Name: expanding nanopatterned substrates using stitch technique for nanotopographical modulation of cell behavior
Uploaded: 2016-12-08T14:00:00.000+00:00
Duration: 9 min 6 s
Description: Watch this Scientific Journal Video about Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior at JoVE.com

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><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> 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

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.

Stencil micropatterning de células-tronco pluripotentes humana para sondar organização espacial de diferenciação Fates

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

Biologia do Desenvolvimento

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.

Capilar Força Litografia de Engenharia tecido cardíaco

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

Bioengenharia

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.

Microfabricação de Padrões de Ouro Nanoporosos para celular-primas estudos de interacção

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.

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 ...

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.

Alongamento Células micropatterned num PDMS Membrana

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.

Micropadronização simples de células únicas sem litografia usando estêncil cortado a laser

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.

Um método versátil de padronização proteínas e células

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.

Fabricação de gradiente Nanopattern pela técnica de Nanoimprinting térmica e rastreio da resposta das células formadoras endoteliais humanas

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.

Controle da Geometria Celular através de Micropatterning Assistido por Laser Infravermelho

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.

Geração de Padrões para Microscopia de Tração de Micropattern

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...