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 border="0" cellpadding="0" cellspacing="0">
	<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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropost array&#160;</td>
			<td rowspan="2">MicroDuits</td>
			<td rowspan="2">MPA-col1/FN</td>
			<td>Micropost dimensions:</td>
		</tr>
		<tr>
			<td>pre-coated with Collagen I/Fibronectin</td>
			<td>&#216;= 6.4 &#181;m, l=18.2 &#181;m; grid=&#160;13 &#181;m, kcorr= 2.87 nN/&#181;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&#160;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

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

不对称受体模式研究细胞滚动轨迹

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

科研

生物工程

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

高效率，站点特定的贴壁细胞转染siRNA的微电极阵列（MEA）

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

密封飞升商会阵列的无细胞生物学

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

在支撑脂双层配体纳米簇阵列

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

微电极阵列接口微流体研究神经元通信和轴向信号传播

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

用于循环作用电位记录的多井微电极阵列上的人类 iPSC 衍生心肌细胞网络

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

用于Vivo神经记录的 Ti3C2 MXene 微电极阵列的制造

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

子结构分析仪：用于荧光显微镜图像中细胞体快速探索和精确分析的用户友好型工作流程

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

高维流式细胞术用于解剖种植体组织的免疫功能分析

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

使用人iPSC衍生的心肌细胞，心脏成纤维细胞和内皮细胞制造3D心脏微组织阵列

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...