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PD...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>CO2 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&#8217;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&#160;</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。?...

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

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

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

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 ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

微流体技术，探讨细胞变形

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

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.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#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 ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

从混合三维培养使用近红外Photoimmunotherapy技术选择性细胞消除

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

用 Widefield 荧光显微镜研究软硅基美国匹茨堡的刚度测量

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

二维和三维 i 型胶原蛋白基质角质形成细胞增殖的评价

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These microbubbles can be condensed or formulated as perfluorocarbon nanodroplets (PFCnDs) that are small enough to extravasate and then be triggered acoustically at the target site. These nanoparticles can be further enhanced by including an optical absorber such as near infrared organic dye or nanoparticles (e.g., copper sulfide nanoparticles or gold nanoparticles/nanorods). Optically tagged PFCnDs can be vaporized through laser irradiation in a process known as optical droplet vaporization (ODV). This process of activation enables the use of high boiling point perfluorocarbon cores, which cannot be vaporized acoustically under the maximum mechanical index threshold for diagnostic imaging. Higher boiling point cores result in droplets that will recondense after vaporization, resulting in "blinking" PFCnDs that briefly produce contrast after vaporization before condensing back into nanodroplet form. This process can be repeated to produce contrast on demand, allowing for the background free imaging, multiplexing, super-resolution, and contrast enhancement through both optical and acoustic modulation. This article will demonstrate how to synthesize optically-triggerable, lipid shell PFCnDs utilizing probe sonication, create polyacrylamide phantoms to characterize the nanodroplets, and acoustically modulate the PFCnDs after ODV to improve contrast.

Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

光学汽化全氟化碳纳米液滴的配方和声学调制

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These ...