Name: isolation and culture of primary cochlear hair cells from neonatal mice
Uploaded: 2023-09-15T12:40:00.000+00:00
Duration: 6 min 7 s
Description: Watch this Scientific Journal Video about Advancements in Cultivating Mouse Hair Cells for Auditory Research at JoVE.com

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

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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><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 &amp;micro;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&amp;nbsp;</td><td></td><td></td><td>provided by Xi&#039;an Jiaotong University</td></tr><tr><td>Dulbecco&amp;rsquo;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&amp;nbsp;</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。

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

作者聚焦：培养小鼠毛细胞用于听觉研究的进展 

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。

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 ...

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

2.2K 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 

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

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

神经科学

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

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

在Ciliopathy鼠标突变耳蜗平面 - 细胞极性表型的评价

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

医学

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

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 ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

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 ...

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 ...

新生小鼠原代耳蜗毛细胞的分离和培养

副本

摘要

探索更多视频

此视频中的章节

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

原代培养及质粒电穿孔尔蒂小鼠器官。

全山解剖和成年小鼠耳蜗的免疫

在Ciliopathy鼠标突变耳蜗平面 - 细胞极性表型的评价

脊髓神经元从新生小鼠中分离和培养

新生儿鼠耳蜗外科手术技术

分离新生小鼠心肌文化和

成年小鼠中的科利耳表面制备

整个新生儿耳蜗外植体作为体外模型

新生小鼠原代耳蜗毛细胞的分离和培养

副本

摘要

探索更多视频

此视频中的章节

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

原代培养及质粒电穿孔尔蒂小鼠器官。

全山解剖和成年小鼠耳蜗的免疫

在Ciliopathy鼠标突变耳蜗平面 - 细胞极性表型的评价

脊髓神经元从新生小鼠中分离和培养

新生儿鼠耳蜗外科手术技术

分离新生小鼠心肌文化和

成年小鼠中的科利耳表面制备

整个新生儿耳蜗外植体作为 体外 模型

整个新生儿耳蜗外植体作为体外模型