Name: assessment of the anticoagulant and anti-inflammatory properties of endothelial cells using 3d cell culture and non-anticoagulated whole blood
Uploaded: 2017-09-05T13:00:00
Description: Watch this Scientific Journal Video about Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood at JoVE.com

这项研究得到了瑞士国家科学基金会 (SNSF, 格兰特 No. 320030_156193) 的支持。作者感谢 Dr. Benoît Werlen 提供 Biosilon 载体珠。我们还感谢 Prof. 和 Prof. 安德烈 Haeberli 帮助建立载体珠模型。

德国长白猪 (野生型养殖在当地的农舍和转基因育种研究所的分子动物繁殖和生物技术学院, 德国慕尼黑, 路德维希-马西米兰大学, 重量介于30公斤到40公斤之间, 被用于这项研究。所有的动物都被安置在标准条件下的水和食物 ad lib 。所有动物实验都是按照《英国动物法》 (科学程序) 和《 NIH 指南》对实验动物的照顾和使用以及瑞士动物保护法进行的。所有动物研究符合到达的指南。县兽医...

<ol>
	<li>Rajendran, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+endothelium+and+human+diseases.">The vascular endothelium and human diseases.</a> Int. J. Biol. Sci. 9, 1057-1069 (2013).</li><li>Rajwal, S., Richards, M., O'Meara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+recalcified+citrated+whole+blood+-+a+pragmatic+approach+for+thromboelastography+in+children.">The use of recalcified citrated whole blood - a pragmatic approach for thromboelastography in children.</a> Pediatric Anesthesia. 14, 656-660 (2004).</li><li>Kohler, H. P., Muller, M., Mombeli, M., Mtraub, M. M., Maeberli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Suppression+of+the+Coagulation+of+Nonanticoagulated+Whole-Blood+in-Vitro+by+Human+Umbilical+Endothelial-Cells+Cultivated+on+Microcarriers+Is+Not+Dependent+on+Protein-C+Activation.">The Suppression of the Coagulation of Nonanticoagulated Whole-Blood in-Vitro by Human Umbilical Endothelial-Cells Cultivated on Microcarriers Is Not Dependent on Protein-C Activation.</a> Thromb. Haemost. 73, 719-724 (1995).</li><li>Biedermann, B., Rosenmund, A., Muller, M., Kohler, H. P., Haeberli, A., Straub, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+endothelial+cells+suppress+prothrombin+activation+in+nonanticoagulated+whole+blood+in+vitro.">Human endothelial cells suppress prothrombin activation in nonanticoagulated whole blood in vitro.</a> J. Lab. Clin. Med. 124, 339-347 (1994).</li><li>Banz, Y., Cung, T., Korchagina, E. Y., Bovin, N. V., Haeberli, A., Rieben, 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+cell+protection+and+complement+inhibition+in+xenotransplantation:+a+novel+in+vitro+model+using+whole+blood.">Endothelial cell protection and complement inhibition in xenotransplantation: a novel in vitro model using whole blood.</a> Xenotransplantation. 12, 434-443 (2005).</li><li>Cowan, P. J., d'Apice, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+activation+and+coagulation+in+xenotransplantation.">Complement activation and coagulation in xenotransplantation.</a> Immunology and Cell Biology. 87, 203-208 (2009).</li><li>Reitsma, S., Slaaf, D. W., Vink, H., van Zandvoort, M. A. M. J., oude Egbrink, M. G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+glycocalyx:+composition,+functions,+and+visualization.">The endothelial glycocalyx: composition, functions, and visualization.</a> Pflugers Arch. 454, 345-359 (2007).</li><li>Wiegner, R., Chakraborty, S., Huber-Lang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement-coagulation+crosstalk+on+cellular+and+artificial+surfaces.">Complement-coagulation crosstalk on cellular and artificial surfaces.</a> Immunobiology. 221, 1073-1079 (2016).</li><li>Bongoni, A. K., 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=Transgenic+Expression+of+Human+CD46+on+Porcine+Endothelium:+Effect+on+Coagulation+and+Fibrinolytic+Cascades+During+Ex+Vivo+Human-to-Pig+Limb+Xenoperfusions.">Transgenic Expression of Human CD46 on Porcine Endothelium: Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions.</a> Transplantation. 99, 2061-2069 (2015).</li><li>Miyazaki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+shear+stress+can+initiate+both+platelet+aggregation+and+shedding+of+procoagulant+containing+microparticles.">High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles.</a> Blood. 88, 3456-3464 (1996).</li><li>Wuensch, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+Sequences+of+the+Porcine+THBD+Gene+Facilitate+Endothelial-Specific+Expression+of+Bioactive+Human+Thrombomodulin+in+Single-and+Multitransgenic+Pigs.">Regulatory Sequences of the Porcine THBD Gene Facilitate Endothelial-Specific Expression of Bioactive Human Thrombomodulin in Single-and Multitransgenic Pigs.</a> Transplantation. 97, 138-147 (2014).</li></ol>

该模型适用于凝血相关研究, 可以分析凝血的不同方面及其与 EC 的相互作用。8在异种移植的研究中, 用人血9对不同基因修饰的猪 ECs 的抗凝血性能进行检测是一个有用的系统。
该协议最关键的步骤是确保微在开始实验之前的完整的细胞覆盖率 (70-100), 以及那些关于仔细收集和处理 non-anticoagulated 全血以避免过早血小板活化由于高剪切应力, ?...

血管内皮由一单层内皮细胞 (EC) 组成, 它能使血管的腔管排成一线。在生理状态下, 静止 EC 负责维持一个抗凝和抗炎环境。1这是由 EC 表面的抗凝和抗炎蛋白的表达所介导的。例如, 由异移植器官的缺血/再灌注损伤或血管排斥引起的 EC 活化导致内皮表面从抗凝和抗炎状态转变为 pro-coagulant 和炎国家.1
为了研究内皮和凝血因子之间的奇妙和复杂的相互作用,在体外模型, 模仿尽可能密切的在体内情况是非常可取的。传统的体外凝血分析的一个共同的局限是使用抗凝血液, 使得凝血介导的作用复柠檬酸全血不能重现结果可获得新鲜 non-anticoagulated 血。2此外, 在传统的平板细胞培养系统中, 不可能利用内皮的抗凝血特性, 因为不能达到每个血液体积的足够的内皮细胞表面。该模型通过在球形载体珠表面培养 ec, 克服了这些局限性, 使 ec surface-to-blood 比 #62; 16 厘米2/毫升可以达到, 这是类似的情况下小动脉或静脉,被描述为足以使血液在 EC 表面进行 "自然" 抗凝。3,4全血可以在不添加抗凝剂的情况下使用。血液标本可在实验过程中采集, 细胞因子、凝血因子和可溶性补体活化标志物均可检测和量化。此外, 还可以通过共聚焦显微镜对补体和免疫球蛋白的沉积以及 ec 活化标记物的表达进行分析, 对 ec 包覆的载体珠进行研究。另一个有趣的应用包括测试药物, 这应该是防止内皮细胞活化, 从而, 凝血。5虽然这个模型不能完全取代动物实验, 它提供了一种方法来测试特定的功能假说,前体使用细胞, 从而减少了动物的数量, 用于基础研究的缺血/再灌注损伤或 (异) 移植。
用该模型模拟了猪主动脉内皮细胞 (PAEC) 在载体念珠上生长并与整个 non-anticoagulated 人血培养的异种移植环境。不同的转基因 PAEC, 携带了一些人类基因, 如 CD46 调节的补充系统和/或血栓调节蛋白 (hTBM) 的调节凝血系统, 分析了他们的抗凝血性能。内皮细胞活化、补体和凝血系统受到严密控制和相互连接。6因此, 重要的是要了解不同的转基因细胞在接触人体血液后的行为与黏附分子的表达和细胞因子的释放、糖的脱落以及抗凝蛋白的丧失有关。7

<table>
	<tbody>
		<tr>
			<td>PAEC</td>
			<td></td>
			<td></td>
			<td>Isolated from pig aorta in our Lab</td>
		</tr>
		<tr>
			<td>Microcarrier beads (Biosilon)</td>
			<td>Thermo Fisher Scientific</td>
			<td>160-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I, Bovine</td>
			<td>Gibco</td>
			<td>A10644-01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>21885-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma</td>
			<td>M7528</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>32404-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral tubes</td>
			<td>Sarstedt</td>
			<td>02.1726.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tubes</td>
			<td>Simport</td>
			<td>T406-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stirrer flasks</td>
			<td>Tecnomara</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biological stirrer</td>
			<td>Brouwer</td>
			<td>MCS-104S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti CD31</td>
			<td>R&#38;D Systems</td>
			<td>MAB33871</td>
			<td>Dilution 1:100</td>
		</tr>
		<tr>
			<td>Goat anti-rat IgG-Cy3</td>
			<td>Jackson Immuno Research</td>
			<td>112-166-003</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Goat anti VE-cadherin</td>
			<td>Santa Cruz</td>
			<td>sc-6458</td>
			<td>Dilution 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti vWF</td>
			<td>DAKO</td>
			<td>A0082</td>
			<td>Dilution 1:100</td>
		</tr>
		<tr>
			<td>Dk anti Gt IgG &#8211; Alexa 488</td>
			<td>Molecular Probes</td>
			<td>A11055</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Goat anti Rabbit - FITC</td>
			<td>Southern Biotech</td>
			<td>4050-02</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>32670-25MG-F</td>
			<td>Dilution 1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>Concentration: 10% in cell culture medium</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Becton Dickinson</td>
			<td>367286</td>
			<td>Size: 0.8x19 mm (21&#610; 3/4&#34;)</td>
		</tr>
		<tr>
			<td>Adapter</td>
			<td>Sarstedt</td>
			<td>14.1205</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM 710</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Image J</td>
			<td></td>
			<td>Available for free at: https://imagej.net</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium SupplementMix</td>
			<td>PromoCell</td>
			<td>C-39216</td>
			<td>2mL in 500mL of DMEM</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Concentration: 1% in cell culture medium</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td>Concentration: 1% in cell culture medium</td>
		</tr>
		<tr>
			<td>Heparinun natricum</td>
			<td>Drossa Pharm AG</td>
			<td></td>
			<td>Stock concentration: 5000 U.I./ mL</td>
		</tr>
		<tr>
			<td>Chamberslides</td>
			<td>Bioswisstec AG</td>
			<td>30108</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 x 2H2O</td>
			<td>Merck</td>
			<td>2382.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 x6 H2O</td>
			<td>Merck</td>
			<td>1.5833.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Axon Lab AG</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycergel mounting medium</td>
			<td>Dako</td>
			<td>C0563</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of the anticoagulant and anti-inflammatory properties of endothelial cells using 3d cell culture and non-anticoagulated whole blood

在体内, 内皮细胞是血液循环的自然抗凝血的关键。因此, 内皮细胞活化导致凝血。这种现象在许多临床情况下被观察, 象器官移植在存在预先 anti-donor 抗体, 包括异种移植, 并且在缺血或再灌注损伤。为了减少动物实验根据3R 标准 (减少, 替换和提炼),在体外模型研究内皮细胞活化作用对凝血是非常可取的。然而, 普通的平板系统的内皮细胞培养提供了一个表面积的比例为 1-5 厘米2的内皮每毫升的血液, 这是不足够的自然, 内皮介导的抗凝。在载体珠上培养内皮细胞可使表面积率提高到 40-160 cm2/毫升。这种增加的比例足以确保 "自然" 的全血抗凝, 从而可以避免使用抗凝剂。本文介绍了一种体外microcarrier-based 系统, 研究了猪内皮细胞的遗传修饰对全 non-anticoagulated 人血液凝固的影响。在所述的测定方法中, 原发猪主动脉内皮细胞, 无论是野生型或转基因的人 CD46 和血栓调节蛋白, 都生长在载体珠上, 然后暴露在新绘制的 non-anticoagulated 人血中。该模型可以测量和量化的细胞因子释放, 以及活化标志的补充和凝聚在血浆中。此外, 通过共聚焦显微镜对活化的内皮细胞进行成像, 并在 endothelialized 珠上进行免疫球蛋白、补体和凝血蛋白的沉积。这种化验也可以用来测试药物, 这是应该防止内皮细胞活化, 从而, 凝血。在它的潜力, 以减少动物的数量用于此类调查, 所述的化验是容易执行和一贯重现性。

在 7-10 天的文化在微调瓶 (图 1) 的细胞是汇合的, 覆盖整个表面的载体珠 (图 2)。70-100 状态的验证是一个重要的步骤, 因为 non-confluent 单层的 EC 在微将导致明显减少凝血时间, 因为载体珠的表面强烈 pro-coagulant (凝固时间: 4 ± 1 min) (图 3)。
另一个需要注意的重要...

Watch this Scientific Journal Video about Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood at JoVE.com

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

我们提出了一个体外模型, 允许对整个 non-anticoagulated 血液中的凝血进行研究和分析。该系统的抗凝血依赖于健康的内皮细胞的天然抗凝作用, 内皮细胞活化会导致血栓形成。

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