Name: in vitro assays to assess blood-brain barrier mesh-like vessel formation and disruption
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莱昂泰由芝加哥伊利诺伊大学创业基金资助。

所有实验均遵循伊利诺斯大学芝加哥体育动物护理和使用委员会协议。 一般准备注意:脑微血管内皮细胞系（hCMEC / D3）是广泛表达的永生化人类BEC细胞系14,15,16,18,19。在基底内皮生长基础培养基中培养hCMEC / D3细胞在包被I型胶原（小牛皮，Hank的含有Ca 2+和Mg 2+的 Hank平衡盐溶液（HBSS）中的0.1％溶液1:20稀释）的组织培养瓶上培养（EBM-2），每500 mL培养基中含有2-5％胎牛血清（FBS），10％抗坏血酸，10％硫酸庆大霉素，25％氢化可的松和提供的生长因子补充剂总体积的1/4，血管内皮细胞（VEGF），表皮生长因子（EGF），胰岛素样生长因子1（IGF-1）和人类碱性成纤维细胞生长因子（bFGF），参见材料表 。 注意:具有FBS和生长因子的EBM-2培养基称为"EBM-2完整"。没有FBS和补充剂的EBM-2被称为"EBM-2基础"。在完全融合时，hCMEC / D3细胞为〜1×10 5个细胞/ cm 2 。 <ol><li>通过首先用HBSS孵育3分钟，不含Ca 2+和Mg 2+，然后用5mL胰蛋白酶/ EDTA（0.25％）分离5分钟，以1:5的比例通过hCMEC / D3细胞。使用EBM-2（1:1中和）中和胰蛋白酶，并在4℃下以290 xg离心5分钟;丢弃上清液。将EBM-2中的沉淀物重新悬浮，并以1:5的比例重新平板化。 </li><li>根据供应商的协议[ 材料表 ]培养主要的人周细胞和星形胶质细胞。文化视角e周围细胞在周边细胞基础培养基中与FBS和周细胞生长补充。 </li><li>在包含星形胶质细胞生长因子的星形胶质细胞培养人星形胶质细胞。培养包被3mL聚-L-赖氨酸（PLL）的组织培养瓶中的细胞周围细胞和星形细胞，用于细胞粘附，并利用第2代和第5代之间的细胞。 </li><li>安乐死7只小鼠，用镊子去除脑干和小脑;通过仔细地滚动大脑在纱布上分离脑膜。用无菌剃刀刀片将HEPES（每脑5 mL）在最小基本培养基（MEM）中培养皿中的剩余脑组织。根据参考方案20从2个月大的C57BL / 6J小鼠分离主要小鼠BEC。 </li><li>将切碎的脑组织转移到15 mL锥形管中。在4℃下以290xg离心5分钟。去除上清液并孵育木瓜蛋白酶（每脑833.33μL）和DNase（每脑41.7μL）的组织溶液，在37℃水浴中1小时，消化组织。 </li><li>将匀浆依次通过19和21号针头，用22％牛血清白蛋白（BSA）溶液与1:1比例混合，并在4℃下以1360×g离心10分钟。 </li><li>将所得沉淀重悬于1mL EBM-2中，并在4℃以290×g离心5分钟。再次重悬在1mL EBM-2中，并在涂有胶原蛋白的6孔板（每孔1个脑）上平板培养细胞（1mL /孔）。 </li><li> 24小时后用含4μg/ mL嘌呤霉素盐酸盐的EBM-2更换培养基; 2天后用EBM-2替换完成。对于hCMEC / D3细胞培养初级BEC，并以1×10 5个细胞/ cm 2完全汇合。 </li><li>在测定前24小时准备100μM的oAβ。 注意:oAβ被认为是A的疾病相关形式。对于oAβ，使用Dahlgre描述的良好表征的制剂n et al。 21 。 </li><li>在4℃下将基底膜储存溶液解冻过夜，并将其等分成无菌PCR管条（8管），每管140μL基底膜。每个条带重新冻结，并在-20°C储存。在测定前24小时，在4℃下，每96孔板解冻一条。预先冷却移液器吸头以避免凝固。 注意:所有处理基底膜基质必须在冰上进行，以避免快速凝固。由于在测定当天使用10μL/孔的基底膜，每个条带对于一个96孔板是足够的。 </li><li>在分析前24小时，在EBM-2基础中孵育完全汇合的BEC。 注意:理由是:EBM-2完整包含补充因子和FBS，目的是促进最佳细胞生长;然而，促进细胞生长的相同分子途径对于脑内皮细胞的覆盖和动力学也是重要的，无论是在存在还是脱落一个压力源。因此，我们血清和补充剂使BECs减少细胞信号通路残留激活的混杂效应。值得注意的是，在测定之前，胰蛋白酶化细胞的中和期间细胞短暂（5分钟）暴露于FBS。与BEC相反，由于需要不同的生长因子和这些细胞类型对血清饥饿的反应的不稳定性（Tai实验室，未发表的观察），周细胞和星形胶质细胞不是血清饥饿。 注意:EBM-2基础在测定期间用于单次和三次培养的网状物形成和破碎测定。这对于防止混淆压力或依赖治疗信号至关重要。 </li></ol>网格化形成和中断测定<ol><li> 单次BEC培养测定: 注意:BEC的单一培养物的三种不同范例如图1A所示</STRONG&gt;。每个范例旨在评估BEC在不同船舶覆盖范围内的响应。范式1是网格形成。该范例旨在检查压力源和/或治疗对血管生成和网状形成的影响。 BECs，oAβ和处理均在0小时时间内加入板中。范式2是预防网络中断。在生长因子或药物存在下接种细胞并孵育4小时以形成网状结构。在这种情况下，应激者oAβ在4小时后加入。这种范例评估了治疗预防压力引起的预成形网状结构损伤的能力。范例3同时处理网状物的破裂。将细胞电镀并允许形成网状结构4小时。然后在4小时时间点同时加入治疗和oAβ，评估预先形成的细胞网络响应各种治疗的能力。所有范例除了相似的常规步骤，除了治疗时间的差异添加。 <ol><li>在测定当天，将基底膜基质以10μL/孔移液到96孔板的底部，并允许在37℃下放置1小时。 </li><li>将冻干的绿色细胞跟踪染料（ 参见表材料 ）悬浮于10μLDMSO中以产生10mM储备溶液，并在EBM-2基础中稀释至10μM（1:1000）。从完全汇合的烧瓶中取出培养基，并用含有绿色细胞跟踪染料的EBM-2基底代替（对于75cm 2的烧瓶为5mL）。在开始测定前20分钟，用绿色细胞跟踪染料预加载BEC。 </li><li>在37℃下孵育细胞20-30分钟，用细胞追踪染料除去培养基，并如步骤1.1所述分离细胞。用含有10％FBS的EBM-2中和培养基，并在4℃以240xg离心5分钟。将沉淀重悬于1mL的EBM-2基础中。 </li><li>在EBM-2基底（所有范例的0小时时间点）以10,000个细胞/孔将96孔板中的BEC平板化。 注意:根据正在使用的范例，在指定的时间点添加治疗，压力源或车辆控制。在所有范例中，收集培养基用于随后的分析（ 例如 ELISA分析），并将细胞在PBS缓冲液（PBS）中用新鲜制备的4％多聚甲醛固定24小时。作为确定oAβ对网格形成的术语影响的替代方法，可以修改方案以用oAβ预孵育汇合的细胞培养瓶，然后进行网格形成范例（+/-oAβ）。 注意:所有范例的每个孔的最终体积为70μL。所有处理（oAβ，EGF 等 ）以1:20的稀释度加入其各自的孔中， 即每次处理3.5μL。对于范例1，将细胞以63μL/ w加入细胞悬液中埃尔。立即将oAβ，所需处理和对照加入3.5μL/孔，共计70μL。对于范例2，在0小时时间点加入63μL细胞悬浮液和3.5μL处理（ 例如 100ng / mL EGF）。孵育4小时后，加入3.5μL的oAβ原液（ 例如，对于100nM的最终的oAβ浓度，3.5μL的2μM的oAβ原液）。对于范例3，在0小时时间点加入63μL细胞悬浮液，并在4小时时加入oAβ，并以3.5μL/孔加入所需的处理，共计70μL。这些体积可以根据治疗进行调整。例如，如果只需要一个处理，则可以将细胞悬液体积调节至66.5μL（维持10,000个细胞/孔），并且处理体积可以保持在3.5μL。 </li></ol></li><li> 三重培养试验: 注意:除了BEC的单一培养测定，我们已经开发（ 图1B ），以确定预先形成的网络中BECs，周细胞和星形胶质细胞对相关压力因子（ 如 oAβ）的反应。在0 h时，BECs需要进行所需的治疗。 4小时后，将周细胞轻轻加入板中。在7小时后，将星形胶质细胞加入板中，然后在11小时加入应激物。然后将细胞孵育至24小时时间点。 注意:对于三重培养测定，重要的是考虑时间以确保所有细胞同时融合。人星形胶质细胞和周细胞应在测定前1周进行培养，而hCMEC / D3细胞需要在测定日期前4-5天铺板或传代。此外，使用低通（2-5）原代细胞以避免表型漂移是重要的。 <ol><li>在开始测定前20分钟，将绿色细胞跟踪染色工作溶液加入到hCMEC / D3细胞中。将10cm的hCMEC / D3细胞平板化，000个细胞/孔在EBM-2基底（0小时时间点）。使用的体积是45μL细胞悬浮液和3.5μL处理（ 即最终EGF浓度为50ng / mL，100ng / mL或1,000ng / mL，浓度为20倍）。 </li><li>在37℃下孵育3.5小时后，将蓝细胞跟踪染料工作溶液（EBM-2基质中的10μM细胞追踪蓝）加入到人类初级周细胞中。在4小时的时间点（用细胞追踪染料孵育〜30分钟），以6μL/孔的体积轻轻加入含有hCMEC / D3的平板上的2,000个周细胞/孔。 </li><li>在另外2.5小时孵育（从0小时时间开始总共6.5小时）后，将橙色细胞跟踪染料工作溶液（EBM-2基础中的10μM细胞追踪器橙色）添加到人类原始星形胶质细胞。在7小时的时间点，轻轻地以12μL的体积向板中加入10,000个星形胶质细胞/孔。因此，内皮细胞的总体细胞比例:周细胞:星形胶质细胞s 5:1:5。此外，达到此目的的体积是66.5μL。 </li><li>加入oAβ（3.5μL100μM储备液）4小时后（从0小时时间开始共11小时）。 </li><li>在新鲜制备的4％多聚甲醛PBS中，13小时后将细胞固定。 小心:在处理多聚甲醛时，戴防护服，手套和眼镜。 </li></ol></li></ol>量化<ol><li>使用解剖显微镜以最大功率50％的2秒曝光时间以1.6倍放大倍数捕获96孔板的整个孔的荧光图像。 注意:可以使用等效显微镜;然而，捕获整个井以进行综合分析至关重要。 </li><li> 对于定量分析，使用ImageJ血管生成分析仪插件22处理图像，以量化分支数，网格数和总细胞长度（这些读数由softwa自动列表re），如下所述。 图2中提供了此步骤的详细视觉协议。 <ol><li>点击软件图标打开fiji ImageJ。 </li><li>单击位于工具栏末端的双红色向前箭头，然后单击"血管生成分析仪"。 </li><li>选择带有图像文件的文件夹，单击"打开"。 </li><li>当出现"分析设置"框时，单击"确定"。 </li><li>出现"处理图像"框时，表示要处理的图像数，单击"确定"。 </li><li>当出现"批量进度窗口"框时，表示预计处理时间。在此期间，程序对包括分支数，网格数和总细胞长度的输出进行量化。 注意:一旦所有图像被量化，结果文件将作为电子表格文档显示在原始图像文件夹中。 </li><li>选择电子表格文件包含结果并比较组之间的分支数，网格数和总细胞长度。 注意:在三重培养测定中，ImageJ被进一步用于分析周细胞/星形胶质细胞的覆盖和数量。图像由盲人实验者和用于产生读数的"分析粒子"功能阈值。固定细胞和管状结构也可以用于Aβ17或其他标记物的免疫染色。 </li></ol></li></ol>

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作者没有什么可以披露的。

所描述的方法可用于解决围绕脑血管覆盖的几个基本生物学问题24 。具体来说，他们可以确定哪些受体和信号通路在血管发生，癌症组织中的血管覆盖以及与脑相关的外周内皮细胞中起作用。实例包括血管生成生长因子受体，一氧化氮，丝裂原活化蛋白激酶信号和钙信号传导25,26,27 。 hCMEC / D3和主要BECs可修改为遗传敲打方法，以促进这一努力18 。利用所述方法从关键蛋白质的完全或内皮细胞特异性敲低的小鼠培养分离的BECs可以获得机制的洞察力。此外，三重培养测定能够深入分析星形胶质细胞和周细胞的影响e对脑内皮细胞的功能。例如，周细胞和星形胶质细胞可以通过产生可溶性介质（ 例如血管生成素，细胞因子）以及通过结构支持24来影响网状样血管形成。 模型系统也可应用于疾病研究。例如，该系统可以解决添加的疾病和老化相关压力因素是否外源性促进血管生成和/或网状物中断。压力因子可能与特定疾病有关， 例如 oAβ或更广泛的， 例如过氧化氢，细胞因子。三重文化范式增加了一个额外的复杂性层次，可以提高对影响BBB动态学的关键性疾病相关机制的理解:描述疾病相关的应激反应是否激活星形胶质细胞和周边细胞以破坏BEC功能。对于单次和三次培养测定，可以将原代细胞分离m模拟感兴趣的疾病以进一步描绘功能效应。 将网状样形成和破坏测定适应于不同细胞类型有许多重要考虑因素。第一个是播种密度。对于每个新细胞系，关键的第一步是优化基础膜基质中的单次和三次培养测定的接种密度。网格形成依赖于细胞数量，因为细胞密度过高和过低都导致缺乏网格形成。此外，即使对于本文所述的方法和细胞，谨慎地进行细胞密度比较以解释在进行较大规模实验之前的细胞处理中的任何实验室变化。第二个考虑是网格形成的时间序列。应进行时间实验，以确定网状结构如何形成和降解。通常，强壮的网格在〜4 h观察到ike形成。第三个考虑因素是播种之前和实验期间的培养基的选择。 BECs，周细胞和星形胶质细胞在培养基中培养，该培养基含有FBS和针对细胞生长优化的多种生长因子。尽管血清和生长因子优选在实验前24小时使BECs饥饿，但对于某些细胞类型，这可能是不可行的。例如，具有特异性受体敲低的原代细胞可能需要额外的因素来促进生长。这些考虑也适用于实验过程，媒介的选择取决于正在研究的具体问题。然而，当研究外源性加压因子和潜在的治疗作用，特别是生长因子或相关化合物的作用时，在实验过程中使用血清和无生长因子的培养基是理想的。另外，可以减少BEC血清饥饿的长度，但这取决于信号调查途径。第四个考虑因素是增加压力或治疗的时间。对于中度治疗的选择，时间是以科学问题为基础的。网格形成测定更类似于血管发生，而网状破坏测定可能对于成熟BBB更相关。根据我们的经验，一旦建立，测定在实验之间产生一致的数据。一致性问题通常是由于人员之间的种子密度差异，并且重要的是，如果介质或相关试剂的组分不知不觉地被改变。 所述方法的局限性和进一步发展的领域。该方法的强度是低浓度的应激物（ 即 oAβ）可以用于诱导网状物破坏， 即与μM相比，nM更具生理学相关性。然而，这种力量也可能是一个限制。事实上，更高，b仍然无毒，药物筛选目的可能需要oAβ的浓度来增加敏感性，这将适用于其他疾病相关的压力。该方法的局限性在于所述方案的细胞网格仅在24小时内稳定。事实上，24小时后，细胞网格开始退化。因此，我们在网络形成后相对较短的时间（4小时）后添加oAβ，使得总的oAβ孵育时间为18小时。也许较低的细胞接种密度可能会延长治疗方案。另外一个潜在的限制是， 体外系统可能不会像在体内观察到的模拟稳定的细胞网。从老年鼠分离细胞可能更加模拟体内的情况。

<html>大脑毛细血管的血脑屏障（BBB）是血液与脑接触的最大界面，在中枢神经系统（CNS） 1,2的稳态中起着核心作用。 BBB的动态过程阻止从血液中吸收不需要的分子，从CNS中除去废物，向CNS提供必需的营养物质和信号分子，并调节神经炎症1,2,3,4,5 。 BBB损伤在老化过程中是普遍存在的，并且包括阿尔茨海默病（AD），多发性硬化和中风1，2，3，4，5 ，ass ="xref"&gt; 6。因此，BBB功能障碍可能在神经变性疾病中起关键作用，包括作为治疗靶点。 保持血管覆盖对于BBB的稳态功能是重要的。然而， 在体内和体外数据上是否涉及神经退行性疾病的过程引起更高或更低的BBB覆盖度6,7,8,9,10,11,12,13，特别是在AD中。因此，使用相关细胞类型的体外模型的发展有很强的理由来评估和更全面地了解BBB覆盖的动态。脑毛细血管由星形胶质细胞，周细胞和脑内皮细胞（BECs）组成， 14,15,16之后对在滤器插入物上生长的细胞进行渗透性测定，或评估关键BEC蛋白的水平。虽然重要的是，这些检测不集中在脑血管覆盖。 这里，我们以前的方法17是详细的，以评估使用永生化人类BEC的单一培养物，原代小鼠BEC的单一培养物和人源化三重培养物的BEC覆盖和网状结构BBB的模型（BECs，星形胶质细胞和周细胞）。目标是证明对于BEC覆盖，被认为是AD进展的重要因素的oAβ的有害作用。表皮生长因子（EGF）的保护作用突出了该技术作为治疗筛选工具的潜力。该技术对于基础和应用研究具有几个广泛的应用，包括:1）描述特定途径对血管生成和血管覆盖的作用，2）评估疾病和衰老相关因素对血管生成和血管覆盖的影响，以及3）鉴定药理学目标。

<table><tbody><tr><td>hCMEC/D3 cells</td><td>Milipore</td><td>SCC066</td><td></td></tr><tr><td>EBM-2 basal media</td><td>Lonza</td><td>CC-3156</td><td></td></tr><tr><td>Collagen Type 1</td><td>ThermoFisher</td><td>A1064401</td><td></td></tr><tr><td>HBSS, calcium, magnesium, no phenol red</td><td>ThermoFisher</td><td>14025092</td><td></td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red</td><td>ThermoFisher</td><td>14175095</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>ThermoFisher</td><td>25200056</td><td></td></tr><tr><td>Final concentrations of the SingleQuot growth factor supplements for EBM2 media</td><td>Lonza</td><td>CC-4147</td><td></td></tr><tr><td>5% FBS</td><td>Lonza</td><td>CC-4147</td><td></td></tr><tr><td>10% Ascorbic acid</td><td>Lonza</td><td>CC-4147</td><td></td></tr><tr><td>10% Gentamycin sulphate</td><td>Lonza</td><td>CC-4147</td><td></td></tr><tr><td>25% Hydrocortisone</td><td>Lonza</td><td>CC-4147</td><td></td></tr><tr><td>1/4 volume of the supplied growth factors: fibroblast growth factor, epidermal growth factor, insulin-like growth factor, vascular endothelial growth factor</td><td>Lonza</td><td>CC-4147</td><td></td></tr><tr><td>Puromycin hydrochloride</td><td>VWR</td><td>80503-312</td><td></td></tr><tr><td>MEM-HEPES</td><td>Thermo Scientific</td><td>12360-038</td><td></td></tr><tr><td>Papain cell dissociation system (papain and DNase1)</td><td>Worthington Biochemical</td><td>LK003150</td><td></td></tr><tr><td>Human pericytes</td><td>Sciencell</td><td>1200</td><td></td></tr><tr><td>Pericyte basal media</td><td>Sciencell</td><td>1201</td><td></td></tr><tr><td>Pericyte growth supplement</td><td>Sciencell</td><td>1252</td><td></td></tr><tr><td>Human Astrocytes</td><td>Sciencell</td><td>1800</td><td></td></tr><tr><td>Astrocyte media</td><td>Sciencell</td><td>1801</td><td></td></tr><tr><td>Astrocyte growth supplement</td><td>Sciencell</td><td>1852</td><td></td></tr><tr><td>Basement membrane (Matrigel Growth Factor Reduced)</td><td>Corning</td><td>356231</td><td></td></tr><tr><td>Angiogenesis m-plates (96-well)</td><td>ibidi</td><td>89646</td><td></td></tr><tr><td>Human Epidermal growth factor</td><td>Shenendoah Biotechnology</td><td>100-26</td><td></td></tr><tr><td>CellTracker green</td><td>ThermoFisher</td><td>C7025</td><td></td></tr><tr><td>CellTracker orange</td><td>ThermoFisher</td><td>C34551</td><td></td></tr><tr><td>CellTracker blue</td><td>ThermoFisher</td><td>C2110</td><td></td></tr><tr><td>Poly-l-lysine</td><td>Sciencell</td><td>0403</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>Sigma-Aldrich</td><td>HT5012-60ML</td><td></td></tr><tr><td>C57BL mice</td><td>Jackson Laboratory</td><td>na</td><td></td></tr><tr><td>PCR tube strips</td><td>GeneMate</td><td>T-3014-2</td><td></td></tr><tr><td>Zeiss stereo discover v.8 dissecting microscope</td><td>Zeiss</td><td>na</td><td></td></tr></tbody></table>

in vitro assays to assess blood-brain barrier mesh-like vessel formation and disruption

血脑屏障（BBB）覆盖在中枢神经系统（CNS）的体内平衡中起着核心作用。 BBB由星形胶质细胞，周细胞和脑内皮细胞（BEC）动态维持。在这里，我们详细介绍使用永生化人类BEC的单一培养物，原代小鼠BEC的单一培养物和BBB的人源化三联培养模型（BEC，星形胶质细胞和周细胞）评估BBB覆盖率的方法。为了突出测定对疾病状态的适用性，我们描述了低聚淀粉样蛋白β（oAβ）对阿尔茨海默病（AD）进展的重要影响，对BBB覆盖的影响。此外，我们利用表皮生长因子（EGF）来阐明该技术的药物筛选潜力。我们的研究结果表明，单一和三重培养的BEC在基础条件下形成网状结构，并且oAβ破坏了这种细胞网状结构，并使预成型的网状结构退化，但EGF阻止了这种中断。因此，描述的技术对于解剖调节BBB覆盖的基础和疾病相关过程是重要的。

在单个培养物中，hCMEC / D3细胞（ 图3A ）和主要小鼠BEC（ 图3B ）都在整个孔中形成网状结构。结构的特点是互连节点的网格（ 图3 ）。在所有描述的范例（ 图1 ）中，网状结构在对照组中24小时后相似，形成约20个网状结构，总细胞长度约为10,000个像素。 为了突出该方法对疾病相关应激因子的适用性，将oAβ添加到hCMEC / D3和两个范例中的主要小鼠BECs。在细胞网状细胞形成（Paradigm 1）的破坏中，同时对oAβ和细胞进行电镀。在预先形成的网状物（Paradigm 2）的破坏中，加入oAβ在细胞电镀4 h后（ 见图1A ）。在100nM的oAβ诱导预成型网的网状样形成和退化的破坏（ 图3A ， 3B ）。例如，在两个范例中使用hCMEC / D3细胞，使用100nMoAβ17，总细胞覆盖/长度的定量降低16-20％。此外，在两个范例17中， 100nMoAβ，网孔数减少了40％。因此，疾病相关的应激源对人和小鼠BEC覆盖率产生相似的有害影响。 细胞培养系统的一个主要优点是能够识别预防疾病相关损害的因素或治疗，然后进行体内测试。作为原理实验的证明，主要血管生成生长因子对预防oAβ诱导的结果的影响评估船舶覆盖率17 。 EGF可以预防oAβ对hCMEC / D3细胞的损伤。基于这些数据，EGF在预防模式中使用转基因小鼠模型进行了测试，该模型概括了AD样病理学的关键方面。 EGF治疗可防止认知和BBB缺陷，包括血管变性23 。这些数据支持体外系统体内活动的预测潜力。目前，我们利用所有三种开发的范式进行筛选:1）网格形成，2）预防细胞网络破坏，3）同时处理网状物破坏。 如图3所示 ，EGF可以在永生化和初级小鼠BEC培养物中保护免受oAβ诱导的损伤。 BBB由BEC，周细胞和星形胶质细胞组成，共有助于整体脑血管病变ular覆盖。因此， 体外系统的一个改编是纳入所有三种BBB细胞类型。我们的三重培养测定范例结合了hCMEC / D3细胞，原代人周细胞和原代人类星形胶质细胞，它们依次添加到基底膜基质（ 图1B ）。在三重培养测定范式中，hCMEC / D3细胞形成与单个培养物相似的网状模式，但是周边细胞和星形胶质细胞附着于节点和连接分支（ 图4 ）。加入100nM的oAβ会引起网状物破坏（〜10-15％），并减少与BECs 17接触的周细胞数量。与来自单一培养测定范例的数据相关，通过EGF处理可以预防oAβ诱导的损伤。因此，三重培养BBB模型非常适合研究集中在星形胶质细胞（peric）的交互作用ytes和BECs关于船舶动力学。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55846/55846fig1.jpg" /> 图1:网格形成和破坏测定的概述。 （ A ）单一培养测定范例。范式1，网格形成。细胞，应激物和治疗都在0小时的时间点加到板上。范式2，防止网状破裂。在所需处理的存在下接种细胞，培养4小时，并在4小时时间点加入应激物。范式3，同时治疗网状细胞中断。将细胞电镀并允许形成网状结构4小时，然后在4小时时间点同时加入和/或应激物。所有范例在24小时时间点结束，细胞用4％多聚甲醛固定。 （ B ）三重培养测定范式。 BEC（10,000个细胞/孔）所需治疗0小时。在4小时的时间点，将细胞周长（2,000个细胞/孔）轻轻加入板中。在7小时时，将星形胶质细胞（10,000个细胞/孔）加入板中，随后在11小时加入相关的应激物。然后将细胞孵育至24小时时间点，并用4％多聚甲醛固定。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55846/55846fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55846/55846fig2.jpg" /> 图2:定量分析。所有图像均在Fiji ImageJ上打开，并使用血管生成分析仪22进行批处理。使用总长度和网格数的量化。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55846/55846fig2large.jpg" target="_blank">请点击这里查看更大的版本</a>他的身影<img alt="图3" class="xfigimg" src="/files/ftp_upload/55846/55846fig3.jpg" /> 图3:网纹破坏测定:代表性图像。所有图像均来源于使用范例2，网格中断的实验。 （ A ）用媒介物对照（VC），EGF（100nM），oAβ（100nM）或oAβ（100nM）+ EGF（100nM）铺板并处理的hCMEC / D3细胞的代表性图像。 10倍放大倍数的图像，比例尺= 100μm。 （ B ）用VC，EGF（100nM），oAβ（100nM）或oAβ（100nM）+ EGF（100nM）接种并处理的小鼠BEC的代表性图像。 10倍放大倍数的图像，绿色= BECs，刻度棒=100μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55846/55846fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 图4.三重培养测定:代表性图像。 （ A ）用VC，EGF（100nM），oAβ（100nM）或oAβ（100nM）+ EGF（100nM）接种并治疗的hCMEC / D3细胞，原代人周细胞和原代人星形胶质细胞的代表性图像，根据图1B所述的范例。 10倍放大倍率的图像，绿色= BECs，蓝色=周细胞和红色（假色）=星形胶质细胞。刻度棒=100μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55846/55846fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption at JoVE.com

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

维持血脑屏障覆盖是中枢神经系统稳态的关键。该方案描述体外技术来描绘调节血脑屏障覆盖的基础和病理过程。

评估血脑屏障网状血管形成和破坏的体外测定

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

in-vitro-assays-to-assess-blood-brain-barrier-mesh-like-vessel

Nanyang Technological University

Research

JoVE Journal

Medicine

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

8.4K 次观看.  University of Illinois at Chicago. 维持血脑屏障覆盖是中枢神经系统稳态的关键。该方案描述体外技术来描绘调节血脑屏障覆盖的基础和病理过程。 

视频: In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of brain tumors, the molecular selectivity of BBB also shields the neoplastic cells by blocking the delivery of peripherally administered chemotherapies. The development of novel drugs (including nanoparticles) targeting malignant brain tumors ideally requires the use of preclinical animal models to study the drug&#8217;s transcytosis and antitumor efficacy. In order to comply with the 3R principle (refine, reduce, and replace) to reduce the number of laboratory animals in experimental setup and perform the high-throughput screening of a large library of antitumor agents, we developed a reproducible in vitro human and murine mimic of the blood-brain tumor-barrier (BBTB) using three-layered cultures of endothelial cells, astrocytes, and patient-derived glioblastoma spheres. For higher scalability and reproducibility, commercial cell lines or immortalized cells have been used in tailored conditions to allow the formation of a barrier resembling the actual BBB. Here we describe a protocol to obtain a BBTB mimic by culturing endothelial cells in contact with astrocytes at specific cell densities on inserts. This BBTB mimic can be used, for instance, for the quantification and confocal imaging of the nanoparticle passage through the endothelial and astrocytic barriers, in addition to the evaluation of the tumor cell targeting within the same assay. Moreover, we show that the obtained data can be used to predict the behavior of nanoparticles in preclinical animal models. In a broader perspective, this in vitro model could be adapted to other neurodegenerative diseases for the determination of the passage of new therapeutic molecules through the BBB and/or be supplemented with brain organoids to directly evaluate the efficacy of drugs.

Drug targeting to central nervous system tumors is a major challenge. Here we describe a protocol to produce an in vitro mimic of the blood-brain tumor-barrier using murine and/or human cells and discuss their relevance for the predictability of central nervous system tumor targeting in vivo.

利用血脑肿瘤屏障在盘中进行活体有效载荷传递预测

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of ...

科研

癌症研究

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 ...

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

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

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

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 ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

神经科学

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

源自人类多能干细胞的 3D 血脑屏障模型

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

作者聚焦:使用体外血脑屏障模型研究阿尔茨海默病的个性化方法 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

体外血脑屏障重建，模拟和治疗神经系统疾病

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...

评估血脑屏障网状血管形成和破坏的体外测定

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利用血脑肿瘤屏障在盘中进行活体有效载荷传递预测

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

使用的淋巴细胞外渗分析

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

体外血脑屏障重建，模拟和治疗神经系统疾病

体外血脑屏障重建，模拟和治疗神经系统疾病

体外血脑屏障重建，模拟和治疗神经系统疾病