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

17.2K 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

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned表面透明质酸与肿瘤细胞相互作用的研究

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

科研

生物工程

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

使用动态面膜投影光刻系统的神经文化Micropatterned水凝胶的制备

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

在硅橡胶膜拉伸微图案细胞

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

基因转染向着文化微模式球体板细胞基因修饰的细胞移植

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

高通量自动 Microbioreactor 系统在 CHO 细胞模型 IgG1 生产中的应用

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

使用自动微生物反应器系统从中国仓鼠卵巢细胞中纯化和分析单克隆抗体

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

使用声学分离器对CHO收获的细胞培养液进行初步澄清

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...