Name: in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti
Uploaded: 2020-12-04T14:45:00
Description: Watch this Scientific Journal Video about In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti at JoVE.com

这项工作得到了韩国国家研究基金会（NRF-2018-R1D1A1B07050175，HURF-2017-66）和韩国国家研究基金会（NRF）和哈莱姆大学研究基金的研究赠款的支持。

所有涉及ICR小鼠的研究协议都得到了元州医学院延世大学机构动物护理与使用委员会（IACUC）的批准。实验是根据世界医学协会的道德规范进行的。在本协议中，怀孕的ICR小鼠被保存在12/12小时的光/暗循环中，可以自由获得食物和水。
1. 科克利解剖
<ol><li>通过打开紫外线约30分钟，对层层流动组织培养罩进行消毒，并在使用前用70%乙醇喷洒所有表面。让表面干燥。</li>
	<li>将...

<ol>
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(124), e55704 (2017).</li><li>Pijuan, 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=In+vitro+cell+migration,+invasion,+and+adhesion+assays:+From+cell+imaging+to+data+analysis.">In vitro cell migration, invasion, and adhesion assays: From cell imaging to data analysis.</a> Frontiers in Cell and Developmental Biology. 7, 107 (2019).</li><li>Rask-Andersen, 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=Human+cochlea:+anatomical+characteristics+and+their+relevance+for+cochlear+implantation.">Human cochlea: anatomical characteristics and their relevance for cochlear implantation.</a> The Anatomical Record. 295 (11), 1791-1811 (2012).</li><li>Kamiya, K., 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=Mesenchymal+stem+cell+transplantation+accelerates+hearing+recovery+through+the+repair+of+injured+cochlear+fibrocytes.">Mesenchymal stem cell transplantation accelerates hearing recovery through the repair of injured cochlear fibrocytes.</a> The American Journal of Pathology. 171 (1), 214-226 (2007).</li><li>Lee, H. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+plasmid+electroporation+of+the+murine+organ+of+Corti.">Primary culture and plasmid electroporation of the murine organ of Corti.</a> Journal of Visualized Experiments. (36), e1685 (2010).</li><li>Ogier, J. M., Burt, R. A., Drury, H. R., Lim, R., Nayagam, B. 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对MSC移植到受损部位，促进受损细胞再生进行了广泛的研究，治疗效果明显。据报道，MSC的移植和随后的分化，以恢复大鼠的听力损失引起的3硝基丙酸13。虽然李等人将MSC应用于人类，但他们在听力14方面并没有取得任何显著改善。直到最近，通过MSC移植，还进行了近12项实验，以恢复啮齿动物模型的听力。虽然由于异质性，结果有些不清楚，但有迹象表明，MSC...

听力损失可能是先天发生的，也可以是由几个因素逐渐引起的，包括衰老、药物和噪音。听力损失往往很难治疗，因为一旦负责听力的毛细胞受损，恢复受损的功能是非常具有挑战性的。据世界卫生组织统计，全世界估计有4.61亿人听力损失，占世界人口的6.1%。在听力损失者中，93%是成年人，7%是儿童。
已尝试采取若干方法治疗听力损失：值得注意的是，使用MSC的再生方法已成为一种很有前途的治疗方法。当组织受损时，MSC自然释放到循环系统中，并迁移到损伤部位，在那里它们分泌各种分子，形成促进再生的微环境2。因此，重要的是要开发一种方法，通过外部植入的MSC迁移来治疗受损组织的目标器官及其随后的分子分泌，导致强大的免疫调节，血管生成和抗凋亡，以加强恢复受损细胞功能3，4，5。
MSC 迁移到受损组织的回程过程可能是最重要的障碍。MSC 具有系统性定位机制，具有连续的系绳/滚动、激活、逮捕、迁移/迁移和迁移6、7、8等步骤。目前，正在努力确定改进这些步骤的方法。各种策略，包括基因改造，细胞表面工程，体外启动，磁导，已经测试了6，7。此外，还多次试图通过将MSC吊到受损的科克菌部位来促进听觉毛细胞的保护和再生。然而，在体内跟踪MSC是耗时和劳动密集型的，需要高度专业化的技能9。
为了解决这个问题，开发了一种方法，通过延时共焦显微镜观察耳蜗中MSC的生长情况，该显微镜可拍摄细胞在几个小时内的迁移（图1）。它开发于20世纪初 ，最近已成为研究特定细胞迁移的有力工具。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61947/61947fig01.jpg" style="font-size: 14px;"> 图1：图形摘要。 （A） Corti 的解剖器官使用钳子粘附在塑料盖片上后，盖片被放置在 35 毫米玻璃底的圆角微碟上，并且（B） 玻璃缸被定位。（C） 在玻璃缸内填充中等物后，在气缸外小心添加带有中型的 GFP 标签的 MSC。（E） 在一夜之间孵化后，（F） 玻璃缸被移除，图像用共聚焦显微镜拍摄。缩写：GFP = 绿色荧光蛋白;MSC=中性干细胞。<a href="https://www.jove.com/files/ftp_upload/61947/61947fig01large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本。</a>

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		<tr>
			<td>10X PBS Buffer</td>
			<td>GenDEPOT</td>
			<td>P2100-104</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Formalin</td>
			<td>T&#38;I</td>
			<td>BPP-9004</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>sigma&#160;</td>
			<td>A5354-10ml</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>sigma&#160;</td>
			<td>A4503-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal dish</td>
			<td>SPL</td>
			<td>200350</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal microscope&#160;</td>
			<td>ZEISS</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr>
			<td>coverslip</td>
			<td>SPL</td>
			<td>20009</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher scientific</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorsheild with DAPI</td>
			<td>sigma&#160;</td>
			<td>F6057</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Dumont</td>
			<td>0508-L5-P0</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Fisher scientific</td>
			<td>14065056</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-iFluor 647 Reagent</td>
			<td>abcam</td>
			<td>ab176759</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage Top Incubator</td>
			<td>TOKAI HIT</td>
			<td>WELSX</td>
			<td></td>
		</tr>
		<tr>
			<td>Strain C57BL/6 mouse messenchymal stem cells with GFP</td>
			<td>cyagen</td>
			<td>MUBMX-01101</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>sigma&#160;</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro time-lapse live-cell imaging to explore cell migration toward the organ of corti

为了研究间质干细胞（MSCs）对细胞再生和治疗的影响，该方法跟踪MSC迁移和与声蜗上皮共同培养后的形态变化。Corti 的器官通过按压解剖过程中产生的里斯纳膜的一部分，在塑料盖上固定。当气缸被移除时，被玻璃缸限制的 MSC 迁移到高音表皮。它们的主要定位是在科尔蒂器官的修饰中观察到的，其方向与神经纤维的方向相似。然而，一些MSC在limbus区域被本地化，并显示出水平拉长的形状。此外，进入毛细胞区的迁移增加，在卡那霉素治疗后，MSC的形态转变为各种形式。最后，这项研究的结果表明，MSC与人工增生上皮的共生将有助于通过细胞移植和细胞再生研究，可以检查各种条件和因素的治疗发展。

在三维模式下，MSC的体外迁移已通过Transwell系统或传统的伤口愈合方法进行评估，以观察二维（2D）模式11中的迁移。科尔蒂的器官是一个复杂的结构，由各种细胞组成，如博彻细胞，克劳迪斯细胞，神细胞，支柱细胞，汉森的细胞，外毛细胞，内毛细胞，神经纤维，罗勒膜，和网状拉米纳12。当MSC被移植到由这种复杂细胞组成的组织中?...

Watch this Scientific Journal Video about In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti at JoVE.com

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

在这项研究中，我们提出了一种实时成像方法，使用共聚焦显微镜观察细胞通过与含有Corti器官的耳蜗上皮的外活体孵化向受损组织移动。

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 ...

Existing in vitro method to investigate migration of MSCs include Transwell or wound healing protocols. The method described here can be used to track migration of MSCs to explant in real time. In terms of culture of explant, the advantage of this method compared to others is that it achieves a reduction in time and cost, as well as a simplification of experimental steps.This technique can be used to observe stem cell homing to target in real time leading to development of models with optimized homing efficiency. Removal of tectorial membrane is difficult for unexperienced researchers. Depending on the development stage, understanding the right time to remove the tectorial membrane is important for the success of this protocol.Demonstrating the procedure will be Dr.Jeong-Eun Park and Dr.Su Hoon Lee, researcher professors from my lab. Begin by sterilizing the laminar flow tissue culture hood by turning on the ultraviolet light for approximately 30 minutes. Spray all surfaces with 70%ethanol prior to use and allow them to dry.Place dissection instruments in 70%ethanol for 10 minutes, then dry them before using. After decapitating a three to four-day-old mouse, soak the ahead in 70%ethanol and place it under a serial microscope in the laminar flow hood. Quickly soak the tissue in tissue dissection solution to remove the ethanol.Cut the center line of the skull with the surgical blade, then expose the skull by pulling down the skin anteriorly and cutting the external auditory canal of the ear. Cut from the anterior to the posterior part of the cranium across the eye line, then open the skull and remove the forebrain, cerebellum, and brainstem with blunt forceps. Using microforceps, separate the cochlea from the temporal bone and transfer it to a Petri dish containing tissue dissection solution.Carefully dissect all of the cochlear otic capsule leaving only the internal cochlear soft tissue. Hold the modiolus of the cochlea with forceps and the cochlear duct with another pair of forceps, then slowly separate the two tissues. Remove the stria vascularis and tectorial membrane by gently peeling them away.Place a sterilized plastic coverslip in new tissue dissection solution, then place the organ of Corti on the coverslip making sure that the basilar membrane faces downward. Immobilize the tissue by pressing the Reissner's membrane and the remaining modiolus tissue onto the coverslip with forceps. Transfer the coverslip with the embedded tissue to the center of a 35 millimeter confocal dish.Place the glass cloning cylinder on the dish with the cochlear explant positioned in the center of the dish and add 100 microliters of explant culture medium into the cylinder. Place 5, 000 bone marrow derived GFP-tagged mesenchymal stem cells or MSCs in two milliliters of culture medium outside the glass cylinder and put the dish in the incubator. After culturing the cells as described in the text manuscript, aspirate all medium inside and outside the cylinder and remove the glass cylinder from the confocal dish.Add two milliliters of fresh culture medium to the dish. Place it in a humidified incubator until ready for analysis. Use a confocal microscopy system with a stage top incubator.Turn on the confocal microscope, fluorescent light and computer. Set the conditions of the stage top incubator to 37 degrees Celsius and 5%carbon dioxide atmosphere. Place the sample dish on the dish fixing vessel, cover it with the dish fixing lid and close the chamber with the top heater lid.Adjust the zoom and focus to localize the organ of Corti and MSCs in the field of view. Open the image processing software. Under the locate option, select a 20X plan-apochromat objective or numerical aperture 0.8 and a 0.5X crop area.Under acquisition, click on smart setup and select EGFP. Open the channel tab under acquisition and set the laser power to 0.2%the pinhole to 44 micrometers, the master gain to 750 volts, and the digital gain to 1.0. Click on ESID under imaging setup and set the ESID gain to four and the digital gain to 7.5.Click on tiles and stake to produce 210 tiles. Open focus strategy and select the focus mode. Under time series, set the duration to 24 hours and the interval to 10 minutes.Under acquisition, set the frame size to 512 by 512 pixels, scan speed to eight, the direction to bidirectional, the averaging to 4X, and the bits per pixel to 16. Click on start experiment to begin the experiment. The two-dimensional path of mesenchymal stem cells or MSCs was tracked in a wound healing method by using a glass cylinder as a barrier between the MSCs and the organ of Corti.When the organ of Corti was cultured, fibroblasts grew very quickly outward from the outer hair cells in the outermost layer. Most GFP-labeled MSCs were pushed out by fast-growing fibroblasts. Nonetheless, some penetrated the layer of fibroblasts and successfully migrated to the organ of Corti.The results from 72 hours of incubation showed that MSCs migrated to the organ of Corti and were mainly localized in the modiolus and the area with the cochlear nerve fibers separated from the modiolus. The morphology of the MSCs was altered to a radial shape similar to that of nerve fibers. The cells were transformed into a linear shape along the limbus line, rather than the hair cell area.The MSCs were able to migrate and grow in the nerve fiber collection area by penetrating the various cell layers and physical barriers. When damage to hair cells was induced by a 16-hour treatment with kanamycin and cells were cultured for a further 72 hours after the removal of kanamycin, MSCs were localized not only in the modiolus, but also in the outer hair cells. Immobilizing the tissue is an important step in this protocol was the organ of Corti is attached.It does not fall off easily. This is a trivial, but helpful tip for successful explant culture. This method may be adapted to study the efficacy of exosomes secreted from MSCs.

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The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

使用反时相蛋白的阵列（RPPA）探索在个人的肾细胞癌蛋白表达的变化

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

焦时间推移成像为牙科复合材料的细胞相容性评价的有效方法

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

心导​​管在小鼠测量压力体积的关系：调查的影响鲍迪奇

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

瘦素受体<sup&gt;分贝</sup&gt; 2型糖尿病小鼠模型：胰岛分离和活细胞双光子成像完好胰岛

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

透过玻璃看：时间推移显微镜和单细胞的纵向跟踪研究抗癌治疗

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...