bone that limits access to biological tissue. Traditional temporal bone histopathology methods rely on lengthy, expensive decalcification protocols that take 9-10 months and reduce the types of tissue analysis possible due to RNA degradation. There is a critical need to develop methods to access fresh human inner ear tissue to better understand otologic diseases, such as M&#233;ni&#232;re's disease, at the cellular and molecular level. This paper describes a technique for the harvest of human vestibular end organs from a living donor under physiologic conditions. An individual with M&#233;ni&#232;re's disease and 'drops attacks' that were refractory to intratympanic gentamicin injection underwent labyrinthectomy. A traditional mastoidectomy was first performed, and the horizontal and superior semicircular canals (SCC) were identified. The mastoid cavity was filled with a balanced salt solution so that the labyrinth could be opened under more physiologic conditions to preserve cellular integrity. A zero-degree endoscope fit with a lens-cleaning sheath irrigation system was used to visualize the submerged mastoid cavity, and a 2 mm diamond burr was used to skeletonize and open the horizontal and superior SCCs, followed by the vestibule. The ampullae and portion of the canal ducts for the superior and lateral SCCs were harvested. The utricle was similarly harvested. Harvested tissue was immediately placed in an ice-cold buffer and then fixed for one hour in 4% paraformaldehyde in phosphate-buffered saline (PBS). The tissue was rinsed several times in 1x PBS and stored for 48 h at 4 &#176;C. The tissue samples underwent immunostaining with a combination of primary antibodies against tenascin-C (Calyx), oncomodulin (streolar hair cells), calretinin (Calyx and Type II hair cells), synaptic vesicle protein 2 (efferent fibers and boutons), &#946;-tubulin 1 (Calyx and afferent boutons), followed by incubation with fluorophore-conjugated secondary antibodies. The tissue samples were then rinsed and mounted for confocal microscopy examination. Images revealed the presence of ampullar and macular hair cells and neural structures. This protocol demonstrates that it is possible to harvest intact, high-quality human inner ear tissue from living donors and may provide an important tool for the study of otologic disease.

Here, we describe a technique for harvesting human vestibular end-organs under physiologic conditions during labyrinthectomy and their analysis using immunostaining.

迷路切除术中生理条件下前庭终末器官的收获

The living human inner ear is challenging to study because it is encased within dense otic capsule bone that limits access to biological tissue. ...

小鼠耳蜗毛细胞的高效体外培养

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.

作者聚焦：培养小鼠毛细胞用于听觉研究的进展 

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.

新生小鼠原代耳蜗毛细胞的分离和培养

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 ...

三维脊髓前庭和听觉感官器官的培养

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此视频中的章节

Vibratome切片增强保尔蒂哺乳动物器官的细胞构筑

一个孤立的半完整的上下游鼠标前庭上皮细胞电生理和高分辨率的双光子显微镜

原代培养及质粒电穿孔尔蒂小鼠器官。

老鼠耳蜗外植体的长期时间推移成像

在蛋和卵外研究中研究鸟类内耳发育的方法

体外延时活细胞成像探索细胞向科尔蒂器官迁移

从新生儿啮齿动物中分离和培养前庭和螺旋神经节 Somata 以进行膜片钳记录

整个新生儿耳蜗外植体作为体外模型

迷路切除术中生理条件下前庭终末器官的收获

新生小鼠原代耳蜗毛细胞的分离和培养

三维脊髓前庭和听觉感官器官的培养

摘要

探索更多视频

此视频中的章节

Vibratome切片增强保尔蒂哺乳动物器官的细胞构筑

一个孤立的半完整的上下游鼠标前庭上皮细胞电生理和高分辨率的双光子显微镜

原代培养及质粒电穿孔尔蒂小鼠器官。

老鼠耳蜗外植体的长期时间推移成像

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

体外延时活细胞成像探索细胞向科尔蒂器官迁移

从新生儿啮齿动物中分离和培养前庭和螺旋神经节 Somata 以进行膜片钳记录

整个新生儿耳蜗外植体作为 体外 模型

迷路切除术中生理条件下前庭终末器官的收获

新生小鼠原代耳蜗毛细胞的分离和培养

在蛋和卵外研究中研究鸟类内耳发育的方法

整个新生儿耳蜗外植体作为体外模型