这项研究是由美国国家科学基金会职业奖，哈特韦尔基金会，和美国心脏协会（＃0830384N）的支持。

1。制备<ol><li>在有盖的仪器高压灭菌托盘下列事项： <ol><li>锯齿组织钳 - 对于处理单张组织</li><li>组织剪（8厘米） - 修剪单张组织和尖</li><li>棉签 - 从单张或风口浪尖上的内皮细胞层隔离</li></ol></li><li>设为无菌胶原酶溶液<ol><li>添加4.0克奶粉的DMEM 250毫升18MΩ的水。 </li><li>添加碳酸氢钠1.11克。 </li><li>新增（600 U / mL）的18万单位的胶原酶。 </li><li>添加1％（3ML）青霉素/链霉素。 </li><li>调整pH值至7.2的解决方案。 </li><li>带来的解决方案，270毫升。 </li><li>穿过一个0.2微米的过滤器消毒的解决方案。 </li><li>添加无菌胎牛血清的10％（30毫升）。 </li></ol></li><li>设为无菌内皮细胞的培养基<ol><li>添加6.7克奶粉的DMEM 400毫升18MΩ的水。 </li><li>添加碳酸氢钠1.85克。 </li><li>添加（50 U / mL）的25,000单位的肝素。 </li><li>添加1％（5毫升）青霉素/链霉素。 </li><li>调整pH值至7.2的解决方案。 </li><li>带来的解决方案，450毫升。 </li><li>穿过一个0.2微米的过滤器消毒的解决方案。 </li><li>添加无菌胎牛血清的10％（50毫升）。 </li></ol></li><li>使无菌间质细胞培养基<ol><li>新增13.37克的粉末状的DMEM 800毫升18MΩ的水。 </li><li>添加碳酸氢钠3.7克。 </li><li>添加1％（10毫升）青霉素/链霉素。 </li><li>调整pH值至7.2的解决方案。 </li><li>带来的解决方案，900毫升。 </li><li>穿过一个0.2微米的过滤器消毒的解决方案。 </li><li>添加无菌牛血清的10％（100毫升）。 </li></ol></li></ol> 2。心脏瓣叶的分离<ol><li>海关立即在无菌条件下从后牺牲的心脏主动脉根部。 </li><li>彻底冲洗用冷水无菌DPBS主动脉根部的血液。限制血管内皮细胞死亡和细菌污染，当务之急是尽快删除所有的血液成分。抗生素和antimycotics不建议在这个阶段，因为他们是潜在的有害的血管内皮细胞。 </li><li>直接从根，并在无菌的15 mL锥形管充满了12毫升的冷DPBS隔离瓣叶（阀3％）。摇动数次，以消除碎片，然后重新用新鲜的DPBS。 </li><li>运回实验室在冰上。 </li><li>到达实验室后，将与组织的无菌罩下的容器。 </li></ol> 3。内皮细胞层的分离<ol><li>填写每阀冷胶原酶溶液（3传单）3ML无菌的35mm菜。 </li><li>放入充满胶原酶溶液的菜从15 mL试管中的所有三个单张。 </li><li>组织孵育5-10分钟，在37 ° C。 </li><li>旋转到单张表面干燥无菌棉签，轻轻地取出的内皮细胞层。旋转和剪切应用量的方向是对样品的纯度至关重要。棉签旋转应在相反的方向直线运动你的手，创造一个控制剪切。这剪升降机从组织的血管内皮细胞。应用武力的金额应足以感受到组织的阻力，但无法穿透基底膜。 </li><li>偶尔，民建联棉签逐出从尖端纤维细胞内的胶原酶溶液。擦拭完成后，内皮细胞层的质地应该感到稍微比以前更顺畅。 </li><li>收集细胞悬液/胶原酶，并转移到一个新的无菌15ML管。 </li><li>在1000 rpm离心管5分钟颗粒任何分离的细胞，吸出上清液。如果隔离间质细胞，以及执行该协议，而这些细胞正在离心。 </li><li>加入3毫升15毫升管内皮猪中等，离心机，第二次和吸媒体。这第二个离心帮助过滤一些不需要的材料，如尖端纤维。 </li><li>重新暂停离心内皮细胞内皮猪的介质和板细胞在5毫升预涂层的T - 25与胶原瓶（使用1瓶每离心管）。 </li><li>让细胞生长改变内皮介质之前，至少有2-3天。这可以帮助细胞恢复分裂以来的隔离过程是相当苛刻和细胞产量可能低。这是附近汇合的传代细胞的关键，因为接触抑制，可能导致细胞转化。 </li></ol> 4。 60毫米培养皿侧具体隔离的制备<ol><li>线60毫米的玻璃培养皿中，用铝箔（2％leafle玻璃菜T）。铝箔有助于消除石蜡，使玻璃培养皿中可用于其他隔离重用。 </li><li>石蜡珠广场内的菜肴，约半满，蒸压盖。 </li><li>一旦完成高压灭菌是石蜡熔化，将菜合奏阴凉平坦的表面。由于石蜡冷却时，它就会变硬，并创建一个层，将支持针头刺破。 </li><li> 30分钟后，消毒盘可以作为一个隔离室，固定单张组织。 </li></ol> 5。买方指定的内皮细胞层的分离<ol><li>从15ML管和准备方具体隔离60毫米培养皿中删除的传单。 </li><li>操纵的小册子，使ventricularis面朝离开的fibrosa一面暴露的表面上的石蜡。为单张的边缘暴露的内皮细胞层。 </li><li>放置在每一个（向上的）内皮表面冷胶原酶几滴孵育5-10分钟的组织，在37 ° C。 </li><li>一如以往，由旋转到单张的表面干燥无菌棉签轻轻取出的内皮细胞层。旋转和剪切应用量的方向是对样品的纯度至关重要。棉签旋转应在相反的方向直线运动你的手，创造一个控制剪切。这剪升降机从组织的内皮细胞，别无其他。应用武力的金额应足以感受到组织的阻力，但不穿透内组织。 </li><li>偶尔，民建联棉签逐出从尖端纤维细胞内的胶原酶溶液。擦拭完成后，内皮细胞层的质地应该感到稍微比以前更顺畅。 </li><li>收集细胞悬液/胶原酶，并转移到一个新的无菌15ML管。标签上注明方的特殊性（可以汇集来自同一阀单张）。 </li><li>单张转移到一个新的培养皿中，使ventricularis方现在已经公开，并重复步骤（5.3-5.6）。 </li><li>一旦所有的细胞悬液/胶原酶的收集，在1000 rpm离心管5分钟颗粒任何分离的细胞和吸出上清液。如果隔离间质细胞，以及执行该协议，而这些细胞正在离心。 </li><li>加入3毫升猪血管内皮中管，离心机，第二次和抽吸介质。这第二个离心帮助过滤一些不需要的材料，如尖端纤维。 </li><li>重悬离心内皮细胞预涂层的T - 25瓶与拼贴（1％使用离心管瓶）内皮猪的介质和板细胞在5毫升。 </li><li>让细胞生长改变内皮介质之前，至少有2-3天。这可以帮助细胞恢复分裂以来的隔离过程是相当苛刻。如果您发现产量很低，考虑到一个较小的瓶或孔板，因为细胞 - 细胞黏附，促进细胞生长。请记住，通过附近汇合，因为接触抑制，可能导致细胞转化。 </li></ol> 6。间质细胞的分离<ol><li>填写胶原酶每个阀解决方案（3传单）10毫升无菌的15 mL离心管中。 </li><li>擦拭内皮细胞的传单后，立即将在适当的15ML管胶原酶溶液。 </li><li>孵育约12至18小时（如果需要的话，鼓动轻轻）。 </li><li>退化的组织与血清学移液器轻轻混合直到细胞悬液/胶原酶变得同质化。这种同质化，有助于打破了组织和间质细胞释放。 </li><li>在1000 rpm离心5分钟的消化组织，吸出上清液。 </li><li>添加5毫升间质性猪液15ML管，离心第二次，吸出上清液。 </li><li>重新暂停5毫升间质性猪的介质和板细胞离心内皮细胞的T - 75瓶（使用1％的离心管瓶）。 </li><li>让细胞生长改变的间质细胞液之前，至少有1-2天。将远远超过组织的内皮细胞，但预计的碎片。细胞也应该更快地成长，以汇合比的内皮细胞，因为细胞的产量和其性质。 </li></ol> 7。代表性的图像<img src="/files/ftp_upload/2158/2158fig1.jpg" alt="图1" /> 图1：在2-3天，后隔离分离出的细胞形态。 （一）血管内皮细胞表现出典型的内皮细胞的形态和形成集群，促进经济增长。 （二）维也纳国际中心的形态是类似的肌纤维母细胞，一般主轴形和均匀传遍烧瓶。 <pCLASS =“jove_content”&gt; <img src="/files/ftp_upload/2158/2158fig2.jpg" alt="图2" /> 图2：在汇合分离的细胞形态。 （一）血管内皮细胞表现出典型的内皮形态，这是一般的鹅卵石和生长接触抑制。 （二）维也纳国际中心的形态是类似一般主轴形和不生长接触抑制肌纤维母细胞。 <img src="/files/ftp_upload/2158/2158fig3.jpg" alt="图3" /> 图3。分离的细胞的功能特点6。 （一）血管内皮细胞与乙酰化低密度脂蛋白摄取的高级别（红色）。 （二）电话与乙酰化低密度脂蛋白的摄取水平低。 <img src="/files/ftp_upload/2158/2158fig4.jpg" alt="图4" /> 图4 6分离的细胞的细胞标记。 （一）血管内皮细胞表型血管性血友病因子（绿色），蓝色（细胞核）染色阳性。 （二）电话表型血管性血友病因子的负染色。 <img src="/files/ftp_upload/2158/2158fig5.jpg" alt="图5" /> 图5分离的细胞的细胞标记。 （一）血管内皮细胞的表型是阿尔法SMA（绿色），蓝色（核）负染。 （二）电话表型为αSMA的阳性染色。 <table border="1"><tbody><tr><td> 解离剂 </td><td> 解离技术 </td><td> 细胞采集 </td><td> 细胞数量 </td><td> 细胞 纯度 </td><td> 污染 </td></tr><tr><td> EDTA（3毫米），不含氯化钙 2 </td><td> 5，20，60分钟。 氯化钙加前</td><td> 20，60，120分钟。收藏前</td><td> - </td><td> + / - </td><td> + + </td></tr><tr><td> EDTA（6mm）的无氯化钙 </td><td> 5，20，60分钟。 氯化钙加前</td><td> 20，60，120分钟。收藏前</td><td> + / - </td><td> + </td><td> + + </td></tr><tr><td>胰蛋白酶EDTA（0.5 g / L的） </td><td> 5，10，15分钟。 前停用</td><td>中等收集立即</td><td> + </td><td> + </td><td> + </td></tr><tr><td>胶原酶II（300 U / mL的） </td><td> 5，10，15分钟。 前停用</td><td>中等收集立即</td><td> - + + + </td><td> - + + + </td><td> + </td></tr><tr><td>胶原酶Ⅱ（600单位/毫升） </td><td> 5，10，15分钟。 前停用</td><td>中等收集立即</td><td> + + + + + </td><td> + + + + - </td><td> - </td></tr></tbody></table> 表1初步瓣膜内皮细胞的分离协议的结果。

<ol>
	<li>Thompson, R. P., Fitzharris, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+the+truncus+arteriosus+of+the+chick+embryo+heart:+the+formation+and+migration+of+mesenchymal+tissue.">Morphogenesis of the truncus arteriosus of the chick embryo heart: the formation and migration of mesenchymal tissue.</a> Am J Anat. 154, 545-556 (1979).</li><li>Johnson, C. M., Fass, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+cardiac+valvular+endothelial+cells+in+culture:+A+relative+deficiency+of+fibronectin+synthesis+in+vitro.">Porcine cardiac valvular endothelial cells in culture: A relative deficiency of fibronectin synthesis in vitro.</a> Lab Invest. 49 (5), 589-598 (1983).</li><li>Manduteanu, I., Popov, D., Radu, A., Simionescu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calf+cardiac+valvular+endothelial+cells+in+culture:+production+of+glycosaminoglycans,+prostacyclin+and+fibronectin.">Calf cardiac valvular endothelial cells in culture: production of glycosaminoglycans, prostacyclin and fibronectin.</a> J Mol Cell Cardiol. 20 (2), 103-118 (1988).</li><li>Cheunyg, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+isolating+and+purifying+porcine+aortic+valve+endothelial+cells.">Techniques for isolating and purifying porcine aortic valve endothelial cells.</a> JHVD. 17 (6), 674-681 (2008).</li><li>Paranya, G., Vineberg, S., Dvorin, E., Kaushal, S., Roth, S. J., Rabkin, E., Schoen, F. J., 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=Aortic+valve+endothelial+cells+undergo+transforming+growth+factor-beta-mediated+and+non-transforming+growth+factor-beta-mediated+transdifferentiation+in+vitro.">Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro.</a> Am J Pathol. 159 (4), 1335-1343 (2001).</li><li>Butcher, J. T., Penrod, A., Garcia, A. J. M., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+morphology+and+focal+adhesion+development+of+valvular+endothelial+cells+in+static+and+fluid+flow+environments.">Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments.</a> Arterioscler Thromb Vasc Bio. 24 (1), 1429-1434 (2004).</li><li>Simmons, C. A., Grant, G. R., Manduchi, E., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+heterogeneity+of+endothelial+phenotypes+correlates+with+side-specific+vulnerability+to+calcification+in+normal+porcine+aortic+valves.">Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves.</a> Circ Res. 96, 792-799 (2005).</li><li>Butcher, J. T., Tressel, S., Johnson, T., Turner, D., Sorescu, G., Jo, H., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Profiles+of+Valvular+and+Vascular+Endothelial+Cells+Reveal+Phenotypic+Differences:+Influence+of+Shear+Stress.">Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences: Influence of Shear Stress.</a> Arterioscler Thromb Vasc Biol. 26, 69-69 (2006).</li><li>Parachuri, S., Yang, J. H., Aikawa, E., Melero-Martin, J. M., Khan, Z. A., Loukogeorgakis, S., Schoen, F. J., 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+Pulmonary+Valve+Progenitor+Cells+Exhibit+Endothelial/Mesenchymal+Plasticity+in+Response+to+Vascular+Endothelial+Growth+Factor-A+and+Transforming+Growth+Factor-&#946;2.">Human Pulmonary Valve Progenitor Cells Exhibit Endothelial/Mesenchymal Plasticity in Response to Vascular Endothelial Growth Factor-A and Transforming Growth Factor-&#946;2.</a> Circ Res. 99 (8), 861-869 (2006).</li><li>Shi, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+circulating+bone+marrow-derived+endothelial+cells.">Evidence for circulating bone marrow-derived endothelial cells.</a> Blood. 92, 362-367 (1998).</li><li>Rehman, J., Li, J., Orschell, C. M., March, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+blood+endothelial+progenitor+cells+are+derived+from+monocyte/macrophages+and+secrete+angiogenic+growth+factors.">Peripheral blood endothelial progenitor cells are derived from monocyte/macrophages and secrete angiogenic growth factors.</a> Circulation. 107, 1164-1169 (2003).</li></ol>

技术隔离的困难和心脏瓣膜内皮细胞的培养纯人口已经受损的瓣膜生物学的理解。典型的隔离技术涉及的底层基础矩阵内皮胶粘剂债券2,3或化学分解酶消化。初步分离实验进行了定性评估，不同的解离剂和潜伏期。这些实验结果表明，EDTA（或胰蛋白酶- EDTA）的潜伏期长达60分钟撞出细胞不成功。然而，检索纯瓣膜内皮细胞（表1）的可用数量为5-10分钟collagense消化证明是有效的。其他酶解?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dulbecco&#x2019;s Modified Eagle Medium</td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>50-103-PB</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>26140</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200</td>
<td>&nbsp;</td>
<tr>
<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>H4784-1G</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase Type 2</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21300-058</td>
<td>&nbsp;</td>
<tr>
<td>Rat Tail Collagen</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>Critical Swabs</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89031-270</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>55761</td>
<td>&nbsp;</td>
<tr>
<td>T25 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353018</td>
<td>&nbsp;</td>
<tr>
<td>T75 Flasks</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>353136</td>
<td>&nbsp;</td>
<tr>
<td>24 Well Plate</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353047</td>
<td>&nbsp;</td>
<tr>
<td>60x15 mm Dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>25384-092</td>
<td>&nbsp;</td>
<tr>
<td>60x15 Glass Dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89000-310</td>
<td>&nbsp;</td>
<tr>
<td>Paraffin Embedding Wax</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>19304-01</td>
<td>&nbsp;</td>
<tr>
<td>Precision Glide Needles</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>305165</td>
<td>&nbsp;</td>
<tr>
<td>500 mL Nalgene Filters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>73520-985</td>
<td>&nbsp;</td>
<tr>
<td>1L Nalgene Filters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>73520-986</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11023-15</td>
<td>&nbsp;</td>
<tr>
<td>FSC Tweezers #5</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11295-00</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of valvular endothelial cells

心脏瓣膜是全权负责维持心血管系统的单向血液流动。这些薄，纤维组织受到显着的机械应力，因为他们打开和关闭一个寿命超过数十亿倍。这些组织的令人难以置信的耐力是，由于居民瓣膜内皮细胞（VEC）和间质细胞（VIC）的不断响应当地的机械和生物信号的修复和改造。直到最近，我们开始认识到这些细胞的独特的行为，在体外实验中起到了关键作用。尤其是具有挑战性的是血管内皮细胞的分离和文化。必须特别小心使用的那一刻起，该组织是由主机，最后通过电镀删除。在这里，我们目前的直接隔离，边具体隔离，文化，和核查的VEC纯种群的协议。我们使用温柔棉签刮技术打跑只是表面细胞的酶消化。这些细胞，然后收集到管到一个沉淀离心。然后再悬浮颗粒和镀到培养瓶中，与Ⅰ型胶原基质预涂。血管内皮细胞的表型是由接触证实，抑制细菌的生长和PECAM1（CD31），如内皮细胞特异性标志物的表达，血管性血友病因子（vWF），α-平滑肌肌动蛋白（α- SMA）的表达阴性。血管内皮细胞的功能特点与乙酰化低密度脂蛋白水平高。与血管内皮细胞，血管内皮细胞有独特的能力，转化为间质细胞，它通常发生在胚胎阀形成 1 。在显着延长在体外培养后融合，这也可能发生，所以应达到或接近汇合通行。维也纳国际中心纯种群的VEC隔离后，然后可以很容易获得。

Watch this Scientific Journal Video about Isolation of Valvular Endothelial Cells at JoVE.com

Isolation of Valvular Endothelial Cells

我们提供了隔离和培养心脏瓣膜内皮细胞（VEC）的纯种群的方法。血管内皮细胞可以分离出了风口浪尖或单张两侧及紧随，底层的间质细胞（VIC）的隔离很简单。

瓣膜内皮细胞的分离

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Research

JoVE Journal

Biology

20.2K 次观看.  Cornell University. 我们提供了隔离和培养心脏瓣膜内皮细胞（VEC）的纯种群的方法。血管内皮细胞可以分离出了风口浪尖或单张两侧及紧随，底层的间质细胞（VIC）的隔离很简单。

视频: Isolation of Valvular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

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, 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 often do not resemble vessels in vivo. Here we demonstrate an optimized 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. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

分离人脐静脉内皮细胞（HUVEC）

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

科研

生物学

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

禽流感胚胎瓣膜祖细胞的分离和培养

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

胚胎心室血管内皮细胞的分离

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

鼠标冠状动脉内皮细胞的分离

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

微型猪原主主动脉内皮细胞的隔离与培养

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

小鼠原发性肝正弦内皮细胞的分离与表征

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...