Name: 作者聚焦：通过将流动动力学集成到体外模型中来推进溶栓测试
Uploaded: 2024-04-19T13:40:00
Description: Read this Scientific Article Protocol about Author Spotlight: Advancing Thrombolytic Testing by Integrating Flow Dynamics in In-Vitro Models at JoVE.com

本出版物中报告的研究得到了美国国立卫生研究院国家心肺血液研究所的支持，奖项编号为 R01HL167877。内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。

下面提到的所有方法均符合机构审查委员会 （IRB） 协议和机构人类研究伦理委员会。所有健康志愿者在献血前都提供了书面和知情同意书。值得注意的是，协议中引用的所有材料都可以在 材料表中找到。虽然在整个协议中讨论了人WB和血浆，但可以购买和替代研究动物血液和因子耗尽的血液制品的使用。
1. 全血采集
<ol><li>使用标准放血技术从同意...

结合 Chandler Loop 和 RT-FluFF 进行离体血凝块裂解监测

<ol>
	<li>Ali, M. R., 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=Aspect+of+thrombolytic+therapy:+a+review.">Aspect of thrombolytic therapy: a review.</a> Scientific World Journal. 2014, 586510 (2014).</li><li>Bhogal, P., Andersson, T., Maus, V., Mpotsaris, A., Yeo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+thrombectomy-A+brief+review+of+a+revolutionary+new+treatment+for+thromboembolic+stroke.">Mechanical thrombectomy-A brief review of a revolutionary new treatment for thromboembolic stroke.</a> Clin Neuroradiol. 28 (3), 313-326 (2018).</li><li>Fluri, F., Schuhmann, M. K., Kleinschnitz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ischemic+stroke+and+their+application+in+clinical+research.">Animal models of ischemic stroke and their application in clinical research.</a> Drug Des Devel Ther. 9, 3445-3454 (2015).</li><li>Kaiser, E. E., West, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+animal+ischemic+stroke+models:+replicating+human+stroke+pathophysiology.">Large animal ischemic stroke models: replicating human stroke pathophysiology.</a> Neural Regen Res. 15 (8), 1377-1387 (2020).</li><li>Elnager, 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=In+vitro+whole+blood+clot+lysis+for+fibrinolytic+activity+study+using+d-dimer+and+confocal+microscopy.">In vitro whole blood clot lysis for fibrinolytic activity study using d-dimer and confocal microscopy.</a> Adv Hematol. 2014, 814684 (2014).</li><li>Prasad, S., 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=Development+of+an+in+vitro+model+to+study+clot+lysis+activity+of+thrombolytic+drugs.">Development of an in vitro model to study clot lysis activity of thrombolytic drugs.</a> Thromb J. 4, 14 (2006).</li><li>Robbie, L. A., Young, S. P., Bennett, B., Booth, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombi+formed+in+a+Chandler+loop+mimic+human+arterial+thrombi+in+structure+and+RAI-1+content+and+distribution.">Thrombi formed in a Chandler loop mimic human arterial thrombi in structure and RAI-1 content and distribution.</a> Thromb Haemost. 77 (3), 510-515 (1997).</li><li>Mutch, N. 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=Model+thrombi+formed+under+flow+reveal+the+role+of+factor+XIII-mediated+cross-linking+in+resistance+to+fibrinolysis.">Model thrombi formed under flow reveal the role of factor XIII-mediated cross-linking in resistance to fibrinolysis.</a> J Thromb Haemost. 8 (9), 2017-2024 (2010).</li><li>Blinc, A., Kennedy, S. D., Bryant, R. G., Marder, V. J., Francis, 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=Flow+through+clots+determines+the+rate+and+pattern+of+fibrinolysis.">Flow through clots determines the rate and pattern of fibrinolysis.</a> Thromb Haemost. 71 (2), 230-235 (1994).</li><li>Mutch, N. 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=The+use+of+the+Chandler+loop+to+examine+the+interaction+potential+of+NXY-059+on+the+thrombolytic+properties+of+rtPA+on+human+thrombi+in+vitro.">The use of the Chandler loop to examine the interaction potential of NXY-059 on the thrombolytic properties of rtPA on human thrombi in vitro.</a> Br J Pharmacol. 153 (1), 124-131 (2008).</li><li>Herbig, B. A., Yu, X., Diamond, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+microfluidic+devices+to+study+thrombosis+in+pathological+blood+flows.">Using microfluidic devices to study thrombosis in pathological blood flows.</a> Biomicrofluidics. 12 (4), 042201 (2018).</li><li>Jigar Panchal, H., Kent, N. J., Knox, A. J. S., Harris, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+in+haemostasis:+A+review.">Microfluidics in haemostasis: A review.</a> Molecules. 25 (4), 833 (2020).</li><li>Zeng, Z., 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=Fluorescently+conjugated+annular+fibrin+clot+for+multiplexed+real-time+digestion+analysis.">Fluorescently conjugated annular fibrin clot for multiplexed real-time digestion analysis.</a> J Mater Chem B. 9 (45), 9295-9307 (2021).</li><li>Zeng, Z., Christodoulides, A., Alves, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+tracking+of+fibrinolysis+under+constant+wall+shear+and+various+pulsatile+flows+in+an+in-vitro+thrombolysis+model.">Real-time tracking of fibrinolysis under constant wall shear and various pulsatile flows in an in-vitro thrombolysis model.</a> Bioeng Transl Med. 8 (3), e10511 (2023).</li><li>Christodoulides, A., Zeng, Z., Alves, N. J. <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+thromboelastographic+characterization+of+reconstituted+whole+blood+utilizing+cryopreserved+platelets.">In-vitro thromboelastographic characterization of reconstituted whole blood utilizing cryopreserved platelets.</a> Blood Coagul Fibrinolysis. 32 (8), 556-563 (2021).</li><li>Zeng, Z., Nallan Chakravarthula, T., Christodoulides, A., Hall, A., Alves, N. J. <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+Chandler+loop+shear+and+tubing+size+on+thrombus+architecture.">Effect of Chandler loop shear and tubing size on thrombus architecture.</a> J Mater Sci Mater Med. 34 (5), 24 (2023).</li><li>Touma, H., Sahin, I., Gaamangwe, T., Gorbet, M. B., Peterson, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+investigation+of+fluid+flow+in+a+chandler+loop.">Numerical investigation of fluid flow in a chandler loop.</a> J Biomech Eng. 136 (7), (2014).</li><li>Wojdyla, M., Raj, S., Petrov, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorption+spectroscopy+of+single+red+blood+cells+in+the+presence+of+mechanical+deformations+induced+by+optical+traps.">Absorption spectroscopy of single red blood cells in the presence of mechanical deformations induced by optical traps.</a> J Biomed Opt. 17 (9), (2012).</li><li>Wu, J. H., Diamond, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence+quench+and+dequench+assay+of+fibrinogen+polymerization,+fibrinogenolysis,+or+fibrinolysis.">A fluorescence quench and dequench assay of fibrinogen polymerization, fibrinogenolysis, or fibrinolysis.</a> Anal Biochem. 224 (1), 83-91 (1995).</li></ol>

凝块形成和标记 钱德勒环已被证明提供了一种简单有效的方法，可以可重复生成模拟 体内 血栓16 的凝块。通过微调管道尺寸、转速、转鼓直径和凝血时间等参数，可以在不同的剪切条件下快速生成凝块，从而捕获在模拟动脉和静脉源的血栓范围内所欣赏的结构特征。正如我们在 RT-FluFF 系统中所展示的那样，能够引入 FITC-Fg 等标记物的额外灵活性扩大了这?...

从根本上说，由血栓栓塞病因引起的疾病是当今社会发病和死亡的主要来源。血栓栓塞发病机制的表现包括但不限于心肌梗死、缺血性卒中、深静脉血栓形成和肺栓塞1。正在进行的大量研究，跨越多个学科，围绕着开发安全有效的方法来处理致病性血栓形成。血栓形成的动脉和静脉表现的差异以及不同的解剖位置导致了不同治疗方法的发展。然而，急性治疗通常依赖于通过纤溶酶原激活剂进行药物溶栓，在某些临床情况下有可能进行机械血栓切除术2。
新型药物治疗策略的开发从根本上依赖于体内动物模型和用于临床前测试的体外消化模型 3,4。体内模型自然受益于它们能够捕捉各种生理参数对治疗效果的复杂相互作用，包括药物的清除以及细胞与药物的相互作用。然而，这种相同的复杂性往往使此类模型相当昂贵，并且在试图分离与人类生理学显着不同的动物的潜在药效学/动力学时，会带来额外的问题。体外模型的发展有助于促进蒸馏测试环境，在该环境中可以进行药物开发和筛选，但通常缺乏概括所研究疾病状态所需的保真度。
通常发现的用于测试新型溶栓剂的体外方案依赖于利用在静态条件下形成和裂解的凝块，其中残留的凝块质量作为主要终点 5,6。不幸的是，这些技术无法解释凝块溶解的机械方面，例如湍流和经血栓压降，这些方面会显着改变测试药物的药效学。此外，在静态条件下形成的凝块包含与生理性凝块不同的微结构。血凝块形成过程中剪切力的存在已被证明会影响由此产生的血栓特性，例如血小板活化和纤维蛋白交联。在剪切流下产生的凝块从尖端到尾部表现出复杂的非均质性，这在静态形成的凝块中不存在7,8。这种对生理凝块结构的偏离可能会影响重要的药物开发表征，包括药物在血栓内的渗透和随后的裂解效率9。
为了解决与使用静态凝血/凝块溶解模型相关的一些限制，在存在剪切的情况下采用钱德勒环进行凝块形成和凝块溶解已经重新出现10.尽管与相对静态的测定相比，此类系统可以更好地表示流动动力学并产生具有更生理相关结构的凝块，但它们简化的流动条件仍然代表着与生理条件的偏差。最后，由于微流体方法易于成像和均匀的流动模式，也被采用;然而，它们仍然显着消除了主要受大多数临床相关血栓栓塞疾病影响的大血管内预期的生理条件11,12。
考虑到上述讨论，我们开发了一种高保真度的 体外 溶栓模型，用于临床前溶栓药物筛选。该模型旨在解决上述新型溶栓疗法筛选领域的一些当前陷阱，并经过验证了在不同浓度的组织纤溶酶原激活剂 （tPA） 下的可重复性和敏感性。本文描述的系统利用蠕动泵、压力阻尼器、加热储液罐、两个压力传感器、在线荧光计和荧光标记的钱德勒环剪切形成凝块模拟物提供生理剪切流，以促进纤维蛋白溶解的实时跟踪13。总而言之，整个系统被称为 实时荧光流动纤维蛋白溶解测定 （RT-FluFF Assay）14 ，本文将讨论在这种高保真 体 外溶栓模型中成功设置和运行测定的复杂性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 G&#160;Disposable Hypodermic Needles</td>
			<td>Exel International&#160;</td>
			<td>26439</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>6 mm HSS Lathe Bar Stock Tool 150 mm Long</td>
			<td>uxcell</td>
			<td>B07SXGSQ82</td>
			<td>Chandler loop,&#160;</td>
		</tr>
		<tr>
			<td>96-Well Clear Flat Bottom UV-Transparent Microplate</td>
			<td>Corning</td>
			<td>3635</td>
			<td>Other Consumables, Non-treated acrylic copolymer, non-sterile</td>
		</tr>
		<tr>
			<td>Air-Tite Luer-lock Unsterile 60 mL Syringes</td>
			<td>Air-Tite</td>
			<td>MLB3</td>
			<td>RT-FluFF Apparatus , dampeners</td>
		</tr>
		<tr>
			<td>Arium Mini Plus Ultrapure Water System</td>
			<td>Sartorius</td>
			<td>NA</td>
			<td>DI water source</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Millipore Sigma</td>
			<td>C5670</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>Disposable BP Transducers</td>
			<td>AD Instruments</td>
			<td>MLT0670</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Drager Siemans HemoMed Pod</td>
			<td>Drager</td>
			<td>5588822</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Drager Siemans Patient Monitor</td>
			<td>Drager</td>
			<td>SC 7000</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Drum (cylinder, diameter 120 mm, width 85 mm)</td>
			<td></td>
			<td></td>
			<td>Chandler loop,</td>
		</tr>
		<tr>
			<td>Face Shield</td>
			<td>Moxe</td>
			<td>SHIELDS10</td>
			<td>Chandler loop,&#160;</td>
		</tr>
		<tr>
			<td>Fibrinogen From Human Plasma, Alexa Fluor 488 Conjugate</td>
			<td>Thermo Scientific</td>
			<td>F13191</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>Fitting, Polycarbonate, Four-Way Stopcock, Male Luer Lock, Non-Sterile</td>
			<td>Masterflex</td>
			<td>30600-04</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Fluorescein (FITC)</td>
			<td>Thermo Scientific</td>
			<td>119245000</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>General-Purpose Water Bath</td>
			<td>Thermo Scientific</td>
			<td>2839</td>
			<td>Chandler loop,&#160;</td>
		</tr>
		<tr>
			<td>Hotplate 4 &#215; 4</td>
			<td>Fisher Scientific</td>
			<td>1152016H</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Human Source Plasma Fresh-Frozen</td>
			<td>Zen-Bio</td>
			<td>SER-SPL</td>
			<td>Other Consumables, CPDA-1 anticoagulant</td>
		</tr>
		<tr>
			<td>Human Whole Blood&#160;</td>
			<td>Zen-Bio</td>
			<td>SER-WB-SDS&#160;</td>
			<td>Other Consumables, CPDA-1 anticoagulant</td>
		</tr>
		<tr>
			<td>L/S Easy-Load II Pump Head for High-Performance Precision Tubing, PPS Housing, SS Rotor</td>
			<td>Masterflex</td>
			<td>77200-62</td>
			<td>RT-FluFF Apparatus, Pump Head</td>
		</tr>
		<tr>
			<td>L/S Variable-Speed Digital Drive Pump with Remote I/O, 6 to 600 rpm; 90 to 260 VAC</td>
			<td>Masterflex</td>
			<td>7528-10</td>
			<td>RT-FluFF Apparatus, Pump</td>
		</tr>
		<tr>
			<td>Motor Speed Controller</td>
			<td>CoCocina</td>
			<td>ZK-MG</td>
			<td>Chandler loop,&#160;</td>
		</tr>
		<tr>
			<td>Nalgene Tubing T-Type Connectors</td>
			<td>Thermo Scientific</td>
			<td>6151-0312</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Peristaltic pump tubing&#160;</td>
			<td>Masterflex</td>
			<td>06424-15&#160;</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Millipore Sigma</td>
			<td>P3813</td>
			<td>Other Consumables, Powder, pH 7.4, for preparing 1 L solutions</td>
		</tr>
		<tr>
			<td>SpectraMax M5 multi-detection microplate reader system (or other fluorescence detection)</td>
			<td>Molecular Devices</td>
			<td>M5</td>
			<td>RT-FluFF Apparatus</td>
		</tr>
		<tr>
			<td>Switching Power Supply</td>
			<td>SoulBay</td>
			<td>UC03U</td>
			<td>Chandler loop,&#160;</td>
		</tr>
		<tr>
			<td>Thermo Scientific National Target All-Plastic Disposable Syringes 10 mL</td>
			<td>Thermo Scientific</td>
			<td>S751010</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>Tissue plasminogen activator, human</td>
			<td>Millipore Sigma</td>
			<td>T0831</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>Tubing ID 1/4&#39;&#39;, OD 3/8&#39;&#39;</td>
			<td>Fisher Scientific</td>
			<td>AGL00017</td>
			<td>Other Consumables, cut into 1.5cm sections use to connect tubing to T-type connectors</td>
		</tr>
		<tr>
			<td>Tubing ID 5/32&#34;, OD 7/32&#34;</td>
			<td>Tygon</td>
			<td>ND-100-65, ADF 00009&#160;</td>
			<td>Other Consumables</td>
		</tr>
		<tr>
			<td>V3 365 nm Mini - Black Light UV Flashlight</td>
			<td>uvBeast</td>
			<td>uvB-V3-365-MINI</td>
			<td>Chandler loop, used to check completed clots</td>
		</tr>
		<tr>
			<td>ZGA37RG ZYTD520 DC Motor, 12 V, 100 rpm</td>
			<td>Pangyoo</td>
			<td>ZGA37RG</td>
			<td>Chandler loop,&#160;</td>
		</tr>
	</tbody>
</table>

tracking fibrinolysis of chandler loop-formed whole blood clots under shear flow in an in-vitro thrombolysis model

血栓栓塞和相关并发症是全球发病和死亡的主要原因，并且已经开发了各种测定法来测试溶栓药物 在体外 和 体内的效率。由于与动物模型相关的复杂性和成本，以及它们通常缺乏对人类生理学的可转化性，因此对用于药物开发的更具生理相关性的 体外 凝块模型的需求越来越大。流量、压力和剪切速率是循环系统的重要特征，在流动下形成的凝块与静态形成的凝块表现出不同的形态和消化特性。这些因素在传统的 体外 凝块消化测定中通常没有得到体现，这可能具有影响药物转化成功率的药理学意义。
Real-T ime Fluorometric Flowing Fibrinolysis （RT-FluFF） 测定被开发为一种高保真溶栓检测平台，它使用在剪切流下形成的荧光标记凝块，然后在存在或不存在纤溶药物制剂的情况下使用循环血浆进行消化。通过改变血栓形成和血栓消化步骤的流速，系统可以在高度多样化的实验设置中模拟动脉、肺部和静脉状况。可以使用在线荧光计连续进行测量，也可以通过获取离散时间点以及传统的终点凝块质量测量进行测量。RT-FluFF 检测是一种灵活的系统，允许在更准确地代表体内生理条件的流动条件下实时跟踪凝块消化，同时保持体外检测系统的控制和可重复性。

Diseases fundamentally stemming from thrombo-embolic etiologies present a major source of morbidity and mortality in present-day society. Manifestations of thrombo-embolic pathogenesis include, but are not limited to, myocardial infarctions, ischemic strokes, deep venous thromboses, and pulmonary emboli1. A tremendous amount of ongoing research, spanning multiple disciplines, revolves around the development of safe and effective methods for dealing with pathogenic thrombosis. Variations in arterial and venous manifestations of thrombosis and varying anatomic locations have resulted in the development of different treatment approaches. However, acute treatment generally relies on the use of pharmacologic thrombolysis via plasminogen activators with the potential for mechanical thrombectomy under certain clinical circumstances2.
The development of novel pharmacologic treatment strategies fundamentally relies on both in-vivo animal models and in-vitro digestion models for preclinical testing3,4. In-vivo models naturally benefit from their ability to capture the complex interaction of various physiologic parameters on treatment efficacy that include clearance of pharmaceutical agents as well as cellular interactions with drugs. However, this same complexity often makes such models quite costly and introduces additional issues when attempting to isolate underlying pharmaco-dynamics/kinetics in animals that significantly differ from human physiology. The development of in-vitro models has helped by facilitating a distilled testing setting in which drug development and screening can be performed but often lacks the fidelity necessary to recapitulate the disease state being studied. 
Commonly found in-vitro protocols for testing novel thrombolytics rely on the utilization of clots formed and lysed under static conditions whereby the residual clot mass serves as the primary endpoint5,6. Unfortunately, such techniques fail to account for the mechanical aspects of clot lysis such as turbulent flow and trans-thrombus pressure drops that can significantly alter the pharmacodynamics of test drugs. Additionally, clots formed under static conditions contain microarchitecture that differs from physiologic clots. The presence of shear during clot formation has reproducibly been shown to impact the resulting clot characteristics such as platelet activation and fibrin-crosslinking. Clots being produced under shear flow exhibit complex heterogeneity from tip-to-tail that is absent in statically formed clots7,8. Such departures from physiologic clot architecture may impact important drug development characterization that includes drug penetration within a thrombus and subsequent lysis efficiency9. 
To address some of these limitations associated with the use of static clotting/clot-lysis models, the adoption of the Chandler loop for both clot formation and clot lysis in the presence of shear has seen a resurgence10. Although such systems allow for a better representation of flow dynamics and generate clots with more physiologically relevant architecture compared to relatively static assays, their simplified flow conditions still represent a deviation from physiologic conditions. Lastly, microfluidic approaches have also been undertaken due to their ease of imaging and uniform flow patterns; however, they remain a significant removal from the physiologic conditions expected within the larger vessels primarily affected in most clinically relevant thrombo-embolic disorders11,12. 
With the above discussion in mind, we developed a high-fidelity, in-vitro thrombolysis model for preclinical thrombolytic drug screening. The model aims at addressing some of the current pitfalls detailed above in the realm of novel thrombolytic therapy screening and was validated for reproducibility and sensitivity at varying concentrations of tissue plasminogen activator (tPA). The system described herein offers physiological shear flows utilizing a peristaltic pump, a pressure dampener, a heated reservoir, two pressure sensors, an in-line fluorometer, and a fluorescently labeled Chandler loop shear-formed clot analog to facilitate real-time tracking of fibrinolysis13. Taken together, the overall system is called the Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF Assay)14 and this manuscript will discuss the intricacies of successfully setting up and running assays in this high-fidelity in-vitro thrombolysis model.

作者聚焦：通过将流动动力学集成到体外模型中来推进溶栓测试

在体外溶栓模型中跟踪剪切流下钱德勒环形成的全血凝块的纤维蛋白溶解

Thrombosis is associated with numerous medical conditions and current treatments risk severe bleeding. Circulatory system flow significantly impacts blood clot structure. Incorporating flow into in-vitro models may improve their physiological relevance, enhancing the selection of better candidates for in-vivo testing.The assays for the study of fibrinolytics predominantly utilize static clot or microfluidic systems to assess properties such as lysis time, clot structure, or viscoelasticity. Static clot lysis assays typically rely on spectrophotometric measurements or clot mass determination as endpoints to evaluate fibrinolysis efficiency. Current thrombolytic testing uses statically-formed clots, which differ structurally from those formed under flow conditions.Microfluidic systems consider flow dynamics, but their smaller scales create unique hemodynamics and clot structures. The currently developed RT-FluFF assay provides ease in monitoring fibrinolysis in real time under physiological flow conditions. This means we now have a platform that allows control over multiple parameters to mimic conditions closer to in-vivo study models for better translation when screening new thrombolytics.

钱德勒环凝块形成 在形成血栓时，我们通常以四重血栓为目标，以确保如果存在任何血凝块异常值（基于大体形态和质量），我们仍然有能力进行一式三份溶栓测定。假设最佳加载条件，凝块的长度 （~3.3 cm）、重量 （~100 mg） 和外观都应该相当均匀，如图 3 所示。在使用FITC-Fg时，我们还旨在在紫外光下检查凝块，以确保荧光相对均匀的色散，而不是凝块内?...

Read this Scientific Article Protocol about Author Spotlight: Advancing Thrombolytic Testing by Integrating Flow Dynamics in In-Vitro Models at JoVE.com

体外 溶栓测定通常难以复制 体内 条件，无论是在正在消化的模型血栓中还是在发生溶栓的环境中。在此，我们探讨了如何将钱德勒环和实时荧光流动纤维蛋白溶解测定 （RT-FluFF） 偶联用于高保真、 离体、凝块溶解监测。

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test ...

血栓形成与许多疾病有关，目前的治疗有严重出血的风险。循环系统流量显着影响血凝块结构。将流动纳入体外模型可以提高其生理相关性，从而增强对体内测试更好候选者的选择。用于研究纤维蛋白溶解剂的测定主要利用静态凝块或微流体系统来评估裂解时间、凝块结构或粘弹性等特性。静态凝块溶解测定通常依靠分光光度法测量或凝块质量测定作为评估纤维蛋白溶解效率的终点。目前的溶栓测试使用静态形成的凝块，其结构与在流动条件下形成的凝块不同。微流体系统考虑了流动动力学，但其较小的尺度会产生独特的血流动力学和凝块结构。目前开发的 RT-FluFF 检测便于在生理流动条件下实时监测纤维蛋白溶解。这意味着我们现在有一个平台，可以控制多个参数，以模拟更接近体内研究模型的条件，以便在筛选新的溶栓剂时更好地翻译。

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Bioengineering

Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates.
The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.

In-vitro thrombolysis assays have often struggled to replicate in-vivo conditions whether in the model thrombus being digested or in the environment in which thrombolysis is occurring. Herein, we explore how coupling the Chandler loop and Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF) is used for high-fidelity, ex-vivo, clot lysis monitoring.