Name: strategic endothelial cell tube formation assay: comparing extracellular matrix and growth factor reduced extracellular matrix
Uploaded: 2016-08-14T14:00:00.000+00:00
Duration: 8 min 46 s
Description: Watch this Scientific Journal Video about Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix at JoVE.com

The work was funded by the Fund for Medical Research and Education, Wayne State University, Detroit, MI.

1.细胞培养<ol><li>种子3×10 5的HUVEC细胞在10ml完全生长培养基10的一个75厘米烧瓶中。 </li><li> 的 CO 2孵育细胞在37℃，5％至70-80％汇合。 </li></ol> 2.肾小管形成分析<ol><li>在4℃下解冻要么生长因子降低（GFR）的胞外基质或正常细胞外基质过夜冰上。 注意:为简便起见&#39;细胞外基质&#39;的缘故将从此用于引用两GFR和正常细胞外基质。让他们做好准备的过程是相同的。 </li><li>保持在冰上的96孔培养板，每使用预冷的技巧以及添加50微升冷却的细胞外基质。制备仅含正常细胞外基质5井一式三份。准备只含GFR细胞外基质的5口井的另一个一式三份。 </li><li>孵育96孔板在37℃下30分钟。 </li><li>洗内皮细胞一旦与没有钙和镁（PBS）的磷酸盐缓冲的溶液，并加入1 ml 0.05％胰蛋白酶-EDTA。孵育在37℃下的菜1-5分钟，检查每分钟细胞直至大多数细胞围捕。通过点击一次瓶打跑细胞。 <ol><li>加入5 ml基础培养基（ 即，内皮细胞生长介质中无任何内容（ 例如 ，补充剂）加）有1％胎牛血清（FBS）。收集在烧瓶中的细胞，通过移液并转移到15毫升管。离心细胞，在200×g离心5分钟并弃上清。 </li><li>重悬用2ml基础培养基，使用血球计数细胞，并调整细胞浓度为2-3×10 5个细胞/ ml。 </li></ol></li><li>制备100微升以下的10倍浓度CXCR2抑制剂SB225002的在基础培养基:11μM，5.6μM，1.1μM，0.56μM和0μM（对照）。 </li><li>吸管300微升的HUVEC悬浮液到每5 MICRocentrifuge管。添加抑制剂浓度的33微升（11μM，5.6μM，1.1μM，0.56μM和0μM）成一个管各自含有内皮细胞，和旋涡。 </li><li>吸移管将50μl各细胞悬浮液（1-1.5×10 4个细胞）的细胞外基质的各个孔中的。板各悬浮液一式三份，并在37℃，5％的CO 2孵育4-16小时。 </li><li> 4-16小时后，取4张照片每使用倒置显微镜相机11（原来的放大倍数×100）以及形成的管子。 </li></ol> 3.肾小管破坏试验<ol><li>根据相同的规格制备细胞外基质和板作为管道形成试验（步骤2.1至2.3）。 </li><li>如在步骤中管道形成试验和等分2.4.2以每孔50微升所述2.4-到含有细胞外基质的96孔板制备的HUVEC悬浮液。 注:板足够井小号Ø有每个药物治疗3口井。 </li><li>在37℃下生长的细胞外基质4小时，5％ 的 CO 2。药物处理开始之前拍摄图像。 </li><li>如在管道形成试验中的步骤2.5所述制备CXCR2抑制剂SB225002的2倍的浓度，并添加50微升每含有内皮细胞的96孔板的各浓度。板一式三份每个浓度。 </li><li>于37℃孵育另外2-12小时的板，5％ 的 CO 2。以形成在每个使用以及倒置显微镜照相机（原放大倍数×100）11管的4张图片。 </li></ol> 4.量化数据<ol><li>通过使用显微镜照相机软件测量4个随机显微镜视野管的总管长度评估所述图像中捕获管形成。 </li><li>打开显微镜摄像头软件图像，然后单击"注释和测量9 ;.根据"长度"部分中，选择"简单的线"工具。点击图片，然后绘制沿管长度的线，然后用鼠标点击。 注意:软件会自动计算在像素线的长度，并把数据转换成一个文件。 </li><li>用同样的方法通过选择"简单的线"工具在图像中的所有管系。点击图片，然后绘制沿管长度的线，然后用鼠标点击。 注意:该软件将记录每个线的长度并在屏幕上显示的数据。 </li><li>在图像测量每个管后，点击"导出"，然后选择"长到电子表格"。 注:长度数据将被导出到电子表格。 </li><li>添加所有的管长度从电子表格得到总的管长度。计算和记录管的平均长度。 注:四个重复推荐使用均值和确定的标准偏差。 </li></ol> 5.从细胞外基质培养细胞中恢复<ol><li>培养，并执行一个管形成试验在更大的范围（见部分2），为96孔板将不会产生足够的细胞数。 12孔板中，使用细胞外基质500微升和500微升的HUVEC细胞悬浮液（1.5×10 5个细胞）。 </li><li>孵育4-16小时的细胞，以便形成管。然后，吸出细胞培养基。冲洗细胞用1ml冷的1×PBS中的无钙和镁。 </li><li>加入1ml冰冷PBS-2.5毫摩尔EDTA缓冲液到培养，并保持在冰上10分钟。 </li><li>逐出使用1000微升枪头与尖端切断菜细胞和细胞外基质混合物，然后转移到冷15ml试管，洗井用4ml冰冷的PBS-2.5 1mM EDTA和放入管。 </li><li>雪藏管1-4小时，几次翻转试管，直到所有的外基质溶解。 </li><li>在1,620 xg离心离心10分钟在0℃下</li><li>重悬用1ml冷的PBS中的细胞沉淀在冰上一个1.ml管。 </li><li>离心再次在3000 xg离心在0℃下5分钟，然后丢弃上清液。 </li><li>存放在-80℃的细胞。 </li></ol>

<ol>
	<li>Sharma, B., Nawandar, D. M., Nannuru, K. C., Varney, M. L., Singh, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+CXCR2+enhances+chemotherapeutic+response,+inhibits+mammary+tumor+growth,+angiogenesis,+and+lung+metastasis.">Targeting CXCR2 enhances chemotherapeutic response, inhibits mammary tumor growth, angiogenesis, and lung metastasis.</a> Mol Cancer Ther. 12 (5), 799-808 (2013).</li><li>Birbrair, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type-2+pericytes+participate+in+normal+and+tumoral+angiogenesis.">Type-2 pericytes participate in normal and tumoral angiogenesis.</a> Am J Physiol Cell Physiol. 307 (1), 25-38 (2014).</li><li>Ferrara, N., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+as+a+therapeutic+target.">Angiogenesis as a therapeutic target.</a> Nature. 438 (7070), 967-974 (2005).</li><li>Speyer, C. L., Hachem, A. H., Assi, A. A., Johnson, J. S., DeVries, J. A., Gorski, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabotropic+glutamate+receptor-1+as+a+novel+target+for+the+antiangiogenic+treatment+of+breast+cancer.">Metabotropic glutamate receptor-1 as a novel target for the antiangiogenic treatment of breast cancer.</a> PLoS One. 9 (3), 88830 (2014).</li><li>Gu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Apurinic/Apyrimidinic+Endonuclease+siRNA+Inhibits+the+Angiogenesis+Induced+by+X-Ray+Irradiation+in+Lung+Cancer+Cells.">Human Apurinic/Apyrimidinic Endonuclease siRNA Inhibits the Angiogenesis Induced by X-Ray Irradiation in Lung Cancer Cells.</a> International Journal of Medical Sciences. 10 (7), 870-882 (2013).</li><li>Manjunathan, R., Ragunathan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+chorioallantoic+membrane+as+a+reliable+model+to+evaluate+osteosarcoma-an+experimental+approach+using+SaOS2+cell+line.">Chicken chorioallantoic membrane as a reliable model to evaluate osteosarcoma-an experimental approach using SaOS2 cell line.</a> Biol Proced Online. 17, 10 (2015).</li><li>Nidhyanandan, S., Boreddy, T. S., Chandrasekhar, K. B., Reddy, N. D., Kulkarni, N. M., Narayanan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphodiesterase+inhibitor,+pentoxifylline+enhances+anticancer+activity+of+histone+deacetylase+inhibitor,+MS-275+in+human+breast+cancer+in+vitro+and+in+vivo.">Phosphodiesterase inhibitor, pentoxifylline enhances anticancer activity of histone deacetylase inhibitor, MS-275 in human breast cancer in vitro and in vivo.</a> Eur J Pharmacol. 764, 508-519 (2015).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper+improves+the+anti-angiogenic+activity+of+disulfiram+through+the+EGFR/src/VEGF+pathway+in+gliomas.">Copper improves the anti-angiogenic activity of disulfiram through the EGFR/src/VEGF pathway in gliomas.</a> Cancer Lett. Aug. , (2015).</li><li>Park, H. J., Zhang, Y., Georgescu, S. P., Johnson, K. L., Kong, D., Galper, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+umbilical+vein+endothelial+cells+and+human+dermal+microvascular+endothelial+cells+offer+new+insights+into+the+relationship+between+lipid+metabolism+and+angiogenesis.">Human umbilical vein endothelial cells and human dermal microvascular endothelial cells offer new insights into the relationship between lipid metabolism and angiogenesis.</a> Stem Cell Rev. 2 (2), 93-102 (2006).</li><li>de Wit, C., Fautz, C., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+PCR+for+retrovirus-like+particle+quantification+in+CHO+cell+culture.">Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture.</a> Biologicals. 28 (3), 137-148 (2000).</li><li>Jaffe, E. A., Nachman, R. L., Becker, C. G., Minick, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+human+endothelial+cells+for+derived+from+umbililcal+veins.+Identification+by+morphologic+and+immunologic+criteria.">Culture of human endothelial cells for derived from umbililcal veins. Identification by morphologic and immunologic criteria.</a> J Clin Invest. 52 (11), 2745-2756 (1973).</li></ol>

The authors declare that they have no competing financial interests.

当进行该测定，必须在任何时候都保持在冰上或4℃下的细胞外基质，除非另有规定。如果细胞外基质温热以上4℃，将聚合，并测定将被破坏。同样重要的是保证任何与之接触（ 例如，枪头，板）与胞外基质是预冷却用于上述原因。在每个孔中接种细胞的数量是关键的，过少的细胞不会产生预期的纸幅的对照样品中，过多的细胞会形成大的细胞团或单层和测定将是无效的。在该测定中的成功的另一个重要因素是血管内皮细胞的细胞传代次数。该通道数量始终应为十下，否则可能不会出现强劲的管形成。此外，应该确保所用的细胞培养基还没有过期，或者细胞将是不可行的。 Alternatively，可以分装在介质及其组件，并将它们存储在-20℃保存用于在到期日之后的一段时间。当然，它仍然是优选事先使用介质的有效期限像所有的程序，也有一些缺点，在进行内皮细胞管形成测定。一个主要的问题是，由于有不同类型的内皮细胞和支持矩阵的，测定的结果可能会因该细胞与基质的类型用于上差异很大。所用的内皮细胞（HUVECs，HAECs或HMVECs）是原代细胞，它们是昂贵相比永生化细胞获得，并有可变性。主细胞具有使用有限的通道，并且因此不适合于长期的血管生成的实验14。对于可靠的数据，每个人都应该使用相同类型的分析。与体外测定其它，一个管形成测定的结果应该是CON坚挺体内 ，因为从二维组织培养的控制并人工条件的结果可能不总是在生物体的复杂生物系统反映。同样重要的是要记住，这种类型的测定法可以仅用于演示内皮细胞管的形成，并且不应当被用来测试其它非内皮，管形成细胞。 该测定是量化的化合物的血管生成潜力的比较简单而快速的方法。它也形成了进一步的实验平台，与​​单独，它不会产生关于由该化合物实际上影响了容器形成过程的特定机制的信息。然而，使用不同的细胞外基质的是如何进一步创造研究问题的框架是不常用的一个例子。有研究的化合物的特定的生化机制，包括靶向组分特异性抑制剂的方法血管生成途径。另一方法可以是从血管内皮细胞中提取的RNA，并使用RT-PCR（实时PCR）12来分析基因表达的改变可能导致在测定中观察到的生长行为。这种方法的未来应用包括转染的内皮细胞，以创建用于血管生成途径的具体步骤击倒测定。因此有可能将这种方法用于研究淋巴管途径。通过改变细胞外基质成分，基质和细胞过程中癌症支承血管发生的相互作用，可以进一步阐明。 RNA和蛋白质从这些特定条件下的内皮细胞中提取附加量化的数据对应于成像数据提供。

为了获得养分生存所必需的，恶性肿瘤需要访问病人的血液流。为了得到该访问，肿瘤细胞释放，刺激的新血管的生长的肿瘤的化学信号，从而劫持称为血管2的正常的生理过程。它也可通过经由血管发生该转移（ 即，癌症到其他器官的扩散），可能会发生在血管。因为血管生成的过程是这样的多种癌症的进展至关重要，它是在抗癌治疗的研究3有吸引力的目标。 用来量化血管生成的一种方法是当放置在细胞外基质以测量内皮祖细胞的形成管的能力。由于这些形成管是关键的一步早在血管生成，试验环境条件（ 例如，一个给定的共同存在或缺乏mpound），可以刺激或抑制管道形成提供了深入了解，可以有针对性地抑制血管生成的具体步骤。内皮细胞管形成测定是测量细胞的形成管的能力最广泛使用4 体外方法之一。这是一个简单的分析，需要相对较少的部件和一个短培养周期5。或许最重要的是，然而，就是从这种类型的测定法中获得的数据是量化。 对使用管形成测定法的实例包括从生长在细胞外基质与生长因子降低（GFR）的胞外基质的血管内皮细胞进行比较管的发展。细胞外基质是从肉瘤细胞和其主要部件分离的基底膜样材料是层粘连蛋白，IV型胶原蛋白，生长因子和蛋白多糖。一些化合物对细胞的能力不同的效果的时候，以形成管具有降低的生长因子和降低的硫酸乙酰肝素蛋白多糖（聚糖）的环境。聚糖是细胞外基质的组分其在GFR外基质显著降低。作为一个例子，如果假设是血管生成抑制剂，如SB225002，其阻断CXCR2受体，具有相当弱的中断管形成时聚糖丰富能力，然后在细胞外基质和GFR外基质管生成活性之间的比较是在探索通过聚糖的控制抑制血管生成的可能性重要。 通常用于确定对血管生成化合物的作用的其他测定法体内方法。这种显着的例子是鸡胚尿囊膜（CAM）测定6使用鸡胚，并在体内基底膜利用老鼠堵塞血管生成测定7。尽管体内方法测量在T血管生成HREE尺寸和较有代表性人体相比， 体外管形成测定中，它们遭受需要显著更多时间的缺陷，并且相当多的困难来执行。两者CAM分析和细胞外插头测定至少需要8周做，而在比较，该管形成测定可以在一天完成，它不需要动物的使用。 要注意，本报告中所使用的血管内皮细胞是人脐静脉内皮细胞（HUVEC）是重要的。这些细胞在血管萌芽和血管生长的关键作用，并有足够的类似于内皮细胞中的癌症也可以应用于在体外和体内实验9以评价抗血管生成活性。也可以使用其它细胞如初级微血管内皮细胞。 同样重要的是要注意，除了具有低聚糖的水平，比较正常的细胞外基质时GFR细胞外基质也降低了许多组分的水平。这包括，但不限于:表皮生长因子，IGF-1，PDGF，和TGF-β。那些进行实验研究的血管生成的关系这些化合物的意义可能有兴趣使用GFR细胞外基质。

<table><tbody><tr><td>EGM-2 complete growth medium (EGM-2 bullet kit CC-3162 )</td><td>Fisher</td><td>NC9525043</td><td>HUVEC complete growth medium: EGM-2 base medium mixed with BBE (Bovine Brain Extract), hEGF (Human Epidermal Growth Factor), Hydrocortisone, GA-1000 (Gentamicin, Amphotericin-B), 2% FBS (Fetal Bovine Serum), VEGF (Vascular Endothelial Growth Factor), hFGF-&amp;beta; (Human Fibroblast Growth Factor Beta), R3-IGF-1 (Long R Insulin-like Growth Factor One), Ascorbic Acid, and Heparin. Aliquot the medium and its components and store them at -20&amp;nbsp;&amp;deg;C, warm in 37&amp;nbsp;&amp;deg;C before use.</td></tr><tr><td>MATRIGEL MATRIX 10ML Growth Factor Reduced (GFR); 10&amp;nbsp;ml; LDEV-Free</td><td>Fisher</td><td>CB-40230</td><td>Growth Factor Reduced (GFR) extracellular matrix, keep in -20&amp;nbsp;&amp;deg;C, warm in 4&amp;nbsp;&amp;deg;C before use.</td></tr><tr><td>MATRIGEL MATRIX 10ML; 10&amp;nbsp;ml; LDEV-Free</td><td>Fisher</td><td>CB-40234</td><td>extracellular matrix, keep in -20&amp;nbsp;&amp;deg;C, warm in 4&amp;nbsp;&amp;deg;C before use.</td></tr><tr><td>SB225002</td><td>Fisher</td><td>559405</td><td>CXCR2 inhibitor, keep in -20&amp;nbsp;&amp;deg;C, warm in room temperature before use.</td></tr><tr><td>Primary human umbilical vein endothelial cells (HUVEC)&amp;nbsp;</td><td>ScienCell Research Laboratories&amp;nbsp;</td><td>8000</td><td>culture it according reference&amp;nbsp; 10.</td></tr><tr><td>Trypsin 0.05%-EDTA phenol red</td><td>Invitrogen</td><td>25300-054</td><td>keep in 4&amp;nbsp;&amp;deg;C, warm in 37&amp;nbsp;&amp;deg;C before use.</td></tr><tr><td>Nikon ECLIPSE Ti</td><td>Nikon</td><td></td><td>Inverted Research Microscope.&amp;nbsp;</td></tr><tr><td>NIS-Elements AR 4.20.02 64 bit</td><td>Nikon</td><td></td><td>Inverted Research Microscope software</td></tr><tr><td>Graph-pad Prism 5</td><td>GraphPad Software, Inc.</td><td></td><td>scientific 2D graphing and statistics software, to calculate IC&lt;sub&gt;50&lt;/sub&gt;.</td></tr></tbody></table>

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.

相当多的，健康的内皮管形成可以很容易地进行对比，以抑制血管形成的显微​​图像。健康管形成显示为毛细管状结构（ 图1）的一个有组织的纤维网。相比较而言，抑制血管形成本身表现为散射的细胞（ 图2）。管形成测定数据是通过测量毛细管（ 表1）的总管长度定量。除了总管长度，平均管长度，管的总数，或分支点总数可以被测量。结构管形成产生更大的净管长度不是分散抑制花粉管生长。在图3中 ，剂量反应曲线允许内皮细胞对GFR外基质和CXCR2抑制剂SB225002的实验条件下的IC 50的计算。 对细胞外基质和生长因子降低（GFR）的细胞外基质 图1. 内皮吨UBE形成两个图像显示在细胞外基质（A）和GFR细胞外基质（B）的管形成成功的。管的互连网络清楚地表明，管的生长是在这些内皮健康。比例尺= 10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54074/54074fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54074/54074fig2.jpg" /> 图2. 抑制对细胞外和生长因子降低（GFR）由CXCR2抑制剂SB225002（5.6μM）细胞外基质。管形成两个图像显示管形成已经由一个CXCR2抑制剂SB225002（5.6μM）对细胞外基质（A）和GFR细胞外基质（B）的抑制。在该图像显示看到HUVEC细胞的分离的团块，这些细胞无法以形成必要的相互连接的管中。比例尺= 10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54074/54074fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54074/54074fig3.jpg" /> 上生长因子降低（GFR）的细胞外基质 图3 的剂量反应曲线 ，X轴示出了管的长度与Y轴表示CXCR2抑制剂的浓度。剂量反应曲线表明，对GFR外基质的CXCR2抑制剂的HUVEC反应是剂量依赖性的。的IC 50（半最大抑制ÿ浓度）用软件13和可与细胞外基质的剂量响应曲线进行比较，例如计算。集成电路50是下降到50％的响应的抑制剂的浓度。误差棒代表标准偏差。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 生长因子降低（GFR）的细胞外基质和抑制剂 （4个重复的每个） </td><td> 总管长度平均（像素）（1 像素= 0.34微米） </td><td> 标准偏差 </td><td> 中P值 </td></tr><tr><td> GFR细胞外基质控制</td><td> 2447 </td><td> 168.174 </td><td></td></tr><tr><td> GFR细胞外基质+ 0.56μMSB225002 </td><td> 1422.5 </td><td> 185.4218 </td><td> 0.000395 </td></tr><tr><td>GFR细胞外基质+ 1.1μMSB225002 </td><td> 1004 </td><td> 126.1784 </td><td> 2.14×10 -5 </td></tr><tr><td> GFR细胞外基质+ 5.6μMSB225002 </td><td> 519.25 </td><td> 129.6368 </td><td> 4.19×10 -6 </td></tr><tr><td> GFR细胞外基质+ 11μMSB225002 </td><td> 393.75 </td><td> 212.6857 </td><td> 1.21×10 -5 </td></tr></tbody></table> 表1在不同条件下的量化生长因子降低（GFR）的细胞外基质总管的长度。利用记录在生长于GFR的细胞外基质与不同浓度的CXCR2抑制剂的样品总导管段的表的一部分。数据表明CXCR2抑制剂不抑制管道形成。包括的是例如平均的四次重复，标准偏差，和P值相关联的值。测量像素，显示要导出的数据。集成电路50可以通过使用统计软件13来计算。 （1个像素= 0.34微米）

Watch this Scientific Journal Video about Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix at JoVE.com

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

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 ...

strategic-endothelial-cell-tube-formation-assay-comparing

Universidad San Sebastian

Research

JoVE Journal

Medicine

13.4K 次观看.  Wayne State University School of Medicine. 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.

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

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 ...

科研

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 ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

生物工程

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 ...

生物学

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

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.

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.

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

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 ...

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.

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

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

使用划痕伤口迁移和球状体发芽测定法研究血管生成

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

作者聚焦：通过检测开发中的挑战和创新研究血管生成

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

利用划痕伤口迁移测定和球状体发芽测定在功能和分子水平上研究血管生成

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...