Name: a novel in vitro model for studying the interactions between human whole blood and endothelium
Uploaded: 2014-11-21T13:00:00.000+00:00
Duration: 6 min 27 s
Description: Watch this Scientific Journal Video about A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium at JoVE.com

这项研究是由瑞典研究理事会（90293501，A0290401，A0290402），欧盟第七框架计划资助下的赠款协议支持N°602699（的DirEKt），该NovoNordisk基金会，Gurli和爱德华Brunnberg基金会，干疗法，Vleugel基金会和阿克维贝格基金会。

注:血是从健康人通过审批，在乌普萨拉伦理审查委员会（许可证号二百六十四分之二千○八）开放式系统静脉穿刺抽。 1.种子细胞<ol><li>培养原代人脐静脉内皮在T75烧瓶细胞（HUVEC），在CO 2的37℃，5％涂布在PBS pH 7.4的1％明胶。 </li><li>除去从T75烧瓶中含有内皮细胞（或其它类型的原代人内皮细胞）培养液，用2.1毫摩尔EDTA洗细胞在pH 7.4的PBS。在37℃下3分钟 - 加入1ml 0.25％胰蛋白酶-EDTA，并孵育2分离细胞。 </li><li>通过漂洗用9ml 10％FBS的烧瓶中在pH 7.4的PBS收集细胞，将细胞悬浮液转移到15毫升管。降速细胞在735×g离心5分钟。 </li><li>除去上清液，小心重悬细胞沉淀在血管内皮细胞生长培养基微血管（EC GMMV）和细胞计数在Bürker室。种子21000çELLS /厘米2 1孔细胞培养玻片孵育在37℃下。培养2天 - 在HUVEC后，将进行1汇合。监控在光学显微镜下的细胞形态和汇合每日。 </li></ol> 2.制备全血<ol><li>制备按照生产商的说明所有人工表面，就可以在接触血液与固定化肝素（HS）的双层。从防尘CHS涂覆材料可以在室温下保存6个月而不丧失功能。 </li><li>使用开放系统静脉穿刺在开始血液内皮室实验前抽血从健康供体不久（&lt;30分钟）。夫妇皮下注射针头（18G的×50毫米），以CHS涂覆的硅管（内径:2mm）的前仔细收集血液中的CHS涂覆50ml管中。补充血液与肝素达到0.75单位/ ml的终浓度。通过英伟混血轻轻rting管2 - 3次。 </li></ol> 3.血液内皮商会型号<ol><li>制造血腔（ 图1A;顶视图）中的丙烯酸聚甲基丙烯酸甲酯（PMMA）组成的两个汽缸（高度:8mm和半径9毫米）被粘到一个聚甲基丙烯酸甲酯滑动件（25×75毫米）中。适合朝上用橡胶O形环，所述滚筒的边缘以密封血室对载玻片与培养的内皮细胞（ 图1B）。旋转连接到具有内皮细胞的培养载玻片此后在垂直位置（ 图1C）的血液腔室。 </li><li>使用CHS涂枪头转移1.5毫升血涂成室，注意不要激活血液中的每个社区卫生服务。 </li><li>转让1毫升血入含有30微升0.34 MK 3 EDTA离心管使用达到10毫米K 3 EDTA终浓度测定初始血浆蛋白浓度酶联免疫吸附测定（ELISA）。小心地混合血液和保持管在冰上。 </li><li>除去根据制造商的说明将细胞培养玻片的塑料壁，并仔细在pH 7.4的PBS洗涤细胞。确保随后从载玻片除去尽可能多的液体成为可能。不要让细胞干出来的。 </li><li>广场上的血室顶部的幻灯片，面临的血细胞。通过将夹具周围的血液内皮细胞的腔室固定在滑动。使用塑料滑动以获得更多支持放置夹具时，为了避免细胞培养载玻片破裂。 </li><li>固定血内皮细胞室到一个旋转轮（40厘米直径）发生在37℃的水浴中。旋转的轮子每个室，在0.5×g离心30分钟。 </li><li>培养结束后，拆除血液内皮细胞室。洗PBS pH值7.4的细胞培养玻片和固定细胞冰冷的1％多聚甲醛（PFA）F或10分钟。转印取血1ml，从每个室含有30微升0.34 MK 3 EDTA的微量离心管中，以达到10mM的K 3 EDTA终浓度和混合管轻轻。置于冰上管。 </li><li>离心管在4560 XG在4℃下10分钟。转让血浆，以新的离心管中，并存储在-70°C，直到进一步的分析。 </li></ol>血液内皮细胞相互作用4.分析<ol><li>用小鼠抗人CD16，再进行二次山羊抗小鼠的Alexa 488和鬼笔环肽 - TexasRed进行免疫荧光染色。进行1小时，在室温下每染色步骤，并在每一步之间的pH 7.4的PBS冲洗载玻片。染液用DAPI对细胞核，在室温下10分钟。分析结合到内皮细胞通过荧光显微镜结合使用适当的图像分析软件，图像分析诸如CellProfiler血液成分。 </li><li>孔定量FY在血液舱与凝血酶 - 抗凝血酶（TAT），酶联免疫吸附反应按照生产商的血浆样品的说明书。 </li><li>用适当的统计工具， 如:，非配对t检验，对数据进行分析。 </li></ol>

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	<li>Mestas, J., Hughes, C. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+Mice+and+Not+Men:+Differences+between+Mouse+and+Human+Immunology.">Of Mice and Not Men: Differences between Mouse and Human Immunology.</a> J Immunol. 172 (2), 2731-2738 (2004).</li><li>Seok, J., 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=Genomic+responses+in+mouse+models+poorly+mimic+human+inflammatory+diseases.">Genomic responses in mouse models poorly mimic human inflammatory diseases.</a> Proc Natl Acad Sci U S A. 110 (9), 3507-3512 (2013).</li><li>Hong, J., Ekdahl, K. N., Reynolds, H., Larsson, R., Nilsson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+in+vitro+model+to+study+interaction+between+whole+blood+and+biomaterials.+Studies+of+platelet+and+coagulation+activation+acid+the+effect+of+aspirin.">A new in vitro model to study interaction between whole blood and biomaterials. Studies of platelet and coagulation activation acid the effect of aspirin.</a> Biomaterials. 20 (7), 603-611 (1999).</li><li>Doctor, A., Spinella, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Processing+and+Storage+on+Red+Blood+Cell+Function+In.">Effect of Processing and Storage on Red Blood Cell Function In.</a> Vivo. Semin Perinatol. 36 (4), 248-259 (2012).</li><li>Andersson, J., 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=Optimal+heparin+surface+concentration+and+antithrombin+binding+capacity+as+evaluated+with+human+non-anticoagulated+blood+in+vitro.">Optimal heparin surface concentration and antithrombin binding capacity as evaluated with human non-anticoagulated blood in vitro.</a> J Biomed Mater Res A. 67 (2), 458-466 (2003).</li><li>Ekdahl, K. N., Hong, J., Hamad, O. A., Larsson, R., Nilsson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+blood+compatibility+of+materials,+cells,+and+tissues:+basic+concepts,+test+models,+and+practical+guidelines.">Evaluation of the blood compatibility of materials, cells, and tissues: basic concepts, test models, and practical guidelines.</a> Adv Exp Med Biol. 735, 257-270 (2013).</li><li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+dynamics+of+the+endothelium.">Spatial and temporal dynamics of the endothelium.</a> J Thromb Haemost. 3 (7), 1392-1406 (2005).</li><li>Cines, D. B., 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=Endothelial+cells+in+physiology+and+in+the+pathophysiology+of+vascular+disorders.">Endothelial cells in physiology and in the pathophysiology of vascular disorders.</a> Blood. 91 (10), 3527-3561 (1998).</li><li>Kirchhofer, D., Tschopp, T. B., Hadvary, P., Baumgartner, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+stimulated+with+tumor+necrosis+factor-alpha+express+varying+amounts+of+tissue+factor+resulting+in+inhomogenous+fibrin+deposition+in+a+native+blood+flow+system.+Effects+of+thrombin+inhibitors.">Endothelial cells stimulated with tumor necrosis factor-alpha express varying amounts of tissue factor resulting in inhomogenous fibrin deposition in a native blood flow system. Effects of thrombin inhibitors.</a> J Clin Invest. (5), 2073-2083 (1994).</li><li>Esmon, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+circuits+in+thrombosis+and+inflammation.">Molecular circuits in thrombosis and inflammation.</a> Thromb Haemost. 109 (3), 416-420 (2013).</li><li>Rosenberg, R. D., Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular-Bed&#8211;Specific+Hemostasis+and+Hypercoagulable+States.">Vascular-Bed&#8211;Specific Hemostasis and Hypercoagulable States.</a> N Engl J Med. 340 (20), 1555-1564 (1999).</li></ol>

The authors have nothing to disclose.

<html>血液和血管壁之间的多重相互作用，通常是由于血管内皮细胞的非粘性和抗血栓形成的性质保持在静止状态。7在涉及炎症的病理条件下，内皮细胞与一个结果增加了粘附受体表达8激活和能力下降，抑制止血9激活的级联系统中的血液又将放大内皮细胞的血栓造成进一步的血栓形成和白细胞招聘10至获得的内皮细胞和全血之间的这种微妙的互动更好地了解，我们有开发的体外方法的新型其中培养的内皮细胞被放置在与全血接触。就我们所知，我们的设置是第一个显示孵育的人全血，没有或很少抗凝剂为更长的时间周期的内皮细胞。 核苷酸"&gt;该系统的灵敏度是由开放系统静脉穿刺术使能结合固定化的肝素对放置在与血液接触的所有表面的保护层。静脉穿刺过程中的开放式系统的过程中血液采购的使用减少了级联系统的激活而允许使用的抗凝血剂的自确定量。市售的真空血液试管可能，但是，可根据不同的研究的目的终点所用。抗凝剂添加到大多数市售的封闭系统管中的最终浓度可以抑制敏感和以其他方式难以学习，相互作用在设定6的保护肝素表面进一步消除了活化的血液通过由比内皮细胞以外的其他表面上的表面接触。5实际上，全血补充有少量肝素的培养室内没有CHS的保护导致血块形成。 <p类="jove_content"&gt;气泡的腔室内部可以使血液的混合培养的内皮细胞中。为了评估气泡尺寸的效果，我们使用两种不同的血液容量。在这个模型中，我们在腔室孵育的HUVEC测定类似的TAT水平不管气泡大小。 TAT是，然而，随着一个小气泡尺寸的充分的CHS处理腔室。此，按照先前所描述的内皮细胞的特性如图11所示 ，表明凝血活性增加由缺乏在惰性CHS室中的内皮细胞提供时的调节作用。气泡中，系统创建于腔室的旋转流。气泡的大小会影响到该旋转系统内的力。这示出由我们在图2D的结果是一个更小的气泡产生代表经血液即下降力更高的TAT值，使血液运动内的腔室是较低并由此激活级联系统的出现，以更高的程度。应当提到，在本系统中，我们创建了一个旋转流，就可以在不同速度沿腔体沿着墙壁和最低的中心的最高速度。这显然​​不是关于流动和剪切应力对内皮细胞的最佳环境，但我们仍然有一个系统产生稳定的和可重复的结果。更理想的情况应包括循环在没有湍流的层流。这是，然而，不可能在这里所表示的模型，并以我们的知识这样的系统不提供更新。虽然有几种微流体系统可商购是不可能用全血，无或低的水平抗凝剂添加到系统中的结合，从而不使相互作用的适当敏感的评价存在于血液和内皮细胞的所有部件之间的。 此外，有一个增加2倍于招聘血细胞对TNFα活​​化的HUVEC结合有加倍形成的TAT独立的血液量。这验证了该模型系统的稳定性，同时还示出了使用活化的内皮细胞与全血组合的可能性。未来上，血内皮细胞腔室可用于通过多种表达于各种条件和活化的阶段下的细胞标志物的研究对活化的内皮细胞的血细胞募集。此外，该模型可以用于在用药剂组合，以评价对炎症的影响。 总之，我们显示结合人血和血管细胞的腔室模型来创建在体外系统中的完整的人来执行血管疾病有关的调查并大有裨益。

方法分析在心血管疾病的血液血管的相互作用通常涉及的动物实验研究。在实验研究的动物模型中创建的结果可能，但是，有很少或没有含意为人类疾病。1,2-因此，有必要对在体外模型中良好和可靠的调查血液舱和血管内皮细胞之间的细胞相互作用一类人制度。因此，我们已经建立了一个血内皮细胞腔模型。这是基于用于研究生物材料和人全血之间的相互作用以前描述的模型。3相反，其他的体外设置在那里通常是有限的纯化组分，对数，即，血小板，白细胞或内皮细胞是可用的，在本模型中包含的所有各组分存在于血管中。 血内皮细胞C中的设置ELL室模型是为了让使用新鲜抽取的人血液中很少或根本没有添加抗凝剂。血液在较长的时间内存储收购所谓的存储病灶，其中红细胞的破裂可能与血液和血管内皮细胞之间的微妙交互干扰。4 为了避免处理的全血离体中凝块的形成需要或高剂量的抗凝血剂，或在与血液接触的所有材料必须是不激活的。作为非活化表面是罕见在实验室环境中，材料可以替代地设有固定化肝素的保护层。固定化肝素的保护层（以下简称为Corline肝素表面（CHS）），其可以被应用到大多数材料创造一个表面，其中血液接触可以发生而不引起凝血级联的活化。5。因此，通过提供所述腔室与CHS，血液中内皮CELL室模型允许使用非常低浓度的抗凝血剂的血液中。避免加入高浓度的抗凝血剂的血液内皮室模型的使得能够研究在血管内，否则可能会被屏蔽敏感的相互作用。6 血液内皮细胞室模型包括通过将塑料圆柱体形成的两个腔室（高度:8毫米;半径:9毫米），一个塑料显微镜载玻片。朝上的气缸的边缘都配有该配有用于密封腔室对细胞培养片橡胶O形环的槽。所述腔室仅部分填充有血液的左腔室中的空气保持血液中运动时，腔室中的垂直位置（ 图1）随后被转动。 为了评估该模型的功能，我们进行了与任一个完全的CHS涂布实验室或人脐静脉内皮细胞（HUVEC）。两个单独的血液量进行测定和凝血酶抗凝血酶（TAT）复合物（凝结的间接标记物）的形成，测定用酶联免疫吸附试验（ELISA）。然后我们评价了血液体积和肿瘤坏死因子α（TNFα），通过图像分析刺激对白细胞募集的内皮细胞的影响。 <img alt="图1" src="/files/ftp_upload/52112/52112fig1highres.jpg" /> 图1.设置血液内皮细胞腔模型。 （A）顶视图血室PMMA制成的示意图。（B）原代人内皮细胞上的1孔玻片培养。人的新鲜全血加入到两个室在显微镜载玻片上。用于处理血液室，所有的材料都被有保护HS涂层。除去策的壁后LL培养载玻片，细胞被置于面向血液和系统被夹紧关闭。（℃）的血液内皮细胞的腔室，然后在旋转下在37℃温育。之后，血液中的隔室中的反应可通过ELISA进行分析，并在内皮细胞中可通过显微镜进行分析。

<table><tbody><tr><td>Human Umbilican Vein Endothelial Cells (HUVEC)</td><td>PromoCell</td><td>C-12200</td><td>Any other type of primary human endothelial cell may be used.</td></tr><tr><td>Endothelial Cell Growth Medium MV (ECGM MV)</td><td>PromoCell</td><td>C-22120</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G-2500</td><td>Prepare 1% gelatin in PBS, incubate 5 ml in a T75 for 15 min and remove before culturing cells.</td></tr><tr><td>TAT ELISA</td><td>Enzyme Research Lab.</td><td>TAT-EIA-C</td><td></td></tr><tr><td>Mouse anti-human CD16</td><td>DAKO</td><td>F7011</td><td>Dilution 1:100</td></tr><tr><td>Goat anti-mouse Alexa 488</td><td>Molecular Probes</td><td>A11001</td><td>Dilution 1:500</td></tr><tr><td>Phalloidin-Texas Red</td><td>Molecular Probes</td><td>A22287</td><td>Dilution 1:200</td></tr><tr><td>DAPI</td><td>Molecular Probes</td><td>D1306</td><td>Dilution 10 &amp;mu;g/ml</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E-6758</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Gibco Life Tecnologies</td><td>25200-056</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco Life Technologies</td><td>10437</td><td></td></tr><tr><td>1 well cell culture slides</td><td>BD Falcon</td><td>354101</td><td></td></tr><tr><td>Blood Chambers</td><td>NA</td><td>NA</td><td>Blood Chamber may be manufactured in PMMA or any other plastic able to bind CHS</td></tr><tr><td>Clamps</td><td>Office Depot</td><td>2052204</td><td></td></tr><tr><td>Corline Heparin Surface (CHS)</td><td>Corline Systems AB</td><td>945-00</td><td></td></tr><tr><td>Hypodermic needle</td><td>Terumo</td><td>NN-1850R</td><td>Size: 18 G x 5 mm</td></tr><tr><td>Unfractionated Heparin (UFH)</td><td>Leo Pharma</td><td>585679</td><td></td></tr><tr><td>K&lt;sub&gt;3&lt;/sub&gt;EDTA</td><td>Alfa Aesar</td><td>1709958</td><td>Prepare a 0.34 M stock solution in milliQ H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>PFA</td><td>Sigma-Aldrich</td><td>P-6148</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon</td><td>80i</td><td></td></tr><tr><td>Light microscope</td><td>Nikon</td><td>TS100</td><td></td></tr><tr><td>Image analysis software</td><td>Broad Institute</td><td>CellProfiler</td><td>Available for free at www.cellprofiler.org</td></tr></tbody></table>

a novel in vitro model for studying the interactions between human whole blood and endothelium

大多数的所有已知的疾病都伴有心血管系统的疾病。研究入期间心血管病理活化的相互作用途径的复杂性，然而，受限于缺乏鲁棒和生理相关的方法。以模型的病理血管事件我们已经开发了一种在体外测定法用于研究内皮细胞和全血之间的相互作用。该测定法包括原代人内皮细胞，其被放置在与人全血接触的。该方法利用天然血液，没有或很少抗凝剂，使存在于血管内的分子和细胞成分之间微妙的相互作用的研究。 我们通过培养具有或不具有人脐静脉内皮细胞（HUVEC）的不同血液体积比较活化凝血研究该测定的功能。而一个更大的血量贡献增加抗凝血酶的形成（TAT）复合物，血管内皮细胞的存在导致凝血的活化减少。此外，我们采用白细胞附着的图像分析，以内皮细胞刺激肿瘤坏死因子α（TNFα），并发现CD16 +细胞的存在是显著更高的TNFα刺激的细胞相比，经血液接触的未刺激的细胞。最后，该分析可用于研究血管病症，其中血管内皮细胞和血液舱之间的相互作用被扰动。

CHS涂料的必要性为了测量特定于那些由内皮细胞引起的反应中的全血，用于血内皮细胞的腔室的所有材料必须被配备在使用前一个CHS涂层。 图2A（左侧）示出了未涂覆的腔室在30分钟后有一条清晰可见的凝块目前血液接触。处理，另一方面与CHS腔室（ 图2A，右）保护血液从不受控制的活化，从而确保其结果是由于内皮细胞-血液相互作用。 在会议厅内血容量的影响凝固的激活更可能在瘀血作为激活的凝血因子之间相互作用的可能性增大。在血液内皮细胞室模型中，血液被保持在运动中，由于气泡的腔室内部的循环。邻的rDer以确定改变血液量的效果，因此也气泡大小，CHS涂覆室连接到已用1.5ml或1.75毫升全血30分钟，测试一个CHS涂载玻片上。的血液的体积，而不由气泡的任何移动是的2.5倍时1.75毫升的血液加入到腔室时相比，将1.5ml血液时（ 图2B）。所测量的TAT-值建议增加的TAT-形成具有增加血液体积（ 图2D）。当HUVEC细胞（ 图2C）一起孵育或者1.5或1.75毫升全血，无区别指出，两组之间，这表明内皮细胞可调节凝血的活化（ 图2D）。 TNFα对血细胞募集的影响及凝血激活为了研究TNFα对白细胞募集的影响，HUVEC在治疗ED与20毫微克/毫升TNFα4小时前血液接触。血液接触-同为1.5 ml或1.75毫升血-再继续进行30分钟，之后将培养载玻片染色的CD16（ 图2F），成像和CD16 +细胞的数量进行定量。 CD16 +细胞的募集与TNFα的治疗（ 图2E），从100到256的CD16 +细胞/ mm 2的（P &lt;0.0001），用1.5ml血液显著增加，从172到378（P &lt;0.0001），与1.75毫升血液。 TAT复合物的形成，测定血液中的相应的血浆样品一起孵育或者TNFα处理或未经处理的细胞（ 图2G）中。 TNFα刺激的细胞中诱导的TAT复合物的近似加倍相比，未经处理的细胞。 <img alt="图2" src="/files/ftp_upload/52112/52112fig2.jpg" width="600" /> 无花果茜2:血内皮细胞室模型的功能。 （A）钱伯斯带或不带CHS培养与全血。未经CHS，30分钟后形成了凝块。凝血酶-抗凝血酶（TAT）复合物，凝血功能的一个间接指标，血培养，而不CHS测量&gt; 6000微克/毫升。（B）的血液量没有跟上的旋转气泡移动将随不同的血量。通过加入1.5毫升的血液，该体积将0.3毫升，而这将增加至0.74毫升，加入1.75毫升（C）的 HUVEC细胞成像与汇合单层的前相差显微镜显示了典型的内皮细胞形态学血液接触。（D）TAT值完全CHS涂层室表现出细微的差别时，添加不同的血量。加入1.5ml的血液导致35±11微克/毫升（7例）相比，1.75毫升，这就造成了8577; 70微克/毫升TAT（N = 8）。这种差异不再存在时的HUVEC孵育以相同的血液量; 66±55微克/毫升为1.5毫升（5例）和66±29微克/毫升为1.75毫升（7例）后的CD16 +细胞的数目。（E）的定量存在于任一未刺激或肿瘤坏死因子α刺激的血管内皮细胞血液接触。 CD16 +细胞的数量显著增加与TNFα刺激的细胞培养或者用1.5ml（基肥:100±40个/ mm;肿瘤坏死因子α:256±121细胞/毫米）或1.75毫升（基肥:172±67个/ mm; TNFα:全血378±185个/ mm）（F）的未刺激和TNFα刺激的HUVEC染色鬼笔环肽（红色代表图像），CD16（绿色）和DAPI（蓝色）。比例尺=250μm的（G）中的TAT复合物的形成，大致是由TNFα的一倍刺激的细胞相比，未处理的细胞培养用血液（1.5毫升:2。1±0.1倍以上的TAT，n = 3时; 1.75毫升:1.7±1.0倍以上的TAT，n = 4时）。所有值表示为平均值±标准差; P值的计算采用非配对t检验， <a href="https://www.jove.com/files/ftp_upload/52112/52112fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium at JoVE.com

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

一种新型<em&gt;在体外</em&gt;模型研究人类全血细胞间的相互作用

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

a-novel-vitro-model-for-studying-interactions-between-human-whole

Research

JoVE Journal

Immunology and Infection

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

9.4K 次观看.  Uppsala University. The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

视频: A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

内皮化微流体研究在血液病的微血管相互作用

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

科研

生物工程

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

免疫与感染

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

在生理体外模型和病理血流应用的血管细胞重塑调查

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

hiPSC 来源的内皮细胞的分化和机械刺激

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

作者聚焦：研究在流体介导的机械刺激下培养的 hiPSC 衍生的内皮细胞 

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

体外研究 胎儿人体血管芯片模型研究发育机械生物学

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides ...

发育生物学

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

研究流动下血管药物靶向的体外3D细胞培养动脉模型

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

3D 细胞培养和非抗凝全血对内皮细胞抗凝及抗炎作用的评价

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

医学

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

实时研究全血-内皮相互作用和血栓动力学的体外微流体疾病模型

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

一种新型

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内皮化微流体研究在血液病的微血管相互作用

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

在生理体外模型和病理血流应用的血管细胞重塑调查

在体外和体内模型来研究细菌粘附于血管壁下流动条件

体外研究胎儿人体血管芯片模型研究发育机械生物学

体外研究胎儿人体血管芯片模型研究发育机械生物学

体外研究胎儿人体血管芯片模型研究发育机械生物学

研究流动下血管药物靶向的体外3D细胞培养动脉模型

3D 细胞培养和非抗凝全血对内皮细胞抗凝及抗炎作用的评价

实时研究全血-内皮相互作用和血栓动力学的体外微流体疾病模型

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