Name: in vitro model of physiological and pathological blood flow with application to investigations of vascular cell remodeling
Uploaded: 2015-11-03T12:00:00
Description: Watch this Scientific Journal Video about In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling at JoVE.com

作者要感谢的资金来源，包括AHA（13GRNT16990019到WT）和NHLBI（HL097246和HL119371到WT）。

1.硅烷化和幻灯片的生物分子官能或聚碳酸酯膜注意：许多本协议中的化学品和解决方案具有高蒸发率（乙醇（乙醇）， 丙酮等）。其他措施带来长时间的保温时间为低蒸发率。石蜡膜，建议以密封容器。注意：多数的化学物质（包括：硫酸，丙酮，（3-氨基丙基）三乙氧基硅烷，戊二醛，乙醇）被认为是有害，或挥发性。请各材料进行适当的储存，处理和处置，然后?...

<ol>
	<li>Scott-Drechsel, D., Su, Z., Hunter, K., Li, M., Shandas, R., Tan, W. <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+flow+co-culture+system+for+studying+mechanobiology+effects+of+pulse+flow+waves.">A new flow co-culture system for studying mechanobiology effects of pulse flow waves.</a> Cytotechnology. 64 (6), 649-666 (2012).</li><li>Tan, 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=Stiffening-Induced+High+Pulsatility+Flow+Activates+Endothelial+Inflammation+via+a+TLR2/NF-&#954;B+Pathway.">Stiffening-Induced High Pulsatility Flow Activates Endothelial Inflammation via a TLR2/NF-&#954;B Pathway.</a> PLoS ONE. 9 (7), e102195 (2014).</li><li>Wexler, L., 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=Coronary+Artery+Calcification:+Pathophysiology,+Epidemiology,+Imaging+Methods,+and+Clinical+Implications+A+Statement+for+Health+Professionals+From+the+American+Heart+Association.">Coronary Artery Calcification: Pathophysiology, Epidemiology, Imaging Methods, and Clinical Implications A Statement for Health Professionals From the American Heart Association.</a> Circulation. 94 (5), 1175-1192 (1996).</li><li>Lan, T. -. H., Huang, X. -. Q., Tan, H. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+fibrosis+in+atherosclerosis.">Vascular fibrosis in atherosclerosis.</a> Cardiovasc Pathol. 22 (5), 401-407 (2013).</li><li>Lee, C. H., 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=Promoting+endothelial+recovery+and+reducing+neointimal+hyperplasia+using+sequential-like+release+of+acetylsalicylic+acid+and+paclitaxel-loaded+biodegradable+stents.">Promoting endothelial recovery and reducing neointimal hyperplasia using sequential-like release of acetylsalicylic acid and paclitaxel-loaded biodegradable stents.</a> Int J Nanomedicine. 9, 4117-4133 (2014).</li><li>Intengan, H. D., Schiffrin, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Remodeling+in+Hypertension+Roles+of+Apoptosis+Inflammation,+and+Fibrosis.">Vascular Remodeling in Hypertension Roles of Apoptosis Inflammation, and Fibrosis.</a> Hypertension. 38 (3), 581-587 (2001).</li><li>Greil, 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=Changes+in+carotid+artery+flow+velocities+after+stent+implantation:+a+fluid+dynamics+study+with+laser+Doppler+anemometry.">Changes in carotid artery flow velocities after stent implantation: a fluid dynamics study with laser Doppler anemometry.</a> J Endovasc Ther. 10 (2), 275-284 (2003).</li><li>Egorova, A. D., van der Heiden, K., Poelmann, R. E., Hierck, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cilia+as+biomechanical+sensors+in+regulating+endothelial+function.">Primary cilia as biomechanical sensors in regulating endothelial function.</a> Differentiation. 83 (2), S56-S61 (2012).</li><li>Liu, S. Q., Goldman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+blood+shear+stress+in+the+regulation+of+vascular+smooth+muscle+cell+migration.">Role of blood shear stress in the regulation of vascular smooth muscle cell migration.</a> IEEE T Bio-Med Eng. 48 (4), 474-483 (2001).</li><li>Scott, D., Tan, Y., Shandas, R., Stenmark, K. R., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+pulsatility+flow+stimulates+smooth+muscle+cell+hypertrophy+and+contractile+protein+expression.">High pulsatility flow stimulates smooth muscle cell hypertrophy and contractile protein expression.</a> AJP: Lung C. 304 (1), L70-L81 (2013).</li><li>Panaritis, V., 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=Pulsatility+Index+of+Temporal+and+Renal+Arteries+as+an+Early+Finding+of+Arteriopathy+in+Diabetic+Patients..">Pulsatility Index of Temporal and Renal Arteries as an Early Finding of Arteriopathy in Diabetic Patients..</a> Ann Vasc Surg. 19 (1), 80-83 (2005).</li><li>Miao, H., 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=Effects+of+Flow+Patterns+on+the+Localization+and+Expression+of+VE-Cadherin+at+Vascular+Endothelial+Cell+Junctions:+In+vivo+and+in+vitro+Investigations.">Effects of Flow Patterns on the Localization and Expression of VE-Cadherin at Vascular Endothelial Cell Junctions: In vivo and in vitro Investigations.</a> J Vasc Res. 42 (1), 77-89 (2005).</li></ol>

这个协议描述再现体外脉动流的方法，并且可以是在确定的流动条件对疾病的病理学贡献器乐第一步。使用此协议之前的研究已经发现，流动条件。此外有助于血管炎症反应。1，10，此协议的目的是为有经验的实验室。正因为如此，无论是在深度流体力学，也不生化分析在本文中描述。对于更高级的流体动力学，1和先进的技术，参考文献2中临界流体下剪切?...

发病率心血管疾病是最大的，在美国，与不健康的血管多所致。健康的动脉包括弹力组织，软腔表面涂有内皮细胞（EC）单层。动脉血流可被建模为具有正的平均流量的振荡波函数。搏动指数（PI）是振荡幅度的商和平均流量（PI =（最大-最小）/平均 ），1和是仿照在体外用可变血管弹性2动脉弹性的存储流量的重要。能量心脏收缩，在收缩压扩张，并起着调节血液流量PI一个显著的作用。因为心脏保持一致，脉动，体积流量，动脉扩张增加截面积，通过减小流速，剪切应力，和PI增强流动稳定性。通常情况下，不健康的动脉目前变化的弹性或遵医嘱，显示从血管重建，疤痕组织或钙化3,4加强。此外，其它血管病症，例如新生内膜增生（NIH），5瘤和高血压6和血管纤维变性4，可收缩血管直径。然而，目前的药物治疗和血管疾病的治疗设备常常忽视在血管疾病血管壁顺应性或血流动力学这常常通过改变容器的形态和性质复杂的重要性。无论是球囊成形术还是支架回答壁弹性7的并发症。因此， 在体外模拟血液流动的动脉疾病和治疗导致在调查疾病病理和治疗疗效未来重要的。在此，我们描述了复制的生理和病理血流设计为确定在血管疾病病理学细胞应答的方法奥希斯。流体流动引起的剪切应力在血管壁，这是在容器健康的重要机械信号，影响脉管系统中的所有单元。在血管内皮细胞的流体剪切几个机械传感器已经确定，包括在最近的研究内皮mechanosensing 8所示初级纤毛。内皮细胞的活性和形态受流速，方向和搏动。此外，平滑肌细胞（SMC）迁移可能会受到低流速的机械性信号，以通过间质液9，并且也可以是通过从内皮细胞的旁分泌信号通过其响应于流动并通过细胞因子机械性转导流信号的发布10。平均剪切，PI和旁分泌信号的“剂量”的依赖也可以是相互依赖的。为此，血管细胞响应于流体剪切具有变化“给药”的判断中单层培养或共培养体外可以提供机械的见解血管重构，改善疾病和治疗的预测。在这个实验中使用的流动系统包括一个血泵，上游流动阻尼空气存贮器，只有实验装置，下游细胞培养，平行板流动室，及媒体储存器过程中使用的下游流量计。血管流动变量的控制，如平均流动速率，每分钟节拍和PI可以通过控制流率，脉冲频率，和引进了压力阻尼来实现。脉动血泵可具有可变行程位移，在受控冲程频率，直接涉及到平均体积流速，和脉冲频率。流电路内的空气存贮器的引入允许压力阻尼，减小流量振荡幅度。媒体是不可压缩的流体，而衰减室内的空气是可压缩的，从而使过量的压力从流波是通过空气压缩吸收。空气介质的比例可超过多少阻尼发生控制权。定制细胞培养流动腔75毫米，长50毫米，宽是由丙烯酸创建。流进入通过入口端口和膨胀通过进气歧管，提供穿过流动腔室的全部一致的流动。类似的流动和结构存在于室出口。将细胞接种于官能幻灯片，并随后附连到流动室。这使得大量人口，在研究之后很容易地检索。共培养实验可以使用多孔聚碳酸酯膜，以消除培养物之间的细胞 - 细胞接触，同时允许细胞因子/流传输。这一系统先前已经用于模拟高PI流动及其对内皮细胞单层培养和EC / SMC共培养1，10的效果，来研究细胞反应病理高PI疾病。通过描述来模拟这些流动CON协议ditions，我们希望能帮助他人确定流量信号的贡献细胞反应。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>34850</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>320501</td>
			<td></td>
		</tr>
		<tr>
			<td>(3-Aminopropyl)trethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>440140</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde Solution</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>459844</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Slide (70mm x 50mm)</td>
			<td>Sigma-Aldrich</td>
			<td>CLS294775X50</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate Membrane</td>
			<td>Millipore Corp.</td>
			<td>HTTP09030</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Gasket</td>
			<td>Grace Bio-Labs</td>
			<td>RD 475464</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin (25 &#956;g/mL)</td>
			<td>Sigma-Aldrich</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type-I</td>
			<td>Sigma-Aldrich</td>
			<td>C3867</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fluka</td>
			<td>36486</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Damping Chamber</td>
			<td></td>
			<td></td>
			<td>This chamber is custom made, and may be requested using the engineering drawing of Figure 3.</td>
		</tr>
		<tr>
			<td>Blood Pump</td>
			<td>Harvard Apparatus</td>
			<td>529552</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Vinyl Carbonate Tubing</td>
			<td>US Plastic</td>
			<td>65066, 65063, 65062</td>
			<td>Various sizes may be required</td>
		</tr>
		<tr>
			<td>Luer Connections</td>
			<td>Nordson Medical</td>
			<td>Various</td>
			<td>Various sizes will be required, and a number of parts should be purchased for replacement use.</td>
		</tr>
		<tr>
			<td>Culture Chamber</td>
			<td>Machined in-house</td>
			<td>Custom</td>
			<td>Acrylic may be purchased in sheets and machined for intended use. The engineering drawing shown in Figure 2 may be used to recreate this chamber</td>
		</tr>
		<tr>
			<td>Square Petri Dish</td>
			<td>Cole-Parmer</td>
			<td>EW-14007-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Slide Holder</td>
			<td>Capitol Scientific</td>
			<td>WHE-900303</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Mediatech, Inc.</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Mediatech, Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Meter</td>
			<td>Sonotec, GmbH</td>
			<td>Sonoflow co.55/060</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard Elastomer Kit</td>
			<td>Sigma-Aldrich</td>
			<td>761036-5EA</td>
			<td></td>
		</tr>
		<tr>
			<td>14 G Steel Cannula</td>
			<td>General Laboratory Supply</td>
			<td>S8365-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro model of physiological and pathological blood flow with application to investigations of vascular cell remodeling

血管疾病是死亡的在美国的常见原因。在此，我们提出了一个方法来检查血流动力学的走向血管疾病病理的贡献。不健康的动脉壁硬化，疤痕，或局部狭窄可能都会影响流体流速，和脉动流的幅度，或搏动指数经常存在。各种流动条件复制是调谐流压力衰减室血液泵的下游的结果。一个封闭的流动系统内的空气的引入允许一个可压缩的介质以吸收来自泵脉动压力，因此改变搏动指数。本文所描述的方法被简单地再现，具有高度可控输入，和容易测量的结果。某些局限性是复杂的生理脉冲波形，这是只能由系统近似的娱乐。内皮细胞，平滑肌细胞和成纤维细胞都受血液流动THRough动脉。血流的动态分量由心输出量和动脉壁顺应性来确定。血管细胞机械性传导流动动力学的可触发的细胞因子释放和动脉内的细胞类型之间的串扰。血管细胞的共培养是在血管壁和机械信令血管反应的更精确的图像反射的细胞 - 细胞相互作用。流动动力学，包括细胞响应于流的动态和平均值（或稳定）成分的贡献，因此，在确定疾病的病理和治疗疗效的重要指标。通过引入体外共培养模型和压力阻尼产生模拟心输出量的血泵的下游，各种动脉疾病的病理可以进行调查。

的流动条件是维持依赖于所述流动回路（图1）的正确装配。管直径是一个重要的选择在装配，具有较大直径减少前和培养室后的流动阻力和随后的压降。为了确保预期的压力和流速，实验目的管前组装系统，流量计。培养室的真空通道（图2），以在硅垫片（未示出）的穿孔对齐，保持流动回路内密封。为了确保密封，弹簧夹具可以用来施加在垫圈额外的压力。载玻片应?...

Watch this Scientific Journal Video about In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling at JoVE.com

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

该协议将复制的生理或病理血流体外在确定疾病的病理细胞应答提供帮助。通过引入压力衰减室血液泵的下游，穿过脉管的血流，可以概括和强加于血管内皮或模拟物共培养的单层。

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 ...

Research

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Bioengineering

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

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

科研

生物工程

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

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3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

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对于中吸入有毒化学测试体外细胞培养模型

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

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一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...