Name: three-dimensional angiogenesis assay system using co-culture spheroids formed by endothelial colony forming cells and mesenchymal stem cells
Uploaded: 2019-09-18T12:30:00
Description: Watch this Scientific Journal Video about Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells at JoVE.com

这项研究得到了食品药品安全部（17172MFDS215）的资助、韩国政府资助的韩国国家研究基金会（MSIP）（2017R1A2B4005463）以及基础科学研究计划的支持。由教育部资助的韩国国家研究基金会（2016R1A6A1A03007648）。

人类氟氯烃与人类外周血分离，如27日的报告所述。简单地说，单核细胞层使用Ficoll-Paque Plus从整个血液中分离出来，并在适当的培养基培养，直到内皮状菌落出现。收集菌落，并使用CD31涂层磁珠分离ECFC。MSCs从人类成人骨髓的粘附单核细胞（MNC）部分中分离出来。研究协议获得杜松女子大学机构审查委员会（IRB No. 2017-002-01）的批准。
1. 细胞培养
<ol><li>
<st...

<ol>
	<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 (7070), 932-936 (2005).</li><li>Goodwin, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assays+of+angiogenesis+for+assessment+of+angiogenic+and+anti-angiogenic+agents.">In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents.</a> Microvascular research. 74 (2-3), 172-183 (2007).</li><li>Staton, C. A., Reed, M. W., Brown, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+analysis+of+current+in+vitro+and+in+vivo+angiogenesis+assays.">A critical analysis of current in vitro and in vivo angiogenesis assays.</a> International journal of experimental pathology. 90 (3), 195-221 (2009).</li><li>Lutolf, M. P., Gilbert, P. M., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+materials+to+direct+stem-cell+fate.">Designing materials to direct stem-cell fate.</a> Nature. 462 (7272), 433-441 (2009).</li><li>Fina, L., 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=Expression+of+the+CD34+gene+in+vascular+endothelial+cells.">Expression of the CD34 gene in vascular endothelial cells.</a> Blood. 75 (12), 2417-2426 (1990).</li><li>Delia, D., 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=CD34+expression+is+regulated+reciprocally+with+adhesion+molecules+in+vascular+endothelial+cells+in+vitro.">CD34 expression is regulated reciprocally with adhesion molecules in vascular endothelial cells in vitro.</a> Blood. 81 (4), 1001-1008 (1993).</li><li>Korff, T., Augustin, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+forces+in+fibrillar+extracellular+matrices+control+directional+capillary+sprouting.">Tensional forces in fibrillar extracellular matrices control directional capillary sprouting.</a> Journal of Cell Science. 112 (19), 3249-3258 (1999).</li><li>Korff, T., Augustin, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+endothelial+cells+in+multicellular+spheroids+prevents+apoptosis+and+induces+differentiation.">Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation.</a> The Journal of cell biology. 143 (5), 1341-1352 (1998).</li><li>Gaengel, K., Genov&#233;, G., Armulik, A., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-mural+cell+signaling+in+vascular+development+and+angiogenesis.">Endothelial-mural cell signaling in vascular development and angiogenesis.</a> Arteriosclerosis, thrombosis, and vascular biology. 29 (5), 630-638 (2009).</li><li>Hutmacher, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+offer+cancer+research+the+third+dimension.">Biomaterials offer cancer research the third dimension.</a> Nature Materials. 9 (2), 90-93 (2010).</li><li>Korff, T., Kimmina, S., Martiny-Baron, G., Augustin, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessel+maturation+in+a+3-dimensional+spheroidal+coculture+model:+direct+contact+with+smooth+muscle+cells+regulates+endothelial+cell+quiescence+and+abrogates+VEGF+responsiveness.">Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness.</a> FASEB Journal. 15 (2), 447-457 (2001).</li><li>Gumkowski, F., Kaminska, G., Kaminski, M., Morrissey, L. W., Auerbach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+mouse+vascular+endothelium.+In+vitro+studies+of+lymphatic,+large+blood+vessel+and+microvascular+endothelial+cells.">Heterogeneity of mouse vascular endothelium. In vitro studies of lymphatic, large blood vessel and microvascular endothelial cells.</a> Journal of Vascular Research. 24 (1-2), 11-23 (1987).</li><li>Asif, A. R., Oellerich, M., Armstrong, V. W., Hecker, M., Cattaruzza, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-786C+polymorphism+of+the+NOS-3+gene+and+the+endothelial+cell+response+to+fluid+shear+stress-a+proteome+analysis.">T-786C polymorphism of the NOS-3 gene and the endothelial cell response to fluid shear stress-a proteome analysis.</a> Journal of Proteome Research. 8 (6), 3161-3168 (2009).</li><li>Melero-Martin, J. M., 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=In+vivo+vasculogenic+potential+of+human+blood-derived+endothelial+progenitor+cells.">In vivo vasculogenic potential of human blood-derived endothelial progenitor cells.</a> Blood. 109 (11), 4761-4768 (2007).</li><li>Ingram, D. 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=Identification+of+a+novel+hierarchy+of+endothelial+progenitor+cells+using+human+peripheral+and+umbilical+cord+blood.">Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.</a> Blood. 104 (9), 2752-2760 (2004).</li><li>Murasawa, S., Asahara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+progenitor+cells+for+vasculogenesis.">Endothelial progenitor cells for vasculogenesis.</a> Physiology (Bethesda). 20, 36-42 (2005).</li><li>Rafii, S., Lyden, D., Benezra, R., Hattori, K., Heissig, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+and+haematopoietic+stem+cells:+novel+targets+for+anti-angiogenesis+therapy.">Vascular and haematopoietic stem cells: novel targets for anti-angiogenesis therapy.</a> Nature Review Cancer. 2 (11), 826-835 (2002).</li><li>Shi, S., Gronthos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+niche+of+postnatal+mesenchymal+stem+cells+in+human+bone+marrow+and+dental+pulp.">Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp.</a> Journal of Bone and Mineral Research. 18 (4), 696-704 (2003).</li><li>Crisan, M., 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=A+perivascular+origin+for+mesenchymal+stem+cells+in+multiple+human+organs.">A perivascular origin for mesenchymal stem cells in multiple human organs.</a> Cell Stem Cell. 3 (3), 301-313 (2008).</li><li>Lee, H., Kang, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+tube+formation+assay+using+human+endothelial+colony+forming+cells+for+in+vitro+evaluation+of+angiogenesis.">Advanced tube formation assay using human endothelial colony forming cells for in vitro evaluation of angiogenesis.</a> Korean Journal of Physiology &amp; Pharmacology. 22 (6), 705-712 (2018).</li><li>Melero-Martin, J. M., 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=Engineering+robust+and+functional+vascular+networks+in+vivo+with+human+adult+and+cord+blood-derived+progenitor+cells.">Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells.</a> Circulation Research. 103 (2), 194-202 (2008).</li><li>Kang, K. T., Allen, P., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+human+vascular+networks+transplanted+into+secondary+mice+reconnect+with+the+host+vasculature+and+re-establish+perfusion.">Bioengineered human vascular networks transplanted into secondary mice reconnect with the host vasculature and re-establish perfusion.</a> Blood. 118 (25), 6718-6721 (2011).</li><li>Kang, K. T., Coggins, M., Xiao, C., Rosenzweig, A., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+vasculogenic+cells+form+functional+blood+vessels+and+mitigate+adverse+remodeling+after+ischemia+reperfusion+injury+in+rats.">Human vasculogenic cells form functional blood vessels and mitigate adverse remodeling after ischemia reperfusion injury in rats.</a> Angiogenesis. 16 (4), 773-784 (2013).</li><li>Kang, K. T., Lin, R. Z., Kuppermann, D., Melero-Martin, J. M., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+colony+forming+cells+and+mesenchymal+progenitor+cells+form+blood+vessels+and+increase+blood+flow+in+ischemic+muscle.">Endothelial colony forming cells and mesenchymal progenitor cells form blood vessels and increase blood flow in ischemic muscle.</a> Scientific Reports. 7 (1), 770 (2017).</li><li>Paupert, J., Sounni, N. E., Noel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphangiogenesis+in+post-natal+tissue+remodeling:+lymphatic+endothelial+cell+connection+with+its+environment.">Lymphangiogenesis in post-natal tissue remodeling: lymphatic endothelial cell connection with its environment.</a> Molecular Aspects of Medicine. 32 (2), 146-158 (2011).</li><li>Lu, P., Weaver, V. M., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix:+a+dynamic+niche+in+cancer+progression.">The extracellular matrix: a dynamic niche in cancer progression.</a> Journal of Cell Biology. 196 (4), 395-406 (2012).</li><li>Melero-Martin, J. M., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+13.+An+in+vivo+experimental+model+for+postnatal+vasculogenesis.">Chapter 13. An in vivo experimental model for postnatal vasculogenesis.</a> Methods in Enzymology. 445, 303-329 (2008).</li><li>Laib, A. M., 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=Spheroid-based+human+endothelial+cell+microvessel+formation+in+vivo.">Spheroid-based human endothelial cell microvessel formation in vivo.</a> Nature Protocols. 4 (8), 1202-1215 (2009).</li><li>Leuning, D. G., 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=The+cytokine+secretion+profile+of+mesenchymal+stromal+cells+is+determined+by+surface+structure+of+the+microenvironment.">The cytokine secretion profile of mesenchymal stromal cells is determined by surface structure of the microenvironment.</a> Scientific Reports. 8 (1), 7716 (2018).</li><li>Shah, S., Kang, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Cell+Spheroid+Angiogenesis+Assay+System+Using+Both+Endothelial+Colony+Forming+Cells+and+Mesenchymal+Stem+Cells.">Two-Cell Spheroid Angiogenesis Assay System Using Both Endothelial Colony Forming Cells and Mesenchymal Stem Cells.</a> Biomolecules &amp; Therapeutics (Seoul). 26 (5), 474-480 (2018).</li></ol>

本研究采用一种利用两个人类血管细胞祖体、ECFC和MSCs共同培养球体的体外血管生成测定系统改进，共培养球体系统可以模拟体内血管芽的形成，即通过内皮细胞和围细胞之间的相互作用和结合来完成。与其他仅反映ECs介导血管生成的其他体外血管生成测定相比，这种共培养性检测系统更代表生理血管生成多步级联，包括细胞相互作用、发芽、管形成、和船舶成熟。这种新建立的测定系统也类似?...

全世界约有5亿人有望受益于血管畸形相关疾病的血管生成调节疗法，如风湿性关节炎、神经病、心血管疾病和肿瘤转移1。因此，控制血管生成的药物的开发已成为制药业的一个重要研究领域。在药物开发过程中，有必要在体内进行动物研究，以探讨候选药物对生理机能和器官间系统相互作用的影响。然而，伦理和成本问题增加了对动物实验2的关注。因此，需要改进体外测定系统，以获得更准确和可预测的数据，从而在动物实验前做出更好的决策。目前的体外血管生成测定通常测量在二维（2D）培养板3中播种的内皮细胞（ECs）的增殖、入侵、迁移或管状结构形成。这些2D血管生成测定快速、简单、定量、经济高效，为发现血管生成调节药物做出了重大贡献。然而，仍有若干问题有待改进。
这种2D体外检测系统不能反映在体内生理条件下发生的血管生成的复杂多步骤事件，导致不准确的结果，导致体外测定数据与临床试验结果4之间的差异。2D培养条件也诱发细胞表型的变化。例如，在二维培养板中增殖后，EC 具有弱细胞表型，表现为 CD34 表达减少和控制细胞响应5、6的几个信号。为了克服基于2D培养的血管生成测定系统的局限性，开发了三维（3D）球形血管生成测定系统。发芽后，由EC形成的球体形成管状结构，反映了体内新血管化过程7、8。因此，3D球形血管生成测定被认为是一种有效的检测系统，用于筛选潜在的亲或抗血管生成药物。
大多数 3D 球形血管生成测定仅利用 EC，主要是人类脐带静脉内皮细胞 （HUVECs） 或人类皮下微血管内皮细胞 （HDMECs）， 专注于血管生成期间 EC 的细胞反应。然而，血毛细血管由两种细胞类型组成：EC和围细胞。阐述EC和围细胞之间的双向相互作用对于正确的血管完整性和功能至关重要。几种疾病，如遗传性中风，糖尿病视网膜病变，和静脉畸形，都与改变的围观密度或减少的内皮依不舍的附着性附件9相关。围卵体也被称为血管生成过程的关键元素。由EC招募围冰船以稳定新形成的船舶结构。在这方面，单培养球形血管生成测定不包括围细胞7，10。因此，由EC和围细胞形成的共培养球体可能为更密切地模拟生理血管生成事件提供有价值的方法。
本研究旨在开发一种3D共培养球形血管生成测定，结合人类内皮菌群形成细胞（ECFCs）和中位体干细胞（MSCs），以更密切地反映体内血管生成。共培养球体系统作为正常血管的体外表示组装，由Korff等人于2001年建立。他们将HUVECs和人类脐带动脉平滑肌细胞（HUSMC）结合，并证明两个成熟血管细胞的共同培养降低了发芽潜力。众所周知，成熟的ECs（HUVECs）会逐渐失去增殖和分化的能力，这对其血管生成反应12、13产生负面影响。成熟的血管细胞（HUSMC）可以通过废除血管内皮生长因子（VEGF）反应能力11导致内皮细胞失活。Korff 和我们的共培养球形系统的主要区别是所使用的细胞类型。我们应用了两种血管前体，ECFC和MSCs，以建立一个适当的血管生成测定系统，以筛选和调查亲或抗血管原药。氟氯烃是氟氯化碳的前身。与成熟的EC14相比，氟氯烃具有强大的扩散能力，能够克服EC的限制。在产后病理生理条件下，氟氯烃有助于新血管的形成。MSCs是多能干细胞，具有分化成围细胞的能力，从而有助于血管生成18，19。
在以前的报告中，ECFCs和MSCs对体外管形成20、体内新血管化21、22以及缺血组织23、24的改善再灌注表现出协同效应。在本研究中，ECFC和MSCs被用来形成共培养球体，并嵌入I型胶原凝胶中，以反映体内的3D环境。胶原蛋白被认为是围绕ECs25的细胞外基质（ECM）的主要成分。ECM在调节细胞行为26中起着关键作用。这里提出的测定方案可以使用通用实验室技术在两天内轻松快速地执行。为了在发芽过程中进行有效的细胞跟踪，可以使用实时细胞记录器对每种细胞类型进行荧光标记和监测。新建立的3D共培养球形血管生成测定系统旨在提高评估潜在血管生成调节器的灵敏度，并在体内研究之前提供可预测的信息。

<table border="0" cellpadding="0" cellspacing="0" style="width:709px;" width="708">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">0.05 % Trypsin EDTA (1X)</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:161px;">25300-054</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Bevacizumab</td>
			<td style="width:123px;">Roche</td>
			<td style="width:161px;">NA</td>
			<td>Commercial name: Avastin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Dulbecco Modified Eagle Medium</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:161px;">11885-084</td>
			<td>DMEM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Dulbeco&#39;s Phosphate buffered saline (10X)</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:161px;">21600-010</td>
			<td>PBS (10X)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Dulbeco&#39;s Phosphate buffered saline (1X)</td>
			<td style="width:123px;">Corning</td>
			<td style="width:161px;">21-031-CVR</td>
			<td>PBS (1X)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Endothelial cell Growth medium MV2 kit</td>
			<td style="width:123px;">Promocell</td>
			<td style="width:161px;">C-22121</td>
			<td>ECGM-MV2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td style="width:123px;">Atlas</td>
			<td style="width:161px;">FP-0500A</td>
			<td>FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Gelatin</td>
			<td style="width:123px;">BD Sciences</td>
			<td style="width:161px;">214340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">L-Glutamine&#8211;Penicillin&#8211;Streptomycin</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:161px;">10378-016</td>
			<td>GPS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Mesenchymal stem cell growth medium-2</td>
			<td style="width:123px;">Promocell</td>
			<td style="width:161px;">C-28009</td>
			<td>MSCGM-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Methyl cellulose</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:161px;">M0512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">PKH26 Fluorescent Cell Linker Kits</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:161px;">MINI26</td>
			<td>PKH26</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">PKH67 Fluorescent Cell Linker Kits</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:161px;">MINI67</td>
			<td>PKH67</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sodium Hydroxide</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:161px;">S5881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Type I collagen gel</td>
			<td style="width:123px;">Corning</td>
			<td style="width:161px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Vascular endothelial growth factor A</td>
			<td style="width:123px;">R&#38;D</td>
			<td style="width:161px;">293-VE-010</td>
			<td>VEGF</td>
		</tr>
	</tbody>
</table>

three-dimensional angiogenesis assay system using co-culture spheroids formed by endothelial colony forming cells and mesenchymal stem cells

在过去的几十年中，血管生成领域的研究一直在积极增长，人们认识到血管生成是50多种病理疾病的标志，如风湿性关节炎、神经病、心血管疾病，和肿瘤转移。在血管生成药物开发过程中，使用具有适当细胞类型和适当条件的体外测定系统以反映生理血管生成过程至关重要。为了克服目前主要使用内皮细胞的体外血管生成测定系统的局限性，我们开发了一种三维（3D）共培养球体发芽测定系统。共培养球体由两个人类血管细胞前体，内皮菌群形成细胞（ECFCs）和间质干细胞（MSCs）产生，比例为5比1。ECFCs_MSCs球体被嵌入到I型胶原蛋白基质中，以模拟体内细胞外环境。利用实时细胞记录仪连续观察24小时从球体发散血管生成过程，还应用了活细胞荧光标记技术，用于在发芽形成过程中处理每种细胞类型的定位。血管生成电位通过计算芽的数量和测量从单个球体产生的芽的累积长度来量化。每个实验组对五个随机选择的球体进行了分析。比较实验表明，与仅E氟氯化碳的球体相比，ECFCs_MSCs球体具有更大的发芽数和累积发芽长度。Bevacizumab，一种FDA批准的血管生成抑制剂，与新开发的共培养球形检测系统进行了测试，以验证其筛选抗血管生成药物的潜力。与仅ECFCs球体相比，ECFCs_MSCs球体的IC50值更接近于从异种移植肿瘤小鼠模型获得的bevacizumab的有效血浆浓度。本研究表明，3D ECFCs_MSCs球形血管生成测定系统与生理血管生成有关，可在动物实验前预测有效的血浆浓度。

比较实验使用单培养球体（仅限氟氯烃）和共培养球体（ECFCs_MSCs）进行，以检查MSC在氟氯化碳介导的血管生成中是否发挥相当大的作用。实时细胞记录器监测了每个球体的发芽形成24小时，可以捕获从球体中产生血管生成的过程。血管生成电位通过计算芽的数量和测量从单个球体产生的芽的累积长度来量化。对每个实验组随机选择的5个球体进行了分析。对于ECFCs_MSCs球体，发芽数量和累积发芽长?...

Watch this Scientific Journal Video about Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells at JoVE.com

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

三维共培养球形血管生成测定系统设计用于模拟生理血管生成。共培养球体由两个人类血管细胞前体ECFC和MSCs组成，并嵌入胶原蛋白凝胶中。新系统对血管生成调节器的评估是有效的，为体内研究提供了更多的相关信息。

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 ...

这个3D共培养球体系统旨在模仿生理血管生成。它将有效地评估潜在的血管生成调节器，并在体内研究之前提供可预测的信息。该系统利用由两个血管祖细胞形成的共培养球体，可用于研究细胞相互作用、发芽和生理血管发生管成熟。如果我们能使用患者细胞形成共培养球体，我们可以利用这个系统开发个性化的治疗异常血管生成相关疾病，如癌症。该测定系统可提高血管生成和药物开发研究的敏感性和效率。首先使用05%trypsin-EDTA将ECFC和MSC分别从80至90%的汇合细胞培养中分离三到五分钟。用新鲜完整的DMEM培养基灭活尝试曲，并移液细胞几次，产生单个细胞悬浮液。通过离心收集细胞，然后用无血清介质洗两次。第二次洗涤后，将ECFC稀释至每毫升中浓度的3倍10至6个细胞，将MSC稀释至每毫升两毫升微离心管中每毫米中浓度的2倍至6个细胞。通过离心来培养细胞，并小心地从每个细胞样本中吸出除了最后15至25微升的上经剂。接下来，从荧光染料试剂盒中重新悬浮250微升稀释剂C中的每个颗粒，并轻轻移液细胞悬浮液，以确保完全分散。将 250 微升新鲜准备好的 20 微摩尔 PKH67 添加到 ECFC 的管中。在 MSCs 的管中加入 250 微升 12 微摩尔 Pkh26。立即混合两个细胞悬浮液。在室温下5分钟后，在震动下，防止光线，停止染色过程，每管0.5毫升FBS孵育一分钟。在孵化结束时，通过离心收集细胞，并小心地去除上经剂，然后每次洗涤两毫升新鲜完整介质中洗两次。然后将细胞悬浮在15毫升管中。对于球类生成，第二次洗涤后，首先稀释细胞悬浮液在适当浓度内皮细胞生长介质MV2中，辅以20%甲基纤维素溶液和5%胎儿牛血清浓度。接下来，将每个细胞悬浮液添加到灭菌的聚苯乙烯矩形储液罐中。使用多通道移液器将大约 125 微升的细胞液滴添加到 150 毫米培养板的封面上。添加所有细胞后，将盖子倒置在含有 15 毫升 PBS 的培养皿底部，并在细胞培养培养箱中放置板 24 小时。第二天，用五毫升PBS将盘子盖冲洗成每盘50毫升的管子。用额外的五毫升 PBS 冲洗盖，收集任何剩余的球体，然后通过离心对球体进行颗粒状。小心丢弃所有，但最后100至200微升的上流液，并轻轻地敲按管壁，使球体自由悬浮在剩余的上流液。添加适当的体积内皮细胞生长介质MV2，辅以5%胎儿牛血清和40%甲基纤维素溶液到每个管，以每毫升100球体中等浓度重新分配球体。使用直径为 3 到 5 毫米的一毫升移液器尖端轻轻混合球形悬浮液。使用新的宽尖、一毫升移液器，将球状悬浮液与中和 I 型胶原蛋白凝胶混合，在冰上以一对一的比例使用。将每种球体悬浮胶原蛋白凝胶溶液的 900 微升添加到预热的 24 孔板的孔中。允许悬浮液在细胞培养培养箱中聚合30分钟，然后用100微升内皮细胞生长介质MV2覆盖球体，每毫升VEGF补充2.5%FBS和50毫微克，仅ECFC球类和中微量仅对MSC和ECFC+MSC球体培养剂进行补充。然后将每个板放在安装在细胞培养培养箱中的实时细胞记录器中，并随机聚焦在 10 倍放大的 5 到 10 个球形上，以每小时 24 小时监控每个荧光标记球体的发芽形成。要量化球体发芽，请将图像文件导入 ImageJ，并测量每个实验组从五个随机选择的球体中表达适当荧光信号的芽的数量和长度。对于ECFC加MSC球体，芽数和累积芽长度明显高于所有时间点的仅ECFC球类。仅 MSC 球体不会形成芽，但演示了 MSC 在球体之外的单独迁移。ECFC与绿色荧光染料和MSC与红荧光染料在结合产生ECFC-plus-MSC球体之前的标签表明，ECFC中端的芽结构被MSC覆盖，表明结合的MSC在芽形成过程中作为血管周围细胞发挥作用，通过两个血管细胞之间的紧密关联来增强芽的稳定性和耐久性。与控制ECFC加-MSC球体相比，使用血管生成抑制剂预处理的ECFC加MSC球体以剂量依赖的方式显示芽数和累积芽长度的减少。在平行实验中，ECFC只用抑制剂预处理的球体，然后用VEGF刺激，也表明生长因子引起的芽数和累积芽长度以剂量依赖的方式减少。值得注意的是，与仅ECFC的球类相比，需要更高的血管生成抑制剂浓度来抑制ECFC加MSC球体。与控制治疗动物和低剂量血管生成抑制剂治疗动物相比，在高剂量血管生成抑制剂治疗的动物中，肿瘤生长在人类异种移植肿瘤小鼠模型中受到显著抑制。高剂量治疗小鼠的贝瓦西祖马布血浆浓度为每毫升568正负40.62微克，接近贝瓦西祖马布的IC50值，用于抑制ECFC-plus-MSC球体的累积发芽长度，而不是仅ECFC类球体。这表明ECFC加-MSC共培养球类系统适合在动物研究之前预测有效的血浆浓度。使用宽尖、一 mL 移液器在冰上胶原蛋白凝胶中轻轻混合球类，对于保护球体的完整性非常重要。我们可以修改这个系统来引入其他细胞类型，如免疫细胞和其他细胞外基质，以研究血管生成期间的细胞-矩阵串扰。

Research

JoVE Journal

Developmental Biology

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

科研

发育生物学

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

从小鼠羊水细胞多能干细胞使用转座子系统的生产

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

通过细胞外囊泡/外来体转移乳腺基质上皮细胞和乳腺腔细胞之间的乳腺形成能力

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

主动脉环共培养法: 一种方便的工具, 可以评估骨髓间质细胞的血管生成潜能体外

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

水平整体安装：厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

犬卵巢间充质干细胞的提取与分化

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...