Name: easy and accurate mechano-profiling on micropost arrays
Uploaded: 2015-11-17T15:00:00.000+00:00
Duration: 10 min 25 s
Description: Watch this Scientific Journal Video about Easy and Accurate Mechano-profiling on Micropost Arrays at JoVE.com

This work was supported by the Committee Technology and Innovation (CTI) Switzerland grant 14796-PFLS-LSCTI. MicroPost arrays were generously provided by MicroDuits GmbH.

在Micropost阵列1.培养细胞注意:所有步骤必须在生物安全柜中进行，以确保无菌。这里给出的容积为T25细胞培养瓶。细胞培养基配方和细胞接种密度为骨癌细胞系HuO9和M132优化。 <ol><li>准备播种的细胞培养到micropost阵列。 <ol><li>通过将培养瓶在标准光学显微镜下检查细胞培养质量。确保细胞显示出典型的生长和形态通过估计如何培养瓶底部的许多被覆盖。 70％-80％A覆盖率反映了健康的增长速度。因为它们代表死细胞和/或一个杂草丛生培养注意漂浮细胞的量。 </li><li> Trypsinize细胞通过除去介质和洗涤用4ml 1×磷酸盐缓冲盐水（PBS）缓冲液中。添加0.8 ml的1×胰蛋白酶/乙二胺四乙酸（EDTA）和在室温下孵育，直到电池小号打跑，大约需要2-4分钟。检查过程与显微镜，偶尔挖掘瓶轻轻地支持撞出。 </li><li>加入5 ml的细胞培养液（Dulbecco改良的Eagle培养基（DMEM）/ F12，用10％胎牛血清（FBS），1％青霉素/链霉（青霉素/链霉素）），使反应停止，并以洗掉残留细胞仍然坐在瓶的底部。接着吸取此悬浮上下数次以获得均匀的混合物。一定要吸取横跨烧瓶的原始生长区域中的所有解决方案以获得一个有效的松脱。 </li><li>在细胞悬液转移到15毫升管中，用台式离心机以0.5×g离心3分钟离心它。 </li><li>除去上清液并且通过移液器上下吹吸5次再悬浮在5ml培养基中的细胞沉淀。请一定要避免泡沫的产生，而这样做。 </li><li>通过使用细胞计数室和一个光microsco确定细胞密度PE。检查细胞聚集体的细胞悬浮液中细胞计数室和确保仅具有单个细胞用于后续实验。吸管再次上下数次，如果细胞倾向于形成聚集体将它们分开。 </li><li>稀释细胞悬浮液以足够的介质获得的25000个细胞/ ml的细胞液。颠倒封闭管数次拌匀。 </li></ol></li><li>准备micropost阵列细胞种植。 关键注意:不要吸取到micropost阵列直接，因为这可能会损害microposts，尤其是当不想要的泡沫进行了介绍。此外，请确保micropost阵列永不干涸的发生毛细作用力导致崩溃的microposts的。 <ol><li>放置与micropost阵列玻璃衬底面朝上在12孔板的孔中通过使用一对镊子的，并通过添加1毫升乙醇中（99％）湿它。在室温下孵育20-30秒。 </li><li>稀释乙醇逐步通过在井侧加入约1毫升无菌去离子水（DI水）和吸移用移液管或抽吸设备大致1毫升重复此步骤，至少3次。 </li><li>通过添加和吸移约1 ml的取代离子水用PBS缓冲液中相同的方式。重复此步骤3次。 </li><li>通过添加和吸约为1毫升培养基替换PBS缓冲带中。重复此步骤3次。 </li></ol></li><li>在每个制备的micropost阵列的顶端吸移管1 ml的细胞液（25,000个细胞）。靠近多孔板，并将其转移到恒温箱（CO 2 5％，37℃）。让细胞粘附和生长在microposts为6-7小时之上。 </li><li>通过使用光显微镜偶尔检查的粘附过程。检查大部分细胞出现全面铺开多个microposts。 </li></ol> 2.固定和染色细胞注意:所有的步骤，并这里给出容积为12孔板的单个孔。建议处理不多十二井超过四个在一个时间，以避免一个晒出孔中的任何液体，这将导致在microposts的崩溃后吸出。 <ol><li>从井抽吸介质和洗涤细胞2×1毫升的PBS缓冲液。确保使用温和的力量，同时用PBS洗涤以去除累积的孵化过程中micropost阵列上的细胞碎片和分离死细胞。 </li><li>固定细胞以5分钟0.5 ml的3.7％缓冲的甲醛溶液。 </li><li>通过洗涤micropost阵列2×1毫升无菌DI水代替甲醛溶液。 </li><li>染色（在50％的水，40％乙醇和10％乙酸0.05％考马斯亮蓝）将细胞与染料为约90秒。洗净多余染色液过2次用1毫升无菌去离子水。加入1毫升去离子水至micropost阵列。 </li><li>使用光学显微镜检查染色的结果。重复吨HE染色步骤，如果单元格的身体太微弱待观察。 </li><li>商店micropost阵列染色细胞之上的冰箱中在4℃下。确保让他们在水下时刻。 </li></ol> 3.细胞成像注:第4步仅适用于显微镜没有无限校正光学系统。 <ol><li>加2ml去离子水至培养皿用薄玻璃底，高分辨率成像。 </li><li>用一对镊子到成像盘与microposts朝上传送micropost阵列。 </li><li>放置在成像培养皿上的光学显微镜的可移动的阶段。 </li><li>转用于成像的透镜的补偿环，直到在标尺上的数字表示沿着光路的所有材料的总厚度，以补偿在玻璃基板上，有机硅弹性体和顶部的液体的折射率的不匹配。这将导致一个明亮的外观的microposts的核心。 </li><li>关闭光圈在照明侧下降到50％，并从光路在允许一个普通明视场模式除去任何相衬环。 </li><li>对齐micropost阵列的microposts形成一条水平线的观察视野。确定起点（ 如左上角），移动培养皿逐步在舞台上扫描micropost阵列同时服用大量的图像。 </li><li>以最高的分辨率设置使用20X或40X的目标相机中的所有图像。希望能发展为在图像的中心的单细胞。 </li><li>通过基板沿z轴扫直到micropost提示是聚焦。使用显微镜的微调调焦轮，使之朝着注重micropost底部约2-3微米。 </li><li>通过采取一系列一个阵列部通过摄像参数之一清扫测试图像在一个时间（建立标准成像过程例如，曝光时间，光圈设置，灯设置等）。与MechProfiler分析图像并确定一个快速和可靠的分析的合适的参数组。 </li></ol>与开放源代码软件"MechProfiler"4.图像分析注意:所有软件功能可与使用鼠标左键指点设备被激活。然而，受过训练的操作将使用设计为在键盘上，以使一个有效的两手的图像处理的左半边的记录快捷键。 <ol><li>启动图像分析软件MechProfiler并打开一个范围，需要通过点击"打开"被分析的图像。 </li><li>将所有参数到由软件开发商提供的"设置"部分。确定必须根据由软件手册中详细描述的程序进行自定义配置（即，轮廓阈值和最小距离）的参数。 </li><li>分析图像■一个接一个通过使用"剪裁"他们种植到感兴趣的区域（单击并拖动鼠标按钮按下）。双击绘制的矩形内完成这个动作。 </li><li>通过点击"画细胞轮廓"，使用十字光标绘制包括细胞附着到所有microposts标记每个可见细胞轮廓一个接酮。 </li><li>通过点击"放弃帖子"放弃任何不必要的microposts并使用十字光标绘制。括属于一个单元的图象部分以外的或者被偏转因其他理由而不是由所关注的小区中的所有microposts。 </li><li>启动软件子程序"查找质心"通过鼠标点击。调整过滤器设置旁边的"查找质心"按钮，直到所有microposts注册，这是可见的红叉的中心。如果过滤器的设置过低microposts将被忽略，如果它太高亩ltiple职位将被标记为一个单一的micropost。保持过滤器的分析会话期间设置不变。 </li><li>使用手动编辑功能"手动编辑"的质心与多个或遗漏micropost位置。使用鼠标十字通过单击并拖动整个选择有问题的micropost。双击矩形内，并将丢失的红色标记放大的图像部分，它会自动关闭研究。放弃这样的micropost如果是绘制细胞外轮廓（见步骤4.5），然后再继续。 </li><li>找到理想的micropost电网用鼠标点击激活"生成网格"功能。确保位置对应的真实micropost头，用蓝色圈所示，绘制细胞轮廓内。 </li><li>更正其中使用相应的"曼努埃尔编辑"功能，用鼠标点击所需要的细胞区域内的任何错位的电网设施。使用十字来选择曲的micropostestion通过点击和拖动它跨越。双击出现的矩形内，并将丢失的蓝色标记的放大图像部分，它会自动关闭研究。 </li><li>使用按钮"计算挠度"通过鼠标点击来获得基于所述图像段中的micropost的位置和产生的理想电网之间的差计算出的偏转值的直方图。 </li><li>保存完整的分析，包括价值观，通过鼠标点击"保存"的表。 </li><li>通过点击"重置视图"和分析"下一步形象"，它可以方便地使用正确的键盘光标键来完成另一个图像部分或鼠标点击继续图像分析。 </li></ol> 5.数据分析 - Mechanoprofiling <ol><li>从与所分析的图像文件夹中的Office电子表格程序打开文件"results.xls"。它包含了人的数据计算L值，包括所有micropost偏转和标准衍生的措施，如工作（ 即基底变形能量）的分析单元。 </li></ol>

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The lead author (NG) is co-founder of MicroDuits GmbH, the commercial supplier of the micropost arrays used in this study. No other authors have any potential conflicts of interest with this work.

这项工作旨在通过大幅降低技术和实用的准入门槛，以推进牵引力显微镜领域。这些障碍来自两个方面。首先是必须克服可重复地制造和实验委托micropost阵列的许多非平凡的技术挑战。第二，是同样的非平凡需要单个细胞的力的可靠半自动分析 - 牢记典型的实验中，可能需要几百甚至几千个细胞的分析。上述技术被开发，以使不具有驻地工程专门细胞生物学实验室内micropost基于牵引力显微镜访问?...

机械性敏感基于细胞的测定允许调查粘附细胞，具有广泛的，反映了力学可以在细胞生物学发挥核心作用的应用程序。这些应用程序通常专注于推动亚细胞过程或全细胞行为的基本机制。一方面，外部环境因素，如细 ​​胞外基质组合物或基质硬度可显着地影响细胞的机械和生物反应。1同样可以使用许多类的药物活性化合物的后观察到，其中，效果是常其特征在于使用细胞培养模型2另一方面基因型性能，例如那些引起自发或实验诱导的基因突变，可诱导了与细胞骨架结构和功能的改变相关的细胞表型的显着的变化。3这些实施例短短数的许多可能的题目为哪些细胞机械表型是相关的，并且所有的这些都被有效地研究了micropost阵列。 在写这篇文章的时候，大约有200多篇已经发表了描述细胞micropost互动。这些作品讨论micropost偏转原理的理论问题，以及在其制造的实用说明。第一篇文章中描述的细胞和灵活micropost阵列的相互作用是出 ​​版的谈及其同事在2003年4相反经典牵引力显微镜（TFM），其中连续的软衬底被用于估计nanonewton规模细胞收缩，Tan等人。描述使用由有机硅弹性体的多个紧密隔开的垂直梁的方法。该技术的主要优势出现两个主要的功能。首先，为了改变小区表观基板刚度1只需要改变micropost尺寸，同时保持基板组合物，否则恒定，从而避免了在表面拓扑结构和化学的差异。第二microposts像可以与力和空间分辨率个别粘着斑的数量级上离散地分析，并且可以减少所固有的由标准的TFM类似分析的分析挑战个人弹簧。 今天的micropost阵列的应用范围大大超过了力量的只是少数单个细胞的映射。例如，秋山报告使用的分离的背脉管组织从一个蛾毛虫作为致动器的一个micropost阵列，为了开发一种昆虫肌肉供电自主微型机器人。5 然而，microposts大部分发布的应用程序都集中在医疗条件，比如感染或癌症的研究。例如，micropost阵列已经用于研究力产生的奈瑟gonorrh捆绑IV型菌毛是与信号级联增强感染有关。6其他已经使用microposts研究并靶向细胞骨架的药物化合物治疗乳腺癌细胞OEA菌落。7 一个micropost的偏转采用经典梁理论常常被描述为与最终负载假设电池的悬臂只重视对micropost的最顶端。这里所施加的力 F引起的偏转δ取决于micropost的"抗弯刚性"k和由下式计算: <img alt="式（1）" src="/files/ftp_upload/53350/53350eq1.jpg" /> （1） 与E，I，和L是杨氏模量，转动惯量和梁的长度分别面积矩。然而，从这个公式的结果只能给力的，因为梁剪切加工和弯曲以及一般近似为底物变形者不被交流计数。考虑到microposts通常由从软材料如聚二甲基硅氧烷（PDMS）为基础的硅橡胶需要这些因素包括在内。 。舒恩等人证实，有这样的基础上micropost（L / D）和相应的聚合物的泊松比 V的纵横比的校正因子8它由下式给出。: <img alt="公式（2）" src="/files/ftp_upload/53350/53350eq2.jpg" /> （2） 以 T 倾斜 （ⅴ）作为一个倾斜系数，其包括嵌合参数a = 1.3作为可以在同一篇文章中找到: <img alt="公式3" src="/files/ftp_upload/53350/53350eq3.jpg" /> （3） 这意味着一个micropost的校正刚度ķ 压改正是纯弯曲刚度k = k 弯曲的产物和校正因子压改正计算公式如下: <img alt="公式4" src="/files/ftp_upload/53350/53350eq4.jpg" /> （4） 因此，细胞力的计算应采用公式（1）的更精致的变化，现在读来进行: <img alt="公式5" src="/files/ftp_upload/53350/53350eq5.jpg" /> （5） 修正的影响，只要是用于micropost尺寸典型值变得更为明显。例如，一个15微米长micropost具有圆形横截面，并且直径为5μm制成的基于PDMS的硅橡胶导致0.77的校正因子，因此，未校正的计算将23％高估施加的细胞的力。这变得更加严重为microposts具有较小的纵横比。 传统上，micropost图像分析也基于理想化的梁弯曲理论。在2005年率先使用MICR的组OPOST阵列出版适合micropost分析的图像分析软件9所述的软件需要软件许可证及用户必须采取三种图像的每个位置。分别来自micropost的顶部和底部的平面中的传输模式，另一种在荧光模式与染色的细胞。比较所述顶部和底部的位置为每个后micropost的软件确定的力矢量场，并计算相关的参数，如每交力。其它软件包存在，他们的分析原则在描述它们的相应条款简要地提到，但这些分析软件包通常是不公开的。10,11 设计用于映射细胞力的micropost阵列可分类为在正交micropost布局或六边形的，后者具有的所有邻居microposts之间等距的间隙的优点是。典型microposts公顷已经圆形截面和它们的尺寸范围从1.0微米至10微米的直径和2到50微米，长度4然而，microposts与椭圆形或正方形的横截面也有报道。12,13 使用基于PDMS的硅氧烷混合物作为micropost材料允许用于将纳米颗粒引入该混合物中。例如添加钴纳米棒使micropost的磁性活化，从而提供了潜在的实验设计的另一个自由度。14大部分组上产生平的刚性基板像盖玻璃或培养皿内其micropost阵列。然而，Mann和同事最近报道上形成可拉伸膜一个micropost阵列15这允许在研究活细胞的亚细胞动态响应在细胞收缩的术语细胞拉伸力到贴壁细胞的应用。 目前广泛使用的，最ESTAB用于制造micropost阵列lished过程中Sniadecki和同事。16-18的见地协议总之标准洁净室处理中描述被用于产生关于使用SU8光致抗蚀剂的硅晶片的顶部的微结构是基于软光刻。这之后是复制方法，其中在硅橡胶被浇铸在将它们转移到模具中的结构。在第二步骤中这些模具被用于复制使用的硅橡胶上的选择的衬底顶部的初始显微结构。然而，尽管大，越来越多的与他们申请公开，建立一个制造过程microposts的花费相当长的时间，即使对微工程专家;存在需要优化和适应特定实验室环境和micropost布局，得到可接受的质量水平的许多工艺步骤。 商业micropost阵列现已就绪-T邻的使用（"现成的架子"）格式具有一贯的高品质。因此，它们是替代所需的现场制作的复杂和冗长的制造工艺。本文的市售micropost阵列用于使用单明场显微术图像映射蜂窝力。更重要的是本文介绍和文件名为MechProfiler一个全功能的开源软件，可以下载作为补充材料，这份手稿。该软件的一个比较活跃的版本也可以在http://www.orthobiomech.ethz.ch找到。 一个"关闭的，现成的"检测和兼容的开源分析软件相结合，显着降低了进入障碍，以实现精确的TFM实验。研究人员无法获得任何无尘室设备或软件开发专业知识成功地分析细胞的力量。它使用户能够集中于mechanosensitivity分析输出，而不是技术本身，使之能够为更广泛的社区牵引力测量。此外，这是对铺平阵列micropost全自动筛选的方式迈出的重要一步。 该MechProfiler分析软件处理的文件格式TIFF，PNG，BMP和JPG图片。这些图像可以用荧光，相衬或明视场光学显微镜拍摄。该独立程序与游离Matlab的编译器运行时（见: 图12）一起运行，并底层算法允许简化图像处理，从而使用户能够处理具有单个或多个细胞的图像中约1分钟。此外，这些细胞既可以活的或"固定"。 该MechProfiler软件能够通过靠质量商业micropost阵列的再现性，大大提高了数据吞吐量分析，更具体地，默认＆＃8220;非偏转"的阵列中的每个交位置可以推定针对理想格（制造偏差为网格在用于该研究的阵列均小于100纳米）。 在短的一个打开选择的用于分析的图像文件，它们作物到感兴趣的区域中，定义覆盖细胞的职位或需要被丢弃，确定后的位置，计算该挠度/对理想格力，最后节省出口有可能所有的细胞特异性的数据，其中包括一个标准的办公电子表格。

<table><tbody><tr><td>T25 cell culture flasks</td><td>Nunc</td><td>156367</td><td></td></tr><tr><td>1x PBS-buffer</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>0.5% Trypsin-EDTA</td><td>Life Technologies</td><td>15400054</td><td></td></tr><tr><td>Medium DMEM/F12</td><td>Sigma</td><td>D8437</td><td></td></tr><tr><td>FBS South America</td><td>Life Technologies</td><td>10270106</td><td></td></tr><tr><td>Penicillin-Streptomycin 100x</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>15 ml centrifuge vials</td><td>Sarstedt</td><td>62.554.502&amp;nbsp;</td><td></td></tr><tr><td>Micropost array pre-coated with Collagen I/Fibronectin</td><td>MicroDuits</td><td>MPA-col1/FN</td><td>Micropost dimensions: &amp;Oslash;=6.4&amp;#160;&amp;micro;m, l=18.2 &amp;micro;m; grid=13 &amp;micro;m, k&lt;sub&gt;corr&lt;/sub&gt;=2.87&amp;#160;nN/&amp;micro;m</td></tr><tr><td>Ethanol abs p.A.</td><td>Merck</td><td>100.983</td><td></td></tr><tr><td>12-well plate</td><td>Nunc</td><td>150628</td><td></td></tr><tr><td>Formalin solution, neutral buffered 10%</td><td>Sigma</td><td>HT5011</td><td></td></tr><tr><td>Brilliant Blue G-250</td><td>Sigma</td><td>27815</td><td>Coomassie blue</td></tr><tr><td>Methanol ACS p.A.</td><td>Merck</td><td>1.06009</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>695092</td><td></td></tr><tr><td>Glass bottom dish</td><td>WillCo Wells BV</td><td>GWSb-3522</td><td>35 mm diameter, aperture 22&amp;nbsp;mm</td></tr><tr><td>T-100 Eclipse</td><td>Nikon</td><td>n/a</td><td>Inverted microscope</td></tr><tr><td>D3-L3</td><td>Nikon</td><td>n/a</td><td>Camera controler</td></tr><tr><td>DS Fi2</td><td>Nikon</td><td></td><td>Camera</td></tr></tbody></table>

easy and accurate mechano-profiling on micropost arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

所描述的技术的主要优点在于它的简单性和潜在的快速和有效地纳入常规实验室工作。搭配开源软件的高品质的商业传感器阵列的组合提供了否则将需要获得洁净室设施和深入的图像分析和软件开发知识机械力敏感的信息。 图1说明了该方法的工作流程。它开始准备和种子细胞到micropost阵列。固定和染色细胞后的micropost阵列准备成像。该技术的核心部分是一个使用MechProfiler软件的图像...

Watch this Scientific Journal Video about Easy and Accurate Mechano-profiling on Micropost Arrays at JoVE.com

Easy and Accurate Mechano-profiling on Micropost Arrays

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

在Micropost阵列方便，准确机械力分析

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

easy-and-accurate-mechano-profiling-on-micropost-arrays

Research

JoVE Journal

Bioengineering

11.1K 次观看.  Balgrist University Hospital. Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost. 

视频: Easy and Accurate Mechano-profiling on Micropost Arrays

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

科研

生物工程

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

微阵列后探测器（mPADs）：机械力的方法来隔离

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

生物学

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

复透皮电化学传感的空心微针为基础的传感器

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

变凹形形状 Microwells 的配对磁珠和磁体阵列

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

AFMS 中的并联有源悬臂阵列可实现高通量检测

Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays for High-Throughput Large-Scale Sample Inspection

An Atomic Force Microscope&#160;(AFM) is&#160;a powerful and versatile tool for nanoscale surface studies to capture 3D topography images of samples. However, due to their limited imaging throughput, AFMs have not been widely adopted for large-scale inspection purposes. Researchers have developed high-speed AFM systems to record dynamic process videos in chemical and biological reactions at tens of frames per second, at the cost of a small imaging area of up to several square micrometers. In contrast, inspecting large-scale nanofabricated structures, such as semiconductor wafers, requires nanoscale spatial resolution imaging of a static sample over hundreds of square centimeters with high productivity. Conventional AFMs use a single passive cantilever probe with an optical beam deflection system, which can only collect one pixel at a time during AFM imaging, resulting in low imaging throughput. This work utilizes an array of active cantilevers with embedded piezoresistive sensors and thermomechanical actuators, which allows simultaneous multi-cantilever operation in parallel operation for increased imaging throughput. When combined with large-range nano-positioners and proper control algorithms, each cantilever can be individually controlled to capture multiple AFM images. With data-driven post-processing algorithms, the images can be stitched together, and defect detection can be performed by comparing them to the desired geometry. This paper introduces principles of the custom AFM using the active cantilever arrays, followed by a discussion on practical experiment considerations for inspection applications. Selected example images of silicon calibration grating, highly-oriented pyrolytic graphite, and extreme ultraviolet lithography masks are captured using an array of four active cantilevers (&#34;Quattro&#34;) with a 125 &#181;m tip separation distance. With more engineering integration, this high-throughput, large-scale imaging tool can provide 3D metrological data for extreme ultraviolet (EUV) masks, chemical mechanical planarization (CMP) inspection, failure analysis, displays, thin-film step measurements, roughness measurement dies, and laser-engraved dry gas seal grooves.

作者聚焦:使用 Quattro-Parallel 悬臂阵列的主动探针原子力显微镜简介

Large-scale sample inspection with nanoscale resolution has a wide range of applications, especially for nanofabricated semiconductor wafers. Atomic force microscopes can be a great tool for this purpose, but are limited by their imaging speed. This work utilizes parallel active cantilever arrays in AFMs to enable high-throughput and large-scale inspections.

使用Quattro-Parallel 悬臂阵列的主动探针原子力显微镜，用于高通量大规模样品检测

An Atomic Force Microscope&#160;(AFM) is&#160;a powerful and versatile tool for nanoscale surface studies to capture 3D topography images of samples. ...

工程学

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

制造和使用微环境芯片（MEArrays）

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

在Micropost阵列方便，准确机械力分析

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使用Quattro-Parallel 悬臂阵列的主动探针原子力显微镜，用于高通量大规模样品检测

使用Quattro-Parallel 悬臂阵列的主动探针原子力显微镜，用于高通量大规模样品检测

使用Quattro-Parallel 悬臂阵列的主动探针原子力显微镜，用于高通量大规模样品检测

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

多路人工细胞微环境阵列的制备

制造和使用微环境芯片（MEArrays）