Name: on-chip endothelial inflammatory phenotyping
Uploaded: 2012-07-21T18:00:00
Description: Watch this Scientific Journal Video about On-Chip Endothelial Inflammatory Phenotyping at JoVE.com

这项工作得到了国立卫生研究院/ NHLBI赠款以R01 HL082689一西蒙斯科特和安东尼·G. Passerini。

1。细胞培养和基材的准备<ol><li>削减3英寸100×20 mm组织培养皿（BD猎鹰）使用车床的圆形基板。浸入70％乙醇消毒基板。 4毫升型胶原在室温下为1小时（100微克/毫升）在培养皿和大衣的地方，然后用4毫升1×PBS冲洗。 </li><li>申请1毫升直接基板，暂停在6.5x10 5细胞/ ml和种子的人主动脉内皮细胞（HAEC，通过4-6）。放置在37℃，5％CO 2的孵化器，使细胞坚持以基材为1小时。 </li><li>添加9...

<ol>
	<li>Schaff, U. Y., Xing, M. M., Lin, K. K., Pan, N., Jeon, N. L., Simon, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+mimetics+based+on+microfluidics+for+imaging+the+leukocyte--endothelial+inflammatory+response.">Vascular mimetics based on microfluidics for imaging the leukocyte--endothelial inflammatory response.</a> Lab Chip. 7, 448-456 (2007).</li><li>Tsou, J. K., Gower, R. M., Ting, H. J., Schaff, U. Y., Insana, M. F., Passerini, A. G., Simon, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+regulation+of+inflammation+by+human+aortic+endothelial+cells+in+a+linear+gradient+of+shear+stress.">Spatial regulation of inflammation by human aortic endothelial cells in a linear gradient of shear stress.</a> Microcirculation. 15, 311-323 (2008).</li><li>Ting, H. J., Stice, J. P., Schaff, U. Y., Hui, D. Y., Rutledge, J. C., Knowlton, A. A., Passerini, A. G., Simon, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triglyceride-rich+lipoproteins+prime+aortic+endothelium+for+an+enhanced+inflammatory+response+to+tumor+necrosis+factor-alpha.">Triglyceride-rich lipoproteins prime aortic endothelium for an enhanced inflammatory response to tumor necrosis factor-alpha.</a> Circ. Res. 100, 381-390 (2007).</li><li>Wang, Y. I., Schulze, J., Raymond, N., Tomita, T., Tam, K., Simon, S. I., Passerini, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+inflammation+correlates+with+subject+triglycerides+and+waist+size+after+a+high-fat+meal.">Endothelial inflammation correlates with subject triglycerides and waist size after a high-fat meal.</a> Am. J. Physiol. Heart Circ. Physiol. 300, H784-H791 (2011).</li><li>Deverse, J. S., Bailey, K. A., Jackson, K. N., Passerini, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress+modulates+rage-mediated+inflammation+in+a+model+of+diabetes-induced+metabolic+stress.">Shear stress modulates rage-mediated inflammation in a model of diabetes-induced metabolic stress.</a> Am. J. Physiol. Heart Circ. Physiol. , (2012).</li><li>Dai, G., Kaazempur-Mofrad, M. R., Natarajan, S., Zhang, Y., Vaughn, S., Blackman, B. R., Kamm, R. D., Garcia-Cardena, G., Gimbrone, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+endothelial+phenotypes+evoked+by+arterial+waveforms+derived+from+atherosclerosis-susceptible+and+-resistant+regions+of+human+vasculature.">Distinct endothelial phenotypes evoked by arterial waveforms derived from atherosclerosis-susceptible and -resistant regions of human vasculature.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 14871-14876 (2004).</li><li>Young, E. W., Wheeler, A. R., Simmons, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-dependent+adhesion+of+vascular+and+valvular+endothelial+cells+in+microfluidic+channels.">Matrix-dependent adhesion of vascular and valvular endothelial cells in microfluidic channels.</a> Lab Chip. 7, 1759-1766 (2007).</li><li>Davies, P. F., Civelek, M., Fang, Y., Guerraty, M. A., Passerini, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+heterogeneity+associated+with+regional+athero-susceptibility+and+adaptation+to+disturbed+blood+flow+in+vivo.">Endothelial heterogeneity associated with regional athero-susceptibility and adaptation to disturbed blood flow in vivo.</a> Semin Thromb Hemost. 36, 265-275 (2008).</li><li>Burdge, G. C., Calder, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+cytokine+response+during+the+postprandial+period:+A+potential+causal+process+in+vascular+disease.">Plasma cytokine response during the postprandial period: A potential causal process in vascular disease.</a> Br. J. Nutr. 93, 3-9 (2005).</li><li>Hotamisligil, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+metabolic+disorders.">Inflammation and metabolic disorders.</a> Nature. 444, 860-867 (2006).</li><li>Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+atherosclerosis.">Inflammation in atherosclerosis.</a> Nature. 420, 868-874 (2002).</li><li>Nigro, P., Abe, J., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+shear+stress+and+atherosclerosis:+A+matter+of+site+specificity.">Flow shear stress and atherosclerosis: A matter of site specificity.</a> Antioxid Redox Signal. 15, 1405-1414 (2011).</li><li>Chiu, J. J., Lee, P. L., Chen, C. N., Lee, C. I., Chang, S. F., Chen, L. J., Lien, S. C., Ko, Y. C., Usami, S., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress+increases+icam-1+and+decreases+vcam-1+and+e-selectin+expressions+induced+by+tumor+necrosis+factor-[alpha]+in+endothelial+cells.">Shear stress increases icam-1 and decreases vcam-1 and e-selectin expressions induced by tumor necrosis factor-[alpha] in endothelial cells.</a> Arterioscler. Thromb. Vasc. Biol. 24, 73-79 (2004).</li><li>Li, Y. S., Haga, J. H., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+effects+of+shear+stress+on+vascular+endothelial+cells.">Molecular basis of the effects of shear stress on vascular endothelial cells.</a> J. Biomech. 38, 1949-1971 (2005).</li><li>Usami, S., Chen, H. H., Zhao, Y., Chien, S., Skalak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+construction+of+a+linear+shear+stress+flow+chamber.">Design and construction of a linear shear stress flow chamber.</a> Ann. Biomed. Eng. 21, 77-83 (1993).</li><li>Helmke, 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=Molecular+control+of+cytoskeletal+mechanics+by+hemodynamic+forces.">Molecular control of cytoskeletal mechanics by hemodynamic forces.</a> Physiology (Bethesda). 20, 43-53 (2005).</li><li>Gower, R. M., Wu, H., Foster, G. A., Devaraj, S., Jialal, I., Ballantyne, C. M., Knowlton, A. A., Simon, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cd11c/cd18+expression+is+upregulated+on+blood+monocytes+during+hypertriglyceridemia+and+enhances+adhesion+to+vascular+cell+adhesion+molecule-1.">Cd11c/cd18 expression is upregulated on blood monocytes during hypertriglyceridemia and enhances adhesion to vascular cell adhesion molecule-1.</a> Arterioscler. Thromb. Vasc. Biol. 31, 160-166 (2011).</li><li>Wu, H., Gower, R. M., Wang, H., Perrard, X. Y., Ma, R., Bullard, D. C., Burns, A. R., Paul, A., Smith, C. W., Simon, S. I., Ballantyne, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+cd11c++monocytes+in+atherogenesis+associated+with+hypercholesterolemia.">Functional role of cd11c+ monocytes in atherogenesis associated with hypercholesterolemia.</a> Circulation. 119, 2708-2717 (2009).</li><li>Bussolari, S. R., Dewey, C. F., Gimbrone, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparatus+for+subjecting+living+cells+to+fluid+shear+stress.">Apparatus for subjecting living cells to fluid shear stress.</a> The Review of scientific instruments. 53, 1851-1854 (1982).</li></ol>

我们描述了微硅橡胶设备的使用片上，通过实时成像CAM的表达和单核细胞粘附内皮炎症型评估。我们的方法的一个主要优势在于能够量化细胞暴露炎症介质，如饮食中的脂肪和定义的水动力条件下的细胞因子，反映在动脉粥样硬化的血管剪应力的血管内皮功能障碍的相关成果。的VMMC方便使用少量的试剂或材料，可能是在有限的供应，并有增加的优点是价格低廉，可定制的，适合高吞吐量。 <p c...

<table border="1"><tbody><tr><td> 项目 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> 100 x 20mm的培养皿</td><td>屋宇署猎鹰</td><td> 353003 </td></tr><tr><td>乙醇95％ </td><td>电解二氧化锰化学品</td><td> EX0290-1 </td></tr><tr><td> DPBS </td><td> cellgro </td><td> 21-031-CV </td></tr><tr><td> I型鼠尾源性胶原蛋白</td><td> Gibco公司</td><td> A10483-01 </td></tr><tr><td>人类主动脉内皮细胞</td><td> genlantis </td><td> PH30405A </td></tr><tr><td>抗生素抗霉菌解决方案</td><td> Invitrogen公司</td><td> 15240-062 </td></tr><tr><td>内皮BulletKit </td><td>龙沙</td><td> CC-4176 </td></tr><tr><td>内皮细胞基底媒体2 </td><td>龙沙</td><td> CC-3156 </td></tr><tr><td> 10毫升注射器</td><td>屋宇署猎鹰</td><td> 309604 </td></tr><tr><td> PU管</td><td>聚乙烯</td><td> ABW0001 </td></tr><tr><td>莱博维茨-15媒体</td><td> Gibco公司</td><td> 11415-069 </td></tr><tr><td> sylgard 184硅橡胶基地</td><td>道康宁</td><td> 184 </td></tr><tr><td> sylgard 184硅橡胶固化剂</td><td>道康宁</td><td> 184 </td></tr><tr><td> SU8光刻胶主晶片</td><td>加州大学戴维斯分校的泛实验室</td><td> N / A </td></tr><tr><td>的Eclipse TE200倒置显微镜</td><td>尼康</td><td>日食TE200 </td></tr><tr><td>注射泵</td><td>哈佛仪器</td><td> PHD2000 </td></tr><tr><td> 19计注射器针头</td><td>肯德尔</td><td> 8881 </td></tr><tr><td> THP-1单核细胞株</td><td> ATCC </td><td> TIB-202 </td></tr><tr><td> HBSS中（汉克斯缓冲液）与Ca 2 + /镁2 + </td><td&gt; Gibco公司</td><td> 14025-092 </td></tr><tr><td>肿瘤坏死因子α（肿瘤坏死因子-α） </td><td> R＆D系统</td><td> 210-TA-010 </td></tr><tr><td>基质细胞衍生因子 - 1（SDF-1） </td><td> R＆D系统</td><td> 350-NS-010 </td></tr><tr><td> RPMI 1640培养</td><td> cellgro </td><td> 10-040-CV </td></tr><tr><td>人血清白蛋白（HSA） </td><td> ZLB Behring公司</td><td> NDC 0053-7680-32 </td></tr></tbody></table> 表2。特定的试剂和设备。

on-chip endothelial inflammatory phenotyping

动脉粥样硬化是potentiated高度的全身性炎症的状态，造成血管内皮功能障碍，有助于代谢异常。然而，早在血管内皮细胞的功能变化，意味着个人的风险程度是不直接评估临床治疗策略，以帮助指导。此外，调节局部血流动力学炎症导致动脉粥样硬化的非随机分布，但机制是难以界定， 在体内 。我们描述了实验室在一个芯片为基础的方法，以定量检测人血管内皮细胞（EC）和精确的流量条件下的单核细胞炎症活动的代谢扰动。软光刻技术的标准方法，用于1这些器件具有microfabricate 模仿血管微商会（VMMC）的 ，这必将直接培养欧共体膜。使用小体积，同时试剂的优势直接成像在接触到一个明确的剪切场欧共体膜炎症事件提供了一个平台。我们已成功地应用于这些设备，以探讨细胞因子，脂质3，4和RAGE诱导人类主动脉EC（HAEC）炎症。在这里，我们记录使用的检测单核细胞（THP-1）轧制和逮捕条件下差剪切特性及炎性细胞因子肿瘤坏死因子-α激活的HAEC单层VMMC。这类研究到atherosusceptibility提供的代谢危险因素在机械洞察力。

Watch this Scientific Journal Video about On-Chip Endothelial Inflammatory Phenotyping at JoVE.com

On-Chip Endothelial Inflammatory Phenotyping

应用光刻和蚀刻的PDMS制作的微流室探测与欧共体功能障碍和炎症相关的功能性结果。在有代表性的实验，能力差剪应力调节单核细胞粘附因子激活欧共体单层证明。

片上内皮炎症表型

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

Research

JoVE Journal

Bioengineering

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

对芯片等速电泳离子的分离和纯化核酸

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

科研

生物工程

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

lensfree芯片断层用人多角度的光照和像素超高分辨率的显微镜

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

量化的内皮细胞糖复合物的力学性能，用原子力显微镜

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

铁磁金属裸支架对血管内皮细胞捕获和保留

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

干膜 Photoresist-based 电化学微流控生物传感器平台: 器件制备、片上检测制备及系统运行

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

高通量酵母菌株，带基于滴滴的RNA测序

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

将3D多细胞乳房球体直接生物打印到内皮网络

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood

Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood

猪外周血血液生长内皮细胞（BOEC）的表征

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

一种研究炎症期间白细胞-内皮细胞相互作用的微生理系统

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...