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>

<ol>
	<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 border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:91px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:272px;">Comments/Description</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Human Umbilican Vein Endothelial Cells (HUVEC)</td>
			<td style="width:91px;">PromoCell</td>
			<td style="width:119px;">C-12200</td>
			<td style="width:272px;">Any other type of primary human endothelial cell may be used.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Endothelial Cell Growth Medium MV (ECGM MV)</td>
			<td style="width:91px;">PromoCell</td>
			<td style="width:119px;">C-22120</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Gelatin</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:119px;">G-2500</td>
			<td style="width:272px;">Prepare 1% gelatin in PBS, incubate 5 ml in a T75 for 15 min and remove before culturing cells.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">TAT ELISA</td>
			<td style="width:91px;">Enzyme Research Lab.</td>
			<td style="width:119px;">TAT-EIA-C</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Mouse anti-human CD16</td>
			<td style="width:91px;">DAKO</td>
			<td style="width:119px;">F7011</td>
			<td>Dilution 1:100</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Goat anti-mouse Alexa 488</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">A11001</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Phalloidin-Texas Red</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">A22287</td>
			<td>Dilution 1:200</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">DAPI</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">D1306</td>
			<td>Dilution 10 &#956;g/ml</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">EDTA</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">E-6758</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">0.25% Trypsin-EDTA</td>
			<td style="width:91px;">Gibco Life Tecnologies</td>
			<td style="width:119px;">25200-056</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:91px;">Gibco Life Technologies</td>
			<td style="width:119px;">10437</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">1 well cell culture slides</td>
			<td style="width:91px;">BD Falcon</td>
			<td style="width:119px;">354101</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Blood Chambers</td>
			<td style="width:91px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:272px;">Blood Chamber may be manufactured in PMMA or any other plastic able to bind CHS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Clamps</td>
			<td style="width:91px;">Office Depot</td>
			<td style="width:119px;">2052204</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Corline Heparin Surface (CHS)</td>
			<td style="width:91px;">Corline Systems AB</td>
			<td style="width:119px;">945-00</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Hypodermic needle</td>
			<td style="width:91px;">Terumo</td>
			<td style="width:119px;">NN-1850R</td>
			<td style="width:272px;">Size: 18 G x 5 mm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Unfractionated Heparin (UFH)</td>
			<td style="width:91px;">Leo Pharma</td>
			<td style="width:119px;">585679</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">K3EDTA</td>
			<td style="width:91px;">Alfa Aesar</td>
			<td style="width:119px;">1709958</td>
			<td style="width:272px;">Prepare a 0.34 M stock solution in milliQ H20</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">PFA</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-6148</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fluorescence microscope</td>
			<td style="width:91px;">Nikon</td>
			<td style="width:119px;">80i</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Light microscope</td>
			<td style="width:91px;">Nikon</td>
			<td style="width:119px;">TS100</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Image analysis software</td>
			<td style="width:91px;">Broad Institute</td>
			<td style="width:119px;">CellProfiler</td>
			<td style="width:272px;">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 ...

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Research

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Immunology and Infection

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

9.3K 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

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

科研

免疫与感染

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

复发性单纯疱疹性角膜基质性角膜炎小鼠模型研究人类HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

研究微生物群落<em&gt;在体内</em&gt;：主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

开发和鉴定人的中性粒细胞源性巨噬细胞新型亚群<em&gt;体外</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...