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. A replicação de PDMS moldes a partir de um molde EBL <ol><li> Fabricar molde de silicone 29 <ol><li> revestimento da rotação de 200 ul de polimetil metacrilato (PMMA) solução sobre um substrato 1 x 1 cm de silício (Si) a 2.500 rpm durante 1 minuto para formar uma película fina. </li><li> Cozer a película de PMMA sobre o substrato de Si a 180 ° C durante 2 min. </li><li> Escreva o nanopattern projetado no filme PMMA usando um feixe de electrões focado em uma dose de 300 uC / cm 2 de área. </li><li> Desenvolver o nanopattern PMMA no desenvolvedor para 80 seg. </li><li> Depositar o nanopattern de PMMA com uma camada de níquel de 50 nm de espessura usando um evaporador de feixe de electrões a uma tensão de 10 kV, corrente de emissão de 0,5 mA com uma velocidade de deposição de 0,5 Ã / seg saída. </li><li> Levantar a parte de PMMA em 20 ml removedor a 80 ° C durante 20 min. </li><li> etch iónico reactivo (RIE) a nanopattern para o substrato de Si para obter um molde de Si a profundidade desejada. NOTA: mistura gasosa de tetrafluoromethane (FC 4) / oxigénio (O2) (90% / 10%) com uma potência de plasma indutivamente acoplado (ICP) de 400 W de potência e RIE de 150 W é usado para gravar Si substrato a uma profundidade de 560 nm. </li></ol></li><li> molde silanizar Si <ol><li> Coloque uma lamela de vidro e o molde Si em um 100 milímetros PS placa de Petri e transferi-los num exsicador de vidro localizada em um exaustor. </li><li> Gota de 10 ul de 1H, 1H, 2H, 2H-Perfluorooctyltrichlorosilane sobre a lamela. Cuidado: 1H, 1H, 2H, 2H-Perfluorooctyltrichlorosilane pode causar corrosão da pele e lesões oculares graves. Use equipamento de proteção pessoal adequado (PPE). </li><li> Cobrir a placa de Petri parcialmente. </li><li> Manter o exsicador sob vácuo durante 5 horas num exaustor de fumos para completar a silanização do molde Si. </li></ol></li><li> Prepare PDMS pré-polímero <ol><li> Pesar 10 g de resina PDMS e 1,05 g agente de cura em um barco de pesagem descartáveis. </li><li> Misturar o pré-polímero de PDMS cuidadosamente usando uma colher de plástico. </li><li> Desgasificar o pré-polímero de PDMS-se num exsicador de plástico sob vácuo durante cerca de 20 minutos até uma mistura clara é observado. </li></ol></li><li> Replicar moldes PDMS <ol><li> Coloque o molde Si silanizada em uma placa de Petri de 60 milímetros. </li><li> Verter o pré-polímero de PDMS no molde Si na placa de Petri. </li><li> Coloque a placa de Petri num exsicador de plástico e degas por cerca de 10 min até que todas as bolhas desapareçam. </li><li> Transferir a placa de Petri a uma placa de aquecimento e cura do pré-polímero de PDMS, a 70 ° C durante 4 h. </li><li> Retire o molde PDMS do molde Si com cuidado usando uma pinça. NOTA: moldes PDMS pode ser armazenado em condições ambientes durante até uma semana. Após a cura, há algumas moléculas de PDMS não reticuladas resina e agente de endurecimento residual nos moldes 30 PDMS. As moléculas de baixo peso molecular vai gradualmente se difundem para fora e se acumulam na superfície ao longo do tempo. Isto afecta as propriedades topográficas e mecânicas da superfície do PDMS 31. o diffusão não é significativa dentro de uma semana. </li></ol></li></ol> 2. Costura de PDMS Moldes em um grande, Mold Individual <ol><li> Prepare moldes múltiplos PDMS repetindo o passo 1.4. NOTA: Pesar mesma quantidade de mistura de PDMS de cada vez para obter moldes de PDMS mesma espessura. </li><li> Determinar a orientação de anisotrópicos PDMS nanopatterns tais como nanogratings sob um microscópio óptico e marcá-lo na parte traseira dos moldes PDMS com uma caneta marcadora. NOTA: Não é necessário marcar a orientação de nanotopografia isotrópica, tais como nanopillars. </li><li> Limpar um substrato de Si com etanol em um exaustor e seque-o com ar comprimido. </li><li> Aparar as áreas unpatterned de cada molde PDMS com uma lâmina. NOTA: Para os moldes de PDMS que serão colocados na periferia do molde costurado, apenas as áreas unpatterned em contacto com os outros deve ser aparada. </li><li> Coloque o molde PDMS aparadas com a nanopattern face para baixo sobreo lado do espelho do substrato de Si e alinhe outros moldes perto mas não tocando o molde circundante (s). </li><li> Prepara-se uma camada adesiva de PDMS <ol><li> Fundido um pré-polímero de PDMS g desgaseificada (PDMS resina e relação de agente de cura: 10: 1,05) numa lâmina de vidro limpo (7,5 cm x 2,5 cm) para formar uma camada de espessura de 0,5 mm. </li><li> Cozer a camada de PDMS, a 100 ° C numa placa de aquecimento durante 3-5 minutos. Utilize uma agulha para tocar na camada e assegurar que a camada é parcialmente mas não completamente curada. NOTA: PDMS parcialmente curada não pode fluir como pré-polímero de PDMS não curado, mas é pegajoso em comparação com PDMS curados. </li></ol></li><li> Coloque a camada de PDMS na parte traseira de moldes PDMS alinhados e rapidamente inverter esta montagem e transferi-lo para a placa de aquecimento. </li><li> Aplicar uma força de compressão (5 kPa) usando um bloco metálico na parte superior do conjunto para assegurar um bom contacto entre a camada adesiva e a parte traseira de PDMS de moldes de PDMS, e curar o PDMS camada de adesivo a 100 ° C durante 1 h. &lt;br /&gt; NOTA: Ajuste cuidadosamente a posição do bloco de metal para evitar a inclinação da montagem. </li><li> Remova o bloco de metal e retire a, molde PDMS costurado single do substrato de Si. </li></ol> 3. Geração de um molde mestre no PS Substratos Nota: O molde PDMS costurado imobilizado sobre uma lâmina de vidro pode ser usado para gerar um molde mestre em uma placa de PS ou uma película fina de PS, a partir do qual podem ser produzidos substratos nanopatterned de trabalho. <ol><li> Gerar um molde mestre em uma placa PS <ol><li> Prepare uma placa PS <ol><li> Secam-se os peletes de PS em uma estufa de vácuo a 80 ° C durante dois dias. </li><li> Pré-aqueça uma máquina de imprensa a 230 ° C. </li><li> Montar uma placa de alumínio, uma folha de politetrafluoretileno (PTFE) e o espaçador de alumínio em uma ordem de baixo para cima. </li><li> Carregar 3,5 g de PS em pelotas o espaçador de alumínio com uma abertura quadrada de 3 cm (L) x 3 cm (W) x 0,3 cm (H). NOTA: O espaçador é de aproximadamente 0.1 cm mais espessos que os moldes de PDMS, e, assim, o substrato final nanopatterned PS é cerca de 0,1 cm de espessura. </li><li> Coloque outra folha de PTFE e, em seguida, uma outra placa de alumínio no espaçador de alumínio. </li><li> Coloque o conjunto na máquina da imprensa. </li><li> Pré-aqueça os peletes Ps a 230 ° C durante 30 min. </li><li> Aplicar uma pressão de compressão (0,1 MPa) no conjunto durante 5 minutos. </li><li> Solte a pressão e, em seguida, voltar a aplicar uma pressão de compressão de 0,5 MPa na montagem. </li><li> Repita o passo 3.1.1.9, com um aumento de pressão de 0,5 MPa até à pressão de 1,5 MPa desejável é atingida. </li><li> Desligue o aquecedor da máquina da imprensa e esfriá-la abaixo de 70 ° C a uma pressão constante de 1,5 MPa. </li><li> Tomar o conjunto para fora e armazenar a placa de PS num forno de vácuo a 80 ° C para impedir a humidade de entrar novamente a placa PS. </li></ol></li><li> Nanoimpressão o molde PDMS costurado na placa PS <ol><li> Coloque a placa PS em um espaçador de alumíniodefinido em uma de 3 polegadas lâmina de silício. NOTA: As dimensões interiores do espaçador são as mesmas que as da placa de PS de modo que a placa PS cabe para a direita no espaçador. </li><li> Aquecer a placa PS numa placa de aquecimento a 250 ° C durante 30 min. </li><li> Coloque o molde PDMS costurado com nanopatterns face para baixo sobre a placa PS fundido. Nota: Um dos lados do molde PDMS é posto em contacto com a superfície da placa de PS em primeiro lugar e o outro lado é gradualmente reduzido em contacto com a superfície de PS para evitar a formação de bolhas de ar na interface. Cuidado: A superfície da placa de aquecimento é quente. Desgaste thermogloves durante o processo de nanoimpressão. </li><li> Coloque uma placa de alumínio na lâmina de vidro do molde PDMS costurado. </li><li> Aplicar uma pressão de compressão (12,5 kPa) usando blocos de metal sobre a chapa de alumínio e esperar durante 3 min. NOTA: Certifique-se de que a placa de alumínio não está inclinado. </li><li> Levante e substituir o bloco de metal da placa de alumínio e increase a pressão de compressão a 25 kPa. </li><li> Repita o passo 3.1.2.6 com a pressão aumentada para 50 kPa. NOTA: Este passo é remover o ar aprisionado entre o molde e a placa de PDMS PS. </li><li> Manter a temperatura da placa de aquecimento entre 240 e 250 ° C, sob a pressão constante de 50 kPa durante 15 minutos. </li><li> Desligue o fogão e esfriar a instalação inteira. NOTA: Um ventilador pode ser usado para acelerar o processo de resfriamento. </li><li> Remover os blocos de metal após a temperatura esteja abaixo de 50 ° C, e cuidadosamente descascar para fora do molde PDMS costurada a partir da placa PS. NOTA: O substrato PS tem os nanopatterns reversa e pode ser utilizado como um molde mestre para produzir substratos de PDMS de trabalho. </li></ol></li></ol></li><li> Gerar um molde mestre em uma película fina PS <ol><li> Prepara-se uma película fina PS <ol><li> Dissolve-se 1 g de PS em 10 mL de tolueno num exaustor de fumos. Atenção: O tolueno pode causar irritatio pelen e lesões oculares graves, e pode causar dano aos órgãos através da exposição repetida ou prolongada. Utilizar EPI adequado. </li><li> Spin-coat solução PS 1 ml em um 2-em wafer a 2.500 rpm por 1 min para formar ~ 1 mm de espessura PS película fina. </li><li> Evaporar o tolueno a partir do filme, definindo o filme PS no bolacha de Si num exaustor de fumos durante 3 dias. </li><li> Recozer a película fina PS num forno de vácuo a 80 ° C durante a noite. </li></ol></li><li> Nanoimpressão o molde PDMS em uma película fina PS <ol><li> Coloque o molde PDMS costurado com nanotopografia face para baixo sobre a película fina PS, que é definido numa placa de aquecimento. </li><li> Aplicar uma pressão de compressão de 12 kPa no molde PDMS usando blocos de metal do lado do vidro do molde PDMS. </li><li> Aumentar a temperatura da placa de aquecimento a 180 ° C e mantê-lo durante 15 min. Cuidado: A película fundida PS pode funcionar como um lubrificante. Preste atenção para evitar que os blocos de metal de deslizar para fora. </li><li> Desligue a placa de aquecimentoe arrefecer a instalação inteira. NOTA: Um ventilador pode ser usado para acelerar o processo de resfriamento. </li><li> Remover os blocos de metal após a temperatura cai abaixo de 50 ° C, e cuidadosamente descascar para fora do molde PDMS cosido a partir do filme de PS. NOTA: O filme nanopatterned PS vai servir como um molde mestre para produzir substratos PDMS de trabalho. </li></ol></li></ol></li></ol> 4. Nanotopographical modulação do comportamento celular Nota: células epiteliais humanas são cultivadas nas nanotopographies representativos para demonstrar modulação nanotopographical da célula se espalhando. <ol><li> Fundido substratos de trabalho PDMS do molde mestre gerado a partir de qualquer Passo 3.1 ou 3.2, dependendo da aplicação. </li><li> Usando um arco de aço oco perfurador, cortar os substratos PDMS nanopatterned em discos para se ajustar a configuração de uma placa multi-poço específico (por exemplo, placa, de 24 poços). </li><li> Utilize uma pinça para colocar os discos PDMS nas cavidades ófa placa multi-poços. </li><li> Esterilizar os substratos PDMS usando 70% de etanol e, em seguida, a exposição de UV, durante 30 minutos cada. </li><li> Lavar o PDMS substratos com 1x de fosfato estéril tamponado salino (PBS) três vezes. </li><li> Revestir os substratos PDMS com proteínas de matriz extracelular (por exemplo, 20 ug / ml de fibronectina) durante 30 min à temperatura ambiente. </li><li> Lavar os substratos PDMS três vezes com PBS estéril, cada uma durante 5 minutos. </li><li> Suspender A549 de células de cancro de pulmão humano em meio de Eagle modificado por Dulbecco com soro bovino fetal a 10% e contar as células utilizando um hemocitómetro. </li><li> Placa as células a uma densidade de sementeira de 2.000 células / cm2 sobre os substratos e cultura PDMS-los a 37 ° C numa atmosfera humidificada contendo 5% de CO 2 durante um dia. </li><li> Lavam-se as células com PBS três vezes. </li><li> Fixar as células em uma mistura de paraformaldeído a 4% e 2% de glutaraldeído em PBS durante 4 h e desidratar as células usando um CO 2 PO críticasecador de int para a digitalização de observação microscópica electrónica 29. Atenção: O paraformaldeído e glutaraldeído pode causar queimaduras na pele e lesões oculares graves. Operar em uma capa química e utilizar EPI adequado. </li></ol>

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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.

Nós apresentamos um método simples, acessível, mas versátil para gerar uma grande área de substrato nanopatterned. Para expandir fielmente nanopatterns altamente definidos, grande atenção deve ser dada às várias etapas críticas. O primeiro é para aparar as PDMS vários moldes. unpatterned áreas dos moldes PDMS precisa ser removido. Além disso, as paredes laterais dos moldes deve ser cortada verticalmente perfeito quanto possível para minimizar as diferenças entre os moldes. Colectivamente, a porção de ?...

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> A técnica do ponto pode gerar uma grande área de substratos nanopatterned com alta fidelidade. A Figura 1a e 1b exibir a grande área de nanopatterns transferidos a partir do molde PDMS costurada a placa PS e PS película fina sobre um substrato de Si, respectivamente. A comparação entre o molde original é EBL-escrita (Figura 1C) e o PDMS finais de substrato de trabalho (Figura 1D), confirma que os nanopattern...

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.

Expansão Nanopatterned Substratos usar a técnica do ponto de Nanotopographical modulação do comportamento celular

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 visualizações.  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.

Vídeo: 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.

Criando Two-Dimensional Substratos modelado para Confinamento de proteína e celular

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

Bioengenharia

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.

Padronização celular em photolithographically Definido Parylene-C: SiO<sub&gt; 2</sub&gt; Substratos

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.

A técnica de microfluídica para sondar Deformabilidade celular

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.

Fabricando substratos de cultura complexos usando Robotic microcontact Printing (R-μCP) e substituição nucleofílica Sequential

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.

A técnica de livre-Label para a imagem espaço-temporal do Secreções única célula

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).

Eliminação celular seletiva de Cultura 3D Mixed usando uma técnica de infravermelho próximo Photoimmunotherapy

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.

Medição da rigidez de substratos de Silicone macio para estudos de Mechanobiology, usando um microscópio de fluorescência Widefield

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.

Avaliação da proliferação queratinócito em duas e três dimensões tipo I colágeno substratos

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.

Padronização da geometria de colônias de células-tronco embrionárias humanas em substratos compatíveis para controlar a mecânica de nível de tecido

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.

Formulação e modulação acústica de nanogotículas de perfluorocarbono opticamente vaporizadas

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