Name: assessment of endothelial cell migration after exposure to toxic chemicals
Uploaded: 2015-07-10T13:00:00.000+00:00
Duration: 7 min 7 s
Description: Watch this Scientific Journal Video about Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals at JoVE.com

This work was funded by the German Ministry of Defense (Grant No. M-SAB1-6-A009).

以下协议描述为早期内皮细胞迁移的调查技术。合适的培养的血管内皮细胞的需要的细胞培养瓶中用明胶预涂布，以确保内皮表型的适当扩散和维护。 细胞培养烧瓶的1.预涂层<ol><li>溶解明胶的0.1M PBS中的0.1％的最终浓度。 </li><li>高压釜中的液体灭菌参数解决方案（详见具体的高压釜的说明）。 </li><li>添加蒸压明胶溶液具有足够的体积，以无菌细胞培养瓶（ 例如，至少5毫升一T25细胞培养烧瓶）中。 </li><li>转移烧瓶成培养箱中（37℃，不需要的5％的CO 2，但不干扰）至少30分钟。 </li><li>除去剩余的明胶溶液。使用预涂层烧瓶随后（参见细胞培养）或s无菌条件，直至使用下撕毁。 </li></ol>早期内皮细胞2.细胞培养注:来自胚胎干细胞的早期内皮细胞（EEC）从分化的鼠胚体如前面10,11描述得到由PECAM-1阳性细胞级分的磁性活化细胞分选。 <ol><li>培养EEC在明胶包被的细胞培养皿中的DMEM补充有15％FCS，50U / ml青霉素，50U / ml链霉素，200μM的L-谷氨酰胺，100μM的β-巯基乙醇和1％非必需氨基酸。处理在无菌条件下的细胞。培养的细胞，用5％的CO 2在37℃和95％的湿度，直至亚融合（最大80％）。 </li><li>在亚融合，分裂细胞以1:5的比例。注意:内皮细胞的脱离是关键的一步。 <ol><li>收获细胞，用RT的Accutase。取出介质，冲洗用PBS和每25厘米加入1 ml的Accutase的<sup&gt; 2。 </li><li>孵育烧瓶在RT 2-10分钟，直到细胞脱离。分散细胞，并将它们传送到所期望的应用。注意:因为它发生时，接种的细胞在37℃下保存在培养箱不需要的Accutase的化学中和。但是，的Accutase活性也可以通过添加的DMEM含有FCS的降低。 </li></ol></li></ol> 3. Boyden小室注意:Boyden小室测定法是通过使用光的不透明聚对苯二甲酸乙酯刀片系统具有8微米的孔径进行。 <ol><li>预涂层的过滤器插入件（即适合细胞培养孔因此形成了boyden小室的内部）通过添加500μl的0.1％明胶的溶解在0.1M PBS中至少30分钟。 </li><li>如果细胞暴露于有毒化学品，根据具体的实验设计之前收获细胞暴露。注意:将细胞暴露于12.5微克丁酸氮芥/ ml的DMEM中24小时。关于具体的实验设计，指令可能会发生变化。 </li><li>收获EEC和通过使用计数池室确定的细胞数。注意:自动计数装置都可以使用，但应谨慎使用:手动细胞计数是更准确，强烈建议。 </li><li>加入500微升的细胞培养基进入Boyden小室的下腔室隔室。 </li><li>加准确的10 4欧洲经济共同体在每滤芯500微升的细胞培养基中的上腔室中。消除泡沫。 </li><li>孵化在孵化器的滤芯整整8小时。 </li><li>用PBS冲洗一次，并更换用0.5ml 4％多聚甲醛的介质在两个室25分钟，进行细胞固定。广泛冲洗过滤器，但至少3次用0.1M PBS中。 </li><li>消费用手术刀膜。 </li><li>将两片玻璃盖玻片之间的膜含有DAPI核stainin安装介质G。注意方向。确保只有已迁移朝向膜的下室侧的细胞进行计数。 </li><li>算已迁移朝向膜的下室侧与在放大400倍荧光显微镜的细胞。不要混淆膜孔与迁移细胞（ 图1A，1B）。研究生物复制合理数量（每状态至少3个生物学重复）。 </li></ol> 4.伤口愈合实验<ol><li>根据可用的设备，精心选择了细胞培养皿或板:当使用DIC的显微镜，避免塑料表面基于培养皿或孔板而是用玻璃基础上的，而不是设备。 注意:如果使用相差显微镜，是根据塑料盘也可使用。 </li><li>在合适的细胞培养装置，培养欧洲经济共同体（ 例如，4厘米玻璃底培养皿适于活细胞成像），直到80％汇合。重要提示:不要培养细胞来完成汇合。 </li><li>从头无菌10微升枪头的单层。轻轻推动尖端没有太大的压力到所述餐具表面和用0.1M PBS中移动它在一条直线上平稳地从一侧到other.Wash细胞两次以除去分离的细胞。 </li><li>添加介质的足够体积到培养皿。如果适用，添加要追究化合物。 注意:含有1.5毫升培养基15纳克/毫升的α-亚麻酸的溶液。 </li><li>安装能够活细胞成像显微镜下培养皿。确保5％CO 2，37℃和潮湿气氛。 注意:加湿是特别重要的，以避免介质的蒸发。 </li><li>超过24小时采集时间推移图像以10分钟的间隔。规划大文件大小。注:512×512像素的分辨率通常是足够的;然而，我们建议使用至少1024×1,0图片24。 </li><li>使用该软件的设置有显微镜的长度工具测量间隙宽度在t = 0小时和在t = 24小时，或者使用开放源码软件（ 例如，ImageJ的）。注意:通常特定图像采集和分析软件是由制造商提供。因此，对于使用该软件的技术细节，请参考手册。 </li></ol> 5.细胞跟踪<ol><li>根据可用的设备，精心选择了细胞培养皿或板:当使用DIC的显微镜，避免塑料表面基于培养皿或孔板而是用玻璃基础上的，而不是设备。 注意:如果使用相差显微镜，是根据塑料盘也可使用。 </li><li>种子5×10 4欧洲经济共同体在补充的DMEM在合适的细胞培养装置（ 例如，24多孔板），并培养所述细胞，用5％的CO 2在37℃和95％湿度下1-2天。 </li><li>当细胞已经长大到80％汇合，除去介质和培养细胞与新媒体的相应测试物质的存在（例如，12.5微克苯丁酸氮芥/ ml的DMEM）。始终包括对照细胞（用溶剂， 例如，乙醇处理）在37℃下在一段时间内的时间（24小时，这取决于各个测定系统上）。 </li><li>安装能够活细胞成像（37℃，5％CO 2和95％湿度），在显微镜下的培养皿。收购时间推移图像超过24小时，在预定的时间间隔。获得以10分钟的间隔图像。 </li><li>执行手动跟踪EEC中所使用的插件的ImageJ的MTrackJ。从视场中选择10个细胞，随机，通过加入每点一个数据点在时间上使用"添加"MTrackJ的命令跟踪他们的运动。 </li></ol>注:MTrackJ是免费提供的ImageJ的和是availab [Meijering， Mtrackj"http://www.imagescience.org/meijering/software/mtrackj/"。]在乐[Rasband， ImageJ的。"http://rsbweb.nih.gov/ij/"。关于MTrackJ插件的详细手册可在"http://www.imagescience.org"。 6.活细胞成像/线粒体膜电位评估<ol><li>在合适的细胞培养装置，培养欧洲经济共同体（ 例如，4厘米玻璃底培养皿适于活细胞成像）到80％汇合。 重要提示:不要培养细胞来完成汇合。 </li><li>如果适用，揭露细胞的化学物质。注意:将细胞暴露于12.5微克/毫升苯丁酸氮芥24小时。关于具体的实验设计，指令可能会发生变化。 </li><li>制备四甲（TMRM）在DMSO中的10mM储备溶液。避光。注意:原液可以储存在-20℃。 </li><li>稀释原液在细胞培养基中，以具有10微米的浓度的工作溶液（1:1,000稀释度）。避光并使用尽快。注意:工作溶液可保持在室温下一段时间（&gt; 1小时），但是，制备的新鲜工作溶液是强烈建议。 </li><li>加2μl的工作溶液到1ml新鲜的细胞培养基中（装载溶液）。 </li><li>通过与加载溶液更换细胞培养基加载细胞。孵育15分钟，在37℃，5％CO 2的加湿气氛（孵化）。注意:几乎所有的荧光指标是由活细胞随着时间的推移出口;因此，避免长时间加载或延迟分析。 </li><li>无需水洗，将适用于活细胞成像显微镜下的菜。重要提示:荧光指标对光非常敏感，因此，避免不必要的曝光。 </li><li>获取图像而无需改变采集参数，以确保不同的图像之间的可比性。 </li></ol>

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The authors have nothing to disclose.

皮肤接触有毒化学品往往导致严重的伤口愈合障碍。基本的机制在很大程度上是未知。伤口愈合是一个复杂的过程，它由不同的阶段（止血，发炎，增殖和重塑）的。细胞迁移涉及每个阶段，然而，它是最重要的肉芽组织的形成。这里，新血管通过血管造影或血管生成要么形成。 这两个过程都需要前体和早期内皮细胞不受影响迁移。细胞迁移和明确的干扰细胞迁移的基本机?...

细胞迁移是许多生理和病理过程，包括发展，各种疾病及皮肤受伤后伤口愈合很重要的。 以下皮肤损伤，炎症消除受损或坏死组织及肉芽驱动器初步伤口缝合，并允许通过血管形成新的毛细血管网络的（小说形成）或血管生成（已有的囊泡萌芽）1-3。既vasculo-和血管生成需要内皮细胞的迁移。越来越多的血管网络是必不可少的运输氧和营养物质的角质形成细胞增殖，最终发生角化，形成新的上皮细胞，并提供伤口缝合。 内皮细胞受损迁移是伤口愈合紊乱4,5的一个根本原因。早期的内皮细胞。因此，方法来评估迁移需要探索病理生理的细胞迁移紊乱术，并确定新的策略的治疗干预。 皮肤暴露于烷化剂（ 例如，硫和氮芥子气）导致伤口愈合紊乱6。这种化合物被用作化学战剂的一些冲突，在20世纪，并保持之所以强烈关注，由于在政治上不稳定的地区和相对简单的合成现有库存。虽然硫芥在1822年首次合成，SM暴露的分子和临床病理学不被了解的细节，也没有解药SM曝光已经确定。 几项研究已经进行了解和伤口愈合不良SM曝光后模拟和测试能够保留该效果的潜在候选化合物。 Schmidt 等人（2009）测试了苯丁酸氮芥的效果，与在谋类似的SM性质的烷基化化合物本身胚体模型，发现了戏剧性，有时在血管形成7减少99％以上。这种不利影响是最有发展的哪个，在生理条件下，通过增殖和血管内皮前体细胞的迁移支配阶段明显。因此，这些细胞被鉴定为特别敏感烷化剂。 Steinritz 等人（2010）测试了活性氧簇（ROS），特别是N-乙酰半胱氨酸（NAC）和阿尔法亚麻酸（ALA）清除剂其减少的SM毒性在小鼠胚体模型和具体地，涉及能力恢复血管形成8。观察临时保护作用，表明过度ROS形成是可能有助于SM的对伤口愈合的不利影响。这些影响是不是永久性的，这两个候选化合物可能不能够恢复在长期8血管形成和伤口愈合的。 HoweveR，这些实验在一个复杂的三维模型，确实让细胞迁移的调查进行的。因此，我们随后被测试的NAC和ALA对于具有在血管形成9的过程中起关键作用的欧洲经济共同体的细胞迁移的有益效果。 此外，有证据表明，细胞极性所需的细胞迁移。线粒体功能障碍导致ROS形成被证明损害细胞极性和可因此细胞迁移有负面影响。因此，活细胞成像对于线粒体功能被执行，并且进行了检查的ROS清除剂的作用。以下协议描述为EEC，Boyden小室法，伤口愈合实验，包括细胞跟踪分析和使用TMRM的详细线粒体功能评估的培养一般要求。对于EEC种植和移植实验方案的重要方面突出。

<table><tbody><tr><td>Boyden Chamber&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Corning FluoroBlok Tissue Culture (TC)-treated Inserts, 24 well - 3 &amp;micro;m</td><td>Corning Incorporated</td><td>#351151</td><td></td></tr><tr><td>Corning FluoroBlok Tissue Culture (TC)-treated Inserts, 24 well - 8 &amp;micro;m</td><td>Life Sciences</td><td>#351152</td><td></td></tr><tr><td>(for use with Falcon Insert 24 well Companion Plate (353504)</td><td></td><td></td><td></td></tr><tr><td>Wound&amp;nbsp; healing assay</td><td></td><td></td><td></td></tr><tr><td>Glass bottom dishes</td><td>Word Precision Instruments, Inc.</td><td>#FD35-100</td><td></td></tr><tr><td>Assessment of mitochondrial potential</td><td></td><td></td><td></td></tr><tr><td>TMRM (tetramethylrhodamine methyl ester)</td><td>Life Technologies</td><td>#T669</td><td></td></tr><tr><td>Cell culture</td><td></td><td></td><td></td></tr><tr><td>Accutase</td><td>PAA, Pasching, Austria</td><td># L11-007</td><td></td></tr><tr><td>&amp;alpha;-Linolenic acid</td><td>Fluka (Sigma), Steinheim, Germany</td><td>&amp;nbsp;# L2376</td><td></td></tr><tr><td>Chlorambucil</td><td>Fluka (Sigma), Steinheim, Germany</td><td># 23125</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich, Steinheim, Germany</td><td># G2500-100G</td><td></td></tr></tbody></table>

assessment of endothelial cell migration after exposure to toxic chemicals

接触化学物质（包括如硫和氮芥子气烷化剂化学战剂）会导致过多的临床症状，包括伤口愈合障碍。伤口愈合的生理过程是非常复杂的。肉芽组织的形成是在这个过程中产生了初步伤口闭合和提供的新的毛细血管的网络中的关键步骤 - 无论是通过血管（新形成）或血管生成（出芽现有船只）。无论vasculo-和血管生成需要内皮细胞的功能，定向迁移。因此，早期内皮细胞（EEC）迁移的调查是重要的，了解化学诱导伤口愈合障碍的病理生理学和潜在确定新的战略治疗干预。 我们评估伤口愈合不良烷化剂暴露后测试潜在的候选化合物TReatment。我们使用了一组在此协议中概述的技术。一个改良的Boyden室定量调查EEC的化学激活描述。此外，使用伤口愈合实验，结合轨道分析，以定性评估迁移的说明。最后，我们证明了使用的荧光染料TMRM对于线粒体膜电位的调查，以确定潜在的干扰细胞迁移的机制。下面介绍的协议已被改编为EEC的调查基本技术。

皮肤暴露于烷化剂引发红斑，水疱形成和与伤口愈合病症相关皮肤溃疡。伤口愈合需要其基于内皮细胞迁移的血管造影和血管。定量迁移可以通过使用Boyden小室测定法进行评估。如图欧洲经济共同体的图1C暴露于烷化剂氮芥导致细胞迁移9一显著降低。除了接触苯丁酸氮芥24小时后直接活性氧清除剂的α-亚麻酸（ALA）的显著救出这种表型，几乎控制水平。 ?...

Watch this Scientific Journal Video about Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals at JoVE.com

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

内皮细胞迁移评估暴露于有毒化学品后，

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

assessment-endothelial-cell-migration-after-exposure-to-toxic

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.6K 次观看.  Bundeswehr Institute of Pharmacology and Toxicology. Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

视频: Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

科研

免疫与感染

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

生物工程

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

体外流动下单核细胞亚群的同时研究

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

一种研究炎症期间白细胞-内皮细胞相互作用的微生理系统

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/&#946;-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion.

Here, we describe a protocol, the extended endothelial cell culture method (EECM), that allows differentiation of pluripotent stem cells to brain microvascular endothelial cell (BMEC)-like cells. These cells show endothelial cell adhesion molecule expression and are thus a human blood-brain barrier model suitable to study immune cell interactions in vitro.

人诱导多能干细胞分化为具有成熟免疫表型的脑微血管内皮细胞样细胞

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

神经科学

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

生物学

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

内皮细胞转吞测定作为 体外 模型评估内部血-视网膜屏障通透性

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

医学

Assessing Interneuron Response to Chemoattractive Cues from Periventricular Endothelial Cells

Source: Datta, D., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/60295">Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons.</a> J. Vis. Exp. (2020).
This video shows a method to test interneuron migration in response to chemoattractive cues from periventricular endothelial cells (PVECs). Interneurons are placed in a central compartment, with PVECs and control cells on either side. Increased migration towards PVECs indicates a positive response to their chemoattractive signals.

评估神经元对脑室周围内皮细胞的化学吸引力线索的反应

1. Chemo-attraction Assay

	Place a three-well culture insert in the center of a poly-L-ornithine/laminin-coated 35 mm dish using sterile tweezers.
	Turn ...

内皮细胞迁移评估暴露于有毒化学品后，

摘要

探索更多视频

此视频中的章节

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

体外流动下单核细胞亚群的同时研究

一种研究炎症期间白细胞-内皮细胞相互作用的微生理系统

人诱导多能干细胞分化为具有成熟免疫表型的脑微血管内皮细胞样细胞

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

内皮细胞粘附性定量

内皮细胞转吞测定作为体外模型评估内部血-视网膜屏障通透性

评估神经元对脑室周围内皮细胞的化学吸引力线索的反应

内皮细胞迁移评估暴露于有毒化学品后，

摘要

探索更多视频

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