作者要感谢克里斯托弗先生阮亚伦亨特和什里夫波特的Jumpstart，SMART和Biostart培训计划的援助以及生物物理，什里夫波特，LA的路易斯安那百年学院中文系。

1.直径为150mm剪切戒指建设（图1） 注:剪切环可以被构造通过改变内外培养皿大小，导致与不同的总表面积，细胞产量的设备和开发的剪切力范围以创建许多不同的尺寸。本报告描述了150毫米的菜具有内部100mm培养皿为98 平方厘米（ 图2）的总表面积相结合。 <img alt="图1" src="/files/ftp_upload/54632/54632fig1.jpg" /> 图1.剪切环组件。该图的顶部示出了部分组装的剪切环。通过3毫米的孔用移液管注入加入0.5ml二氯甲烷，如果一个紧密的密封还没有完全的内部和外部的菜肴之间形成。剪切环是通过将硅橡胶粘合剂的1mm厚的珠周围的内EDG密封关剪切环顶部电子。图的底部显示组装剪切环。蓝色代表镀内皮细胞的区域。外部和内部的红色箭头表示当置于轨道摇床剪切环内的剪切圈和媒体的轨道运动。 <a href="http://ecsource.jove.com/files/ftp_upload/54632/54632fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54632/54632fig2.jpg" /> 图2.表面三围为150mm的剪切环（未按比例）。前面板显示对响应的轨道旋转外圈液离心式换档。 <a href="http://ecsource.jove.com/files/ftp_upload/54632/54632fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>通过生成创建剪切环建筑模板在与放置在150 mm直径圈的中心100毫米的内直径的演示软件150毫米外缘轮廓。打印在A4白纸一张的模板。 </li><li>吸管的10ml二氯甲烷（二氯甲烷）至150毫米的玻璃培养皿。至关重要的是，该盘是玻璃，而不是聚苯乙烯（或其它的乙烯基塑料），二氯甲烷溶解大部分塑料并在这里用来加入塑料部件。 注意:使用手套所有建筑有足够的通风罩。二氯甲烷是接触刺激性和是肝毒性。 </li><li>对齐150毫米的塑料培养皿到外切环模板。 </li><li>使用旋转工具100毫米的菜的中心钻3毫米的孔。取下钻探产生的任何塑料刮屑。 </li><li>同时用戴手套的手保持，反转和浸来自前一步骤到二氯甲烷池100毫米的培养皿的顶部边缘约3秒。 </li><li>横贯FER的"湿"100mm皿边缘向下到150毫米盘的中央，仔细调整100毫米的菜到显着的模板。二氯甲烷将保险丝塑料，加入有100 mm和150 mm培养皿。轻轻顺时针旋转碟100mm的旋转和逆时针几次，以确保良好的附着力，以150毫米的菜。 注:应特别小心，以确保内部菜的外盘中央的正确对齐。偏心对准可能导致在在剪切环的不同位置的剪切应力的变化。小心不要让二氯甲烷到100mm皿的放置期间偶然滴到150毫米的培养皿的外轨道部分。这会熔化的底表面，其中的细胞将生长在塑料，产生不平坦的表面，可能会引起干扰。 </li><li>一旦二氯甲烷已经干涸，翻转新绑定的培养皿中，仔细检查的接触点，以确保紧密的密封已被两个培养皿中之间形成。 </li><li>如果一个密封还没有完全形成，通过3毫米的孔用移液管注入加入0.5ml二氯甲烷中。拿起菜并轻轻旋转，以允许二氯甲烷到达边缘。 注:如果旋转太快，二氯甲烷可以蔓延到150毫米的菜的外部分，表面细胞的地方将增长变形，破坏剪切环。泄漏的剪切戒指应该被丢弃。 </li><li>通过采用硅橡胶密封胶的3毫米珠封锁在100毫米的菜孔。 </li><li>使用配备有一台切割头旋转工具，切断一个15毫升的锥形聚苯乙烯组织培养试管前3 cm长的，造成至少1厘米以下帽管。抛光切割管的边缘，直到光滑和平坦使用切削头的平侧面。除去研磨产生的任何塑料刮屑。 </li><li>跟踪切断的15毫升锥形管到150毫米的菜用记号笔，从盘的边缘约为0.5厘米处的盖子。钻一个孔的描画圆内，留下一个余量大致从圆的边缘1-2毫米。 </li><li>将切断锥形管顶在钻孔。使用移液管，施加约250微升的二氯甲烷到锥形管的切断边缘与锥形管的150毫米盖结合。 </li><li>通过施加硅橡胶密封剂中的1毫米厚的珠周围的顶部培养皿的内边缘密封150毫米盖到150毫米的菜。 </li></ol> 2.剪切戒指的消毒<ol><li>吸取约10毫升磷酸盐通过15毫升锥形管口缓冲液进入新成立的剪环。漩涡周围去除剪切​​环内的任何杂物。用巴斯德吸管/真空抽吸器除去磷酸盐缓冲盐水。 </li><li>直到碎片被删除重复上述步骤。 </li><li>要STERI丽泽剪切环，使用用紫外线照射70％的乙醇漂洗的组合。 <ol><li>拧开盖子通风，吸管大约10mL，70％的乙醇通过接入端口，并重新拧紧瓶盖。旋转和剪切环多次翻转，确保剪切环的内部被充分洗涤。 </li><li>在通风橱，吸出多余的70％的乙醇。用喷雾瓶，彻底喷用70％乙醇在盖和进入端口。 </li><li>将下的组织培养罩内的紫外线辐射剪切环和帽直至完全干燥。 </li></ol></li><li>干燥后，拧在室温下回到了帽和消毒店剪戒指，直到在细胞培养电镀。 </li></ol> 3.剪应力研究<ol><li>板下通常使用1标准细胞培养协议消毒剪切环内皮细胞:转化的内皮细胞系4分流比。 注:大鼠视网膜microv在商业上获得ascular内皮细胞。人脑内皮细胞（hCMEC / D3）细胞提供从皮埃尔 - 奥利弗博士Couraud（INSERM，法国）一份厚礼，并在完全内皮基础培养基（EBM）进行培养。 </li><li>允许文化中流动的研究开始之前达到汇合。 USE 30 ml的组织培养基（10％胎牛血清，Dulbecco改良的Eagle与1％青霉素/链霉素/两性霉素培养基）。改变细胞培养基，每3天，并保持细胞在37℃用7.5％的CO 2和92.5％的室内空气。 </li><li>放置在孵化器的轨道摇床。 注:轨道摇床通常是重（&gt; 10公斤）。放置培养箱的底部货架上的轨道摇床，以尽量减少货架应力和振动。 </li><li>将实验剪切环对轨道振动筛。将静态对照组剪切环同样的培养箱内。 </li><li>在SH内估计最大剪应力耳环与方程<img alt="式（1）" src="/files/ftp_upload/54632/54632eq1.jpg" />哪里<img alt="公式（2）" src="/files/ftp_upload/54632/54632eq2.jpg" />是旋转的轨道摇床半径（厘米）， <img alt="公式3" src="/files/ftp_upload/54632/54632eq3.jpg" />为介质的粘度（泊）， <img alt="公式4" src="/files/ftp_upload/54632/54632eq4.jpg" />是介质的密度（以g / ml）和<img alt="公式5" src="/files/ftp_upload/54632/54632eq5.jpg" />是旋转频率（旋转/秒）24。 </li><li>发起于轨道摇床旋转设置到所需的转速（ 例如，90转），而使剪切环上的振动筛剪切应力应用程序（ 例如 ，72小时）的所需的持续时间。 注:轨道振动器能够产生的热量，所以培养箱温度最初应监测，以确保37℃保持整个实验的持续时间。另外，S听到环可转移到环境室（ 例如 ，模块化培养箱室），然后置于旋转培养箱内。 </li><li>后的细胞层被暴露于剪切为所需的时间长度，从孵化器中移除剪切环。拉断的剪切环盖和检索单元和/或媒体进行检查所需端点分析（ 例如，蛋白质印迹，荧光激活细胞分选等 ）25。 </li></ol>

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J.汶禹云已经从吕秀莲尼切洛基础研究基金。 J.亚历山大·史蒂芬拥有神经内科，LSUHSC-S系研究支持。

对暴露的内皮细胞剪切剪切环系统的建设是一个简单的方法来进行剪切应力的研究。然而，有一些是获得卓越的剪切戒指和更好的结果的关键几步之遥。一个完整的密封应在内和外环，以防止介质从泄漏可能创建样品之间不一致的剪切应力之间进行。如果完整的密封不进行，二氯甲烷最小量应通过在内圈的孔被加入到内和外碟用移液管之间的边缘。轻轻转动该环应该允许二氯甲烷以形成一个完整?...

流体剪切应力已经显示出调节内皮基因方案1 -通过顺式调节元件6的活化，组蛋白乙酰转移酶活7和剪切应力响应元件（SSRE）8 5。通过调节组织因子9和组织型纤溶酶原激活剂（tPA）10表达的剪切应力的影响对凝血内皮的贡献。剪应力也影响通过调节PDGF-B的合成和反应8血管生成11和血管重塑的控制权。内皮衍生的血管活性介质肾上腺髓质素，内皮素-1，尾加压素II和松弛素也被剪切机12调节。内皮型一氧化氮合成酶的生产和一氧化氮产生的转录都是剪切依赖性10。剪切还控制内皮的ICAM-1的表达13。流动引起的剪切应力，因此可以powerfuLLY影响种类繁多的内皮细胞反应。重要的是，血管脉动现在也出现在血管性痴呆14的正常血管老化和形式的病理生理学中发挥重要作用，甚至可能导致其它神经变性疾病，如多发性硬化15。 静脉和动脉内皮细胞固有地暴露于不同的血流动力学流动模式在体内 ，许多不同的内皮细胞的表型可以显示出16。取决于幅度和流量的周期性，对内皮细胞的作用可以包括炎性细胞活化和细胞凋亡，其可反映在基因或蛋白质表达17,18变化。研究内皮细胞对剪切现象，因此仍然被困难复杂， 在能够充分产生这样的剪切模式体外模型制作。 许多不同的experimental协议已被开发应用的流体剪切应力内皮细胞单层。一个最常用的系统是平行板流动室，它创建室19内均匀的层流- 21。蠕动泵通常连接到创建的周期性流，这可以概括通常在体内 22的许多地点找到的流动特性。另一种常见的建立使用&#39;锥板"的模式，其中，流体剪切应力是由锥体23的旋转速度确定的。两个系统，和其他安排类似于它们，可能是很乏味的设置和需要，可以是相对昂贵并且不可访问的许多实验室的组件。 这些当前模型的另一个主要限制是，可以同时进行，每一个相对低的表面积复制研究的数量相对较少。这增加了时间和共<html>这种方法的mplexity。因此，诱导单向和周期性剪切的理想模型可能之一，可以很容易地设置研究重复的较高的数值，每个具有相对较大的表面积。此外，上述模式需要一个相当复杂的设置，其可以是成本高昂的许多用户。可使用的基本材料实验室产生的流体剪切扰动模型可能有几个优点。 施加单向的，周期性的剪切应力的一种简单和非常经济的方法涉及的圆形菜定轨振荡器上24的位置。这个协议是很简单的，并且可以按比例增加，以实现高的数字研究复制，每个具有相对较大的表面面积，根据需要。但是，设在盘的中心的细胞暴露于不同的流动模式比沿周细胞，产生在相同的培养皿混合蜂窝的表型应答。 _content"&gt;在当前的报告中，我们通过限制流量描述"剪切戒指&#39;，我们的模型创建单向和定期剪应力的建设和使用，设计的剪切环有效限制"混合"蜂窝剪切引起的表型通过内圈的位置的圆形培养皿至周边内途径。剪切环的结构和操作是简单和经济的，并且可以很容易地扩展使用广泛使用的组织培养供应以适应广泛范围的轨道摇床中。这模型可以在内皮细胞实验应用于生理和病理水平之内提供单向和周期性流动模式。

<table><tbody><tr><td>100 x 20 mm plastic tissue culture dish</td><td>Corning</td><td>430167</td><td>The dishes must be polystyrene</td></tr><tr><td>150 x 25 mm plastic tissue culture dish</td><td>Corning</td><td>430599</td><td>The dishes must be polystyrene</td></tr><tr><td>150 mm glass Petri dish</td><td>Fisher</td><td>3160150BO</td><td></td></tr><tr><td>15 ml polystyrene tissue culture plastic tubes</td><td>Falcon</td><td>352099</td><td></td></tr><tr><td>Methylene chloride</td><td>Sigma-Aldrich</td><td>D65100</td><td></td></tr><tr><td>silicone rubber sealant</td><td>DAP</td><td>7079808641</td><td></td></tr><tr><td>ethanol</td><td>Decon</td><td>2701</td><td></td></tr><tr><td>3 ml transfer pipette</td><td>Becton-Dickinson</td><td>357524</td><td></td></tr><tr><td>printer paper</td><td></td><td></td><td></td></tr><tr><td>scissors</td><td></td><td></td><td></td></tr><tr><td>gloves</td><td></td><td></td><td></td></tr><tr><td>rotary tool and set</td><td>Dremel</td><td>4000-6/50</td><td></td></tr><tr><td>rotary tool cutting head</td><td>Dremel</td><td>EZ476</td><td></td></tr><tr><td>rotary tool drill head</td><td></td><td></td><td></td></tr><tr><td>distilled water</td><td></td><td></td><td></td></tr><tr><td>orbital shaker</td><td>VWR</td><td>57018-754</td><td></td></tr><tr><td>incubator</td><td></td><td></td><td></td></tr><tr><td>Rat retinal microvascular endothelial cells</td><td>Cell Biologics</td><td>RA-6065</td><td></td></tr></tbody></table>

the assembly and application of 'shear rings': a novel endothelial model for orbital, unidirectional and periodic fluid flow and shear stress

通过诱导适应性以及在血管内皮表型和基因表达的病理变化，从正常水平，并在血管生理学和病理生理学的血管流体剪切中发挥重要作用的型态偏差。特别是，周期性的，单向流动引起的剪应力的适应不良效果可以触发各种几个血管细胞类型，尤其是血管内皮细胞的影响。而现在来自不同解剖起源的内皮细胞已经培养的，深入的其对流体剪切反应受到阻碍剪切模型的相对复杂性分析（ 例如，平行平板流动腔，锥板流速模型）。虽然这些都代表着优秀的方法，这种模式在技术上是复杂和缺点，包括相对漫长和复杂的设置时间，低表面积，泵和增压常常需要密封胶和密封垫要求受苦，创建对BOT的挑战^ h维护不育和无法运行多个实验。然而，如果流动和剪切的更高的吞吐量模型可用，对血管内皮的剪切响应更大的进步，在分子水平上尤其周期性剪切研究，可能会更迅速地先进。这里，我们描述了构造和使用剪切环:一种新颖的，简单到组装，和便宜的组织培养模型具有相对大的表面面积，可以轻松地允许在单向的，周期性的剪切应力在研究大量的实验重复的内皮细胞。

在这里，我们提出从两个hCMEC / D3脑内皮细胞和大鼠视网膜微血管内皮细胞单层，在剪切环培养代表性的结果。 允许hCMEC / D3脑内皮细胞单层生长在完整的EBM至汇合后，剪切环被放置在定轨摇床上72小时。从步骤3.5使用等式，计算出的最大剪应力<img alt="公式6" src="/files/ftp_upload/54632/54632eq6.jpg" />约为2.8达因/厘米2（与参数<i...

Watch this Scientific Journal Video about The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress at JoVE.com

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和"剪切环"中的应用:一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

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Research

JoVE Journal

Biology

8.0K 次观看.  Louisiana State University Health Sciences Center in Shreveport. Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

视频: The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

科研

生物工程

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

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 ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

的剪切粘合地图使用SynVivo合成微血管网络代

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

Visualization and Quantification of TGF&#946;/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and homeostasis of the vasculature system Therefore, deregulation in this signaling pathway results in severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, and atherosclerosis. Endothelial cells (ECs), as the innermost layer of blood vessels, are constantly exposed to fluid shear stress (SS). Abnormal patterns of fluid SS have been shown to enhance TGF&#946;/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGF&#946;/BMP signaling while atheroprotective, high laminar SS, diminishes this signaling. To efficiently analyze the activation of these pathways, we designed a workflow to investigate the formation of transcription factor complexes under low laminar SS and high laminar SS conditions using a commercially available pneumatic pump system and proximity ligation assay (PLA).
Active TGF&#946;/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGF&#946; and BMP signaling, respectively) with a common mediator SMAD (co-SMAD; SMAD4). Using PLA targeting different subunits of the trimeric SMAD-complex, i.e., either R-SMAD/co-SMAD or R-SMAD/R-SMAD, the formation of active SMAD transcription factor complexes can be measured quantitatively and spatially using fluorescence microscopy.
The usage of flow slides with 6 small parallel channels, that can be connected in series, allows for the investigation of the transcription factor complex formation and inclusion of necessary controls. 
The workflow explained here can be easily adapted for studies targeting the proximity of SMADs to other transcription factors or to transcription factor complexes other than SMADs, in different fluid SS conditions. The workflow presented here shows a quick and effective way to study the fluid SS induced TGF&#946;/BMP signaling in ECs, both quantitatively and spatially.

Here, we establish a protocol to simultaneously visualize and analyze multiple SMAD complexes using proximity ligation assay (PLA) in endothelial cells exposed to pathological and physiological fluid shear stress conditions.

使用接近-连接-测定法对不同流体剪切应力条件下TGFβ/BMP/SMAD信号传导的可视化和定量

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and ...

生物化学

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

多平行板流室对剪切应力暴露的内皮细胞基因表达分析

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

在机械生物学研究的轨道摇床上的 6 井板中分段内皮细胞的生长

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

模仿神经血管化初始事件的微流体模型

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood flow triggers a signaling cascade resulting in vasodilation thereby alleviating mechanical stress on the vascular wall. The pattern of shear stress is also well known to be a critical factor in the development of atherosclerosis with laminar shear stress being atheroprotective and disturbed shear stress being pro-atherogenic. While we have a detailed understanding of the various intermediate cell signaling pathways, the receptors that first translate the mechanical stimulus into chemical mediators are not completely understood. Mechanosensitive ion channels are critical to the response to shear and regulate shear-induced cell signaling thereby controlling the production of vasoactive mediators. These channels are among the earliest activated signaling components to shear and have been linked to shear-induced vasodilation through promoting nitric oxide production (e.g., inwardly rectifying K+ [Kir] and transient receptor potential [TRP] channels) and endothelium hyperpolarizing factor (e.g., Kir and calcium-activated K+ [KCa] channels) and shear-induced vasoconstriction through an undetermined mechanism that involves piezo channels. Understanding the biophysical mechanism by which these channels are activated by shear forces (i.e., directly or through a primary mechano-receptor) could provide potential new targets to resolve the pathophysiology associated with endothelial dysfunction and atherogenesis. It is still a major challenge to record flow-induced activation of ion channels in real time using electrophysiology. The standard methods to expose cells to well-defined shear stress, such as the cone and plate rheometer and closed parallel plate flow chamber do not allow real time study of ion channel activation. The goal of this protocol is to describe a modified parallel plate flow chamber that allows real time electrophysiological recording of mechanosensitive ion channels under well-defined shear stress.

The goal of this protocol is to describe a modified parallel plate flow chamber for use in investigating real time activation of mechanosensitive ion channels by shear stress.

在定义良好的剪切应力下单细胞电电流的电生理记录

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood ...