1。细胞微图案的制备和播种<ol><li>放置2 2 6孔板独立井CYTOOchips确保您阅读"CYTOO"在右下角的CYTOOchip。微图案可以被荧光标记Cy3或Cy5的染料。本实验所用的CYTOOChips Cy3标记有荧光标记的微图案。 注意:请在黑暗中的芯片。 </li><li>胰蛋白酶消化收集HeLa细胞（〜80％融合）。在显微镜下检查，适当分散的胰蛋白酶处理细胞。收集和离心机300克细胞为4分钟，然后轻轻悬浮细胞沉淀。计数细胞，稀释到15000完成的DMEM/F12培养基中的细胞/毫升的浓度。 </li><li>免除到以及包含每一个CYTOOchip 4毫升（60,000细胞）。 注意:该板块应尽可能少移动，以避免诱导培养基中的任何旋转运动，因为这往往会集中在细胞的中心。 </li><li>让我们为10分钟引擎盖下的细胞沉淀物，然后将它们移动到细胞培养箱。 10-20分钟后，细胞就会开始坚持的微图案。 10分钟后，定期检查，在显微镜下。 </li><li>只要细胞连接，改变细胞培养基，轻轻冲洗盖玻片表面，使用下列程序: </li><li>保持板式平，轻轻地从一侧以及吸管中吸。 注意:绝不能让芯片干出来的，这意味着，从未吸过所有的液体，但留出足够的覆盖在任何时候都CYTOOchip。 </li><li>添加4毫升PBS，首先在芯片的中心开始，然后移动到一侧以及再次吸。吸过的PBS不暴露在空气中的芯片。重复3-4次。 </li><li>浮动细胞在显微镜下检查: <ul><li>如果大量的浮动细胞仍然存在，重复洗涤过程。 </li><li>如果你看到极少数的细胞，吸出的PBS和2毫升的新鲜培养基代替。吸的新鲜培养基中添加4毫升两次。 </li></ul></li><li>在组织培养箱内培养板放置在37℃，5％的CO 2至少三个小时，让细胞来实现全面蔓延。 注意:使用这种方法之间的10-30％（2000-6500）将被占用一个或多个细胞微图案。 注意:坚持之前的洗涤步骤和全细胞微图案的蔓延将具体到每个细胞株所需的时间为细胞所需的时间。 </li></ol> 2。 Blebbistatin治疗<ol><li>在100％DMSO溶解blebbistatin获得一个17毫米的股票解决方案。进一步原液稀释至5毫米，100％DMSO。请在培养基中的最终稀释获得了5微米blebbistatin终浓度在0.1％DMSO。 </li><li>对于控制条件，准备4毫升含0.1％DMSO的唯一媒介。 </li><li>吸细胞上的媒体和替换治疗和控制的条件下编制的4毫升中等。 </li><li>孵育细胞1小时在37 ° C。 </li></ol> 3。肌动蛋白细胞固定和染色<ol><li>细胞固定的解决方案准备 请参阅为骨架缓冲液（CB）和PFA 5％的制备试剂部分。 CB是特别推荐的微丝和微管的荧光标记。 <ul><li>准备1xCB蔗糖:蔗糖加1克至2毫升的CB。 包括蔗糖，有助于保护内部的细胞结构。 </li><li>准备4％PFA - CB -蔗糖:温暖的煤灰5％至4毫升加入1毫升的1xCB蔗糖。 注意:此解决方案可保持在4℃，只几天。 </li></ul></li><li>煤灰固定<ul><li>使用镊子CYTOO标志，并把它拿起CYTOOchip 6井以及含2毫升4％PFA - CB -蔗糖溶液板（不洗） </li><li>在室温下孵育10分钟</li><li>删除PFA - CB -蔗糖，用PBS洗一次</li><li>做了2毫升100毫米的NH 4氯10分钟的第二次洗，以淬火残余煤灰交联活动 注:煤灰固定幻灯片可以在PBS存放数天，在4 °彗星之前，细胞器染色荧光成像。 </li></ul></li><li>与Triton X - 100的细胞膜的通透性， 用洗涤剂的细胞通透性，使抗体或其他探针穿透细胞膜，并消除了一些细胞骨架蛋白的胞质池，否则可以减少使其难以分析其本地化的骨架聚合物的对比。 <ul><li>删除NH 4 CL，并加入2 ml / 0.1％TRITON X - 100 - CB在室温下3分钟</li><li>用2 mL PBS洗两次</li></ul></li><li>肌动蛋白染色 荧光-鬼笔环肽h本土肌动蛋白结合不仅是用来染色的微丝。 <ul><li>添加1.5％BSA 2毫升的PBS 1:2000稀释的FITC标记的鬼笔环肽</li><li>在室温下孵育1小时</li><li>用2 mL PBS洗一次</li></ul></li><li>核染色<ul><li>加入2毫升的赫斯特（1μg/毫升染色的PBS） </li><li>在室温下孵育3分钟</li><li>用2 mL PBS清洗2次</li></ul></li><li>安装幻灯片<ul><li>拿起CYTOO标志和装载CYTOOchip CYTOOchips到一个标准的显微镜使用25-30μLMowiol或任何其他的安装解决方案的幻灯片。 </li></ul></li></ol> 4。显微镜的安装和自动图像采集存在大量的成像软件包，控制快速自动图像采集和显示的显微镜。这个例子使用Metamorph成像软件和配备尼康的Eclipse TI滨松与CCD相机Intensilight汞光纤照明显微镜进行自动采集。获得相应的DAPI（核），CY - 3（微图案）和FITC -鬼笔环肽（肌动蛋白）在三种波长的20倍放大倍率的图像。微图案的数量，可视化的每场相机和目标设置上会有所不同而有所不同。 CYTOO微图案是定位在每个跨越大大方便采集芯片（100或130微米）定义的时间间隔。 <ol><li>选择20倍的放大倍率</li><li>打开多维收购（在菜单栏:应用程序/多维收购），并设置不同的实验参数: <ul><li>波长数:3 </li><li>波长类型:DAPI，FITC，Cy3标记</li><li>每个波长的曝光时间设置，首先在重点领域的细胞</li><li>每个波长可以进行自动对焦</li><li>选择在哪里保存数据</li></ul></li><li>选择Cy3标记的波长和相机的位置，使12微图案可视化，并在相机领域（4列和第3行模式）为中心。 </li><li>创建一个日志，运行多维收购。图1描述创建一个杂志的过程。 </li><li>打开扫描阶段的功能（在菜单栏:设备/舞台/扫描阶段）。要获取24幅图像，每片含12微图案，在20倍的放大倍率，输入-400微米X步骤大小和300微米Ÿ步长和4行6列。在设置杂志执行，上传杂志MDA.JNL。然后按下扫描开始在三种波长的整幢大厦的自动采集。 注:X和Y的步长在此间表示将根据目前在相机领域的微图案。根据你的舞台设置，在X和/或Y方向可能需要在正确的方向运动时的负值。 </li></ol> 5。自动图像处理和分析存在许多图像处理和分析软件包。下面描述的过程的轮廓图像处理和分析步骤2定制，为开源项目ImageJ编写的宏进行。第一个宏CellRef创建引用单元，第二斜边措施刚性/细胞膜的崩溃。两者都是通过请求后可<a href="www.cytoo.com">www.cytoo.com</a> 。 <ol><li>微图案（宏CellRef）为中心的所有三个通道的创建图像堆栈。 微图案的使用，避免细胞集群和聚集，大大简化了在自动化图像处理的第一步，这是单个细胞的本地化和分割。微型图象荧光标记的图像是用于定义和划定的微图案为中心的地区。然后，这些地区调换到其他渠道获得的的图像和裁剪。每个波长堆叠，然后组装成相应的裁剪图像。从三个通道（图2）合并也创造了一个RGB叠。 </li><li>应用单细胞选择过滤器（宏CellRef） 如上所述，10-30％的微图案（2000-6500）被占领的单个单元格或多个单元格，而其余的微图案是空的。要选择占用只是一个单细胞微图案的图像显示，宏观检测每幅图像的原子核的数目，并从所有协议栈（RGB和不同波长的）那些包含多个细胞核或没有细胞核（图3）消除。 </li><li>消除非标准化和细胞不完全拉长整个微型图象使用单元面积截止滤光片（宏CellRef） 要删除分裂的细胞会逃脱原子核的过滤器，另一个过滤器使用FITC标记的肌动蛋白通道应用。单元面积计算低于某一切断（2倍左右的单元格相间细胞面积低于地区）的所有堆栈中删除。格局的大小，是由细胞信封面积。 </li><li&gt;对齐细胞图像（宏CellRef） 从前面的步骤，包含单micropatterned细胞的图像被选中。即使这些图像上微图案为中心，他们从来没有完全对齐彼此。要结束的图像处理，ImageJ插件（MultiStackReg）是适用于重新调整所有收集到的图像和所有渠道的基础上的微型图象的位置。这一步，​​现在可以用于单细胞分析或创建引用单元（图4）个别图像生成对齐栈</li><li>构建参考单元（宏CellRef） 在微图案上生长的细胞，意味着所有的细胞也有类似的形状。这正常化，允许许多细胞图像精确对准和覆盖。总结在整个堆栈每个图像信号允许一个可视化和衡量一个"平均药物作用"。最后的整体形象，或参考细胞是一个独特的代表性和图像分析和有用的工具，只能获得micropatterned细胞（图4）。 ImageJ，引用单元格是通过从堆栈的过滤和对齐图像投影构造（图像&gt;书库&gt; Z项目）。几个投影方法可应用于如平均或最大强度，消除图像堆栈的主要离群，但通常的平均预测是选择。为了便于检查的结果，一个LUT（查找表）的单色图像转换成一种颜色编码的频率地图。 </li><li>测量和量化感兴趣的参数（宏观斜边） 在这个视频文章中，L - 微图案是因为形式组织成一个主要的应力纤维肌动蛋白细胞骨架。突出以这种方式肌动蛋白，blebbistatin的细胞骨架的影响可确定在许多方面:测量的肌动蛋白压力纤维的曲率变化，染色应力纤维的强度，长度或厚度，改变的肌动蛋白细丝的形态特点或细胞的形状等，这些参数可以测量单个细胞的图像，或在最后的参考细胞本身。 在这里，5微米blebbistatin影响的特点是通过使用一个名为斜边（图7）第二ImageJ宏观倒塌细胞计数的数量。简单地说，在L -微型图象的阈值的图像被用来定义一个单元格的三角形状的理论斜边。下，肌动蛋白染色图像进行阈值是为了近似实际的细胞形态。然后测量理论的细胞三角形和实际凹细胞膜的斜边之间的区域。当细胞已经坍塌，下面的理论斜边的面积出现。倒塌细胞的百分比是用来比较的控制和治疗条件。 </li></ol> 6。代表性的成果什么细胞的例子应立即上微图案看起来像几个小时后，关键在1.6-1.8洗中描述的步骤，如图5所示。上CYTOOchips 15分钟后，圆形的HeLa细胞的观察表明，他们坚持的纤维连接蛋白微图案（图5a）的距离相等。尽管轻轻摇动培养容器固定细胞时，附着力完成。为了尽量减少占用微图案由多个细胞的数量，芯片，然后用PBS冲洗，以去除细胞漂浮在cytophobic地区。几个小时后，细胞在微图案完全伸展，如图5b所示的样子。 典型的单细胞blebbistatin，固定和肌动蛋白和核染色的细胞孵化后获得的图像，如图6。后的自动采集多个图像和图像处理使用ImageJ CellRef宏观，颜色编码的频率地图生成，描述了肌动蛋白的强度平均每所有细胞图像（图6C和6F）。引用单元格非治疗和治疗条件的比较，表明这种方法容易观察所研究的表型的潜力，并为探索细胞的药物影响是特别有用。 blebbistatin对细胞的作用可能分析的一个例子是在图7。倒塌的细胞数（即细胞与一个空白的理论斜边下，现有面积）在50个控制细胞检测，显示50个细胞治疗5微米blebbistatin ImageJ斜边宏检测。倒塌细胞blebbistatin处理人口数量增加，反映的应力纤维，因此在膜由于blebbistatin actinomyosin收缩力量抑制细胞内的紧张放松。 <img src="/files/ftp_upload/2514/2514fig1.jpg" alt="图1" /> 图1的过程来创建一个新的杂志与Metamorph 。 </P&gt; <img src="/files/ftp_upload/2514/2514fig2.jpg" alt="图2" /> 图2的自动图像处理程序流程图。 <img src="/files/ftp_upload/2514/2514fig3.jpg" alt="图3" /> 图3，单细胞筛选。 <img src="/files/ftp_upload/2514/2514fig4.jpg" alt="图4" /> 图4。建立一个参考电池。 <img src="/files/ftp_upload/2514/2514fig5.jpg" alt="图5" /> 图5。细胞种植。一）Hela细胞后冲洗的步骤（4倍的放大倍率）坚持以微图案二）HeLa细胞L上的微图案（10倍的放大倍率）经过充分的蔓延。 <img src="/files/ftp_upload/2514/2514fig6.jpg" alt="图6" /> 图6。nonpatterned控制和药物治疗的条件和micropatterned细胞的比较。 HeLa细胞接种3小时和5 1小时微米blebbistatin（下半部）或不及时治疗（上图）的治疗。在控制nonpatterned细胞（A），肌动蛋白组装成多种纤维5微米blebbistatin（四）治疗后潜移默化地拆卸的。对L -微图案，对照组采用一个三角形状（二）发展一个重要的肌动蛋白纤维之间2根尖相比属nonpatterned细胞（D）（应力纤维），blebbistatin诱使一个重要的表型改变对L - micropatterned细胞（E）。迅速与20细胞构建的引用单元格介绍药物作用，控制和治疗条件比较直接的可视化，可可视化。单个细胞相似，一个清晰而强烈的应力纤维是目前控制参考单元（C），而blebbistatin处理细胞崩解和消失的主要应力纤维（F）。 <img src="/files/ftp_upload/2514/2514fig7.jpg" alt="图7" /> 图7。使用宏Hypothenuse细胞blebbistatin影响分析的一个例子。而对照组的25％倒塌，这增加了3倍，75％在5微米blebbistatin接受反映削弱actinomyosin收缩网络（N = 50个细胞）的细胞。

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安妮Béghin以外的所有作者CYTOO SA，产生在这篇文章中精选的试剂和仪器仪表的员工。

选择的微型图象虽然5微米blebbistatin的影响几乎是标准的文化支持检测的，有效的量化是集中到一个主要的应力纤维的肌动蛋白的微图案。这增强了可视化协助对细胞blebbistatin的影响的准确定量的肌动蛋白细胞骨架。微型图象形状的选择是在实验的设计关键，是根据在研究中强调的表型改变设计。 结论微图案方便的药物效果的可视化和量化，特别是在低浓度。与基于细胞的检测胶粘剂微图案同质平面基板的更换，提高了生产数据的准确性，灵敏度和质量。结果，需要较少的细胞进行分析，以实现强大的统计结果 3 。胶粘剂微图案提供了一个真正的机会，改善功能的研究和探索在较低药物浓度的表型变化，以避免诱发有毒或间接的药物效果。如果使用适当的筛选平台，微图案能满足制药公司的要求，为提高基因/药物筛选应用。分析和定量分析细胞的现象，利用微图案的一些额外的例子，最近的出版物被引用低于3-13。

<table border="1"><tbody><tr><td colspan="2"> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td colspan="5" align="center"> 细胞培养 </td></tr><tr><td colspan="2"> CYTOOchips 20X20的LM - FN </td><td> CYTOO </td><td> 10-114 </td><td>您所选择的蛋白质吸附CYTOOchips </td></tr><tr><td colspan="2"> PBS 1X </td><td> Gibco公司</td><td> 14040-091 </td><td></td></tr><tr><td colspan="2">计数室（Kova幻灯片） </td><td> Prolabo </td><td> 71287.01 </td><td></td></tr><tr><td colspan="2"> DMEM/F-12 + Glutamax，我</td><td> GIBCO，Invitrogen公司</td><td> 25300-054 </td><td></td></tr><tr><td colspan="2">胎牛血清（f TAL牛血清） </td><td>临时机场管理局</td><td> 31331-028 </td><td></td></tr><tr><td colspan="2">青霉素和链霉素</td><td>临时机场管理局</td><td> A15 - 101 </td><td></td></tr><tr><td colspan="2"> 6井板</td><td> Dutscher </td><td> 353046 </td><td></td></tr><tr><td colspan="2">离心分离</td><td> Fisher Scientific则</td><td> M65838 </td><td></td></tr><tr><td colspan="2">显微镜</td><td>尼康</td><td></td><td></td></tr><tr><td colspan="5" align="center"> 毒品，固定和免疫 </td></tr><tr><td colspan="2" >骨架缓冲1X（CB） </td><td >西格玛</td><td > MES:M8250 氯化钾:I2636 氯化镁:M2393 EGTA:E3889 </td><td > 10毫米MES pH值6.1，138mM氯化钾， 3MM氯化镁， 2MM EGTA </td></tr><tr><td colspan="2" >蔗糖</td><td >西格玛</td><td > S2395 </td><td></td></tr><tr><td colspan="2">煤灰32％ </td><td>电子显微镜科学</td><td> 15714 </td><td></td></tr><tr><td colspan="2">煤灰5％ </td><td></td><td></td><td>混合54毫升CB 1.185X 10毫升32％煤灰</td></tr><tr><td colspan="2"> TRITON - X100 </td><td>西格玛</td><td> T9284 </td><td></td></tr><tr><td colspan="2"> PBS片</td><td> GIBCO，Invitrogen公司</td><td> 18912-014 </td><td></td></tr><tr><td colspan="2"> Bovin血清白蛋白（BSA） </td><td>西格玛</td><td> A7806 </td><td></td></tr><tr><td colspan="2"> Phallodin - FITC </td><td>西格玛</td><td> P5282 </td><td></td></tr><tr><td colspan="2">赫司特</td><td> Invitrogen公司</td><td> H3570 </td><td></td></tr><tr><td colspan="2"> Blebbistatin </td><td> Euromedex </td><td> BN0640 </td><td></td></tr><tr><td colspan="2">二甲基亚砜</td><td>西格玛</td><td> D8418 </td><td></td></tr><tr><td colspan="2"> NH 4氯0.1M </td><td>西格玛</td><td> M11209 </td><td></td></tr><tr><td colspan="5" align="center"> 图像采集 </td></tr><tr><td colspan="2">啶显微镜</td><td>尼康</td><td> Eclipse的钛</td><td></td></tr><tr><td colspan="2"> CCD相机</td><td>待定</td><td>待定</td><td></td></tr><tr><td colspan="2"> ImageJ软件</td><td> <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> </td><td></td><td></td></tr></tbody></table>

improved visualization and quantitative analysis of drug effects using micropatterned cells

迄今为止，最HCA（高含量的分析）的研究进行了贴壁细胞，在组织文化的同质性治疗微板的衬底上生长。在这些条件下，细胞的扩散，并在所有方向，在细胞的形状，形态和行为的内在变化导致分裂。高整体人口的细胞与细胞之间的差异阻碍了HCA的成功，特别是为药物开发。微图案的能力，每一个单个细胞的形状和内部极性正常化提供了一个巨大的机会， 解决这个关键瓶颈1-2。 为了便于访问和使用的缩微技术，CYTOO已开发出了一系列准备使用微图案，在盖玻片和微孔格式。在这个视频文章中，我们提供详细的协议CYTOOchip微图案，药物治疗，固定和染色的自动采集，自动图像处理和分析，最终的数据，从细胞播种的所有程序。这个例子中，我们说明如何微图案，可以促进细胞检测。细胞骨架的改变是难以量化同质衬底上培养的细胞，但培养细胞中的肌动蛋白小梁由于在每一个细胞的细胞粘附接触系统定位重现性组织的微图案业绩。这种细胞内的结构正常化，使量化的肌动蛋白细胞骨架上，甚至小的影响，证明在这些协议使用blebbistatin，肌动蛋白 - 肌球蛋白相互作用的抑制剂。

Watch this Scientific Journal Video about Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells at JoVE.com

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

可用于胶粘剂的微图案，细胞结构正常化，增加检测药物作用的敏感性，改善的重现性和简化自动化图像采集和分析。这种技术将有利于药物/ siRNA的筛选试验，进行常规的细胞培养支持，并因此遭受过度的细胞 - 细胞变性。

改进的可视化和定量分析，药物使用Micropatterned细胞的影响

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

improved-visualization-quantitative-analysis-drug-effects-using

Research

JoVE Journal

Bioengineering

17.3K 次观看.  Grenoble, France. 可用于胶粘剂的微图案，细胞结构正常化，增加检测药物作用的敏感性，改善的重现性和简化自动化图像采集和分析。这种技术将有利于药物/ siRNA的筛选试验，进行常规的细胞培养支持，并因此遭受过度的细胞 - 细胞变性。

视频: Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

图案的使用生物倒装芯片的胚胎干细胞

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

科研

生物学

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

利用双水相系统的细胞共培养图案

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

生物工程

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

细胞的浸入式印刷到改性表面采用连续流动Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

使用微模式和定量成像绘制哺乳动物细胞的新兴空间组织图

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

发育生物学

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

图案蛋白质和细胞的通用的方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

在微流体进化加速器上生成跨癌症人群的异构药物梯度，用于实时观察

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

使用微图案弹性体对单细胞收缩力进行简化的高通量分析

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

微图案牵引显微镜的图案生成

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...