Name: modified in vivo matrix gel plug assay for angiogenesis studies
Uploaded: 2023-06-30T14:15:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies at JoVE.com

这项工作由浙江省自然科学基金（LY22H020005）和中国国家自然科学基金（81873466）资助。

所有涉及动物受试者的程序均已获得温州医科大学机构动物护理和使用委员会（IACUC）的批准（XMSQ2021-0057,2021 年 7 月 19日）。所有试剂和耗材都列在 材料表中。
1.培养基制备
<ol><li>10x M199 培养基：将 M199 粉末用 90 mL 去离子水溶解至 10 倍浓度，加入 10 mL 胎牛血清 （FBS），然后通过 0.22 μm 过滤器。将培养基储存在4°C长达2个月。</li>
	<li>完全内皮培养基：向 460 mL 内皮细胞培养基 （ECM） 中加入 50 mL FBS、5 mL 青霉素/链霉素和 5 mL 内皮细胞生长补充剂。将培养基储存在4°C长达1个月。</li>
</ol>2. 血管细胞制备
<ol><li>在37°C和5%CO2至70%汇合度的100mm组织培养皿中，用8mL完全内皮培养基培养1×105个血管细胞（原代培养的内皮祖细胞14，内皮细胞或内皮细胞系）。</li>
	<li>取出培养基，用 1x 磷酸盐缓冲盐水 （PBS） 冲洗培养皿 2 次，以去除未附着的细胞和碎屑。去除PBS并加入3mL含有2.21mM EDTA的0.25%胰蛋白酶，并在37°C孵育1分钟。</li>
	<li>用 7 mL 完全内皮培养基中和胰蛋白酶，然后轻轻冲洗培养皿中的细胞。在光学显微镜（40倍放大倍率）下确认细胞分离。</li>
	<li>将细胞悬液收集在 15 mL 管中，并以 400 x g 离心 10 分钟。除去上清液并用 5 mL 完全内皮培养基重悬细胞。</li>
	<li>使用血细胞计数器计数细胞，并将含有 2 x 106 个细胞的悬浮液移至无菌 1.5 mL 管中。每个插头包含 1.5 x 106 个电池，另外 25% 的电池用于浪费。每组至少包括 3 个插头。 注意：经过试验，发现 300 μL 基质凝胶中的 1.5 x 106 个细胞导致血管生成的正常发育，因此选择进行实验。</li>
	<li>将细胞悬浮液以400× g 离心5分钟以沉淀细胞，然后除去上清液。</li>
</ol>3.基质凝胶制备
<ol><li>在4°C水浴中预冷含有细胞沉淀的无菌1.5mL管。在4°C冰箱中预冷30G 1mL胰岛素注射器。</li>
	<li>在4°C水浴中完全解冻基质凝胶。不要混合或涡旋。在4°C水浴中预冷10x M199和测试试剂/目标药物。</li>
	<li>将预冷的基质凝胶与含有10%FBS的10x M199和待测试剂混合，体积比为8.8：1：0.2，得到含有1%FBS的基质凝胶和M199混合物和试剂。</li>
	<li>用 400 μL 基质凝胶混合物重悬细胞，轻轻混合以避免形成气泡。将试管放在冰上，直到小鼠准备注射。将基质凝胶混合物始终放在冰上以避免凝固。 注意：在这里，使用含有 1.5 x 106 个细胞的 300 μL 基质凝胶混合物形成凝胶塞。根据步骤2中25%的额外细胞制备25%的额外凝胶。</li>
</ol>4.小鼠准备
<ol><li>用异氟醚（3%异氟醚在100%氧气中以1 L / min的流速）麻醉6-8周龄的雄性Nu / Nu小鼠（18-25g）。麻醉成功后，通过没有矫正反射和脚趾捏反射确认，将小鼠移出腔室，将小鼠移出腔室并在小鼠上放置麻醉面罩，并将异氟烷的浓度更改为1.5%在100%氧气中以1L / min的流速。 注意：在整个过程中使用加热垫为动物提供热支持。</li>
	<li>在眼睛上使用兽医软膏以防止干燥。将小鼠的四肢以俯卧位粘在操作板上。</li>
</ol>5.基质凝胶混合物注射
<ol><li>用 30G 针头将 300 μL 基质凝胶混合物加载到 1 mL 胰岛素注射器中。避免气泡形成。快速加载基质凝胶（2分钟内），以避免在此期间凝固。立即将装有基质凝胶的胰岛素注射器放在冰上。</li>
	<li>使用75%的酒精棉片清洁小鼠背部的皮肤。皮下注射300μL基质凝胶混合物到小鼠背部的一侧。</li>
	<li>轻轻地从注射部位取下针头，以防止基质凝胶混合物泄漏。检查注射部位是否有小驼峰（图1）。</li>
	<li>将鼠标放在加热垫上2分钟，让基质凝胶混合物凝结并形成塞子。</li>
	<li>重复步骤 5.2。至 5.4.在鼠标背面的另一侧创建插头。用记号笔标记驼峰的边缘。</li>
	<li>观察小鼠，直到它恢复足够的意识以保持胸骨卧位。将鼠标放在单独的笼子中，直到它完全恢复。将小鼠饲养在22±2°C的SPF级实验动物实验室中，光/暗循环12小时，持续7天。</li>
</ol>6.通过尾静脉注射葡聚糖-FITC
<ol><li>基质凝胶注射7天后，将0.5mg葡聚糖-FITC与500μL双蒸水（ddH2O）重悬，然后剧烈涡旋，得到终浓度为1μg/μL的葡聚糖-FITC溶液。 注意：使用分子量为~150 kDa的葡聚糖-FITC进行血管生成测定，并使用分子量为~4 kDa的葡聚糖-FITC进行血管通透性测定。</li>
	<li>将 50 μL 葡聚糖-FITC 溶液加载到 29G 注射器中。</li>
	<li>将鼠标固定在尾静脉注射仪器上。用75%酒精棉球清洁尾部，并通过尾静脉轻轻注射50μL葡聚糖-FITC。</li>
	<li>用棉签按压注射部位1分钟以止血，然后将小鼠放回笼子中30分钟。</li>
	<li>重复步骤 6.3。和 6.4.直到所有小鼠都接受葡聚糖-FITC注射。</li>
</ol>7. 基质凝胶塞收集
<ol><li>腹膜内注射 1% 戊巴比妥钠在 PBS（200 mg/kg 体重）中，并使用 IACUC 批准的程序对小鼠实施安乐死。</li>
	<li>使用手术剪刀沿栓子的标记边缘切割皮肤，然后去除基质凝胶塞上方的皮肤。</li>
	<li>收集塞子并在装有1x PBS的小烧杯中冲洗以洗去多余的血液（图2A）。栓子的颜色可以大致描绘出血液丰度和新血管的内容（图3A）。</li>
</ol>8. 嵌入基质凝胶塞和切片制备
<ol><li>用约0.5 mL的组织包埋凝胶覆盖组织包埋盒的按钮，然后立即将基质凝胶塞以所需方向放置在组织包埋凝胶上，如 图2B所示。然后，用额外的组织包埋凝胶包埋塞子。</li>
	<li>将盒放入-80°C冰箱中12小时凝固。</li>
	<li>对于厚切片制备，取出凝固的塞块，按照说明使用冷冻切片机切割12μm厚的切片，然后安装在显微镜载玻片上。</li>
	<li>从每个插头块上切下 5-10 个部分。</li>
</ol>9.血管生成的量化（图3）
<ol><li>在10倍放大倍率下，使用488nm波长的倒置荧光显微镜从切片的5个独立场获取荧光图像。</li>
	<li>使用 Image J 软件 （https://imagej.en.softonic.com/） 打开图像文件，然后单击 “血管生成分析 ”按钮。然后，单击 “分析 HUVEC 相差 ”按钮，并切换到 “统计结果表” 以收集信息。测量每个切片的所有 5 张采集的荧光图像。</li>
	<li>分析不同组新血管的总长度，以统计评估这些组之间血管生成的差异。 注：总主段长度表示新船的总长度。增加新血管长度的试剂称为促血管生成剂，而减少新血管长度的试剂称为抗血管生成。</li>
</ol>10.血管通透性的量化（图4）
<ol><li>在20倍放大倍率下，使用488nm波长的倒置荧光显微镜从切片的5个独立场获取荧光图像。</li>
	<li>使用 Image J 软件打开图像文件。单击 “手绘选择 ”按钮并圈出泄漏区域，然后单击 “分析 ”菜单并选择“ 测量 ”选项以获取泄漏区域的区域信息。使用渗漏面积和图像面积的比率来指示血管通透性。</li>
	<li>分析不同组的渗漏面积和图像面积的比率，以统计评估这些组之间血管通透性的差异。</li>
</ol>

使用改良基质凝胶塞测定简化血管生成研究

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作者声明没有利益冲突。

我们提出了一种可靠且方便的方法，用于在不染色 的情况下定量评估体内 血管生成。在该方案中，在促血管生成或抗血管生成试剂存在下将血管细胞与基质凝胶混合，然后皮下注射到Nu / Nu小鼠的背部以形成凝胶塞（图1）。凝胶栓形成7天后，静脉注射葡聚糖-FITC循环30分钟。收集凝胶塞并用组织包埋凝胶包埋，并切片 12 μm 用于摄影（图 2）。葡聚糖-FITC无需染色即可指示功能性血管，新血管的长度可用于定量评价试剂的促血管生成或抗血管生成功能。在该方案中介绍的示例中，棕榈酸酯处理减少了新血管的长度，而FGF1增加了新血管的长度（图3），这表明棕榈酸酯具有抗血管生成作用，而FGF1具有促血管生成作用。除此之外，该协议还可用于评估血管通透性（图4）。
在选择受体小鼠，处理和注射基质凝胶，收集凝胶塞以及检测血管长度时应谨慎。推荐使用6-8周龄的免疫缺陷小鼠，尽管C57BL / 6小鼠也有效。考虑到插头的可变性，建议每组 3-5 个插头。不同组之间的血管细胞数量应均匀。基质凝胶应按照说明小心处理，如果凝固或含有颗粒物或大量气泡，则不应使用。在基质凝胶制备步骤中，基质凝胶的最终浓度不应低于80%，并且不允许涡流，因为可能会形成气泡。需要注意的是，凝胶塞的形状可能会影响结果的重现性，因此采用加热垫来加速基质凝胶塞的凝固。也可以使用37°C的动物培养箱。此外，在凝胶塞包埋和切片制备过程中，应保护塞子和切片免受强光照射，并应尽快在荧光显微镜下拍照。
在各种 体内 血管生成测定中，基质凝胶塞测定是使用最广泛的方法之一，因为它用途广泛、成本较低且易于执行11。然而，普通基质凝胶塞测定中的染色过程容易出错。插头易碎且含水量高。染色过程中包含的脱水和固定步骤可能会使凝胶塞变形，并进一步影响对血管长度的评估。除此之外，在普通的基质凝胶塞测定中，可以对塞子中分散的血管细胞进行染色并被识别为功能性血管。这种改良的基质凝胶塞测定法最重要的优点之一是可以在不染色的情况下可视化功能血管。FITC标记的葡聚糖用于指示血管，并且只能指示具有血流的功能性血管，这有助于该测定的便利性和可靠性。
除了新血管的长度外，渗透性还影响新血管的功能20。低分子量（~4,400 Da）的荧光标记葡聚糖可以穿过血管中的间隙，可用于指示新血管的渗透性。如果需要，研究人员可以通过使用低分子量和高分子量的荧光标记葡聚糖来评估同一凝胶塞中的血管生成和血管通透性21。
血管生成可受 体内 环境22的影响，如糖尿病13、14、19等。这种 体内 环境很难在这些 体外 模型中重现。然而，在这种修改后的方案中，通过选择合适的受体小鼠可以很容易地复制 体内 环境。除此之外，不同血管细胞之间的相互作用也会影响血管生成的过程。不同种类的血管细胞，包括内皮细胞，平滑肌细胞和周细胞，可以在该协议的插件中加入以形成新血管，以研究不同血管细胞在血管生成中的作用。
但是，这种方法也有一些局限性。新血管的形成会受到凝胶塞形状的影响。此外，凝胶塞中形成的新血管不均匀。凝胶塞边缘的功能性血管比中心的功能性血管多（图2A）。因此，结果可能会受到表演者熟练程度的影响。

血管生成是从预先存在的血管形成新血管的过程，在许多生理和病理过程中起着至关重要的作用，例如胚胎发育、伤口愈合、动脉粥样硬化、肿瘤发展等1,2,3,4,5。这种动态过程涉及几个步骤，包括基质的降解、血管细胞增殖、迁移和自组织以形成管状结构以及新血管的稳定 6。促进血管生成已被证明在治疗心肌梗塞、中风和其他类型的缺血性疾病中至关重要7，而抑制血管生成被认为是治疗癌症8和类风湿性疾病的有前途的策略9。血管生成被认为是药物发现的组织原则10.因此，构建一种可靠且方便的方法来评估血管生成的程度对于血管生成依赖性疾病的机械研究或药物发现至关重要。
已经开发了几种体外和体内模型来评估血管生成11。其中，二维（2-D）模型，如基质凝胶管形成测定12，不能形成功能性管状结构。动物模型，如后肢缺血模型13,14，可以再现血管生成过程，但很复杂，需要激光散斑血流成像系统。血管形态发生的 3D 模型，如基质凝胶塞测定，提供了一个简单的平台，可以模拟体内血管生成的过程 15，但血管生成的检测需要免疫组化或免疫荧光染色 16,17,18，这些方法可变且可视化不佳。
在这里，我们描述了一种改良的基质凝胶塞测定方案，其中将血管细胞与基质凝胶混合并皮下注射到小鼠背部以形成塞子。在塞子中，血管细胞需要降解基质、增殖、迁移和自组织，最终形成内部环境中有血流的功能性血管。此后，通过尾静脉注射荧光标记的葡聚糖，流经塞子，并将标记可视化以指示新血管。血管生成的内容可以通过血管的长度来定量评估。该方法可以形成在二维血管生成模型12中无法产生的功能性血管，并且不需要像普通基质凝胶塞测定11那样复杂的染色过程。它也不需要昂贵的特定仪器，如后肢缺血模型13、14、19 中的激光散斑血流成像系统。该方法用途广泛、成本低、可量化且易于执行，可用于确定药物的促血管生成或抗血管生成能力，或用于涉及血管生成的机械研究。

<table><tbody><tr><td>Adhesion Microscope Slides</td><td>CITOTEST</td><td>188105</td><td></td></tr><tr><td>Anesthesia System</td><td>RWD</td><td>R640-S1</td><td></td></tr><tr><td>Cell Counter</td><td>Invitrogen</td><td>AMQAX1000</td><td></td></tr><tr><td>Cell Culture Dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>Cryoslicer</td><td>Thermo Fisher</td><td>CryoStar NX50</td><td></td></tr><tr><td>Dextrans-FITC-150kDa</td><td>WEIHUA BIO</td><td>WH007N07</td><td></td></tr><tr><td>Dextrans-FITC-4kDa</td><td>WEIHUA BIO</td><td>WH007N0705</td><td></td></tr><tr><td>Embedding Cassettes</td><td>CITOTEST</td><td>80203-0007</td><td></td></tr><tr><td>Endothelial Cell Medium</td><td>ScienCell</td><td>35809</td><td></td></tr><tr><td>Endothelial Growth Supplements</td><td>ScienCell</td><td>1025</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10100147C</td><td></td></tr><tr><td>Fibroblast Growth Factor 1</td><td>AtaGenix</td><td>9043p-082318-A01</td><td>FGF1</td></tr><tr><td>Fluorescence Microscope</td><td>Nikon</td><td>ECLIPSE Ni</td><td></td></tr><tr><td>Heating Pad</td><td>Boruida</td><td>30-50-30</td><td></td></tr><tr><td>Insulin Syringe</td><td>BD</td><td>300841</td><td></td></tr><tr><td>Isoflurane</td><td>RWD</td><td>R510-22-10</td><td></td></tr><tr><td>Laboratory&amp;nbsp;Balance</td><td>Sartorius</td><td>BSA124S-CW</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>356234</td><td>Matrix gel</td></tr><tr><td>Medium 199 powder</td><td>Gibco</td><td>31100-035</td><td></td></tr><tr><td>Microtubes</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>Optimal Cutting Temperature (OCT) Compound</td><td>SUKURA</td><td>4583</td><td>Tissue embedding gel</td></tr><tr><td>Palmitate Acid</td><td>KunChuang</td><td>KC001</td><td></td></tr><tr><td>Penicillin-Streptomycin Liquid</td><td>Solarbio</td><td>P1400</td><td></td></tr><tr><td>Phosphate Buffer Saline</td><td>Solarbio</td><td>P1022</td><td></td></tr><tr><td>Surgical Instruments</td><td>RWD</td><td>RWD</td><td></td></tr><tr><td>Tail Vein Injection Instrument</td><td>KEW BASIS</td><td>KW-XXY</td><td></td></tr><tr><td>Trypsin-EDTA Solution</td><td>Solarbio</td><td>T1320</td><td></td></tr><tr><td>Ultra-Low Temperature Freezer</td><td>eppendorf</td><td>U410</td><td></td></tr><tr><td>Vascular Endothelial Growth Factor</td><td>CHAMOT</td><td>CM058-5HP</td><td>VEGF</td></tr></tbody></table>

modified in vivo matrix gel plug assay for angiogenesis studies

已经开发了几种模型来研究 体内血管生成。然而，这些模型大多复杂且昂贵，需要专门的设备，或者难以进行后续定量分析。在这里，我们提出了一种改良的基质凝胶塞测定法来评估 体内血管生成。在该方案中，在存在或不存在促血管生成或抗血管生成试剂的情况下，将血管细胞与基质凝胶混合，然后皮下注射到受体小鼠的背部。7天后，通过尾静脉注射含有葡聚糖-FITC的磷酸盐缓冲盐水并在血管中循环30分钟。收集基质凝胶塞并用组织包埋凝胶包埋，然后切割 12 μm 切片用于荧光检测，无需染色。在该测定中，高分子量（~150,000 Da）的葡聚糖-FITC可用于指示功能性血管以检测其长度，而低分子量（~4,400 Da）的葡聚糖-FITC可用于指示新血管的渗透性。总之，该方案可为 体内血管生成的定量研究提供一种可靠、方便的方法。

图1 是描述如何制备基质凝胶、血管细胞、培养基和试剂混合物的流程图。然后将混合物皮下注射到Nu / Nu小鼠的背部，并使用加热垫加热以加速其凝固，最终形成凝胶塞。
图2A 是指示荧光标记葡聚糖的血管的流程图。通过尾静脉和圆圈注射荧光标记的葡聚糖30分钟，使其可以进入凝胶塞中的功能血管。此后，收集凝胶栓，使用组织包埋凝胶包埋（包埋时基质凝胶的方向如 图2B所示）。从栓子上切下 12 μm 厚的切片（每侧 5 片），并拍摄荧光照片以定量分析血管生成和/或血管通透性。
图3 是抗血管生成试剂棕榈酸酯和促血管生成试剂成纤维细胞生长因子1（FGF1）对凝胶塞中血管生成的影响。 图3A 是带有载体、棕榈酸酯或FGF1的凝胶栓的外观。血液可以进入凝胶塞中的功能性新血管，使塞子不同程度地变红。 图3B 是基质凝胶塞的荧光图像，其中功能血管通过高分子量的葡聚糖-FITC可视化。 图3C 是不同组血管长度的定量结果。棕榈酸酯可以显著缩短新血管的长度，而FGF1处理可以明显增加新血管的长度。
图 4 比较了使用或不使用血管内皮生长因子 （VEGF） 治疗的凝胶塞的血管通透性。 图4A 是基质凝胶塞的荧光图像，其中泄漏区域由低分子量的葡聚糖-FITC可视化，并用箭头表示。 图4B 是泄漏面积的定量结果。VEGF治疗组渗漏面积的增加表明VEGF可以增加血管通透性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65567/65567fig01.jpg"> 图 1.显示小鼠凝胶塞形成的流程图。 将单层培养物中培养的维管细胞解离，沉淀，用内皮细胞培养基（ECM）重悬并计数（I-IV）。在基质凝胶混合物（含有8.8体积的基质凝胶，1体积的10x M199补充有10%FBS和0.2体积试剂）中沉淀和重悬后，将300μL凝胶混合物皮下注射到小鼠的背部（V-VII）以形成塞子。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65567/65567fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65567/65567fig02.jpg"> 图2.显示葡聚糖-FITC注射和血管生成定量的流程图。 （A） 通过尾静脉 （I） 注射葡聚糖-FITC。30分钟后，获取凝胶塞，包埋并使用冷冻切片机（II，III）切片。此后，使用荧光显微镜 （IV） 拍摄荧光图像，并使用 Image J 软件 （V） 定量分析血管生成。（B） 嵌入组织包埋盒中的基质凝胶塞取向图。缩写：OCT = 最佳切削温度化合物。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65567/65567fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65567/65567fig03.jpg"> 图3.使用改良的基质凝胶塞测定法评估血管生成。 （A） 具有代表性的基质凝胶塞的外观。（B）葡聚糖-FITC指示的基质凝胶栓中功能血管的荧光图片。（C） 使用单因素方差分析对基质凝胶塞中功能性血管长度进行定量分析。*p<0.05 与车辆相比;P<0.001 与车辆。比例尺为 100 μm。缩写：FGF1 = 成纤维细胞生长因子 1。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65567/65567fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65567/65567fig04.jpg"> 图4.使用改良的基质凝胶塞测定法评估血管通透性。 （A）凝胶塞中血管的代表性荧光图像，箭头表示泄漏部位。（B） 使用学生 t 检验对渗漏区域进行定量分析以评估血管通透性。第 <0.001 页。比例尺为 100 μm。缩写：VEGF=血管内皮生长因子。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65567/65567fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies at JoVE.com

作者聚焦：通过改良基质凝胶塞测定研究血管生成和血管通透性

这里介绍的方法可以在不染色的情况下评估 试剂对体内 血管生成或血管通透性的影响。该方法使用右旋糖酐-FITC通过尾静脉注射来观察新血管或血管渗漏。

用于血管生成研究 的改良体内 基质凝胶塞测定

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...

我们的研究涉及血管生成，即从现有血管形成新血管的过程。它在许多生理和病理过程中起着关键作用，如胚胎发育或治疗动脉粥样硬化、肿瘤发展等。构建一个可靠的模型来评估血管生成的程度对于心肌研究或发现治疗血管生成依赖性疾病的药物至关重要。然而，大多数现有模型都复杂、昂贵，并且需要专门的设备。在这里，我们提出了一种改良的基质凝胶塞测定法，可用于以简单、更直观和有效的方式研究血管生成和血管通透性，而无需染色。

modified-in-vivo-matrix-gel-plug-assay-for-angiogenesis-studies

Nanyang Technological University

Research

JoVE Journal

Biology

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

836 次观看.  Pingyang Affiliated Hospital of Wenzhou Medical University. 我们的研究涉及血管生成，即从现有血管形成新血管的过程。它在许多生理和病理过程中起着关键作用，如胚胎发育或治疗动脉粥样硬化、肿瘤发展等。构建一个可靠的模型来评估血管生成的程度对于心肌研究或发现治疗血管生成依赖性疾病的药物至关重要。然而，大多数现有模型都复杂、昂贵，并且需要专门的设备。在这里，我们提出了一种改良的基质凝胶塞测定法?...

视频: 作者聚焦：通过改良基质凝胶塞测定研究血管生成和血管通透性

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

克里斯托弗休斯：在体外血管生成的研究模型（面试）

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

科研

生物学

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

优化的纤维蛋白凝胶珠检测血管生成的研究

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

角膜微囊含量：血管生成在小鼠眼模型

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

神经科学

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

生物工程

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

战略内皮细胞形成实验:比较外基质和生长因子，细胞外基质

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

医学

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

大鼠肠系膜血管生成法

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

标准化和可扩展的测定，以研究iPSC衍生内皮细胞在Vitro的渗透3D血管生成发芽

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

监控功能和血管形态由快速增长的肿瘤产生细胞分泌的因素招募

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

用于血管生成研究的改良体内基质凝胶塞测定

副本

摘要

探索更多视频

此视频中的章节

克里斯托弗休斯：在体外血管生成的研究模型（面试）

优化的纤维蛋白凝胶珠检测血管生成的研究

角膜微囊含量：血管生成在小鼠眼模型

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

战略内皮细胞形成实验:比较外基质和生长因子，细胞外基质

大鼠肠系膜血管生成法

标准化和可扩展的测定，以研究iPSC衍生内皮细胞在Vitro的渗透3D血管生成发芽

内皮细胞小管形成实验的

监控功能和血管形态由快速增长的肿瘤产生细胞分泌的因素招募

在人类内皮周细胞的相互作用使用基质凝胶插件检测小鼠的体内研究

用于血管生成研究 的改良体内 基质凝胶塞测定

副本

摘要

探索更多视频

此视频中的章节

克里斯托弗休斯：在体外血管生成的研究模型（面试）

优化的纤维蛋白凝胶珠检测血管生成的研究

角膜微囊含量：血管生成在小鼠眼模型

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

战略内皮细胞形成实验:比较外基质和生长因子，细胞外基质

大鼠肠系膜血管生成法

标准化和可扩展的测定，以研究iPSC衍生内皮细胞在Vitro的渗透3D血管生成发芽

内皮细胞小管形成实验的

监控功能和血管形态由快速增长的肿瘤产生细胞分泌的因素招募

在人类内皮周细胞的相互作用使用基质凝胶插件检测小鼠的体内研究

用于血管生成研究的改良体内基质凝胶塞测定