This study has been supported by the Collaborative Research Centre CRC TR128 "Initiating/Effector versus Regulatory Mechanisms in Multiple Sclerosis-Progress towards Tackling the Disease" (Project A9 to H.W. and C.C.G., project B1 to N.S.).

1.细胞人脑微血管内皮细胞的培养（HBMEC） <ol><li>细胞培养烧瓶的涂层<ol><li>为了制备纤连蛋白溶液，加入10毫升PBS至15mL离心管中。加入150μL纤维连接蛋白拌匀。 </li><li>以覆盖所述底部的T-25细胞培养瓶添加2毫升纤连蛋白溶液。在孵化器中孵育的细胞培养瓶为至少3个小时在37℃下。纤连蛋白包被的烧瓶可以在37℃/ 5％CO 2中可以储存2周。 </li></ol></li><li>播种和HBMEC的细胞培养<ol><li>从细胞培养烧瓶的底部吸出纤连蛋白溶液。添加7.2×10 4 HBMEC /平方厘米悬浮于6mL ECM-B培养基（= ECM-B补充有5％胎牛血清，1％青霉素/链霉素和1％的内皮细胞生长补充物）。孵育在37℃/ 5％CO 2。每天检查细胞生长用显微镜。 </li><li>改变介质每3天。收获或分裂的细胞，当HBMEC达到大约80％汇合。 HBMEC应通道1和15之间使用，以避免生理特性的损失。 </li></ol></li><li>收获HBMEC。 <ol><li>制备通过在1:1的比例混合，用PBS ACCUTASE（1×）ACCUTASE溶液:1。保持在水浴中在37℃下ACCUTASE溶液直至进一步使用。 </li><li>从细胞培养烧瓶转移ECM-B培养基至15mL离心管中。通过添加5mL PBS到细胞培养瓶的底部洗HBMEC。吸PBS和重复洗涤步骤两次以上。 </li><li>加入2毫升预热ACCUTASE溶液。孵育在37℃下2分钟。此后，HBMEC被牢牢地拍打细胞培养瓶数次重悬。细胞剥离用显微镜控制</li><li>预先存储在一个15毫升管中的ECM-B-介质只要HBMEC开始分离加回到细胞培养瓶。冲洗瓶的底部，直到大部分是HBMEC重新悬浮。 </li><li>细胞悬液转移到15毫升离心管中。离心机中以300×g于室温下10分钟。弃上清，重悬在1mL ECM-B培养基的细胞。计数细胞并稀释细胞悬浮液，以实现每毫升ECM-B培养基3×10 5的HBMEC终浓度。 </li></ol></li></ol> 2.细胞培养插入的制备<ol><li>细胞培养插入物的涂层 重要提示 :避免接触细胞培养插入的膜。 <ol><li>添加100μL的纤连蛋白溶液（参见1.1.1），以每个细胞培养插入物（ 图1A）和96孔平底平板的一个孔（光控制孔）。孵育在37℃下至少3个小时。孵育后，吸出纤维连接蛋白的解决方案。 </li><li>加入100μLHBMEC悬浮到细胞培养插入和光控制井。 600μl的ECM-B培养基添加到该单元的下部隔室培养插入。孵育3 - 4日，在37℃/ 5％CO 2，直到屏障完整性（ 图1B）到达，检查由HBMEC的显微镜评价细胞生长在光控制井。注意:不建议细胞生长超过四天。 </li><li>可选:为了模仿炎性病症抽吸从下部隔室中的培养基，并用补充有500U / mL的IFN-γ/ TNF-α前24个小时的迁移测定ECM-B培养基更换。 </li></ol></li></ol> 3.质量控制的伊文思蓝在轮回试验的当天<ol><li>伊文思蓝溶液的制备<ol><li>使用15mL离心管用200μlB27补充剂制备PBS / B27溶液混合10毫升PBS中。稀伊文思蓝原液（20毫克/毫升PBS）1:1000用PBS / B27。 </li></ol></li><li>伊文思蓝的渗透性测定<ol><li>从下部隔室，随后上部隔室吸出介质的含有汇合的单层HBMEC一个细胞培养插管。加入100μL伊文思蓝解决细胞培养插管。 </li><li>添加600μLPBS / B27到下部隔室，并在37℃/ 5％CO 2孵育60分钟。小心使用镊子取出细胞培养插管。 </li></ol></li><li>伊文思蓝测量<ol><li>从下部室中取出PBS / B27以各100μL转移到黑色聚苯乙烯96孔平底板的两个孔。在Tecan无限临M200读板器插入板和确定最佳的z位置。 </li><li>的伊文思蓝使用相应的设置测量的激发（例如:激发:620纳米，发射:680纳米，激发带宽:9 nm，发射带宽:20纳米，175x的增强，25个闪烁，积分的时间:20微秒）。 </li><li>为了确定HBMEC屏障功能比较所获取的数据与标准曲线描绘跨越HBMEC伊文思蓝渗透在种子后不同的时间点荷兰国际集团的细胞（ 图1B，右侧）。 </li></ol></li></ol> 4.迁移分析<ol><li>外周血单核细胞（PBMC）的制备。 <ol><li>加入10 mL RPMI到15mL离心管中，加入200μL的B27补充剂。计数PBMC和离心细胞以300×g离心5分钟。重新悬浮PBMC以5×10 6个细胞/ mL RPMI / B27以终浓度。 </li></ol></li><li>移民法的建立<ol><li>从下部隔室，接着含有汇合的单层HBMEC（ 图1A）的细胞培养插入物的上部隔室吸出培养基。每个供体添加每个100μL的PBMC悬浮液与细胞培养插入并且还每一个24孔板的一个孔中（ 体外控制）。 </li><li>添加600μLRPMI / B27的细胞培养插入和500微升的体外控制的PBMC的下部隔室，并在37℃/ 5％CO 2孵育6小时。 </l我&gt; </ol></li><li>迁移PBMC的收获<ol><li>使用镊子取出细胞培养插管并仔细冲洗用400微升PBS底部不接触膜。弃细胞培养插管。 </li><li>加入20μL流数荧光微（大约1,000珠/μL）向细胞培养物的下部隔室插入以及到体外控制拌匀。吸取1 mL所得PBMC悬浮液至流式细胞仪管。 </li></ol></li></ol> 5.流式细胞仪<ol><li>样品制备<ol><li>离心机PBMC在300×g下在室温下5分钟。 </li><li>通过加入荧光标记的抗体，以100μL的制备抗体溶液流式细胞仪缓冲液（PBS / 1％BSA / 2mM EDTA）中，每个样品。对于下面给出1μLCD4-FITC的结果，1μLCD3-PerCP / Cy5.5的，1μL的CD56-PC7，1μL的CD8-A700，和1μlCD16-A750每个样品使用。 </li><li>重新暂停PBMC在100μL的抗体溶液中，并在4℃下温育30分钟。 </li><li>加入250μL流式细胞仪缓冲液中并离心，在300×g离心5分钟。 </li></ol></li><li>样品采集<ol><li>以所需的量再悬浮PBMC（取决于流式细胞仪中使用的流）流式细胞仪缓冲液中。 </li><li>使用流式细胞仪用525和700nm的波长之间的有源检测器，检测流数荧光微获得染色PBMC（激发488 nm，发射525 - 700纳米）。 （下面的步骤是，如果使用GALLIOS流式细胞仪用克鲁札G软件操作的示例:（1）启动计算机（2）当操作系统被完全加载时，通过按"上流式细胞仪"按钮血细胞计数开始流动通过按下"开放协议"按钮。（3）装入相应的获取协议。（4）选择所需的协议并选择"打开"。（5）由与右点击复制为每个样本的协议在协议中虚拟转盘和在球场上"重复"左击鼠标可见按钮。 （6）标签在样品列表中的每个样本。 （7）传送所述样本以转盘的指示位置和开始采集。） </li></ol></li><li>样品分析<ol><li>使用相应的软件开所得的流式细胞术数据。确定的来自体外对照孔为transmigrated PBMC兴趣以及细胞亚群的数目，并使用相应的分析软件计数流动荧光微。 （门控策略的一个例子在结果部分中给出（ 图1 C:为了分析NK细胞亚群的轮回，首先在一个侧向散射信道（SSC）相对于前向散射信道（FSC）情节选择淋巴细胞淋巴细胞是。然后在CD3对CD56情节和CD56 + CD3显示- NK细胞被选择为NK细胞亚群之间进行区分，NK细胞都显示在一个CD56与CD16的情节和CD56 明亮的 CD16 暗/ -以及CD56 暗淡 CD16 + NK细胞被选择。此外，流数荧光微是从FSC相对于SSC情节选择并随后在信道的带525个700纳米与时间的关系，以确定它们的数目之间的发射的曲线图显示。） </li><li>来计算每个样品的总细胞数，使用流数荧光微正常化的细胞的检测数: <img alt="方程" src="/files/ftp_upload/55390/55390eq1.jpg" /></li><li>确定迁移的细胞作为总迁移的细胞和总细胞在体外控制之间比率的百分比。 </li></ol></li></ol>

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: A.S.-M. and U.B. have no financial disclosures. T. S.-H. received travel and conference expenses from Biogen. N.S. received speaker and advisory board honoraria from Biogen and Novartis Pharma, as well as travel expenses from Biogen. H.W. received compensation for serving on Scientific Advisory Boards/Steering Committees for Bayer Healthcare, Biogen, Merck Serono, Novartis, and Sanofi-Genzyme. He also received speaker honoraria and travel support from Bayer Vital GmbH, Bayer Schering AG, Biogen, CSL Behring, Fresenius Medical Care, Glaxo Smith Kline, GW Pharmaceuticals, Lundbeck, Merck Serono, Omniamed, Novartis, and Sanofi-Genzyme. He received compensation as a consultant from Biogen, Merck Serono, Novartis, and Sanofi-Genzyme. H.W. received research support from Bayer Vital, Biogen, Genzyme, Merck Serono, Novartis, Sanofi-Aventis Germany, and Sanofi US. C.C.G. received speaker honoraria and travel expenses for attending meetings from Genzyme, Novartis Pharma GmbH, and Bayer Health Care.

在这里，我们提出了一种技术，调查淋巴细胞跨越人类血脑屏障的轮回。 在淋巴细胞迁移至CNS的体外分析是很重要的学习淋巴细胞渗出，潜在的疾病相关的变化，以及新的治疗方法的基本过程。 血 - 脑屏障模型的若干修改是可能的。例如，从上室细胞可以解析，调查了未迁移细胞群的组合物。此外，治疗的HBMEC单层与测定前24小时IFN-γ和TNF-α的可以用来模仿发炎...

从血液进入组织的细胞迁移是免疫监视的关键。特定分子相互作用的序列确保位点特异性外渗到小肠，皮肤，淋巴结，中枢神经系统（CNS），以及其他组织1。在淋巴细胞迁移的改变都参与了许多的广泛传播疾病2的病理生理学。迁移到免疫豁免CNS是被严格调控，因此，该过程的改变参与CNS相关的疾病，如脑脊髓炎3，视神经，中风和多发性硬化（MS）2，4，5，6，7。因此，研究淋巴细胞渗出，以更好地了解疾病的病理生理机制，并制定一个工具，它是非常重要的<html>疾病负担8，9，10，11，12的土壤改良。 淋巴细胞通过不同的路线迁移进入CNS。外渗通过毛细血管小静脉成可经由脉络丛内和跨血脑屏障血-脑脊液屏障蛛网膜下腔已经描述1，13，14，15。跨过血-脑屏障迁移是由淋巴细胞的内皮细胞14的相互作用进行。与此相反，以内皮细胞在外围，CNS的内皮细胞表达大量紧密连接的分子，从而严格限制细胞和蛋白质能够穿过血脑屏障的量小姑娘= "外部参照"&gt; 16。炎症导致紧密连接的松动，并且诱导的粘附分子的表达;因此，增强淋巴细胞迁移进入CNS 1，17，18。 通过血 - 脑屏障外渗是一个多步骤过程。淋巴细胞系链对内皮细胞，然后沿着主要由选择素1，15介导的过程的内皮上滚动。随后，在淋巴细胞上表达由内皮分泌的趋化因子和相应的趋化因子受体之间的相互作用诱导整联的构象变化，从而促进牢固粘附到内皮细胞1。最后，无论是淋巴细胞针对沿着血流内皮屏障抓取transmigrating进入血管周围空间中之前，或立即和直接传动和失速igrate在牢固粘附1，19，20的部位。淋巴细胞外渗所有这些步骤都可以在体外使用不同技术21进行分析。延时录像显微镜用于研究的初步圈养及压延15。粘附分析提供关于牢固停滞的详细信息内皮障碍22。如这里演示轮回测定允许免疫细胞轮回21，23，24，25，26，27，28，29的分析。 使用体外血脑屏障模型的人，我们可以最近表明，较高的MIGRCD56 明亮的 CD16的atory容量暗淡/ - NK细胞相比，他们的CD56 暗淡 CD16 +同行通过在鞘内室21这NK细胞子集的优势体现。因此，我们的实验装置似乎是适用于模拟体内的情况。

<table><tbody><tr><td>PBS</td><td>Gibco</td><td>14190-094</td><td>without CaCl&lt;sub&gt;2&lt;/sub&gt; or MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Fibronectin 1 mg/mL</td><td>Sigma</td><td>F1141-5MG</td><td>from bovine plasma</td></tr><tr><td>T-25 cell culture flask</td><td>Greiner BioOne</td><td>690160</td><td></td></tr><tr><td>HBMEC</td><td>ScienCell</td><td>1000</td><td></td></tr><tr><td></td><td>Pelobiotech</td><td>PB-H-6023</td><td></td></tr><tr><td>Accutase</td><td>Sigma</td><td>A6964-100ML</td><td></td></tr><tr><td>ECM-b</td><td>ScienCell</td><td>1001-b</td><td></td></tr><tr><td>FBS</td><td>ScienCell</td><td>1001-b</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>ScienCell</td><td>1001-b</td><td></td></tr><tr><td>Endothelial cell growth supplement</td><td>ScienCell</td><td>1001-b</td><td></td></tr><tr><td>Transwell</td><td>Corning</td><td>3472</td><td>clear, 6.5 mm diameter, 3.0 &amp;micro;m pore size</td></tr><tr><td>96-well flat bottom plate</td><td>Corning</td><td>3596</td><td></td></tr><tr><td>Evans blue</td><td>Sigma</td><td>E2129-10G</td><td>stock solution: 1 g/50 mL PBS</td></tr><tr><td>B27</td><td>Gibco</td><td>17504-044</td><td>50x concentrated</td></tr><tr><td>Infinite M200Pro</td><td>Tecan</td><td></td><td></td></tr><tr><td>96-well black flat bottom plate</td><td>Greiner BioOne</td><td>675086</td><td></td></tr><tr><td>48-well plate</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>61870-010</td><td></td></tr><tr><td>Flow Count Fluorospheres</td><td>Beckman Coulter</td><td>7547053</td><td></td></tr><tr><td>Na-EDTA</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>Gallios 10-color flow cytometer</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Kaluza 1.5a</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>TNF-&amp;alpha;</td><td>Peprotech</td><td>300-01A</td><td></td></tr><tr><td>IFN-&amp;gamma;</td><td>Peprotech</td><td>300-02</td><td></td></tr><tr><td>CD3-PerCP/Cy5.5</td><td>Biolegend</td><td>300430</td><td>clone UCHT1</td></tr><tr><td>CD56-PC7</td><td>Beckman Coulter</td><td>A21692</td><td>clone N901</td></tr><tr><td>CD16-A750</td><td>Beckman Coulter</td><td>A66330</td><td>clone 3G8</td></tr><tr><td>CD4-FITC</td><td>Biolegend</td><td>300506</td><td>clone RPA-T4</td></tr><tr><td>CD8-A700</td><td>Beckman Coulter</td><td>A66332</td><td>clone B9.11</td></tr></tbody></table>

analysis of lymphocyte extravasation using an in vitro model of the human blood-brain barrier

淋巴细胞外渗到中枢神经系统（CNS）是免疫监视的关键。淋巴细胞渗出的疾病相关的变化，可能会导致在中枢神经系统的病理生理改变。因此，淋巴细胞迁移的调查中枢神经系统重要的是要了解炎性中枢神经系统疾病和开发新的治疗方法。在这里，我们提出了人类血脑屏障的体外模型来研究淋巴细胞渗出。人脑微血管内皮细胞（HBMEC）的汇合生长在多孔聚对苯二甲酸乙酯的Transwell插入到模拟血脑屏障的内皮。屏障功能是由紧密连接免疫组织化学，跨内皮电阻（TEER）的测量以及伊文思蓝渗透的分析验证。这种模式使罕见的淋巴细胞亚群血细胞渗出的调查，如CD56 亮 CD16 暗淡/ - NK细胞。 Furtherm矿石，其他细胞，细胞因子和趋化因子，疾病相关的改变，以及对淋巴细胞迁移能力不同治疗方案的效果进行研究。最后，炎性刺激对内皮屏障的影响，以及不同的治疗方案可以被分析。

示出NK细胞和使用人的血脑屏障模型（ 图1A）的T细胞亚群的轮回代表性的结果示。的HBMEC单层的完整性是由紧密连接分子ZO-1，跨内皮电阻（TEER）的测量，和伊文思蓝渗透（ 图1B）的染色验证。以下3 -第4天的培养HBMEC表达的紧密连接分子ZO-1（ 图1B，左）。此外，HBMEC增长在表现出跨内皮电阻（ 图1B中）以及降低的渗透为伊?...

Watch this Scientific Journal Video about Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier at JoVE.com

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

analysis-lymphocyte-extravasation-using-an-vitro-model-human-blood

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

20.3K 次观看.  University Hospital Münster. Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro. 

视频: Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

科研

生物工程

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

设置了一个<em&gt;体外</em大鼠血脑屏障&gt;模型（BBB）：在BBB抗渗性和受体介导的运输为中心

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

医学

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

一个<em&gt;体外</em&gt;血 - 脑屏障使用阻抗谱型号:对T细胞，内皮细胞的相互作用为中心

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

免疫与感染

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of non-essential ions and toxic substances. Tight junctions (TJ) effectively seal the paracellular space in the monolayers resulting in an intact barrier. This study describes a LY-based fluorescence assay that can be used to determine its apparent permeability coefficient (Papp) and in turn can be used to determine the kinetics of the formation of confluent monolayers and the resulting tight junction barrier integrity in hCMEC/D3 monolayers. We further demonstrate an additional utility of this assay to determine TJ functional integrity in transfected cells. Our data from the LY Papp assay shows that the hCMEC/D3 cells seeded in a transwell setup effectively limit LY paracellular transport 7 days-post culture. As an additional utility of the presented assay, we also demonstrate that the DNA nanoparticle transfection does not alter LY paracellular transport in hCMEC/D3 monolayers.

We present a fluorescence assay to demonstrate that Lucifer Yellow (LY) is a robust marker to determine the apparent paracellular permeability of hCMEC/D3 cell monolayers, an in vitro model of the human blood-brain barrier. We used this assay to determine the kinetics of a confluent monolayer formation in cultured hCMEC/D3 cells.

路西法黄 - 人类血脑屏障细胞模型中一个健壮的细胞渗透标记

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

外迁移系统中淋巴细胞迁移的评估

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

通过免疫荧光染色分析血脑屏障模型

Use of the MicroSiM (&#181;SiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the&#160;&#181;SiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between &#39;blood&#39; and &#39;brain&#39; chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the &#181;SiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

作者聚焦：推进组织芯片技术在人体组织中的应用 

This report provides protocols for assembly, cell culture, and assays on the &#181;SiM platform for the construction of blood-brain barrier models.

使用 MicroSiM （μSiM） 屏障组织平台对血脑屏障进行建模

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, ...

<meta charset="utf8"></head><body>基于荧光染料的小鼠血脑屏障分解可视化

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is a complex process influenced by several factors, such as the concentration of dyes in the blood, permeability of brain vessels, duration of dye extravasation, and reduction in dye concentration in the tissue due to degradation and diffusion. In a mild traumatic brain injury model, exposure to blast-induced shock waves (BSWs) triggers BBB breakdown within a limited time window. To determine the precise sequence of BBB breakdown, Evans blue, and fluorescein isothiocyanate-dextran were injected intravascularly and intracardially into mice at various time points relative to BSW exposure. The distribution of dye fluorescence in brain slices was then recorded. Differences in the distribution and intensity between the two dyes revealed the spatiotemporal sequence of BBB breakdown. Immunostaining of the brain slices showed that astrocytic and microglial responses correlated with the sites of BBB breakdown. This protocol has broad potential for application in studies involving different BBB breakdown models.

<meta charset="utf8"></head><body>轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

A method was developed to visualize dye extravasation due to blood-brain barrier (BBB) breakdown by administering two fluorescent dyes to mice at different time points. The use of glycerol as a cryoprotectant facilitated immunohistochemistry on the same sample.

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is ...

使用的淋巴细胞外渗分析

摘要

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此视频中的章节

改进方法对血脑屏障的人体细胞为主，接触模型的制备

设置了一个

一个

路西法黄 - 人类血脑屏障细胞模型中一个健壮的细胞渗透标记

外迁移系统中淋巴细胞迁移的评估

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

使用的淋巴细胞外渗分析

摘要

探索更多视频

此视频中的章节

改进方法对血脑屏障的人体细胞为主，接触模型的制备

设置了一个

一个

路西法黄 - 人类血脑屏障细胞模型中一个健壮的细胞渗透标记

外迁移系统中淋巴细胞迁移的评估

使用 MicroSiM （μSiM） 屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM） 屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM） 屏障组织平台对血脑屏障进行建模

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

轻度创伤性脑损伤小鼠模型中血脑屏障破坏的评价

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模

使用 MicroSiM （μSiM）屏障组织平台对血脑屏障进行建模