This work was supported by National Institutes of Health Grant to A.S.M. (EY021784).

下面的协议符合托马斯·杰斐逊大学机构动物护理和使用委员会的指导方针，并与ARVO声明为动物的视觉研究使用。 1.安装镜头，并准备于离体培养伤口<ol><li>将3座100 25mm培养皿在无菌的层流罩。填充两个的中途用Tris /葡萄糖缓冲培养皿（TD缓冲器; 140 mM氯化钠，5mM的氯化钾，0.7毫的Na 2 PO 4，5mM的D-葡萄糖，8.25毫摩尔Tris碱，pH值至7.4，用HCl）在RT ，留下第三空。预暖培养基（媒体199补充有1％L-谷氨酰胺和1％青霉素/链霉素）至37℃。 注意：标准伤口愈合的培养基是无血清，如发生在体内 ;然而，在伤口修复培养可以成功地在确定的培养基条件包括血清或其他因素生长。 </li><li>沃取出胚胎ðAY从孵化器15白来航鸡鸡蛋（37.7℃，温和的摇摆举行） </li><li>放置在层流罩和清洁外壳用70％的乙醇从洗瓶选择蛋。进行在层流罩下面在无菌条件下所有的程序，用无菌溶液和仪器。 </li><li>裂纹蛋和地点的内容为空百毫米培养皿。使用标准镊子和剪刀精胚斩首。将鸡胚头中含有的TD缓冲培养皿并适当处理的胚胎的其余部分。任选保持于TD缓冲鸡胚磁头的短时间内，不超过15分钟。 </li><li>将鸡胚头部上的培养皿盖。采用高精度镊子，随着从眼及其连接玻璃体以下顺序取下镜片。捏眼睛后部与镊子创建一个小的开口在眼睛的后部。 </li><li>然后，把握玻璃体W¯¯第i镊子轻轻用力拉玻璃体与滚动运动，附着在透镜玻璃体将从眼移位。地方镜头/玻璃体中含有TD缓冲区剩余的培养皿。让镜头留在TD缓冲的时间不能超过30分钟。 </li><li>移动透镜到一个新的培养皿盖子在解剖显微镜下。从这点上执行在解剖显微镜下的所有步骤。以高精度镊子小心刷掉任何睫状体（色素细胞），这些使用镊子的边缘，同时小心不要损坏晶状体组织脱落与透镜。 注意：卸下睫状体确保那些不是内源的透镜的细胞类型不包括在伤口修复培养。 </li><li>通过从与后晶状体囊关联夹断玻璃体分开玻璃体以高精度镊子镜头。 </li><li>采用高精度镊子镜头转移到小降TD的缓冲液（约200微升）在一个35mm的组织培养皿。 </li></ol> 2.执行模拟白内障手术<ol><li>定向在TD缓冲在35mm培养皿与透镜的朝上的前方面的下降的透镜。 注意：该透镜的前部很容易被致密环在该注意到的晶状体上皮的前部和赤道区域之间的边界的组织的存在识别。与此相反，存在于所述晶状体囊到的晶状体纤维细胞附着的后部不存在标记。 </li><li>使用两个高精度镊子作一小切口（大约850μm）的前晶状体囊中，粗基底膜围绕晶状体组织，和其相关联的前晶状体上皮的中心，通过抓住与一个钳组织中的每个手轻轻揪着相反方向。 </li><li>取出纤维细胞块，它构成了大部分的晶状体组织，dislod的从附件晋它的晶状体上皮和水力洗脱（经典白内障手术使用的方法，仿照图1A）周围的晶状体囊。 </li><li>填充1ml注射器用27.5ģ针尖与300微升的TD缓冲液中。插入针头尖端插入在晶状体前囊制成的切口，和大约一半到透镜。 </li><li>轻踏注射器注入TD缓冲区进入晶状体纤维细胞团。注入微升50至200 TD，且绝不超过300微升。观察纤维细胞团从上皮和晶状体囊放松自己。 </li><li>采用高精度镊子，通过前切口部位取下镜头松动的纤维细胞团。 注：此过程留下晶状体后基底膜囊到纤维细胞已附着在剥蚀细胞，和一个受伤的晶状体上皮只是毗邻这个网站。 </li></ol> 3. Preparin克受伤的镜头体外培养<ol><li>平坦化透镜囊袋而导致的上述的培养皿的白内障手术，细胞面朝上，通过使5削减囊袋的前方面。 </li><li>通过在透镜的赤道切割垂直于原始切口部位。弄平晶状体囊所得五“护翼”与培养皿胶囊侧附上皮下来，细胞侧朝上。注意体外受伤的镜头，现在需要一个明星或花形的形状（ 见图1B）。 </li><li>为了保证胶囊的菜，请用钳子轻轻向下在每个点的明星。这将使一个小凹部，外植体的五个最外的技巧，并导致持续附着到培养皿中。 注意：可以在此过程中损坏的胶囊，因此它以固定胶囊的菜接近的FLA的前端是重要ps的越好，以及使最少量的固定点的作为可能的（一般两，最多三个每瓣）。 </li><li>从35毫米培养皿中取出的TD缓冲器，并将其与1.5预热介质毫升替换。覆盖35mm的培养皿与其盖并置于培养箱（37℃，5％CO 2）。 </li></ol> 4.分离中央迁移区（CMZ），在那里再上皮后囊的创面的发生，从原始附件晶状体上皮细胞的区（OAZ），定量分析。 注：细胞开始响应损伤立即移动到CMZ区域。通过在培养一天足够的细胞迁移横跨CMZ分子和生化分析，继CMZ和OAZ通过显微解剖23分离。该协议涉及除去一个翼片（OAZ）中的从胶囊的受伤区域中的时间。 <ol><li>观察德玛尔阳离子，在解剖显微镜下清晰可见，在OAZ和CMZ之间（ 见图2A）。使用两个高精度镊子，两手抓，钳子在OAZ / CMZ线，一条仅仅相邻的其它的边缘，上线的两侧（参见图2A，B，箭头）。 </li><li>用一只手/在CMZ一边一个镊子继续守住伤员文化，同时用另一只手/镊子，轻轻一拉沿着OAZ / CMZ线OAZ。该CMZ容易分开沿着这条线的OAZ。沿围绕整个文化这条线继续下去，直到两个地区是完全分开的。 </li><li>研究分离OAZ和CMZ分数进行分子分析，例如RNA测序24或生化分析，如蛋白印迹或免疫共沉淀23,25。 </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

Here is described a technique for preparing a culture model of wound repair that involves performing an ex vivo cataract surgery on chick embryo lenses after their removal from the eye. The lens epithelium responds to this clinically relevant wounding with a repair process that closely mimics that which occurs in vivo, and shares features with wound repair in other epithelial tissues2,4. While the protocol is straightforward and simple to follow, performing mock cataract surgery with embryoni...

临床相关，模拟白内障手术，这里所描述的体外上皮伤口愈合模型的开发是为了提供调查，在响应于损伤调节修复上皮组织的机制的一种工具。在创建该模型旨在为关键特征包括1）提供条件精密地复制在体内响应于伤人在培养设置，2）缓和调制修复的调控元件，以及3）能力图像的修复过程中，其整体，在实际时间。我们面临的挑战，因此，是在细胞的天然微环境创造一种文化模式，即有可能学习和操作，上皮创面修复。这种创伤修复模型的有效性开辟了新的可能性，标识来自调节修复过程基质蛋白，细胞因子和趋化因子的内源性信号提示。另外，该模型是理想的研究如何一Ñ ​​上皮能够移动作为集体片重新epithelialize伤口区域2,3，和用于确定在伤口边缘起作用的引导受伤上皮4的集体迁移间充质领袖细胞的谱系。这种模式还提供了一个用以标识疗法，可以有效的促进伤口愈合，防止异常创面修复5的平台。 目前已经有许多可用的创伤修复模式，无论是在文化和体内 ，它提供了当今大多数所谓对伤口修复过程。在动物损伤模型，如角膜6-12和皮肤13-17，有研究的组织的响应伤人的所有修复介质是可以参与的过程中，包括来自所述的上下文中，机会血管和神经系统。但是，也有限制操纵experi体内的精神的条件下，它是目前无法进行体内修复反应的成像研究，随时间连续。与此相反，大多数体外伤口修复培养模型，如划痕，可以很容易地操纵和随后随着时间的推移，但缺乏研究伤口愈合中的体内组织的环境背景。虽然体外模型提供了细胞的微环境加上调节修复的分子调节的过程中的任何时间点的能力范围内不断研究损伤修复过程中随着时间的优势，很少有车型适合这些参数。 这里描述的方法，以产生高度可重复的离体上皮的伤口愈合是再现上皮组织的响应于生理伤人培养物。使用鸡胚镜头作为组织源， 体外 MOCķ白内障手术被执行。该透镜是一种理想的组织用于这些研究，因为它是自包含在一个厚的基底膜胶囊，无血管，没有神经支配，且没有任何相关联的基质18,19。在人类疾病，白内障手术涉及视力丧失，由于透镜的混浊，并且包括除去晶状体纤维细胞块，其包含大量的透镜组成。白内障手术后视力是通过人工眼内透镜的插入恢复。在白内障手术过程中，通过除去纤维细胞，诱导在相邻透镜上皮，这是为了响应通过再上皮晶状体囊的后部区域已被占用的纤维细胞的损伤的反应。在白内障手术中，因为在大多数伤口修复反应，还有，有时会发生的异常纤维化结果向伤口愈合反应，与肌纤维母细胞的出现，这在透镜被称为后验Capsu相关联乐混浊20-22。为了产生白内障手术伤口愈合的模型，一个白内障手术的过程是模仿从鸡胚眼中除去产生生理损伤镜头。显微手术切除晶状体纤维细胞导致的晶状体上皮细胞包围着一个非常一致的圆形伤口面积。该细胞群保持牢固地附着在透镜基底膜胶囊和由外科手术过程中受伤。上皮细胞迁移到内源性基底膜的裸露面积愈合伤口的带动下，在维修过程中被称为领导者细胞1波形丰富的间充质细胞群。用该模型的上皮损伤的响应可容易地可视化并随后随时间在细胞的微环境的上下文中。细胞是易于接触到的表达或活化有望发挥在伤口修复中的作用的分子的修饰。日的一项强大功能是模型是分离和研究在伤口愈合的框架迁移特异性改变的能力。准备大量老年匹配体外伤口愈合培养物用于研究的能力是该模型的另一优点。因此，这个模型系统提供了一个独特的机会，梳理出创面修复机制和试验治疗为他们的伤口愈合过程中的作用。预计在体外模拟白内障手术模式，具有广泛的适用性，为研究损伤修复机制的重要资源。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Use at 140mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Use at 5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td>Use at .7mM in TD Buffer</td>
		</tr>
		<tr>
			<td>D-glucose (Dextrose)</td>
			<td>Fisher Scientific</td>
			<td>D16-500</td>
			<td>Use at 0.5mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Use at 8.25mM in TD Buffer</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td>Use to pH TD buffer to 7.4</td>
		</tr>
		<tr>
			<td>Media 199</td>
			<td>GIBCO</td>
			<td>11150-059</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning/CellGro</td>
			<td>25-005-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Corning/CellGro</td>
			<td>30-002-CI</td>
			<td>Use at 1% in Media199</td>
		</tr>
		<tr>
			<td>100mm petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875711Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Filter Unit</td>
			<td>Millipore</td>
			<td>SCGPU01RE</td>
			<td>Use to filter sterilize Media</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps (need 2)</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm Cell Culture Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>27 Gauge 1mL SlipTip with precision glide needle</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Forceps</td>
			<td>Fine Science Tools</td>
			<td>91100-12</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Other Items Needed: General dissection instruments,&#160; fertile white leghorn chicken eggs,&#160;</td>
		</tr>
		<tr>
			<td colspan="4">check egg incubator (humidified, 37.7&#176;C), laminar flow hood, binocular stereovision dissecting&#160;</td>
		</tr>
		<tr>
			<td>microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a clinically relevant ex vivo mock cataract surgery model for investigating epithelial wound repair in a native microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

离体模型创建研究伤口愈合过程中的细胞的天然微环境调查涉及细胞的微环境原生内调节上皮伤口愈合机制，创建了一个临床相关的体外模拟白内障手术模式。这个模型是从晶状体组织提供了许多优点，由于其内在特性创建：1）透镜是一个自包含的器官由粗基底膜称为晶状体囊包围; 2）它是无血管，3）没有神经支配和4）免费相关基质。因此，检查了修复过程?...

Watch this Scientific Journal Video about Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment at JoVE.com

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

建立临床相关<em&gt;离体</em&gt;调查上皮创伤修复的微环境本地人模拟白内障手术模型

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

establishment-clinically-relevant-ex-vivo-mock-cataract-surgery-model

Research

JoVE Journal

Developmental Biology

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

6.5K 次观看.  Thomas Jefferson University. Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment. 

视频: Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

科研

发育生物学

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

心外膜生长培养测定和<em&gt;离体</em&gt;心外膜源性细胞迁移的评估

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

腺病毒基因治疗对糖尿病性角膜病变：对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

水下滤纸三明治长期<em&gt;防爆大毛</em&gt;早期鸡胚时间推移成像

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

戒毒治疗和<em&gt;在体内</em&gt;在青鳉鱼骨质疏松模型成骨破骨细胞相互作用的成像

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

一种在96孔板表演细胞迁移/伤口愈合设备

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch&#039;s Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

老年人刷膜的体外模型对视网膜色素上皮细胞的培养

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...