这项研究由美国国家癌症研究所在美国国立卫生研究院院内研究计划的一部分得到了支持，并通过新华保险给予UA5CA152907。

1，收集条件培养基来测试潜在的血管生成<ol><li>成长伯或永生化细胞可以用于血管生成或抗血管生成潜力的天然的或低血清培养基中进行测试，并收集条件培养基。 <ol><li>或者，使用立即条件培养基，或等分并储存在-80℃下几个月。使用nonconditioned天然或低血清培养基作为阴性对照，并使用非条件完全生长培养基（10％FBS，或适当浓度）作为阳性对照。可替代地，测试血管生成的潜在刺激剂，补充剂木炭剥离介质缺乏生长因子与已知浓度的感兴趣的特定蛋白质，并比较单独的生长因子的培养基。 </li></ol></li></ol> 2，准备内皮细胞在测定前的<ol><li>两天来测定之前，通过3B-11细胞变成一个新的T-75瓶。细胞的传代次数是重要。该测定时效果最好的细胞是在第二和第六通道之间。该测定将无法正常工作，如果之前的第二或10次传代后的内皮细胞被使用。 </li><li>生长细胞24小时，在补充有10％FBS，2mM的L-谷氨酰胺，1mM丙酮酸钠，100单位/ ml青霉素和100微克/毫升链霉素。 </li><li>前一天测定，血清饥饿3B-11细胞中，如下所示： <ol><li>吸媒体从3B-11细胞。 </li><li> DMEM中添加降低血清培养基补充有0.2％FBS，2mM的L-谷氨酰胺，1mM的丙酮酸钠，100单位/ ml青霉素和100微克/毫升链霉素。 </li><li>生长细胞另外24小时。 </li></ol></li></ol> 3，试剂的配制在测定前注：BME浓度变化很大取决于很多。 BME不能很好地工作，如果浓度小于10毫克/毫升，所以BME制造商应购买，以确保现有接触收购一个足够浓很多。 <ol><li>除霜低生长因子BME在冰箱中。 BME温暖，当凝固，所以它必须保持低温才能使用。 BME能在冷藏条件下可保存数周，或分装冷冻在-80℃。 </li><li>减小的生长因子BME优于传统的生物医学工程。血管生成因子在BME缺乏会更容易地看到，因子从条件培养基对管形成的影响。 </li><li>要测试的解冻介质。 </li></ol> 4，准备内皮细胞前立即测定的<ol><li>洗3B-11细胞的DPBS。 <ol><li>如果可视荧光或共聚焦显微镜是期望的，洗涤3B-11细胞之前，钙黄绿素AM增加内皮细胞中的介质，以2微克/毫升的最终浓度。放置钙黄绿素标记的细胞在黑暗中，在37℃和5％CO 2之前测定的开始。溶解钙黄绿素的股票AM在DMSO中，并一次钙黄绿素稀释在DMSO中的媒体最终浓度应不超过0.1％。后从细胞30-45分钟洗净钙黄绿素AM（步骤4.1以上），并继续与实验如下。 </li></ol></li><li>加入1ml胰蛋白酶-EDTA至T-75烧瓶中Trypsinize细胞。 </li><li>胰蛋白酶消化完成后，中和胰蛋白酶通过再悬浮细胞至10ml的终体积在DMEM，10％FBS，2mM的L-谷氨酰胺，1mM的丙酮酸钠，100单位/ ml青霉素和100微克/毫升链霉素。 </li><li>通过100微米的细胞过滤过滤单元以去除团块。 </li><li>计数细胞，重悬细胞，以7.5×10 6个细胞/在DMEM毫升，10％FBS，2mM的L-谷氨酰胺，1mM丙酮酸钠，100单位/ ml青霉素的终浓度，和100微克/毫升链霉素。 </li></ol> 5，小管形成实验<ol><li>放置在冰箱或在冰上20-30分钟24孔板和1ml提示加载板，使得BME不PREM前aturely凝固。 </li><li>移液管加入250μl低生长因子BME成24孔板的每个孔中。避免未踩下吸管的最后一站，而配药产生气泡。注意使足够BME保持在中心孔的弯月面的形式时;如果矩阵是太薄，则细胞将只形成单层。 </li><li>孵育24孔板，在37℃和5％CO 2的30分钟以固化BME。 </li><li>一旦BME已成立，混合约300微升条件培养基用10微升悬浮3B-11细胞（约75,000细胞）。 <ol><li>调整的条件培养基和细胞取决于所用的内皮细胞类型镀的数量，并测试该条件培养基中的连续稀释液以证明活性。如果在不同条件下生长的比较单元，规范化条件培养基体积对细胞进行用于调节所述介质的数量。 </li></ol></li><li>管西港岛线升开始形成在2-4小时。根据在条件培养基中的血管生成因子的浓度，峰值管形成，可能会出现小时，并在测定的时间在3和12将需要被优化。如果用原代内皮细胞，而不是永生化细胞的工作，初始管形成可以通过几个小时延迟。 <ol><li>管经常会开始恶化内18小时，内皮细胞会进行细胞凋亡。 </li></ol></li><li>在高峰管形成时，从阱小心吸出介质，以避免破坏在BME管网络。 <ol><li>每1毫升的DPBS，然后吸出好洗。 <ol><li>立即可视化，使用连接到一台数码相机，或荧光/共聚焦显微镜，如果细胞用钙黄绿素AM如4.1.1倒置显微镜加入1 ml新鲜的DPBS到每个孔中，图像中的管网。 </li><li>要解决管网可视化后，加入4％多聚甲醛为每吸出，并培育15分钟，在室温下进行。 <ol><li>除去多聚甲醛固定，用DPBS冲洗两次，然后加入1ml新鲜的DPBS到每个孔中。 </li><li>显微镜下的图像之前固定的几个管会在此过程中断裂。 </li></ol></li></ol></li></ol></li></ol> 6，定量管网<ol><li>量化的管网中的几个不同的方式根据研究者的偏好，和几个计算机程序有能力来测量以下参数：管子的数目;中环/目数;的分支站点/节点数目;长管。 </li><li>使用有代表性的计算机程序，例如与血管生成分析器插件16，用于管网的定量的Image J。 </li></ol>

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	<li>Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+life,+disease+and+medicine.">Angiogenesis in life, disease and medicine.</a> Nature. 438, 932-936 (1038).</li><li>Carmeliet, P., Jain, R. 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作者什么都没有透露。

血管生成是涉及在生理和病理过程。研究参与血管生成的机制要求使用概括在血管生​​成中的重要步骤测定的。内皮细胞管形成测定提供了几个优点优于其它试验。很容易设置，是比较廉价，生产小时内小管，并且是可量化的。此外，它可以在24位或96孔板内完成，因此可用于高通量筛选，以确定该刺激因子或抑制血管生成。虽然我们用3B-11小鼠内皮细胞为我们的实验中，测定可与各种人或鼠内?...

血管生成，新血管从先前存在的血管的发展，是各种过程，包括器官发育，胚胎发育和伤口愈合1-3至关重要。新开发的血管，内皮细胞，供应氧气和营养物质，内衬以组织，促进造血细胞的免疫监视和清除废物的2,4。血管生成是至关重要的胚胎和胎儿发育过程中。然而，这种方法保持休眠期间除外伤口愈合，骨生长，怀孕次数或月经周期1-3期间在成体中。 在过去的二十年中，调节血管生成的关键分子机制已经开始出现。血管新生是一个严格监管的情况下，通过亲和抗血管生成信号，包括整合，趋化因子，血管生成素，氧传感代理商，交界性分子和内源性抑制剂5平衡。一旦proangiogenic信号，如碱性成纤维细胞生长因子（bFGF），血管内皮生长因子（VEGF），血小板衍生生长因子（PDGF），和表皮生长因子（EGF），激活内皮细胞受体的内皮细胞释放的蛋白酶降解基底膜。内皮细胞，然后增殖和迁移，以每6,7天数毫米的速度形成豆芽。 血管生成与多种病理状况，包括癌症，牛皮癣，糖尿病性视网膜病，关节炎，哮喘，自身免疫性疾病，感染性疾病，动脉粥样硬化和8-10相关联。由于血管生成的各种疾病的重要性，了解调节这一过程是更好的治疗设计的关键基因和途径。 在管道形成试验是一种快速和定量的方法，用于确定参与血管生成基因或通路。于1988年首先描述，此测定法的基本原则是内皮细胞保持分裂和迅速地响应于血管生成的信号11-13迁移的能力。此外，血管内皮细胞被诱导分化和形成管样结构，当上的基底膜提取物（BME）的基质中培养。这些管道包括通过连接复合体连在一起的内皮细胞围成的管腔。管形成很快发生最管在该测定中形成内2-6小时，这取决于数量和血管生成的刺激的类型。 几种类型的内皮细胞可用于该测定中既包括原代细胞和永生化细胞系14,15。本文使用的细胞系是小鼠3B-11，但是相同的方法可以与其他内皮细胞系如SVEC4-10（小鼠）或原代内皮细胞，例如血管内皮细胞（人）的细胞被应用。这取决于细胞系用于与是否内皮策LLS转化或未转化，优化将需要被进行，以确定所需要的适当的管道形成了理想的时间。

<table><tbody><tr><td>Costar 24-well tissue culture-treated plate</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>BD Matrigel basement membrane matrix, growth factor reduced</td><td>BD Biosciences</td><td>354230</td><td></td></tr><tr><td>Gibco 0.05% trypsin-EDTA</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Gibco L-glutamine 200 mM (100x)</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>Gibco pen strep</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Gibco DMEM (1x)</td><td>Life Technologies</td><td>11965-092</td><td></td></tr><tr><td>Gibco DPBS (1x)</td><td>Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlas Biologicals</td><td>F-0500-A</td><td></td></tr><tr><td>Calcein AM</td><td>Life Technologies</td><td>C3100MP</td><td></td></tr><tr><td>DMSO</td><td>ATCC</td><td>4-X</td><td></td></tr></tbody></table>

endothelial cell tube formation assay for the in vitro study of angiogenesis

血管生成是用于正常组织发育和伤口愈合的重要方法，而且还与多种病理状况相关联。通过此协议，血管生成可在体外快速的，可量化的方式进行测量。初级或永生化的内皮细胞被混合的条件培养基并接种于基底膜基质。内皮细胞形成毛细血管样结构响应于在条件培养基中发现的血管生成信号。该管形成迅速发生与血管内皮细胞开始1小时内和含腔，小管开始出现在2个小时的赞同。管可以使用相衬倒置显微镜进行可视化，或者细胞可以与现有的测定法和管通过荧光或共聚焦显微镜观察钙黄绿素AM被处理。的分支站点/节点，回路的数量/网格，或数字或形成管的长度可以很容易地量化为在VITR措施Ø血管生成。总之，本试验可用于确定基因和参与了促进或抑制血管生成中具有快速，可重复的，和定量的方式通路。

鼠标3B-11内皮细胞接种在固化低生长因子BME - 在该试验中，产品的基质胶，使用 - 和随后随着时间的推移。 如图1中 ，由任一鼠角质细胞或成纤维细胞分泌的血管生成因子能够诱导管的形成随时间的。血管内皮细胞迁移，并开始在1-2小时电镀形成小分支。最大管形成是通过使用先前从角质细胞获得的条件培养基4-6小时到达。 24小时，一些分支机构仍然存在，但许多细胞凋亡成为和管?...

Watch this Scientific Journal Video about Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis at JoVE.com

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

小管形成实验是测量在体外血管生成一种快速的，可量化的方法。内皮细胞结合的条件培养基并接种于基底膜提取物。管形成时小时内新形成的小管很容易量化。

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 ...

endothelial-cell-tube-formation-assay-for-vitro-study

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

59.7K 次观看.  American University. 小管形成实验是测量在体外血管生成一种快速的，可量化的方法。内皮细胞结合的条件培养基并接种于基底膜提取物。管形成时小时内新形成的小管很容易量化。 

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

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 ...

科研

生物学

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ECM also participates in cell migration, proliferation, and differentiation. Fibroblasts are the main type of cells modifying ECM fiber arrangement and production. In cancer, CAFs (cancer associated fibroblasts) are in permanent activation status, participating in ECM remodeling, facilitating tumor cell migration, and stimulating tumor-associated angiogenesis, among other pro-tumorigenic roles. The objective of this method is to create a three-dimensional matrix with a fiber composition that is similar to in vivo matrices, using immortalized fibroblasts or human primary CAFs. Fibroblasts are cultured in pre-treated cell culture plates and grown under ascorbic acid stimulation. Then, fibroblasts are removed and matrices are blocked for further cell seeding. In this ECM model, fibroblasts can be activated or modified to generate different kinds of matrix, whose effects can be studied in cell culture. 3D matrices are also shaped by cell signals, like degradation or cross-linking enzymes that might modify fiber distribution. In this context, angiogenesis can be studied, along with other cell types such as epithelial tumor cells.

The aim of this method is to obtain fibroblast-derived 3D matrices as a natural scaffold for subsequent cellular assays. Fibroblasts are seeded in a pre-treated culture plate and stimulated with ascorbic acid for matrix generation. Matrices are decellularized and blocked to culture relevant cells (e.g., endothelial cells).

纤维细胞衍生的三维矩阵系统适用于内皮管形成测定

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ...

癌症研究

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

HUVEC 管形成测定评估天然产物对血管生成的影响

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

发育生物学

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

基于一个基质胶管形成实验，以评估对肿瘤细胞的血管生成活性

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

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

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 ...

生物工程

<meta charset="utf8"></head><body>分析血管生成：BM 基质上试管形成的体外测定

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

<meta charset="utf8"></head><body>从血管生成-管形成测定中回收滋养层细胞，用于分化标志物表达分析

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

从血管生成-管形成测定中回收滋养层细胞，用于分化标志物表达分析

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

内皮细胞小管形成实验的

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优化的纤维蛋白凝胶珠检测血管生成的研究

纤维细胞衍生的三维矩阵系统适用于内皮管形成测定

HUVEC 管形成测定评估天然产物对血管生成的影响

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

基于一个基质胶管形成实验，以评估对肿瘤细胞的血管生成活性

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

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

从血管生成-管形成测定中回收滋养层细胞，用于分化标志物表达分析

从血管生成-管形成测定中回收滋养层细胞，用于分化标志物表达分析