谢谢孙威利对技术援助和克里斯Gellynck博士有益的意见和三个匿名评审的建设性意见。这项工作是由美国的新宁听力重建倡议

小鼠组织从Sox2的EGFP收获-reporter维护和安乐死按照中国的护理和使用实验动物的动物护理准则加拿大议会小鼠12。 1.培养胚胎耳蜗<ol><li>通过混合8.89毫升DMEM中，加入1ml的胎牛血清（FBS），100微升100×N2补充，和10μl的10mg / ml的环丙沙星补充的Dulbecco改良的Eagle培养基（DMEM）。通过混合495毫升的HBSS，用5毫升100％的HEPES补充Hank氏平衡盐溶液（HBSS）中。 </li><li>准备玻璃底培养皿: <ol><li>在层流罩，悬浮200μl的基底膜提取物衬底的5ml DMEM中。准备直径35 mm的玻璃底培养皿10毫米井。吸取150微升底物/ DMEM在每个玻璃中央见底菜。这些菜可以使用后40分钟温育，或可以存储在CO 2 </sub&gt;培养箱中在35℃下进行至少一周的时间。 注:玻璃有底菜肴用于下列原因:以确保盘的底部是透明的并且适于成像，以良好的，其允许外植体，以解决在一个容易找到区和最后的盘的中心创建一个使一个试验后的样品可用于免疫染色及随后的分析处理。 </li></ol></li><li>准备工作站和工具: <ol><li>打开层流洁净工作台，并喷洒下来用70％乙醇，以创造一个干净的工作空间。浸泡镊子，勺子，和一个黑色184硅酮弹性体培养皿（184硅酮弹性体（10份）混合，固化剂（1份）和木炭粉（2.5克））在70％EtOH中。 </li></ol></li><li>在洁净工作台，收获胚胎的实验。收集在冰适当妊娠冷HBSS中补充有1％HEPES的胚胎。 <ol><li>确定外部或可见机关将展示用荧光立体显微镜报告基因活性并检查胚胎。收集表现出报告基因活性在冰冷的HBSS中补充有1％HEPES，因为这些是在实验的受试者的胚胎。 </li></ol></li><li>收集颞骨: <ol><li>工作迅速，使用冷色光源和细镊子，收集幼崽的头。照顾到夹在颈椎和下颚下方，以避免损坏的颞骨。 </li><li>小心地打开头盖骨。取出脑，修剪过的头部的前部，并且所述后颅骨转移到新鲜的冰冷的HBSS在干净的培养皿补充有1％HEPES。仔细剖析出花生形颞骨注意保持前庭系统的机智。 </li></ol></li><li>解剖耳蜗: <ol><li>传送颞骨到在冰黑硅弹性体涂覆的培养皿冷HBSS中补充有1％HEPES，针骨的前庭区域。插入昆虫针在的倾斜角，以稳定的颞骨和创建​​房间的钳子。钉扎是在这个过程中的一个关键步骤，因为如果颞骨被允许移动至太大，很难收获完整的耳蜗。 </li><li>小心取出围绕耳蜗的软骨。插入钳子的一个齿进入软骨在耳蜗的基底的外边缘和夹子的孔进入软骨。 </li><li>夹的翼片向上侧，插入钳子的齿和轻轻分开从软骨管道的屋顶。剪辑水平穿过顶部和倾斜，并小心地取下胶囊的前部。插入镊子的一个叉头中剩余的软骨与管道之间并轻轻夹关闭最后一节。耳蜗的顶端面现露出。 </li><li>开始在基，抓其中所述导管的屋顶满足耳蜗上皮面积和打开耳蜗管。轻轻撕下屋顶，修剪必要时，直到它被完全除去。剪掉膜左内侧上的导管中的任何部分。清理多余的间叶组织的管道，并从分离的前庭系统的风管。 </li></ol></li><li>文化的外植体: <ol><li>将外植体，腔表面了，在一个基板镀膜玻璃底培养皿的中心，精心编制了所有的液体，留下了两分钟。加入150微升补充DMEM落明智的外植体，注意不要打扰它。应的外植体自由漂浮，定位钳，但要小心，外植体沉淀到培养皿的底部，使得它可以连接到基板上。 </li><li>将玻璃底菜在深直径12cm的培养皿中，用一小碟的无菌水来保持湿度。将培养物在一个35℃的培养箱中过夜，以使外植体附着到基片和压平。 </li></ol></li></ol> 2.现场的ImagING <ol><li>选择植样本。用荧光立体显微镜来评估每个外植耳蜗的条件。只有选择的外植体，其中所述管道是完好并附着在玻璃从基部到顶点。 </li><li>设置培养箱中于35℃，5％CO 2下培养耳蜗组织。把文化进入孵化器的显微镜: <ol><li>轻轻吸过的DMEM补充和替换，至少500微升新鲜培养基。用于成像长达6天，1-1.5毫升为好。在其中植体的连接不牢固的情况下，吸取的媒体的环周围的盘的边缘。这个额外的液体将一个微型"加湿室&#39;而不干扰外植体，而它仍然附着于基质。 <ol><li>可选地，使用铰接盘覆盖，打开盖子，而盘停留在其在显微镜嵌合如果试剂需要在图像集合之间的时间间隔交换。铰链盖菜让盖被开不干扰样品或从它们的设置去除菜肴。这样可保持其对后续的图像捕捉精确位置。 </li></ol></li><li>将包含相应的样品放入样品盘架的玻璃底菜。在本实施例的显微镜具有旋转平台，保持835毫米样品菜肴。 </li><li>在层流罩更换塑料盖用玻璃盖并插入餐具放入样品盘支架。将样品盘支架培养箱照顾内不从培养皿的底部去除所述外植体或扰乱介质。 </li></ol></li><li>成立了成像程序: <ol><li>交换机上的显微镜，紫外灯和摄像头，打开图像处理软件。 </li><li>找到样品，挑的成像区域，重点平面和调整曝光时间顺序每道菜。选择焦点不仅取决于外植体在起动时的视图的平面，但人所以铭记如何组织将移动多久的时间过程会运行。 </li><li>设置为Z堆栈围绕所选焦平面定心在荧光通道。设置采样进行实验的次数和时间长度。采样的频率也由它需要采集图像的时间是有限的，所以这应该在选择视场和设置为Z堆叠后确定。在这种情况下，需要采样的地方，每隔30分钟，超过五天。在实验的持续时间可长达14天。 </li></ol></li><li>生成的电影: <ol><li>在时间流逝期结束打开的图像文件。打开连续采集点在这种情况下的Metamorph软件使用。逐帧选出最佳焦平面为示出感兴趣的细胞群。 </li><li>这些图像转换为.avi文件，或者将其导出为一蒙太奇以生成一组可被打开并分析在图像处理软件或转换为多个预估图像利用视频处理软件TS。 </li></ol></li></ol>

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作者什么都没有透露。

有几种技术要点当培养物被建立并在成立的时间推移显微镜为了长期成像来考虑。 我们用基底膜基质作为基质培养耳蜗植体，但在衬底应匹配于细胞类型。例如，将图像的神经元培养物，它可能是更好的是提供一种纤连蛋白涂层。温育温度和气体组成也应当根据组织类型选择。选择外植体的年龄是很重要的。我们开始对E13在最早因为对E12的生长方向是难以预料。因为它是必?...

哺乳动物耳蜗是高度有序的复杂器官。在小鼠中，从上一天E11耳泡和发育程序完成后，将原始内耳的出现在出生后早期阶段之间，细胞信号转导的多种波和协调的基因表达的变化发生。从基本运行到顶点的蜗管的是声音探测感觉上皮尔蒂的，或器官。柯蒂氏器的发展精美控制，使得通过显影结束时，其将包括内毛细胞的单排，三排外毛细胞的散布有五排支持细胞（两列支柱细胞，三行Deiters&#39;细胞）1。偏离这个精确的顺序会导致听力下降，heighlighting studye的国税发起源和感官epithemlium 2的图案化的重要性。 在胚胎小鼠COCH的体外培养LEA是研究Corti器的发展机制的一个重要工具。该技术成立于1974年，并在过去的40年中，一直被用来阐明许多由感官上皮规定和Corti器建立3的机制。耳蜗是一个复杂的器官的动态发育过程;一个操作可能需要长达7天的表现1。例如，添加GSK3β抑制剂在E13耳蜗植体培养时，最佳的潜伏期，观察该化合物的一个健壮效果为6天4。 显影耳蜗的实时成像可以调查变化的柯蒂氏器的形态，基因表达的变化，跟踪迁移的，增殖或死亡的细胞，并允许实时观察药剂的结果的和信号的中断途径。到现在为止，住耳蜗的摄像已经主要使用共聚焦显微镜对柯蒂氏在短时间内5-8的脏器图像的小区域进行的，但该技术具有由于外植体存活的局限性。在较长期的操作慢发育过程的影响成像，成像环境是至关重要的。典型的共焦实时成像系统采用的加湿塑料箱，它位于所述的显微镜。热和湿度，可以通过在孵育箱那里它满足显微镜台，通过接入窗，通过铰链孔，并通过围绕microscope-各部分的间隙的间隙逸出，如目标或光源。这不是最优的用于维持健康植超过两三天。 我们定义"孵化显微镜&#39;为倒置显微镜内部的标准的CO 2培养箱中密封，而不是在显微镜周围建造一个孵化器。孵化器显微镜前趋于实验的寿命，使得而非成像在两三天，样品可以被成像长达两周。培养箱显微镜提供用于细胞生长和分化良好的环境，以最小的干扰外植体培养物和标准控制的条件。在研究，发生在多天里，通常通过从培养箱中取出，并携带它们到一个倒置荧光显微镜诉诸成像样品，每天。虽然这种方法可以工作，从培养箱中取出盘子对其造成的敏感发展组织应力。由于除去从孵化器中的更改培养用培养基和波动的温度的酸度可导致次优的发展和不健康的组织。成象的区域在同一焦平面上，并在相同的取向，在每一个时间点是非常有挑战性的。通过利用恒温箱中的自动化系统，它能够维持健康组织，采集图像，在多个时间点，并确保在同一区域被捕获在每一帧。近年来几个集成显微镜组织培养箱被开发出来，这些不仅在临床实践9，而且在干细胞和癌症研究10,11是有用的。 在这里，我们提出了一个协议，用于胚胎小鼠耳蜗外植体的长期实时成像。我们使用自动显微镜系统内部的标准的CO 2培养箱具有捕获多个样品的图像在设定的时间点的能力。该系统由一个倒置显微镜孵化器内设定的。样品被放置在一个旋转的讲台，允许多个样品的成像在每个时间点。照明，图像采集和讲台的旋转是通过的Metamorph软件操作的自动化系统控制。通过操作软件设置的成像程序，我们可以设置一个实验，运行长达TWØ周以最少的人工干预。在这个例子中我们使用两个亮场和荧光显示大规模增长和耳蜗的重排，并且具体地，prosensory区域。在该实验中，耳蜗将从Sox2的EGFP报告小鼠解剖胚胎天E13。 在体外培养物将被建立，然后成像五天。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle media</td><td>Gibco</td><td>12430</td><td>Multiple brands manufacture this</td></tr><tr><td>Basement membrane extract&amp;nbsp;</td><td>Corning</td><td>354230</td><td>Matrigel. Alternative similar products are available from other suppliers.&lt;br/&gt; http://catalog2.corning.com/Lifesciences/en-US/Shopping/Product.aspx?categoryname=Cell+Culture+and+Bioprocess%28Lifesciences%29|Extracellular+Matrix+Proteins+ECMs+and+Attachment+Factors%28Lifesciences%29|Matrigel+Basement+Membrane+Matrix+%28Lifesciences%29</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>16000044</td><td>Multiple brands manufacture this&lt;br/&gt; http://www.lifetechnologies.com/search/global/searchAction.action?query=fbs&amp;amp;resultPage=1&amp;amp;results PerPage=15&amp;amp;autocomplete=</td></tr><tr><td>HEPES</td><td>Gibco</td><td>5630080</td><td>Multiple brands manufacture this&lt;br/&gt; http://www.lifetechnologies.com/search/global/searchAction.action?query=hepes&amp;amp;resultPage=1&amp;amp;results PerPage=15&amp;amp;autocomplete=</td></tr><tr><td>100 x N2 supplement</td><td>Gibco</td><td>17502-048</td><td>Multiple brands manufacture this&lt;br/&gt; http://www.lifetechnologies.com/search/global/searchAction.action?query=n2&amp;amp;resultPage=1&amp;amp;results PerPage=15&amp;amp;autocomplete=</td></tr><tr><td>Ciprofloxacin</td><td>Sigma Aldrich</td><td>17850-5G-F</td><td>Multiple brands manufacture this&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/fluka/17850?lang=en&amp;amp;region=CA</td></tr><tr><td>Hank&#039;s balanced salt solution</td><td>Gibco</td><td>14170161</td><td>Multiple brands manufacture this. Should be refidgerated before use.&lt;br/&gt; http://www.lifetechnologies.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/balanced-salt-solutions/hbss-hanks-balanced-salt-solution.html?s_kwcid=AL!3652!3!26107410508!e!!g!!hbss&amp;amp;ef_id=xoFOglw2s UMAAMU8:20140228185720:s</td></tr><tr><td>Fine Forceps</td><td>Fine Science tools</td><td>11254-20</td><td>Size number 5&lt;br/&gt; http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=350&amp;amp;CategoryId=29</td></tr><tr><td>Curette&amp;nbsp;</td><td>Fine Science Tools</td><td>10080-05</td><td>size 1 mm&amp;nbsp;&lt;br/&gt; http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=91&amp;amp;CategoryId=118</td></tr><tr><td>Insect Pins</td><td>Fine Science Tools</td><td>26001-35</td><td>Must be stainless Steel&lt;br/&gt; http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=124</td></tr><tr><td>50 mm plastic dishes</td><td>Corning/Falcon</td><td>351006</td><td>Multiple brands manufacture this</td></tr><tr><td>charcoal</td><td>Sigma Aldrich</td><td>05105-250G</td><td>Multiple brands manufacture similar items&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/fluka/05105?lang=en&amp;amp;region=CA</td></tr><tr><td>184 silicone elastomer</td><td>Dow/Corning&amp;nbsp;</td><td>SYLGARD&amp;reg; 184 SILICONE ELASTOMER KIT</td><td>Dishes are home made several weeks in advance.&amp;nbsp; Silicone elastomer can be from any supplier.&lt;a href=&quot;http://www.dowcorning.com/applications/search/products/Details.aspx?prod=01064291&quot;&gt;http://www.dowcorning.com/applications/search/products/Details.aspx?prod=01064291&lt;/a&gt;</td></tr><tr><td>Glass bottom dishes</td><td>MatTek</td><td>P35G-0-10-C</td><td>The dimensions of the dish are determined by the specifications of the imaging system.&amp;nbsp; 35 mm diameter, 10mm well, number 0 coverslip fits Olympus Vivaview FL.&lt;br/&gt; http://glass-bottom-dishes.com/catalog/index.php?main_page=product_info&amp;amp;cPath= 1_4_15&amp;amp;products_id=2</td></tr><tr><td>Stereomicroscope</td><td>Zeiss&amp;nbsp;</td><td>495101-9804-000</td><td>&amp;nbsp;Stemi 2000 model. multiple brands manufacture similar items&lt;br/&gt; http://microscopy.zeiss.com/microscopy/en_de/products/stereo-zoom-microscopes/stemi-2000.html</td></tr><tr><td>Cold Light Source</td><td>Zeiss</td><td>000000-1063-182</td><td>Multiple brands manufacture similar items&lt;br/&gt; http://microscopy.zeiss.com/microscopy/en_de/products/microscope-components/lightsources.html</td></tr><tr><td>Fluorescent Stereomicroscope&amp;nbsp;</td><td>Leica microsystems</td><td>Contact Leica microsystems</td><td>Leica M165-FC. Multiple brands manufacture similar items&lt;br/&gt; http://www.leica-microsystems.com/products/stereo-microscopes-macroscopes/fluorescence/details/product/leica-m165-fc/</td></tr><tr><td>Incubator Microscope +imaging software</td><td>Olympus</td><td>Contact Olympus</td><td>Inverted microcope sealed inside a Co2 incubator. Vivaview FL incubator microscope with proprietry Metamorpoh imaging software.&lt;br/&gt; http://olympuscanada.com/seg_section/product.asp?product=1055&amp;amp;c=0</td></tr><tr><td>Clean bench</td><td>Thermo Scientific</td><td>51029701</td><td>Multiple brands manufacture similar items&lt;br/&gt; http://www.thermoscientific.com/en/product/heraguard-eco-clean-bench.html</td></tr><tr><td>CO2 incubator</td><td>Thermo Scientific</td><td>3310</td><td>Multiple brands manufacture similar items&lt;br/&gt; http://www.thermoscientific.com/en/products/co2-incubators.html</td></tr><tr><td>Laminar Flow hood</td><td>Thermo Scientific</td><td>51026651</td><td>Multiple brands manufacture similar items&lt;br/&gt; http://www.thermoscientific.com/en/products/biological-safety-cabinets-clean-benches.html</td></tr></tbody></table>

long-term time lapse imaging of mouse cochlear explants

在这里，我们提出了活鼠胚胎的耳蜗植长期时间推移成像的方法。负责建设的哺乳动物耳蜗收益的高度有序的，复杂的结构，大约十天后的发展计划。为了研究在此期间基因表达的变化和它们的响应的药物或基因操作，长期成像是必要的。先前，实时成像已经典型地通过移植组织的存活，在湿润的腔室顶上标准显微镜的限制。难以保持对培养物生长有关的湿度和温度的最佳条件已经放置在成像实验的长度的限制。显微镜集成到修改后的组织培养孵化器提供长期，实时成像的良好环境。在这个方法中，我们演示了如何建立胚胎小鼠耳蜗植，以及如何使用孵化器的显微镜来进行时间的推移意马使用毫微都亮场和荧光显微镜检查的典型胚胎天（E） - 13耳蜗植体和Sox2，耳蜗的prosensory细胞的标记，在5天的行为。

在这里，我们将展示一个蒙太奇（ 图1），看电影（ 图2），这说明一个典型的器官耳蜗植将如何发展，如果镀上E13.5。一个Sox2的记者鼠标采用可视化的prosensory区域。动画示出了耳蜗经历生长和收敛性和扩展，使细胞中的绿色Sox2的域的横向区域似乎没有划分为围绕它展开的组织。这是Corti器的特性;关于E13准感觉上皮退出细胞周期和随后称为防扩散13?...

Watch this Scientific Journal Video about Long-term Time Lapse Imaging of Mouse Cochlear Explants at JoVE.com

Long-term Time Lapse Imaging of Mouse Cochlear Explants

胚胎哺乳动物耳蜗的实时成像是具有挑战性的，因为手头的发育过程超过十天时间梯度进行操作。在这里，我们提出了培养，然后从成像荧光记者鼠标采取了五天的胚胎耳蜗植体组织的方法。

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 ...

long-term-time-lapse-imaging-of-mouse-cochlear-explants

Research

JoVE Journal

Neuroscience

9.6K 次观看.  Sunnybrook Research Institute. 胚胎哺乳动物耳蜗的实时成像是具有挑战性的，因为手头的发育过程超过十天时间梯度进行操作。在这里，我们提出了培养，然后从成像荧光记者鼠标采取了五天的胚胎耳蜗植体组织的方法。 

视频: Long-term Time Lapse Imaging of Mouse Cochlear Explants

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

科研

发育生物学

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

生物工程

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

用于内耳组织工程的 Decellularizing 小鼠耳蜗协议

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET remain unknown. Electrophysiological studies of hair cells revealed that the MET channel has a large conductance and is permeable to relatively large fluorescent cationic molecules, including some styryl dyes and Texas Red-labeled aminoglycoside antibiotics. In this protocol, we describe a method to visualize and evaluate the uptake of fluorescent dextrans in hair cells of the organ of Corti explants that can be used to assay for functional MET channels. We found that 3 kDa Texas Red-labeled dextran specifically labels functional auditory hair cells after 1&#8211;2 h incubation. In particular, 3 kDa dextran labels the two shorter stereocilia rows and accumulates in the cell body in a diffuse pattern when functional MET channels are present. An additional vesicle-like pattern of labeling was observed in the cell body of hair cells and surrounding supporting cells. Our data suggest that 3 kDa Texas-Red dextran can be used to visualize and study two pathways for cellular dye uptake; a hair cell-specific entry route through functional MET channels and endocytosis, a pattern also available to larger dextran.

Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3&#8211;10 kDa can be used to study endocytosis in hair and supporting cells of the organ of Corti.

活体和功能性鼠耳蜗毛细胞中的德特贴标签和吸收

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

医学

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

成年小鼠中的科利耳表面制备

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

神经科学

Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia that are arranged in rows of increasing height. When stereocilia are deflected, they tug on tiny (~5 nm in diameter) extracellular tip links interconnecting stereocilia, which convey forces to the mechanosensitive transduction channels. Although mechanotransduction has been studied in live hair cells for decades, the functionally important ultrastructural details of the mechanotransduction machinery at the tips of stereocilia (such as tip link dynamics or transduction-dependent stereocilia remodeling) can still be studied only in dead cells with electron microscopy. Theoretically, scanning probe techniques, such as atomic force microscopy, have enough resolution to visualize the surface of stereocilia. However, independent of imaging mode, even the slightest contact of the atomic force microscopy probe with the stereocilia bundle usually damages the bundle. Here we present a detailed protocol for the hopping probe ion conductance microscopy (HPICM) imaging of live rodent auditory hair cells. This non-contact scanning probe technique allows time lapse imaging of the surface of live cells with a complex topography, like hair cells, with single nanometers resolution and without making physical contact with the sample. The HPICM uses an electrical current passing through the glass nanopipette to detect the cell surface in close vicinity to the pipette, while a 3D-positioning piezoelectric system scans the surface and generates its image. With HPICM, we were able to image stereocilia bundles and the links interconnecting stereocilia in live auditory hair cells for several hours without noticeable damage. We anticipate that the use of HPICM will allow direct exploration of ultrastructural changes in the stereocilia of live hair cells for better understanding of their function.

Here we present a protocol for the Hopping Probe Ion Conductance Microscopy (HPICM), a non-contact scanning probe technique that allows nanoscale imaging of stereocilia bundles in live auditory hair cells.

立体纤毛束成像在活哺乳动物听觉毛细胞中的纳米级分辨率

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia ...

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 ...

Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy

Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or presbycusis is a primary cause of sensorineural hearing loss and is characterized by damage to hair cells, spiral ganglion neurons (SGNs), and the stria vascularis. These structures reside within the cochlea, which has a complex, spiral-shaped anatomy of membranous tissues suspended in fluid and surrounded by bone. These properties make it technically difficult to investigate and quantify histopathological changes. To address this need, we developed a light-sheet microscope (TSLIM) that can image and digitize the whole cochlea to facilitate the study of structure-function relationships in the inner ear. Well-aligned serial sections of the whole cochlea result in a stack of images for three-dimensional (3D) volume rendering and segmentation of individual structures for 3D visualization and quantitative analysis (i.e., length, width, surface, volume, and number). Cochleae require minimal processing steps (fixation, decalcification, dehydration, staining, and optical clearing), all of which are compatible with subsequent high-resolution imaging by scanning and transmission electron microscopy. Since all the tissues are present in the stacks, each structure can be assessed individually or relative to other structures. In addition, since imaging uses fluorescent probes, immunohistochemistry and ligand binding can be used to identify specific structures and their 3D volume or distribution within the cochlea. Here we used TSLIM to examine cochleae from aged mice to quantify the loss of hair cells and spiral ganglion neurons. In addition, advanced analyses (e.g., cluster analysis) were used to visualize local reductions of spiral ganglion neurons in Rosenthal&#39;s canal along its 3D volume. These approaches demonstrate TSLIM microscopy&#39;s ability to quantify structure-function relationships within and between cochleae.

A light-sheet microscope was developed to image and digitize whole cochlea.

使用光片荧光显微镜对老化耳蜗进行成像

Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or ...

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 ...

老鼠耳蜗外植体的长期时间推移成像

摘要

探索更多视频

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

用于内耳组织工程的 Decellularizing 小鼠耳蜗协议

活体和功能性鼠耳蜗毛细胞中的德特贴标签和吸收

新生儿鼠耳蜗外科手术技术

成年小鼠中的科利耳表面制备

立体纤毛束成像在活哺乳动物听觉毛细胞中的纳米级分辨率

体外延时活细胞成像探索细胞向科尔蒂器官迁移

使用光片荧光显微镜对老化耳蜗进行成像

整个新生儿耳蜗外植体作为体外模型

老鼠耳蜗外植体的长期时间推移成像

摘要

探索更多视频

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

用于内耳组织工程的 Decellularizing 小鼠耳蜗协议

活体和功能性鼠耳蜗毛细胞中的德特贴标签和吸收

新生儿鼠耳蜗外科手术技术

成年小鼠中的科利耳表面制备

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使用光片荧光显微镜对老化耳蜗进行成像

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