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毫升内皮生长中期辅以1X抗生素抗霉菌解决细胞（EGM-2）。改变媒体每2天，直到细胞达到90％汇合。 </li><li>保持在THP-1细胞的RPMI 1640培养基中10％FBS和：1X抗生素抗霉菌的解决方案。当他们到达密度为1的传代细胞0 6细胞/毫升。 </li></ol> 2。细胞剪切协议细胞习惯于在一个自定义板锥细胞剪切设备（CSD）。设备细节和设计规范，记录在表1和图4。 <ol><li>准备惩教署。消毒用乙醇室住房，并用无菌PBS冲洗。住房加热至37°C和水平，以确保锥细胞单层垂直。从外壳底部的细胞组装基板和锥设置到所需的高度。 </li><li>使用莱博维茨-15（L-15的）内皮BulletKit补充中型剪切介质中的CO 2的情况下保持适当的pH值。刺激炎症，肿瘤坏死因子-α（TNF-α0.5毫微克/毫升）加剪切介质和注入设备，通过连接到进气口注射器6毫升，照顾不引进空气气泡室。 </li><li> （15达因/厘米2）或振荡剪切应力为4小时（OSS，0±5达因/厘米2，1赫兹）高速钢，高的幅度稳步剪应力剪细胞。所需的SS为实现以上的细胞，而这恰恰是如前面所述的微步进电机控制旋转锥6墙表面SS（τW）近似。 <img src="/files/ftp_upload/4169/4169eq1.jpg" alt="式（1）" /> 其中，μ为介质的粘度，是圆锥体的角速度ω，α是锥角（0.5°）。锥与基板对齐调整为0.05°内通过精密水准和间隙高度设置在20±10微米，使用深度计，以尽量减少周向和径向在τ 瓦特变化。该设备已被验证，以确保层流特性。删除Ç从设备的ELLS和准备分析单核细胞粘附。 </li></ol> 3。制备的PDMS微流控室<ol><li>获得所需的微通道数量和通道的高度，取决于应用程序的主模具。在本协议中所使用的具体设计的特点，详列于表1。 创建这些方法已在文献记载。1简言之，网络渠道使用CAD软件Autodesk Inventor的设计和印刷于5000 DPI透明度。负光阻（SU8）纺到硅片厚度100微米。覆盖的透明度和暴露在紫外线下。非聚合光刻胶去除产生积极的副本主。 </li><li>使用硅胶固化按重量10:1代理在权衡船的PDMS溶液混合硅基地和5毫升血清吸管搅拌。要小心，不要刮墙壁称重船解决方案，以避免增加塑料片。 </li><li>主晶片放置在一个100x15mm培养皿，倒入菜的PDMS解决方案。 </li><li>覆盖的菜，放入真空容器中，15分钟，以消除气泡。 15分钟后，培养皿，在表面轻轻吹空气中删除任何多余的气泡。 </li><li> 1小时烤箱放置在培养皿，在70°C。取出盘子切出微腔，用锋利的刀片。 </li></ol> 4。大会在显微镜设备<ol><li>打开光源倒置显微镜和设置加热至37°C。连接管10毫升注射器装满蒸馏水等，有没有气泡。将在注射器的注射泵和集流率，诱使1达因/厘米2的墙卫队。沿着通道中心线产生的壁面剪切应力（τW）使用标准的近似平行板流动腔方程为： <img src="/files/ftp_upload/4169/4169eq2.jpg" alt="公式（2）" /> 其中，μ为介质的粘度，Q为体积流量，H通道高度，w为通道宽度注：因为渠道有限宽度的实际τw将随W的通道长宽比，避免测量的25％，在最近的一面墙壁。1,7 </li><li>放置在水中的PDMS室和使用100μL微量消除空气从各个渠道。将适配器真空室制作确保设置为关闭阀门。 </li><li>将培养皿HAEC单层以上的目标。放置在培养皿中的PDMS室和精心设置的HAEC单层，同时确保无气泡形成之间的设备和单层。关闭阀门，使真空室连接牢固。 </李&gt; <li>削减19个表针生硬等，有金属左侧只有5毫米。缓冲区填充塑料水库和投入的PDMS室门口。重复使用的通道的适当数量。附加油管从注射泵的PDMS通道出口。 </li></ol> 5。剪应力下的单核细胞粘附实验<ol><li>暂停在2X10 6个细胞/毫升的HBSS +的Ca 2 + /镁2 + 0.1％HSA的THP-1细胞。激活的THP-1细胞与基质细胞衍生因子1（SDF-1，50微克/毫升）的孵化室温为15分钟。 </li><li>注射泵转动建立流，然后加入50μL细胞悬液，水库没有引入气泡。一旦流充分发展，开始数据采集。 </li><li>运行适当的流速为5分钟，加入缓冲水库解决方案，如果变低。继续流入渠道洗出流流中的任何绑定细胞的缓冲区。 </li></ol> 6。数据采集​​与分析<ol><li>在3帧/秒，2分钟后，沿纵轴通道3通道的位置流充分发展，照顾侧面记录后，立即入口或出境口岸前，或在25％，最近获得的数字图像序列墙壁。 </li><li>每场拘捕细胞的数量计算确定其相明亮的外观，在相同的单层焦平面。 </li><li>细胞周期阻滞是指公司通过运动&lt;半细胞直径在10秒。平均超过3个随机的领域/通道和数据量化使用ImageJ的软件。 </li></ol> 7。代表结果虽然血管内皮细胞均匀地暴露激动剂有助于全身炎症，动脉粥样硬化是一种疫源性疾病动脉流扰动的网站，优先发展，在血管内皮功能的空间异质性暗示。 </suP&gt;循环炎症介质如肿瘤坏死因子-α升高人体血清中的代谢压力下，相关的血管内皮功能受损。9日，10肿瘤坏死因子-α诱导良好的特点，在欧共体的反应，包括上调细胞粘附分子（CAMS如VCAM-1，ICAM-1，E-选择素）和趋化因子，招募单核细胞11血流动力学，从流通，早期动脉粥样硬化的一个标志，是一个重要的调节内皮细胞炎症型12个高幅度剪应力（高速钢。如15达因/厘米2）或脉动剪切应力（PSS;如15±5达因/厘米2）与抗动脉粥样硬化的网站，在动脉下调TNF-α诱导的反应。相反，低幅度剪切应力（LSS，如2达因/厘米2）或振荡剪切（OSS;如0±5达因/厘米2）， 在体内的易感位点的特点，加剧cytokiNE引起的炎症。13，14我们的PDMS微流控设备进行机理研究，测试与流体力学因素在调节血管内皮功能和病理代谢应激叠加的假设提供了一个平台。通过以下的有代表性的成果，大致说明了该方法的实用性和多功能。 <img src="/files/ftp_upload/4169/4169fig1.jpg" alt="图1" /> 图1。剪应力差异调节THP-1细胞粘附肿瘤坏死因子-α发炎的内皮细胞单层。使用上面描述和记录视频的详细协议，这些数据被收购。 HAEC单层同时暴露到低剂量的炎性细胞因子肿瘤坏死因子-α（0.5纳克/毫升，50〜欧共体VCAM-1的上调），要么HSS（15达因/厘米2）或OSS（0±5达因/厘米2，定制惩教署在1赫兹）作为descri 4小时床的详细协议。 THP-1细胞阻滞在PDMS流动腔的剪切流场下的量化。这模仿人类单核细胞表型的细胞系，被选为其重现性和易用性。相比，高速钢抑制炎症​​反应的OSS，导致下降的THP-1细胞逮捕。学生t检验数据进行分析，并表示为平均值±SEM 3个独立的实验适应从DeVerse等权限5。 下面的数据表明我们的方法的通用性，使用的例子，从我们的出版工作。偏离上述协议表示（如适用）。 <img src="/files/ftp_upload/4169/4169fig2.jpg" alt="图2" /> 图2。调节细胞因子诱导的VCAM-1表达和单核细胞在剪应力梯度招聘。一）HAEC单层4小时刺激下FLO与肿瘤坏死因子-α（0.3毫微克/毫升）w在设计基于海伦-肖流理论VMMC 15在SS幅度沿着通道中心线的直线下降。设计室侧壁配合二维停滞流简化和塑造年底实现通道，以符合ISO-电位线。沿着这个通道从入口（X）的轴向距离中心线产生的壁面剪切应力（τW）计算公式如下： <img src="/files/ftp_upload/4169/4169eq3.jpg" alt="公式（3）" /> 其中，μ为介质的粘度，Q为体积流量，h是通道的高度，W 1通道入口的宽度，L是总的通道长度，Q被选为生成从16日在SS幅度梯度达因/厘米，在进口2 0驻点“一个”公正的outle近端ţB）-C），VCAM-1表达的原位免疫荧光显微镜使用抗体共轭量子点和比例中位数的荧光强度（MFI）。无刺激的静态控制测量。卫队没有调节VCAM-1的基础水平，这是在未刺激HAEC（灰色三角形）低。然而，VCAM-1表达增加肿瘤坏死因子-α在LSS的程度，高峰在2达因/厘米2，并返回到TNF-α的基线或以下&gt; 5达因/厘米2（黑色正方形），D剪切程度）单核细胞（10 6 /毫升）灌流2达因/厘米2， 在A）为前提的单层，平行流渠道为5分钟。单核细胞向血管内皮细胞的招聘数量与VCAM-1表达，在一个给定的剪应力。值得注意的是，能够探测与罚款在monolaye空间分辨率的VCAM-1表达和单核细胞招聘ŕ透露了在党卫军幅度狭窄的范围内，不会被应用在不同的实验只有几个离散值剪切赞赏显着的变化。数据进行了分析，重复测量方差分析和纽曼的两两比较后测评估差异，静态条件下，* P &lt;0.05从肿瘤坏死因子-α。数据平均值±3-6独立实验的SEM 改编权限由邹族等。2 <img src="/files/ftp_upload/4169/4169fig3.jpg" alt="图3" /> 图3。富含甘油三酯的脂蛋白（TGRL）调节比例捐助者的血清甘油三酯水平和VCAM-1表达的细胞因子诱导的炎症。HAEC膜与肿瘤坏死因子-α（0.3毫微克/毫升）4个小时与TGRL（10 mg / dL的同时刺激载脂蛋白B）分离，从静态培养高脂肪食物后，人类受试者A）测定VCAM-1表达在暂停欧共体分别用流式细胞仪和单核细胞粘附在详细协议中所述，使用VMMC作为量化。数据以％肿瘤坏死因子-α的变化。单核细胞逮捕不同直接比例内皮VCAM-1表达，从而增加直接与捐助者的血清甘油三酯水平B）单核细胞促炎症捐助者的一个子集逮捕被废止的VCAM-1的阻断抗体的存在。配对t检验确定的意义，这意味着±SEM 3-5独立实验。 改编，王等人的许可。4

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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>

没有利益冲突的声明。

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 ...

on-chip-endothelial-inflammatory-phenotyping

Research

JoVE Journal

Bioengineering

10.8K 次观看.  University of California, Davis. 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。

视频: On-Chip Endothelial Inflammatory Phenotyping

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

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

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

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

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

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

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

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show 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

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

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

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

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

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes 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

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

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

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, inflammation, and homeostasis. The endothelium also plays an important role in pathophysiologies such as atherosclerosis, hypertension, and diabetes. Endothelial cells form the inner lining of blood and lymphatic vessels and display heterogeneity in structure and function. Various groups have evaluated the functionality of endothelial cells derived from human peripheral blood with a focus on endothelial progenitor cells derived from hematopoietic stem cells or mature blood outgrowth endothelial cells (or endothelial colony-forming cells). These cells provide an autologous resource for therapeutics and disease modeling. Xenogeneic cells may provide an alternative source of therapeutics due to their availability and homogeneity achieved by using genetically similar animals raised in similar conditions. Hence, a robust protocol for the isolation and expansion of highly proliferative blood outgrowth endothelial cells from porcine peripheral blood has been presented. These cells can be used for numerous applications such as cardiovascular tissue engineering, cell therapy, disease modeling, drug screening, studying endothelial cell biology, and in vitro co-cultures to investigate inflammatory and coagulation responses in xenotransplantation.

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

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

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 ...