Funding for this work, GTK, TK, and JDM were provided by the Wisconsin Institute for Discovery and the Wisconsin Alumni Research Foundation.

1.生成弹性邮票<ol style="margin-left: 40px;"><li>要生成PDMS印章的硅大师，采用计算机辅助设计软件设计的光罩的功能模式。 <ol><li>设计的第一图案为20×20阵列的环的具有300μm的内径（ID）和600微米的外径与1200微米的中心到中心的间距。 </li><li>设计第二图案作为20×20阵列的环带600微米的ID和900微米外径与1200微米的中心到中心的间距。 </li><li>此外，将1×1 平方毫米平方的参考标记在所有四个角的每个阵列的设计间隔1200微米的中心到中心的距离的角图案中的45°角。 </li><li>制造硅的主人用于本实验中，用1：1的长宽比，使用与微流体铸造别处15或协作详述标准光刻技术关联到一个300微米的特征深度。 注：功能深度小于100微米的可导致与基材表面接触之前，邮票异常变形。 注：此协议首先在已经得到的硅大师与光致抗蚀剂的叙述模式，这需要专门的设备和洁净室创建。这是最好的一个制作者或核心设备来创建这些图案高手请教。 </li></ol></li><li> Silanize硅大师O / N通过孵育（十三-1,1,2,2-tetrahydroocty）三氯硅烷的蒸气。 注：硅烷的蒸气有剧毒，只应在通风橱处理。 </li><li> 1比率的PDMS预聚物和PDMS在60℃下固化的硅烷化硅的主人的顶部剂在培养皿中O / N的：通过固化10创建的硅主人的逆复制品。 </li><li>从硅主人除去PDMS压模，并粘接邮票到丙烯腈 - 丁二烯 - 苯乙烯（ABS）的背衬或任何其他刚性材料S使用胶水。 注意：选择的材料不需要是透明的或生物相容的。但是，它必须与所述机器人的工具和更高的刚性比PDMS的接口提供一个平坦的表面。 </li></ol> 2.准备Coverslides <ol style="margin-left: 40px;"><li>漂洗显微镜coverslides在甲苯，甲醇和超声处理在丙酮中为下一个温和的氮气流中，用乙醇冲洗并干燥前1分钟（1 24毫米×50毫米＃）。 注：只有在通风橱处理甲苯和丙酮。 </li><li>外套coverslides以〜3.5 nm的钛（Ti），然后通过使用聚焦的电子束蒸发器18.0纳米的金（Au）。 注：此步骤是用各种金涂覆的基材包括硅和聚苯乙烯兼容。而有可能在现场产生这些，金涂覆的基材也可通过商业供应商。 注：钛层至少应为3纳米厚，而在衬底上的Au层应该是一个吨至少5纳米厚和范围可以全部在厚度为1mm取决于最终衬底16,17的理想的光学特性的方式。衬底不需要是用于制造光学透明的;然而，它便于制造基板的微观分析。 </li><li>在即将使用前冲洗镀金coverslides温和氮气下乙醇和干燥。 </li></ol> 3. R​​-μCP外环的<ol style="margin-left: 40px;"><li> R-μCP与选择标准的多关节机器人臂（SCARA）系统之前，校准使用系统软件的机器人工具效应和相应的双摄像头系统。 注意：这些工具被描绘在图1中。 </li></ol><img alt="图1" src="/files/ftp_upload/52186/52186fig1highres.jpg" /> 图1. R-μCP系统和机械臂工具。 </s仲&gt;（A）R-μCP系统的所有工装夹具大型看法，（一）真空吸盘，（二）试剂槽，（三）朝下的摄像头，（四）邮票嵌套夹具，（E）的机器人工具。 （B）中的机器人臂的工具描绘（六）金刚石尖蚀刻工具和（g）气动吸尘工具保持将a（h）的ABS支持的PDMS压模。 <ol start="2" style="margin-left: 40px;"><li>手动将600微米的ID和900微米OD突出的环在邮票嵌套夹具朝下一个PDMS印章。手动放置一个刚清洗干净的金涂覆coverslide上的真空吸盘，在图1A中显示的顶部，并使用该连接的实验室真空固定它。 </li><li>使用机械手的控制，对准朝下的相机在镀金coverslide的中心点，如在图2A中 。 注：此点最初可通过目视检查而定义的，并不需要很高的精度，由于PDMS压模得多LARG尔比的涂覆金的滑动。 </li><li>指示机器人臂以蚀刻四个参考的“X”标记，在一个正方形的尺寸为顶点3.8毫米由3.8毫米集中在使用连接到机器人臂的金刚石尖蚀刻工具的镀金coverslide。 （示于图2B;自动化的过程） 注：这将确保所有四个参考点都在朝下的摄像头之一的视觉框架。 </li><li>使用机器人气动吸工装，接机，并保持PDMS印章1毫米上面的邮票嵌套夹具， 如图2C。 （自动化过程） </li><li>利用朝上的摄像头和LED照明环描绘在图2D和E，可视化的印模的正方形的参考标记，并确定它们与照相机软件10倍，以确定所述时间戳的平均的X，Y，和从该中心的角度偏移机器人工装轴。 （自动化过程） </li><li>作为<strong&gt;图2A中，用面朝下的照相机来可视化和检测coverslide的蚀刻的基准标记的10倍，以确定coverslide的平均的X，Y中心位置和角度，从所述机器人的工具轴的中心偏移。 （自动化的过程）。 注意：一个内部计算由所述机器人系统的软件进行精确地对准印模的和盖玻片的X，Y中心位置和角度偏移量中的未来的R-微接触的步骤。 </li><li> 3.8）放置在链烷烃硫醇的ATRP引发剂，ω-mercaptoundecyl溴异丁酸乙酯（2毫摩尔的乙醇浴中的邮票），如在图2F中描绘。 （自动化过程） </li><li>从链烷醇溶液除去印模，并将其放置在加压到0.48巴（5 psi）的氮气流中蒸发乙醇， 如图2G。 1分钟后增加的氮气流的压力为1.03巴（15磅），以确保均匀的干燥。 （自动化流程） 注：不完全干燥会导致图案逼真的全部或部分损失。 </li><li>干燥后，将印模在镀金滑动的所计算出的中心位置，并降低其在100微米为增量，同时监测Z轴马达力， 如图2H所描绘。 注意：此连通的扭矩通过Z轴电机经历和在所述机器人指导软件显示。 （自动化过程） </li><li>停止降低一次的79.2千帕的预定压力已经达到印模，并保持与镀金的滑动，持续15秒的接触。 （自动化过程） 注：该压力值以下是目前邮票的设计和功能的高度优化。如果印模设计改变，特别为高纵横比的邮票，调整这些相应地通过尝试和错误的过程。 </li><li>慢慢地除去从镀金滑动印模和放置印章背面的印章嵌套夹具。 （自动化过程） </li></OL&gt; PEGMEMA对微模式Coverslides 4 SI-ATRP <ol style="margin-left: 40px;"><li>释放真空压力保持在微图案化coverslide，并将其转移到50毫升的Schlenk烧瓶中。用真空泵密封和脱气的Schlenk烧瓶中。 </li><li>添加5.5含大分子单体PEGMEMA（208.75毫摩尔），水（34.4毫升），甲醇（43.8毫升），铜（II）溴化物（1毫摩尔），和2&#39;，2-联吡啶（3毫摩尔）的原子转移自由基聚合反应混合物中的溶液在Schlenk烧瓶中。 </li><li>加入0.5毫升的L-抗坏血酸钠（454.3毫摩尔）的水，使反应开始，并允许它继续16小时，在室温下惰性气体。 注：在加入的L-抗坏血酸钠，反应颜色由浅绿色转变为暗棕色， 如图3A-B中。 注意：该反应可以继续超越16小时，但它不会显著增加的PEG刷子的长度。 </li></ol><imgALT =“图3”SRC =“/文件/ ftp_upload / 52186 / 52186fig3highres.jpg”/&gt; 图3.启动SI-ATRP的。（A）的反应和（B）以后的颜色变化如下添加L-抗坏血酸钠的启动。微图案化coverslide表面以下的SI-ATRP过程中（C）显微图像。比例尺1mm以下。 <ol start="4" style="margin-left: 40px;"><li>从申克烧瓶中取出微图案coverslide并用乙醇，水，以及乙醇和干燥下温和氮气流冲洗。 注：下面的SI-ATRP，表面修饰应肉眼可见的，并在显微镜下进行成像和分析， 如图3C所示 。 注：这是测试试验的精确度，因为它省却了免疫染色基板的最简单的方法。 </li></ol>的微模式PEGMEMA链5.叠氮化物官能<ol style="margin-left: 40px;"><li>放置在20毫升玻璃反应管形瓶中的微图案化coverslide。 </li><li>加入6毫升N，N-二甲基甲酰胺（DMF）中含有100mM氮化钠的反应小瓶。保持该反应在37℃下放置24小时。 注：只有在通风橱处理DMF。称出叠氮化钠时请务必小心。 </li><li>完成后，除去从反应小瓶的微图案化coverslide和冲洗用在氮气流中的乙醇，然后用水和干燥。 </li></ol> 6.钝化溴官能PEGMEMA链<ol style="margin-left: 40px;"><li>放置在20毫升玻璃反应瓶微图案coverslide。 </li><li>加入6毫升含有100毫乙醇胺和300mM的三乙胺的反应小瓶二甲亚砜（DMSO）中的。保持该反应在40℃下放置24小时。 注意：只有在化学通风橱中处理的DMSO，乙醇胺，三乙胺等。 </li><li>完成后删除该微图案化coversl从反应小瓶的ide，冲洗用在氮气流中的乙醇，然后用水和干燥。 </li></ol> 7. R-μCP内环和PEGMEMA的SI-ATRP <ol style="margin-left: 40px;"><li>开展R-μCP步骤如前面步骤3.2和3.6描述 - 3.12，与300微米的ID和600微米OD突出的环，使得具有较小的特点的环都放在前面微图案的环内代以PDMS印章。 注：此邮票的压力阈值是132.0千帕。如前面所提到的，这些压力设置为在该实验中所使用的印模特征优化。 </li><li>开展SI-ATRP步骤如前面的步骤描述4.1 - 4.4。 </li></ol>的微模式PEGMEMA链8.乙炔官能<ol style="margin-left: 40px;"><li>放置微图案coverslide在20毫升玻璃反应瓶中。 </li><li>加6毫升DMSO含100毫米炔丙基胺的反应小瓶。保持此反应混合物在室温放置24小时。 注：仅在通风橱处理DMSO和炔丙基胺。 </li><li>完成后除去从反应小瓶的微图案化coverslide和冲洗用在氮气流中的乙醇，然后用水和干燥。 </li></ol> 9.铜催化的“点击”乙炔的生物素化终止PEGMEMA链<ol style="margin-left: 40px;"><li>放置微图案coverslide在20毫升玻璃反应瓶中。 </li><li>添加6毫升硫酸铜（15毫摩尔）/三[（1-苄基 -1H- 1,2,3-三唑-4-基）甲基]胺（TBTA，30毫摩尔）的（1:1体积/体积的水/ DMF）中含有562μM的叠氮基的PEG 4 -生物素偶联物的反应小瓶。在水中添加加入1.2ml L-抗坏血酸（0.15毫摩尔）至混合物中，以初始化该反应。 </li><li>通过10秒的反应液中的氮泡，用封口膜密封的小瓶，并24小时在室温下反应。 </li><li>当completion个反应瓶中取出微图案化coverslide，用清水冲洗干净，并放置在一个12孔聚苯乙烯菜。 </li></ol> 10.免疫荧光检测生物素化乙炔组<ol style="margin-left: 40px;"><li> 1小时在室温块DPBS（3％驴血清）微图案化coverslide。染色为生物素化的乙炔基团与链霉亲和546结合物（2微克/毫升）在DBPS（3％驴血清的PBS中）处理2小时，在室温。 </li><li>冲洗微图案coverslide 5次用DPBS 10分钟，温和搅拌。 注： 图4B中示出了滑动的图像后，此步骤就完成了。 </li></ol> 11.无铜“，点击”叠氮的生物素化终止PEGMEMA链注：如果需要的话，该底物修饰步骤可以在原位细胞培养过程中进行的。 <ol style="margin-left: 40px;"><li>离开微图案化coverslide在12孔Polystyrene盘以下DBPS​​冲洗。 </li><li>加2 ml DBPS（或细胞培养基）中含有20μM的DBCO-PEG 4 -生物素，并且允许该反应继续进行24小时，在室温（或37℃的培养箱）。 </li><li>完成后，冲洗微图案化coverslide 5次DPBS（或者干脆进行媒体的变化）。 </li></ol> 12.免疫荧光检测生物素标记的叠氮基团的<ol style="margin-left: 40px;"><li>染色为生物素化的叠氮基团与链霉亲和488结合物（2微克/毫升）在2毫升的DPBS（3％驴血清）培养2小时，在室温。 </li><li>冲洗微图案coverslide 5倍于DPBS中10分钟，温和搅拌。 </li><li>图象微图案coverslide使用共聚焦荧光显微镜。 图4A-C示出了该幻灯片的图片在此步骤之后完成。 注意：当进行组织培养试验用基板，跳过第10及该协议第12条。所需degre后ê官能化，通过在氮气流下漂洗两面用100％乙醇和烘干消毒工程coverslide。幻灯片转移到无菌的生物安全柜，并继续进行细胞接种和培养前冲洗幻灯片，用PBS五倍。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+for+micro-+and+nanoscalepatterning.">Soft lithography for micro- and nanoscalepatterning.</a> Nature Protocols. 5 (3), 491-502 (2010).</li><li>Nam, Y., Chang, J. C., Wheeler, B. C., Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold-Coated+Microelectrode+Array+With+Thiol+Linked+Self-Assembled+Monolayers+for+Engineering+Neuronal+Cultures.">Gold-Coated Microelectrode Array With Thiol Linked Self-Assembled Monolayers for Engineering Neuronal Cultures.</a> IEEE Transactions on Biomedical Engineering. 51 (1), 158-165 (2004).</li><li>Ma, H., Wells, M., Beebe, T. P., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-Initiated+Atom+Transfer+Radical+Polymerization+of+Oligo(ethylene+glycol)+Methyl+Methacrylate+from+a+Mixed+Self-Assembled+Monolayer+on+Gold.">Surface-Initiated Atom Transfer Radical Polymerization of Oligo(ethylene glycol) Methyl Methacrylate from a Mixed Self-Assembled Monolayer on Gold.</a> Advanced Functional Materials. 16 (5), 640-648 (2006).</li><li>Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stem-cell+niche+as+an+entity+of+action.">The stem-cell niche as an entity of action.</a> Nature. 441 (7097), (2006).</li><li>Codelli, J. A., Baskin, J. M., Agard, N. J., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-Generation+Difluorinated+Cyclooctynes+for+Copper-Free+Click+Chemistry.">Second-Generation Difluorinated Cyclooctynes for Copper-Free Click Chemistry.</a> Journal of the American Chemical Society. 130 (34), 11486-11493 (2008).</li><li>Debets, M. F., van Berkel, S. S., Schoffelen, S., Rutjes, F. P. J. T., van Hest, J. C. M., van Delft, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aza-dibenzocyclooctynes+for+fast+and+efficient+enzyme+PEGylation+via+copper-free+(3+2)+cycloaddition.">Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition.</a> Chemical Communications. 46 (1), 97 (2010).</li><li>DeForest, C. A., Polizzotti, B. D., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+click+reactions+for+synthesizing+and+patterning+three-dimensional+cell+microenvironments.">Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments.</a> Nature Materials. 8 (8), 659-664 (2009).</li><li>Roth, E. A., Xu, T., Das, M., Gregory, C., Hickman, J. J., Boland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inkjet+printing+for+high-throughput+cell+patterning.">Inkjet printing for high-throughput cell patterning.</a> Biomaterials. 25 (17), 3707-3715 (2004).</li><li>Xu, T., Zhao, W., Zhu, J. M., Albanna, M. Z., Yoo, J. 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The authors declare that they have no competing financial interest.

理想的基质用于组织工程将仿生并由此概括的天然组织中发现的关键的生物活性配体的空间分布。他们也将具有动态属性，使配体的时间调整，并且在它们出现，以允许定向组织形态发生和空间上的限制诱导细胞命运的分布模式。这种基材的制造需要在衬底上复杂和高度有序取向多种生化线索的固定。虽然模拟细胞的小生境所有的内源性因素在体外是不合理的，这里所描述的R-μCP系统允许?...

的PEG接枝表面显示共价结合的配体的生化，同时保持固有的不污性能的能力，使他们为工程定制微观环境对培养基质1,2,3的理想选择。通过配体结合的PEG介导刷的生物特异性相互作用使得在单个细胞的表型体内组织的微环境中的复杂的生物化学发现线索的作用还原论分析。此外，生物正交的“点击”化学方法可用来促进配体的定向固定化，使得它们在天然构象4-6给出。因此，PEG的微观空间的图案笔刷是一个多功能的工具来创建设计师在体外龛调查引起的固定生化线索6,7细胞信号转导。 产生生化立方米的空间格局的常用方法ES限嗣继承微接触印刷（μCP）镀金基板与PEG结合的烷基硫醇模式。然后，PEG-聚乙二醇化复合链烷硫醇的微图案化的自组装单分子层限制了生化分子， 例如，蛋白质的物理吸附，只在基板8,9的非图案区域。然而，通过这种技术产生的自组装是在长期的细胞培养介质中对氧化敏感。因此，μCP&#39;d硫醇自组装膜通常还采用表面引发的原子转移自由基聚合（SI-ATRP），以增加该地区的不污稳定性10接枝的PEG聚合物刷。具体地，微接触的烷基硫醇聚合引发剂，ω-meraptoundecyl溴异丁酸乙酯，对金涂覆的表面，随后由SI-ATRP的聚（乙二醇）甲基醚甲基丙烯酸酯（PEGMEMA）单体生成具有微图案化的长期，稳定，非表面结垢PEG刷。此外，这些<html>能够被进一步修改，以呈现不同的化学部分11。 以这个属性，沙等优势。人 。开发出一种方法来设计培养基质多组分PEGMEMA画笔呈现直角“点击”化学物质。在该方法中，他们使用了一系列的穿插顺序叠氮化钠，乙醇胺微接触/ SI-ATRP的步骤，和炔丙基胺的亲核取代来创建培养基材呈现多种固定化配体6的微型图案。而使用这样的化学品在与人工微接触结合来设计新的培养基材的电位是巨大的，它是由精度和准确度与多个微接触的步骤可以在一个单一的基片对齐的限制。将需要的精度和准确度水平高可再现地制造复杂中使用这些通用的技术体外龛。 e_content“&gt;为了解决此限制，几个自动化和半自动化的微接触系统已经生成。轮穴等人开发出了微接触系统，其中定制邮票被放置在轨道系统和进入共形接触以使用金包被的载玻片一个计算机控制的气动致动器，然而，这种方法需要的定制邮票设计的精确制造，并报告有10微米的精度与执行多个微接触步骤12，当所取得的精确度的报告。最近，利用集成的运动耦合系统的方法小于1微米的精度报告使用单个图案，但是无法准确地对准多个图案由于缺少精确控制从模具印模特征到模具13上 。另外，两者的以前的方法所需要的基底图案形成步骤之间保持固定从而显著限制性的表面改性化学，可以是分集利用。在这里，我们描述了一种自动化的R-微接触系统能够对多个微接触步骤，准确和精确的对准，同时允许在邮票的设计和制造的灵活性最大。此外，图案化衬底可以重复从冲压件之间的体系中除去，从而允许使用各种不同的底物改性化学品，包括顺序的亲核取代。衬底使用这样的化学工程已被用于预先细胞培养由我们两人6,14和其他7。因此，我们已经合并的R-微接触和连续的亲核取代反应来开发用于培养基材的复杂和微图案化的生化线索可伸缩的制造方法。

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Name</td>
			<td style="width:185px;">Company</td>
			<td style="width:185px;">Catalog Number</td>
			<td style="width:185px;">Comments</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">SCARA&#160;</td>
			<td style="width:185px;">Epson</td>
			<td style="width:185px;">LS3-401ST</td>
			<td style="width:185px;">Higher end models with increased precision are available if desired.&#160;</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:185px;">(TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL)TRICHLOROSILANE</td>
			<td style="width:185px;">Gelest</td>
			<td style="width:185px;">SIT8174.0</td>
			<td style="width:185px;">CAUTION, Should only be handled in a chemical fume hood. When silanizing wafers no one should enter the hood until all silane has been evaporated.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:185px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:185px;">Ellsworth Adhesive Co</td>
			<td style="width:185px;">NC9020938</td>
			<td style="width:185px;">Thouroughly degass solutions via vacuum exposure before use. Alternative kits such as Kit 182 are acceptable.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:185px;">24mm X 50 mm #1 Cover Glass Slides</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">48393106</td>
			<td style="width:185px;">These can be purchased from a number of suppliers with varying dimensions to suit need.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">CHA-600 Telemark Electron Beam Evaporator</td>
			<td style="width:185px;">Telemark</td>
			<td style="width:185px;">SEC-600-RAP</td>
			<td style="width:185px;">Requries specialized training.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">EPSON LS3 SCARA</td>
			<td style="width:185px;">EPSON</td>
			<td style="width:185px;">LS3-401ST</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:185px;">&#969;-mertcaptoundecyl bromoisobutyrate</td>
			<td style="width:185px;">Prochimia</td>
			<td style="width:185px;">FT 015-m11-0.2</td>
			<td style="width:185px;">Store at -20&#176;C. Other ATRP initiators may be used as this R-&#956;CP platform is applicable to all micropatterning modalities.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Schlenk Tube Flask 50 mL</td>
			<td style="width:185px;">Synthware</td>
			<td style="width:185px;">60003-078</td>
			<td style="width:185px;">Requires rubber stoppers with diaphram.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">Poly(ethylene glycol) methyl ether methacrylate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">447943</td>
			<td style="width:185px;">Shipped containing MEHQ and BHT free readical inhibitors.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Methanol (Certified ACS)</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">A412-4</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Copper(II) Bromide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">437867</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">2&#39;,2-Bipyridine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">D216305</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Sodium L-Ascorbate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">A4034</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">20mL Borosilicate Glass Scintillation Vials</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">03-340-4E</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Sodium Azide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">S2002</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">N,N-dimethyformamide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">227056</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Ethanolamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">398136</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Triethylamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">T0886</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Dimethylsulfoxide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">276855</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Propargylamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">P50900</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">200 Proof Ethanol</td>
			<td style="width:185px;">University of Wisconsin Material Distribution Services</td>
			<td style="width:185px;">2292</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Azide-PEG3-Biotin</td>
			<td style="width:185px;">ClickChemistryTools</td>
			<td style="width:185px;">AZ104-100</td>
			<td style="width:185px;">Solubilized in DMF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Copper(II) Sulfate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">C1297</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">678937</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">L-Ascorbic Acid</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">A7506</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Phosphate Buffer Saline</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:185px;">14190144</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:185px;">Donkey Serum</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">D9663</td>
			<td style="width:185px;">Donkey serum contaminated items are considered bio-hazardous material and should be disposed of accordingly. Various other compounds (e.g. BSA) are available and serve this purpose.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">12-Well Polystyrene Plate</td>
			<td style="width:185px;">Thermo Scientifit - NUNC</td>
			<td style="width:185px;">07-200-81</td>
			<td style="width:185px;">Plates can be purchased form a number of suppliers with varying dimensions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">DBCO-PEG4-Biotin</td>
			<td style="width:185px;">Clickchemistytools</td>
			<td style="width:185px;">A105P4-10</td>
			<td style="width:185px;">Solubilized in DMF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Streptavidin, Alexa Fluor 488 Conjugate</td>
			<td style="width:185px;">Life Technologies</td>
			<td style="width:185px;">S-11223</td>
			<td style="width:185px;">Solubilized in PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Streptavidin, Alexa Fluor 546 conjugate</td>
			<td style="width:185px;">Life Technologies</td>
			<td style="width:185px;">S-11225</td>
			<td style="width:185px;">Solubilized in PBS</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:185px;">Nikon A1-R Confocal Microscope</td>
			<td style="width:185px;">Nikon</td>
			<td style="width:185px;">Nikon Eclipse Ti, A1R</td>
			<td style="width:185px;">An epifluorescent microscope is sufficient to image functionalized micropatterned substrates.</td>
		</tr>
	</tbody>
</table>

fabricating complex culture substrates using robotic microcontact printing (r-&#181;cp) and sequential nucleophilic substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

使用手动调整μCP技术工程师培养基质与功能化正交PEG接枝画笔数组“点击”化学品已被证明在以往的工作6。然而，这提供的图案取向最小控制和通常导致功能化区域的重叠。这里，一个新颖的R-微接触系统被用于克服此限制，并且它能够准确图案的聚乙二醇刷的环用300微米的ID和600μm的外径呈现末端炔基的聚乙二醇刷的环单独的阵列中与600微米的阵列ID和900微米OD呈现终端叠氮基团被证实<...

Watch this Scientific Journal Video about Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R-ÂµCP and Sequential Nucleophilic Substitution at JoVE.com

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &amp;#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

制造复杂的栽培基质使用机器人微接触印刷（R-μCP）和顺序亲核取代

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

fabricating-complex-culture-substrates-using-robotic-microcontact

Research

JoVE Journal

Bioengineering

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

10.4K 次观看.  University of Wisconsin, Madison. Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands. 

视频: Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

通过微接触印刷的定向功能性心肌组织的产生

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

制造超疏水高分子材料生物医学应用

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

制造棉分析仪器

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

扩大奈米基材使用缝合技术的细胞行为Nanotopographical调制

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Four-Dimensional CT Analysis Using Sequential 3D-3D Registration

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data remains difficult because segmentation in all volumetric frames is time-consuming. We aimed to analyze joint kinematics using a sequential 3D-3D registration technique to provide the kinematics of the moving bone with respect to the fixed bone semiautomatically using 4DCT DICOM data and existing software. Surface data of the source bones are reconstructed from 3DCT. The trimmed surface data are respectively matched with surface data from the first frame in 4DCT. These trimmed surfaces are sequentially matched until the last frame. These processes provide positional information for target bones in all frames of the 4DCT. Once the coordinate systems of the target bones are decided, translation and rotation angles between any two bones can be calculated. This 4DCT analysis offers advantages in kinematic analyses of complex structures such as carpal or tarsal bones. However, fast or large-scale motions cannot be traced because of motion artifacts.

We analyzed joint kinematics from four-dimensional computed tomography data. The sequential 3D-3D registration method semiautomatically provides the kinematics of the moving bone with respect to the subject bone from four-dimensional computed tomography data.

使用顺序 3D-3D 注册进行四维 CT 分析

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

使用滑动运动蛋白运动度测定法制造多组分脂质纳米管网络

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

使用机器人微注射操作脑片中单神经干细胞和神经元

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...