This work was partly supported by NSF CBET 1227766, NSF CBET 1511759, and Byars-Tarnay Endowment. We gratefully acknowledge use of the West Virginia University Shared Research Facilities which are supported, in part, by NSF EPS-1003907.

1.从EBL模具硅橡胶模具的复制<ol><li>制造硅模具29 <ol><li>旋涂200μl的聚甲基丙烯酸甲酯（PMMA）以2,500转1分钟1×1 4cm的硅（Si）的基片上的解决方案，以形成薄膜。 </li><li>烘烤，在180℃的Si衬底2分钟在PMMA膜。 </li><li>通过在300μC/ cm 2的面积的剂量使用聚焦电子束写在PMMA膜中的设计的纳米图案。 </li><li>开发开发商PMMA纳米图案为80秒。 </li><li>沉积的PMMA纳米图案并在厚度使用电子束蒸发器在10千伏，0.5mA的发射电流和0.5埃/秒的沉积速率的输出电压的50纳米的镍层。 </li><li>剥离在20ml卸妆聚甲基丙烯酸甲酯部分在80℃下进行20分钟。 </li><li>反应离子刻蚀（RIE）的纳米图案到Si衬底来获得所需的深度的Si模具。 注意:四面体的混合气体afluoromethane（CF 4），在150瓦400瓦和RIE功率的感应耦合等离子体（ICP）功率/氧（O 2）（90％/ 10％）被用于蚀刻硅衬底，以560纳米的深度。 </li></ol></li><li> Silanize思模<ol><li>放的玻璃盖玻片和Si模具在100毫米的PS培养皿并在位于通风橱的玻璃干燥器转移它们。 </li><li>滴在盖玻片10微升1H，1H，2H，2H-Perfluorooctyltrichlorosilane。 注意:1H，1H，2H，2H-Perfluorooctyltrichlorosilane可能会导致皮肤腐蚀和严重眼损伤。穿戴合适的个人防护装备（PPE）。 </li><li>部分覆盖培养皿。 </li><li>保持在真空下的干燥器5小时在通风橱以完成硅模具的硅烷化。 </li></ol></li><li>准备PDMS预聚物<ol><li>称取10克PDMS树脂和1.05克固化剂在一次性称量舟。 </li><li>用塑料勺子调匀的PDMS预聚物。 </li><li>直到观察到澄清的混合物脱气在真空下一个塑料干燥器的PDMS预聚物为约20分钟。 </li></ol></li><li>复制模具硅橡胶<ol><li>把硅烷化硅模具在60毫米的培养皿。 </li><li>倒在培养皿硅模具中的PDMS预聚物。 </li><li>放置在一个塑料干燥器和脱气培养皿约10分钟，直至所有的气泡消失。 </li><li>转移培养皿到烤盘并在70℃固化的PDMS预聚物4小时。 </li><li>通过仔细用镊子剥离从硅模具的PDMS模具。 注:PDMS模具可以存储在环境条件下长达一周。固化后，也有在与PDMS模具30的一些未交联的PDMS树脂分子和残留的固化剂。低分子量的分子将逐渐扩散出来，并在表面上的时间累积。这会影响PDMS表面31的地形和机械性能。该DIF融合不是一个星期内显著。 </li></ol></li></ol> 2.拼接PDMS模具投入大，单一的模具<ol><li>通过重复步骤1.4准备多个的PDMS模具。 注:称取同样量的PDMS混合物的每次获得相同厚度的PDMS模具。 </li><li>确定的各向异性PDMS纳米图案的取向，如在光学显微镜下nanogratings并标记上的PDMS模具用记号笔的背面。 注意:这是没有必要来标记各向同性纳米形貌的取向，如纳米支柱。 </li><li>在通风橱中清洗用乙醇Si衬底并用压缩空气干燥。 </li><li>修剪过用刀片每个PDMS模具的无图案的区域。 注:对于将被放置在缝合模具的周边的PDMS模具，仅在与他人接触的无图案区应修剪掉。 </li><li>将修剪PDMS模具的纳米图案面朝下的所述Si衬底的镜侧然后对齐靠近但不接触周围的模具（多个）其它模具。 </li><li>准备PDMS粘合层<ol><li>投1克脱气的PDMS预聚物（PDMS树脂和固化剂的比例:10:1.05）在一个干净的载玻片（7.5厘米* 2.5厘米），以形成0.5mm厚层。 </li><li>烘烤，在100℃上3-5分钟热板的PDMS层。用针触摸层和确保该层被部分地而不是完全固化。 注:部分固化的PDMS不能流动像固化PDMS预聚体，但与固化硅橡胶相比，它已被置顶。 </li></ol></li><li>放置在对准的PDMS模具的背侧的PDMS层，并迅速反转此组件，并将其转移到加热板上。 </li><li>上使用组件的顶部的金属块，以确保与PDMS粘合层和PDMS模具的背面之间具有良好的接触，并且固化所述PDMS粘合层在100℃下1小时施加压缩力（5千帕）。&lt;BR /&gt;注意:小心地调整金属块的位置，避免组件的倾斜。 </li><li>去除金属块和剥离从Si基板的单一的，缝合的PDMS模具。 </li></ol> 3. PS基板母模的生成注意:固定在载玻片上的缝合的PDMS模具可被用来生成对一PS板或PS薄膜，从该工作纳米图案基板可以制造母模。 <ol><li>产生于PS版母模<ol><li>准备一个PS版<ol><li>干燥在真空烘箱中在PS粒料在80℃两天。 </li><li>在230℃预热的压机。 </li><li>组装的铝板，聚四氟乙烯（PTFE）片和铝间隔中从底部顺序到顶部。 </li><li>负载3.5克聚苯乙烯粒料于铝垫片具有方形开口3厘米（长）×3厘米（宽）×0.3厘米（高）。 注:该间隔是约0。1厘米比PDMS模具厚，并因此最终纳米图案的PS基底为约0.1厘米厚。 </li><li>广场上的铝间隔另一四氟板，然后又铝板。 </li><li>将组件压机。 </li><li>预热，在230℃下的PS粒料30分钟。 </li><li>应用在组件的压缩压力（0.1MPa）下5分钟。 </li><li>释放压力，然后重新应用0.5兆帕上装配一个压缩的压力。 </li><li>重复步骤3.1.1.9 0.5兆帕的压力增加，直到1.5兆帕的所需压力为止。 </li><li>关闭压机的加热器，并在1.5MPa的压力恒定冷却下来低于70℃。 </li><li>取组件出并在真空烘箱中在PS板存储于80℃，以防止重新进入PS板水分。 </li></ol></li><li>纳米压印缝合PDMS模具放入PS版<ol><li>放置PS版的铝垫片一个3英寸的硅晶片上设置的。 注:隔板的内部尺寸是相同的PS版，使PS版适合直接在间隔物。 </li><li>加热上，在250℃的加热板上30分钟的PS板。 </li><li>把缝合PDMS模具上的熔融PS版纳米图案面朝下。 注意:与PDMS模具的一侧放置在触摸与PS板的表面的第一和另一侧中与PS表面接触逐渐降低，以避免气泡的界面处形成。 注意:烤盘表面是热的。在纳米压印过程中佩戴thermogloves。 </li><li>将铝板上缝合PDMS模具的搏命。 </li><li>通过使用该铝板金属板施加压缩压力（12.5千帕），并等待3分钟。 注:确保铝板没有倾斜。 </li><li>提起并从该铝板代替金属块和我现象越来越多压压到25千帕。 </li><li>重复步骤3.1.2.6与压力提高到50千帕。 注意:此步骤是除去捕集的PDMS模具和PS板之间的空气。 </li><li>保持50千帕的15分钟的稳定的压力下240和250℃之间的加热板的温度。 </li><li>关闭电磁炉和冷却整个安装。 注:风扇可以用来加速冷却过程。 </li><li>除去金属板的温度低于50℃后，小心地剥离从PS板缝合的PDMS模具。 注:PS衬底具有反向纳米图案和可以用作一个母模制作工作的PDMS衬底。 </li></ol></li></ol></li><li>生成一个PS薄膜的母模<ol><li>准备PS薄膜<ol><li>溶解在10ml甲苯1克PS在通风橱中。 注意:甲苯可引起皮肤irritation和严重眼损伤，并可能导致长期或反复接触器官造成伤害。穿戴合适的个人防护装备。 </li><li>在2500转1分钟上的2合晶片旋涂1毫升PS溶液，以形成约1微米厚的聚苯乙烯薄膜。 </li><li>通过在通风橱中3天设置硅晶片上的聚苯乙烯薄膜蒸发从膜甲苯。 </li><li>在80℃退火在真空烘箱中在PS薄膜过夜。 </li></ol></li><li>纳米压印在PS薄膜的PDMS模具<ol><li>把缝合的PDMS模具与PS上的薄膜，它是在电炉上设置纳米形貌面朝下。 </li><li>通过使用上的PDMS模具的玻璃侧金属板施加12千帕的PDMS模具的压缩压力。 </li><li>提高热板的温度升高到180℃，并保持它为15分钟。 注意:熔融聚苯乙烯薄膜可以作为润滑剂的作用。要注意防止金属块滑落。 </li><li>关闭烤盘和冷却整个安装。 注:风扇可以用来加速冷却过程。 </li><li>除去金属板的温度降至50℃以下之后，并小心地剥离从PS膜缝合的PDMS模具。 注:该纳米图案化的聚苯乙烯薄膜将作为母模生产工作PDMS基板。 </li></ol></li></ol></li></ol> 4.细胞行为Nanotopographical调制注意:人的上皮细胞是培养的代表nanotopographies展示的细胞扩散nanotopographical调制。 <ol><li>从任一步骤3.1或3.2取决于应用程序生成的母模铸造PDMS基板工作。 </li><li>使用中空钢拱冲床，剪奈米PDMS基板刻录到光盘上，以适应特定的多孔板（ 例如 ，24孔板）的配置。 </li><li>用镊子将PDMS光盘放入井中Ø发多孔板。 </li><li>通过使用70％的乙醇中，然后UV曝光，每次30分钟进行消毒的PDMS衬底。 </li><li>洗用1×无菌磷酸盐缓冲盐水（PBS）的三倍的PDMS衬底。 </li><li>涂层与细胞外基质蛋白（ 即 ，20微克/毫升纤连蛋白）在室温下30分钟的PDMS衬底。 </li><li>冲洗的PDMS衬底用无菌PBS清洗3次，每次5分钟。 </li><li>暂停人肺癌细胞A549中的Dulbecco用10％胎牛血清改良Eagle培养基和使用血球计数细胞。 </li><li>板以2,000个细胞/ cm 2的对PDMS衬底并进行培养的接种密度将细胞于37℃在含有5％CO 2的一天的潮湿气氛中。 </li><li>洗细胞用PBS洗3次。 </li><li>固定细胞在PBS中的4％多聚甲醛和2％戊二醛的混合物4小时，使用CO 2临界婆脱水细胞INT机扫描电子显微镜观察29。 注意:多聚甲醛和戊二醛可能引起严重的皮肤灼伤和眼睛损伤。工作在化学罩和穿戴适当的个人防护装备。 </li></ol>

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Mater. 20 (16), 3155-3158 (2008).</li><li>Colburn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Step+and+flash+imprint+lithography:+a+new+approach+to+high-resolution+patterning.">Step and flash imprint lithography: a new approach to high-resolution patterning.</a> Proc. SPIE. 3676 ((Pt. 1, Emerging Lithographic Technologies III)), 379-389 (1999).</li><li>Ahn, S. H., Guo, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+roll-to-roll+nanoimprint+lithography+on+flexible+plastic+substrates.">High-speed roll-to-roll nanoimprint lithography on flexible plastic substrates.</a> Adv. Mater. 20 (11), 2044-2049 (2008).</li><li>Vieu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+beam+lithography:+resolution+limits+and+applications.">Electron beam lithography: resolution limits and applications.</a> Appl. Surf. Sci. 164, 111-117 (2000).</li><li>Nagase, T., Gamo, K., Kubota, T., Mashiko, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+fabrication+of+nano-gap+electrodes+by+focused+ion+beam+etching.">Direct fabrication of nano-gap electrodes by focused ion beam etching.</a> Thin Solid Films. 499 (1-2), 279-284 (2006).</li><li>Juodkazis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+lithography+of+nanorods+in+SU-8+photoresist.">Two-photon lithography of nanorods in SU-8 photoresist.</a> Nanotechnology. 16 (6), 846 (2005).</li><li>Yang, Y., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+surfacing+for+regenerative+medicine.">Nanoscale surfacing for regenerative medicine.</a> Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2 (5), 478-495 (2010).</li><li>Yang, Y., Kulangara, K., Sia, J., Wang, L., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+a+Microfluidic+Cell+Culture+Platform+Embedded+with+Nanoscale+Features.">Engineering of a Microfluidic Cell Culture Platform Embedded with Nanoscale Features.</a> Lab Chip. 11 (9), 1638-1646 (2011).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotopographical+modulation+of+cell+function+through+nuclear+deformation.">Nanotopographical modulation of cell function through nuclear deformation.</a> Acs Appl. Mater. Inter. 8 (8), 5082-5092 (2016).</li><li>Lee, J. N., Park, C., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+Compatibility+of+Poly(dimethylsiloxane)-Based+Microfluidic+Devices.">Solvent Compatibility of Poly(dimethylsiloxane)-Based Microfluidic Devices.</a> Anal. Chem. 75 (23), 6544-6554 (2003).</li><li>Yang, Y., Kulangara, K., Lam, R. T. S., Dharmawan, R., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Topographical+and+Mechanical+Property+Alterations+Induced+by+Oxygen+Plasma+Modification+on+Stem+Cell+Behavior.">Effects of Topographical and Mechanical Property Alterations Induced by Oxygen Plasma Modification on Stem Cell Behavior.</a> ACS Nano. 6 (10), 8591-8598 (2012).</li><li>Hua, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+Imprint+Lithography+with+Molecular-Scale+Resolution.">Polymer Imprint Lithography with Molecular-Scale Resolution.</a> Nano Lett. 4 (12), 2467-2471 (2004).</li><li>Delamarche, E., Schmid, H., Michel, B., Biebuyck, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+molded+polydimethylsiloxane+microstructures.">Stability of molded polydimethylsiloxane microstructures.</a> Adv. Mater. 9 (9), 741-746 (1997).</li></ol>

The authors have nothing to disclose.

我们提出了一个简单，经济实惠，还没有通用的方法来生成纳米图案化衬底的大面积。要忠实地扩大高度定义的纳米图案，极具应注意的几个关键步骤。第一个是修剪多个PDMS模具。与PDMS模具的无图案的区域需要被去除。此外，模具的侧壁应切垂直地尽可能完美以最小化模具之间的间隙。总的来说，未图案化的区域中的最后一针模的部分可以减小。其次，需要没有在硅衬底上的任何扭曲对齐这些PDMS模具的纳米图案表面。因为PDMS纳米结构容易变形，这是至关重要的纳米图案的表面放置针对轻轻和均匀的硅基板的镜侧（避开PDMS模具和硅表面之间捕集的空气）。将PDMS模具将尽可能接近对齐，但不触及NEIghboring模具进一步减少最终线圈模具的无图案部分。否则，所触摸的纳米结构将纳米压印期间变形。第三，PDMS模具的厚度可以变化批次，因此至关重要的是使除了厚度均匀，由PDMS铸造之前完全整平小的Si模具使每个模具均匀的厚度。虽然在整个的PDMS模具厚度的变化可以通过调整的PDMS预聚物（粘合剂）的厚度来补偿层浇注在玻璃片上，一个厚的预聚物层可能是有问题的。所述预聚物可以通过与PDMS模具由毛细管力的图案化表面之间的空隙中被拉出，并因此损坏纳米图案。厚度变化可以通过从EBL模具铸造时制备的PDMS混合物的相同量被最小化。其结果是，可以使用一个薄的PDMS预聚物层。另外，部分固化prepolymeR层将会增加其粘度，从而降低其增强和最终消除纳米图案的表面的可能的损害。 线圈技术由PDMS的弹性体性质的限制。虽然软光刻已经被应用到复制的特征尺寸小至2nm的32，并且在原则上，可以达到小于0.5nm 18的分辨率，所述纳米级的PDMS功能不能完美地复制时高度与宽度的纵横比过高（&gt; 2）或过低（&lt;0.2）。当宽高比太高纳米体可能会崩溃，或当&lt;0.2比的PDMS压模使用33导致不足以缓解。此外，多个PDMS模具不能缝合，因为空隙和PDMS模具的不完全修整的无缝连接，并因此有未构图和错位的区域（特别是用于连续纳米图案如nanogratings）。鉴于叛逃的很小比例面积超过总表面积，针迹技术仍然提供了一种简单和经济的方式，以产生纳米图案基板的大面积。另外，当该缝合模具nanoimprinted到聚合物基材，熔融聚合物可能流入缝隙，导致不平整的表面（ 图1a）。在凹凸不平的表面使得它具有挑战性的收集样品细胞或分子生物学分析。在微流控应用中，该升也引起不完全的密封，当微通道对图案化衬底密封。凹凸不平的表面的问题可以容易地通过施加聚合物薄膜技术通过调整膜厚，以减少该升来解决（ 图1b）。 虽然缝技术需要一个限定主模具的膨胀，这是简单且经济实惠与其它技术，如步进闪光光刻和一个辊到辊纳米压印lithogra相比PHY。缝合技术只需要热板，并在拼接和纳米压印过程中施加压缩力的一种方式，而不是昂贵的设备。此外，该缝过程可以在清洁环境中进行，但不一定在洁净室。 线圈技术也通用。除了扩大的相同纳米图案以大面积，针迹技术可应用于包括各种形状，尺寸和安排的微米和/或纳米级特征的模具。要对此，微/ nanotopographies的组合的库建成后可提供高吞吐量平台调查细胞地形的相互作用。这个简单，价格实惠，多功能针技术有可能扩展到创建混合组件微/纳米器件。

A number of recent findings reveal that substrate nanotopography has pronounced influence on cell behavior, from cell adhesion, spreading and migration, to proliferation and differentiation1-6. For instance, a smaller cell size and lower proliferation rate have been observed in cells cultured on deep nanogratings, even leading to apoptosis although the cell alignment, elongation and migration were enhanced, compared with the flat controls2,7-10. Moreover, nanotopography has been shown to facilitate the differentiation of stem cells into certain lineages such as neuron2,11,12, muscle13, and bone3,4. In addition, because of increasing concerns on the toxicity of engineered nanomaterials14,15, there is a need to incorporate nanotopography into physiologically relevant in vitro models for accurate risk assessment of nanomaterials. To fulfil the biochemical and molecular biology analyses, enough cells are needed to be grown on a large area of nanopatterned substrate. However, conventional nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area.
Self-assembly including colloid lithography16 and polymer demixing17 can readily generate large-area nanostructures at low costs. Because self-assembly relies on interactions between the assembling elements such as colloidal particles and macromolecules, and possible interactions between these elements and substrate, it cannot be a stand-alone method of producing nanostructures with precise spatial positioning and arbitrary shapes18. The accompanied high density of defects is also a drawback. Precise spatial control of nanopatterns can be achieved by employing templated self-assembly, which uses top-down lithographic approaches to provide the topographical and/or chemical template to guide the bottom-up assembly of the assembling elements19-21. Alternative nanofabrication techniques such as step-and-flash lithography22 and a roll-to-roll nanoimprinting lithography23 have been developed but have limited use because of their sophisticated process or the requirement of specialized equipment. Nevertheless, a template or a master mold with defined nanoscale patterns is needed for templated or alternative nanofabrication techniques. 
Such templates and master molds are conventionally generated by using focused electron, ion, or photon beam lithography. For instance, electron beam lithography (EBL)24 and focused ion beam lithography25 can generate defined patterns with a sub-5 nm resolution. Two-photon lithography has demonstrated a feature size as small as 30 nm26. Although the focused beam lithography techniques enable generation of well-defined nanoscale structures, the capital investment and the time-consuming, costly process restrict their widespread use in academic research27. Therefore, it is highly desirable to develop enabling yet affordable techniques to produce a large area of nanopatterned surfaces with high fidelity.
We have reported a simple, cost-effective stitch technique for generating a large area of nanopatterned surface from a small well-defined mold28. This protocol provides step-by-step procedure from replication of poly(dimethylsiloxane) (PDMS) molds using an EBL-written pattern, to assembly of multiple PDMS molds into a single large mold, to generation of a master mold on polymeric such as polystyrene (PS) substrates, to production of working substrates. With the expanded nanopatterned substrates, we demonstrated nanotopographical modulation of cell spreading.

<table><tbody><tr><td>JEOL field emission SEM</td><td>JEOL</td><td>JSM-7600F</td><td>EBL</td></tr><tr><td>E-beam evaporator</td><td>Kurt J. Lesker</td><td>Model: LAB 18 e-beam evaporator</td><td>nickel deposition</td></tr><tr><td>Trion Minilock III ICP/RIE</td><td>Trion technology</td><td>Model: Minilock-phantom III</td><td></td></tr><tr><td>Press machine</td><td>PHI Hydraulic Press</td><td>Molde: SQ-230H</td><td></td></tr><tr><td>Spin coater</td><td>Laurell Technologies</td><td>Modle: WS-400A-6NPP-LITE</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; critical dryer</td><td>Tousimis</td><td>Modle: Autosamdri-815</td><td></td></tr><tr><td>Silicon wafer</td><td>University Wafer</td><td>1080</td><td></td></tr><tr><td>Aluminum plates</td><td>McMaster-carr</td><td>9057K123</td><td></td></tr><tr><td>Teflon sheets</td><td>McMaster-carr</td><td>8711K92</td><td></td></tr><tr><td>100 mm Petri dish</td><td>FALCON</td><td>353003</td><td></td></tr><tr><td>60 mm Petri dish</td><td>FALCON</td><td>353004</td><td></td></tr><tr><td>Glass coverslip</td><td>Fisher Scientific</td><td>12-542-B</td><td></td></tr><tr><td>Glass slide</td><td>Fisher Scientific</td><td>12-550-34</td><td></td></tr><tr><td>Disposable weighing boats</td><td>Fisher Scientific</td><td>13-735-743</td><td></td></tr><tr><td>Glass desiccator</td><td>Fisher Scientific</td><td>02-913-360</td><td></td></tr><tr><td>Plastic desiccator</td><td>Bel-Art Products</td><td>F42025-000</td><td></td></tr><tr><td>Hotplate</td><td>Fisher Scientific</td><td>1110049SH</td><td></td></tr><tr><td>Tweezer</td><td>Ted Pella, inc.</td><td>5726</td><td></td></tr><tr><td>Blade</td><td>Fisher Scientific</td><td>S17302</td><td></td></tr><tr><td>Metal blocks</td><td>McMaster-carr</td><td></td><td></td></tr><tr><td>Punch</td><td>Brettuns Village Leather Craft Supplies</td><td>Arch punch</td><td></td></tr><tr><td>Poly(methyl methacrylate)</td><td>MicroChem</td><td>495 PMMA A4</td><td></td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184 kit</td><td></td></tr><tr><td>Polystyrene</td><td>Dow Chemical</td><td>Styron 685D</td><td></td></tr><tr><td>1H,1H,2H,2H-perfluorooctylmethyldichlorosilane</td><td>Oakwood Chemical</td><td>7142</td><td></td></tr><tr><td>Developer</td><td>MicroChem</td><td>MIBK/IPA at 1: 3 ratio</td><td></td></tr><tr><td>Remover</td><td>MicroChem</td><td>Remover PG</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818500</td><td></td></tr><tr><td>Toluene</td><td>Fisher Scientific</td><td>T324-500</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>D8537</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified eagle medium</td><td>Sigma Aldrich</td><td>D5796</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Atlanta Biologicals</td><td>S11550</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microsopy Science</td><td>15712-S</td><td></td></tr><tr><td>Glutaraldehyde&amp;nbsp;</td><td>Fisher Chemical</td><td>G151-1</td><td></td></tr><tr><td>Fibronectin</td><td>Corning</td><td>356008</td><td></td></tr><tr><td>A549 cells</td><td>ATCC</td><td>ATCC CCL-185</td><td></td></tr></tbody></table>

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.

<html>线圈技术可以产生具有高保真纳米图案基板的大面积。 图1a和1b分别显示从缝合的PDMS模具PS板和PS薄膜的Si衬底上转移，纳米图案的大的面积。原始EBL写入模具（ 图1c）和工作基板（ 图1d）的最终的PDMS之间的比较证实，EBL写纳米图案可以忠实地转印到工作衬底。各种几何形状和尺寸的纳米形貌可以用于调节细胞行为。如在图2与A549证明，一个adenocarcinomic基底上皮细胞系作为模型细胞，各向异性nanogratings可伸长细胞随着多极化形态是A549细胞上各向同性纳米柱显示相比nanograting方向。 NT"FO:保together.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/54840/54840fig1.jpg" /> 图 大面积使用缝合技术，纳米图案化衬底的 1 代。 （A，B）转移到分别PS板和PS薄膜，该纳米图案的光学图像。箭头指示在缝合的PDMS模具的空隙中的聚合物加注。 （C，D）上的EBL模具和分别加工基板，最终PDMS中的纳米图案的SEM图像。刻度条是1微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54840/54840fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54840/54840fig2.jpg" /> 图2. Nanotopographical调制单元A549细胞的扩散。 </strong&gt;上生长（a）以线宽nanogratings 500纳米，在间距为500nm并且高度为560nm和（b）的直径为500纳米纳米柱，在边缘到边缘间距450纳米，高度560nm的A549细胞， 分别。规模酒吧10微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54840/54840fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior at JoVE.com

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

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 ...

expanding-nanopatterned-substrates-using-stitch-technique-for

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

6.5K 次观看.  West Virginia University. A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

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

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

人类多能干细胞的模板微图案用于探测分化命运的空间组织

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

科研

发育生物学

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

生物工程

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

细胞物质相互作用研究微细的纳米多孔金模式

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

在光刻技术定义的聚对二甲苯-C细胞图案：二氧化硅<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

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 ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

使用激光切割模具实现简单的无光刻单细胞微模式

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

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 ...

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200&#8239;nm (GP 120/200), 200-280&#8239;nm (GP 200/280), and 280-360&#8239;nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

热 Nanoimprinting 技术制备梯度 Nanopattern 及其对人血管内皮细胞菌落形成的响应筛选

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

通过红外激光辅助微模式控制细胞几何

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

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 ...