Name: hemocompatibility testing of blood-contacting implants in a flow loop model mimicking human blood flow
Uploaded: 2020-03-05T14:00:00
Description: Watch this Scientific Journal Video about Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow at JoVE.com

对于扫描电子显微镜的性能，我们感谢来自图宾根大学医院医学材料科学和技术部分的恩斯特·施魏泽。这项研究得到了国家可持续发展方案二（BIOCEV-FAR LQ1604项目）和捷克科学基金会第18-01163S项目（BIOCEV-FAR LQ1604）中CR的青年和体育部的支持。

血液取样程序经图宾根大学医学院道德委员会批准（项目识别代码：270/2010BO1）。所有科目均提供书面、知情同意，供在参与前加入。
1. 准备肝素装载的单体
<ol><li>将未稀释的肝素 （5，000 IU/mL） 与氯化钠（NaCl，0.9%） 混合溶液，并准备一个溶液，结果浓度为15 IU/mL的肝素。</li>
	<li>在每个中性单体（9 mL）中加入900μL的稀释肝素溶液，在血液取样后获得最终肝素浓度为1.5 IU/m...

<ol>
	<li>ISO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biological evaluation of medical devices. , (2002).</li><li>Weber, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-Contacting+Biomaterials:+In+Vitro+Evaluation+of+the+Hemocompatibility.">Blood-Contacting Biomaterials: In Vitro Evaluation of the Hemocompatibility.</a> Frontiers in Bioengineering and Biotechnology. 6, 99 (2018).</li><li>Li, Y., Boraschi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin+contamination:+a+key+element+in+the+interpretation+of+nanosafety+studies.">Endotoxin contamination: a key element in the interpretation of nanosafety studies.</a> Nanomedicine (Lond). 11 (3), 269-287 (2016).</li><li>Cattaneo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+investigation+of+chemical+properties+and+biocompatibility+of+neurovascular+braided+implants.">In vitro investigation of chemical properties and biocompatibility of neurovascular braided implants.</a> Journal of Materials Science: Materials in Medicine. 30 (6), 67 (2019).</li><li>Stang, 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=Hemocompatibility+testing+according+to+ISO+10993-4:+discrimination+between+pyrogen-+and+device-induced+hemostatic+activation.">Hemocompatibility testing according to ISO 10993-4: discrimination between pyrogen- and device-induced hemostatic activation.</a> Materials Science and Engineering: C Materials for Biological Applications. 42, 422-428 (2014).</li><li>van Oeveren, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obstacles+in+haemocompatibility+testing.">Obstacles in haemocompatibility testing.</a> Scientifica (Cairo). , 392584 (2013).</li><li>Engels, G. E., Blok, S. L., van Oeveren, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+blood+flow+model+with+physiological+wall+shear+stress+for+hemocompatibility+testing-An+example+of+coronary+stent+testing.">In vitro blood flow model with physiological wall shear stress for hemocompatibility testing-An example of coronary stent testing.</a> Biointerphases. 11 (3), 031004 (2016).</li><li>Blok, S. L., Engels, G. E., van Oeveren, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+hemocompatibility+testing:+The+importance+of+fresh+blood.">In vitro hemocompatibility testing: The importance of fresh blood.</a> Biointerphases. 11 (2), 029802 (2016).</li><li>Kaplan, O., 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=Low-thrombogenic+fibrin-heparin+coating+promotes+in+vitro+endothelialization.">Low-thrombogenic fibrin-heparin coating promotes in vitro endothelialization.</a> Journal of Biomedical Materials Research Part A. 105 (11), 2995-3005 (2017).</li><li>. 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该协议描述了一种全面可靠的方法，用于在模拟人类血流的剪切流模型中，根据ISO 10993-4对血液接触植入物进行血液相容性测试。这项研究基于激光切割神经血管植入物的测试，但可以通过各种样本进行。结果表明，该方法能够广泛分析各种参数，如血细胞计数、几种血合性标记的患病率以及血液接触后设备表面的微观可视化。使用此协议，可以检测到不同设备与系统相容性的潜在差异。
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植入物和生物材料与人的血液相互作用，在体内应用，需要严格的临床前测试，重点是调查止血系统的各种标记。国际标准化组织 10993-4 （ISO 10993-4） 规定了血液接触装置（即支架和血管移植物）评估的核心原则，并考虑了设备设计、临床效用和所需材料1。
人血是一种含有各种血浆蛋白和细胞的液体，包括白细胞（白细胞[WBCs]）、红细胞（红血球[RBC]）和血小板，在人体中执行复杂的功能2。异物与血液的直接接触可引起不良影响，如免疫或凝固系统的激活，可导致炎症或血栓并发症和植入后严重问题3、4、5。因此，体外血合一验证提供了在植入前发现和排除血液接触时可能引起的任何血液并发症的机会。
提出的流回路模型是为评估神经血管支架和类似装置的血管相容性而建立的，在管材中应用150 mL/min的流速（直径为3.2毫米），以模拟大脑流动条件和动脉直径2、7。除了需要一个最佳的体外模型外，血液来源是分析生物材料8的血液相容性时获得可靠和不变结果的重要因素。采集的血液应在取样后立即使用，以防止长时间储存引起的变化。一般来说，应使用21 G针进行无停滞的温和采集血液，以尽量减少血小板的预活化和血脉叠。此外，捐赠者排除标准包括吸烟、怀孕、健康状况不佳或在过去14天内服用口服避孕药或止痛药的人。
本研究描述了在流动条件下对支架植入物进行广泛溶容性测试的体外模型。当比较未涂覆的纤维布林-肝素涂层支架时，全面的溶性测试结果反映了纤维蛋白-肝素涂层支架9的溶性增强。相反，未涂层支架诱导凝固级联的激活，如血小板附着到支架表面引起的汤宾-抗血栓三 （TAT） 浓度增加和血小板数损失。总体而言，建议将此血效相容性模型集成到临床前测试中，以检测设备对止血系统造成的任何不利影响。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>aqua ad iniectabilia</td>
			<td>Fresenius-Kabi, Bad-Homburg, Germany</td>
			<td>1088813</td>
			<td></td>
		</tr>
		<tr>
			<td>beta-TG ELISA</td>
			<td>Diagnostica Stago, Duesseldorf, Germany</td>
			<td>00950</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Rotana 460 R</td>
			<td>Andreas Hettich, Tuttlingen, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrat monovettes (1.4 mL)</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>6,16,68,001</td>
			<td></td>
		</tr>
		<tr>
			<td>CTAD monovettes (2.7 mL)</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>367562</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA monovettes (1.2 mL)</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>6,16,62,001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol p.A. (1000 mL)</td>
			<td>AppliChem, Darmstadt, Germany</td>
			<td>1,31,08,61,611</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde (25 % in water)</td>
			<td>SERVA Electrophoresis, Heidelberg, Germany</td>
			<td>23114.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin coating for tubes</td>
			<td>Ension, Pittsburgh, USA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin-Natrium (25.000 IE/ 5 mL)</td>
			<td>LEO Pharma, Neu-Isenburg, Germany</td>
			<td>PZN 15261203</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiplate Reader Mithras LB 940</td>
			<td>Berthold, Bad Wildbad, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0,9%</td>
			<td>Fresenius-Kabi, Bad-Homburg, Germany</td>
			<td>1312813</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral monovettes (9 mL)</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>2,10,63,001</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer (w/o Ca2+/Mg2+)</td>
			<td>Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump ISM444B</td>
			<td>Cole Parmer, Wertheim, Germany</td>
			<td>3475</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (100 &#181;L)</td>
			<td>Eppendorf, Wesseling-Berzdorf, Germany</td>
			<td>3124000075</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (1000 &#181;L)</td>
			<td>Eppendorf, Wesseling-Berzdorf, Germany</td>
			<td>3123000063</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic container (100 mL)</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>7,55,62,300</td>
			<td></td>
		</tr>
		<tr>
			<td>PMN-Elastase ELISA</td>
			<td>Demeditec Diagnostics, Kiel Germany</td>
			<td>DEH3311</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl chloride tube</td>
			<td>Saint-Gobain Performance Plastics Inc., Courbevoie France</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction Tubes (1.5 mL)</td>
			<td>Eppendorf, Wesseling-Berzdorf, Germany</td>
			<td>30123328</td>
			<td></td>
		</tr>
		<tr>
			<td>neurovascular laser-cut implants</td>
			<td>Acandis GmbH, Pforzheim</td>
			<td>01-0011x</td>
			<td></td>
		</tr>
		<tr>
			<td>SC5b-9 ELISA</td>
			<td>TECOmedical, Buende, Germany</td>
			<td>A029</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Cambridge Instruments, Cambridge, UK</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealing tape (96 well plate)</td>
			<td>Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>15036</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 10/12 mL Norm-Ject</td>
			<td>Henke-Sass-Wolf, Tuttlingen, Germany</td>
			<td>10080010</td>
			<td></td>
		</tr>
		<tr>
			<td>TAT micro kit</td>
			<td>Siemens Healthcare, Marburg, Germany</td>
			<td>OWMG15</td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath Type 1083</td>
			<td>Gesellschaft f&#252;r Labortechnik, Burgwedel, Germany</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

hemocompatibility testing of blood-contacting implants in a flow loop model mimicking human blood flow

医疗设备（例如血管移植物、支架和心脏导管）越来越多地用于暂时或永久目的，这些装置仍留在人体的循环系统中，需要一种可靠和多参数化的方法，评估这些设备（即血液成分的激活和销毁）可能造成的造血并发症。血液接触植入物的体外血相容性综合测试是成功实施体内的第一步。因此，在临床应用之前，必须根据国际标准化组织 10993-4 （ISO 10993-4） 进行广泛的分析。所述的流回路描述了一个敏感模型，用于分析支架的止血性能（在本例中为神经血管），并揭示不利影响。使用新鲜的人类全血和温和的血液取样对于避免血液的预活化至关重要。血液通过含测试标本的肝化管中渗透，在37°C下以150 mL/min的速度使用蠕动泵60分钟。灌注前后， 血液学标记（即血细胞计数、血红蛋白、血细胞和血浆标记），指示白细胞（多态核[PMN]-弹性酶）、血小板（β-血栓球蛋白[-TG]）、凝血系统（通宾-抗血栓III[TAT]）和补数级联（SC5b-9）的。最后，在临床应用之前，我们提出了支架和其他血液接触装置广泛血液相容测试的基本可靠模型。

简要总结，人类整个血液被收集在肝素加载的单体，然后汇集，并用于评估细胞计数的基线水平以及血浆血相容性标记。
随后，装有神经血管植入样本的管子被填充，血液在150mL/min和37°C使用蠕动泵被灌注60分钟。再次，对所有组细胞数进行分析，并为ELISA分析制备血浆样本（图1）。血细胞和血液参数的定量，如血红蛋白和血细胞，直接在采血后，以及?...

Watch this Scientific Journal Video about Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow at JoVE.com

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

该协议描述了使用激光切割神经血管植入物对血液接触装置的全面血液相容性评估。采用新鲜肝化人血的流循环模型模拟血流。灌注后，对各种造血标记进行分析，并将与采血后直接获得的值进行比较，对被测装置进行血液相容性评价。

流循环模型中血液接触植入物的血液相容性测试模拟人类血流

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

该协议为根据国际标准组织指南的血液接触装置的血相容性调查提供了一个模型。该模型非常适合模拟与植入物对应的生理条件，因为血栓事件的低背景和低抗凝血剂的浓度使血液参数得到广泛分析。该方法是生物材料研究的理想的方法，因为它提供了一种评估血相容性的简单方法。此外，它可用于临床前研究。对于肝素的装载，首先将5000个国际未稀释的肝素单位与0.9%的氯化钠混合，以获得每毫升肝素浓度15个国际单位。接下来，将稀释的肝素溶液的900微升添加到三个中性单体和三个保留的单体中，并将肝素加载的单体储存在4摄氏度。血液样本采集前30分钟，将单体放在室温下。当单体加热后，在每个捐赠者将所有27毫升血液从捐赠者集中到一个塑料容器中之前，将9毫升血液收集到每个捐赠者的三个单体中。为了准备流环，用感兴趣的神经血管激光切割植入物每个条件至少加载一个管，并在每个管的一端放在充满0.9%氯化钠的储液罐中。将卸载的管子放入储液罐中，将所有管子连接到泵头。然后将每个管的另一端插入测量油缸，并使用计时器将近位泵的设置调整为每分钟 150 毫升的流量，同时检查测量油缸中的填充水平。对于血容测试，使用12毫升注射器向每个管中填充6毫升血液。形成电路后，使用 0.5 厘米长的硅胶连接管紧密地关闭管，将管子放在 37 摄氏度的水浴中。然后启动60分钟的管。对于全血计数分析，在灌注到含有EDTA的单个单体中后，从每个管中加入1.2毫升非灌注血液或1.2毫升血液。小心地反转单体五次，将小瓶装入血液分析仪中，对每个样本进行血液计数分析。在分析结束时，将单体放在冰上 15-60 分钟。对于柠檬酸盐血浆的收集，用1.4毫升的未滴合或泛热血填满含有柠檬酸盐的单体，并小心地反转每个单体五次。通过离心分离血浆，将血浆分数的三个 250 微升等分转移到单独的 1.5 毫升反应管中。然后将血浆样品冷冻在液氮中，并将其储存在零下20摄氏度，直到其分析。对于EDTA血浆采集，在血液计数测量后4摄氏度的孵育后，通过离心分离血浆，将血浆分数的三个250微升等分转移到单独的1.5毫升反应管中。然后将血浆样品冷冻在液氮中，储存零下20摄氏度。对于 CTAD 血浆采集，用 2.7 毫升新鲜抽出或注入的血液填充含有 CTAD 的单体，并小心地反转管内五次。将单体放在冰上15-60分钟，然后通过离心收集等离子体。将每个中间血浆分数的700微升转移到单独的1.5毫升反应管中，然后再次离心填充的反应管。然后将中间馏分的两个100微升等分转移到单独的1.5毫升反应管中，将液氮中的管冷冻在零下20摄氏度的温度下储存。为了测量柠檬酸盐等离子体样品中的TAT水平，在96孔平底板的每个孔中加入50微升ELISA样品缓冲液，将50微升的等离子体标准、等离子体控制和未稀释的等离子样品重复添加到该板的适当孔中。密封板后，在37摄氏度下孵育样品15分钟，轻轻摇动。在孵育结束时，用每井300微升的洗涤溶液洗三次，然后向每孔添加100微升过氧化物酶结合的抗人TAT抗体。在37摄氏度的高温下孵育15分钟后，用每井300微升的新鲜洗涤溶液洗三次。上次洗涤后，每井加入100微升新鲜准备的铬质溶液，在室温下孵育30分钟。在孵化结束时，向每个井添加 100 微升停止溶液，并使用标准曲线数据作为计算每个样品的 TAT 浓度的趋势线，读取 490 至 500 纳米光度计的光学密度。要准备用于扫描电子显微镜的植入物，请使用钳子将植入物从管子中取出，并在每次洗涤的新鲜 0.9% 氯化钠中短暂冲洗每个植入物三次。上次洗涤后，将植入物存放在PBS中2%谷胱甘肽的单独容器中，在4摄氏度下过夜，不含有钙和镁。第二天早上，将植入物孵育到PBS的单个容器中10分钟，然后按指示将植入物在乙醇浓度上升中脱水10分钟。直接在血液采集和灌注后，白血球或红血球的数量或造血量值未检测到任何变化。然而，在流量循环系统中灌注后检测到血红蛋白水平下降。血小板数也会减少，当管内存在未涂覆的支架时，血小板数量会增加。值得注意的是，当血液用纤维蛋白-肝素涂层支架孵育时，这种损失会减少。TAT复合浓度因灌注而温和增加。但是，当添加裸机支架时，检测到 TAT 显著增加，表明凝固系统发生了深刻的激活。使用纤维素肝素涂层支架可防止此激活。灌注导致增加的恭维级联的激活，不受未涂装或纤维蛋白-肝素涂层支架的存在的影响。同样，嗜中性粒细胞被激活，多态核嗜中性粒细胞酶水平的定量就证明了这一点。通过扫描电子显微镜进行可视化后，在灌注后，未涂覆支架表面存在密集的血细胞和蛋白质网络，而灌注后，在纤维蛋白-肝素涂层支架上未观察到。最重要的是确认血液符合获取样本的含水标准，并尽快使用新鲜抽血。人类血液可能含有血液传播的病毒和其他病原体，并带有感染风险，因此在处理样品时一定要佩戴适当的 PPE。

Research

JoVE Journal

Medicine

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

可视化和肝脏微循环血流量和耗氧量分析：急性肝炎模型中的应用

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

科研

医学

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

视网膜血流小鼠活体视频显微镜测量

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

微循环血流速度在大鼠肺转移自动测量

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

收集，分离，和人类宫颈样品的流式细胞仪分析

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

冠脉流量和冠状动脉血流储备用小鼠压力超负荷基于模型的超声评估

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

应用流式细胞仪对人体脂肪组织免疫细胞的表征

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

通过不同的均质化方法从小鼠大脑/脊髓中检索到的多种细胞类型的同步流细胞测量特征

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

使用激光多普勒流测量测量大鼠脑血流自动调节的评估

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

兔子静脉介词模型模仿再血管化手术使用静脉移植评估动脉血压下的影响增生

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...