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. La replicación de PDMS moldes de un molde de EBL <ol><li> Fabricar molde de silicona 29 <ol><li> capa de la vuelta 200 l polimetacrilato de metilo (PMMA) solución sobre un sustrato de 1 × 1 cm de silicio (Si) a 2.500 rpm durante 1 min para formar una película delgada. </li><li> Hornear la película de PMMA en el sustrato de Si a 180 ° C durante 2 min. </li><li> Escribir la nanopattern diseñado sobre la película de PMMA mediante el uso de un haz de electrones enfocado a una dosis área de 300 mu c / cm 2. </li><li> Desarrollar la nanopattern PMMA en el revelador de 80 seg. </li><li> Depositar el nanopattern PMMA con una capa de níquel de 50 nm de espesor utilizando un evaporador de haz de electrones a un voltaje de salida de 10 kV, corriente de emisión de 0,5 mA y una velocidad de deposición de 0,5 Å / s. </li><li> Levante la parte PMMA en 20 removedor ml a 80 ° C durante 20 minutos. </li><li> etch iones reactivos (RIE) la nanopattern en el sustrato de Si para obtener un molde de Si de la profundidad deseada. NOTA: Mezclas de gas de TetRafluoromethane (CF 4) / oxígeno (O 2) (90% / 10%) con una potencia de plasma de acoplamiento inductivo (ICP) de 400 W de potencia y RIE de 150 W se utiliza para grabar sustrato de Si a una profundidad de 560 nm. </li></ol></li><li> Si molde silaniza <ol><li> Ponga un cubreobjetos de vidrio y el molde de Si en un 100 mm placa de Petri PS y transferirlos en un desecador de vidrio situado en una campana de humos. </li><li> Caída de 10 l 1H, 1H, 2H, 2H-Perfluorooctyltrichlorosilane sobre el cubreobjetos. Precaución: 1H, 1H, 2H, 2H-Perfluorooctyltrichlorosilane puede causar la corrosión de la piel y lesiones oculares graves. Use equipo de protección personal adecuado (PPE). </li><li> Cubrir la placa de Petri parcialmente. </li><li> Mantenga el desecador a vacío durante 5 horas en una campana extractora para completar la silanización del molde Si. </li></ol></li><li> Preparar PDMS prepolímero <ol><li> Pesar 10 g de resina de PDMS y 1,05 g de agente de curado en un recipiente pesado desechable. </li><li> Mezclar el prepolímero PDMS a fondo mediante el uso de una cuchara de plástico. </li><li> Desgasificar la prepolímero PDMS en un desecador de plástico al vacío durante unos 20 minutos hasta que se observa una mezcla clara. </li></ol></li><li> Replicar moldes de PDMS <ol><li> Ponga el molde Si silanizada en una placa de Petri 60 mm. </li><li> Verter el prepolímero PDMS sobre el molde de Si en la placa de Petri. </li><li> Coloque la placa de Petri en un desecador de plástico y desgasificar durante aproximadamente 10 minutos hasta que todas las burbujas desaparecen. </li><li> La transferencia de la placa de Petri con una placa calefactora y curar el prepolímero PDMS a 70 ° C durante 4 horas. </li><li> Despegar el molde de PDMS del molde Si cuidadosamente usando pinzas. NOTA: moldes de PDMS se pueden almacenar en condiciones ambientales durante hasta una semana. Después de curar, hay algunas moléculas de resina PDMS no reticuladas y agente de curado residual en los moldes de PDMS 30. Las moléculas de bajo peso molecular se difunden gradualmente y se acumulan en la superficie con el tiempo. Esto afecta a las propiedades topográficas y mecánicas de la superficie PDMS 31. el DIFfusión no es significativo dentro de una semana. </li></ol></li></ol> 2. Costura de PDMS moldes en una grande, solo molde <ol><li> Preparar moldes múltiples PDMS repitiendo el paso 1.4. NOTA: Pesar misma cantidad de mezcla de PDMS cada vez para obtener moldes de PDMS de igual espesor. </li><li> Determinar la orientación de anisotrópicos PDMS nanopatrones como nanogratings bajo un microscopio óptico y se marca en la parte posterior de los moldes de PDMS con un rotulador. NOTA: No es necesario marcar la orientación de nanotopografía isotrópico como nanopilares. </li><li> Limpiar un sustrato de Si con etanol en una campana de humos y se seca con aire comprimido. </li><li> Se quita las áreas sin patrón de cada molde de PDMS con una cuchilla. NOTA: Para obtener los moldes de PDMS que serán colocados en la periferia del molde cosido, sólo las áreas no pautadas en contacto con otras personas deben ser recortados. </li><li> Coloque el molde de PDMS adornado con la nanopattern boca abajo enel espejo lateral del sustrato de Si y luego se suman otros moldes cerca pero sin tocar el molde (s) de los alrededores. </li><li> Preparar una capa adhesiva PDMS <ol><li> Reparto de 1 g desgasifica PDMS prepolímero (resina de PDMS y curado relación de agente: 10: 1,05) en un portaobjetos de vidrio limpio (7,5 cm x 2,5 cm) para formar una capa de espesor de 0,5 mm. </li><li> Hornear la capa de PDMS a 100 ° C sobre una placa caliente durante 3-5 min. Use una aguja a tocar la capa y asegurar que la capa está parcialmente pero no completamente curada. NOTA: PDMS parcialmente curado no puede fluir como prepolímero de PDMS sin curar, pero es pegajoso en comparación con PDMS curados. </li></ol></li><li> Coloque la capa de PDMS en la parte trasera de moldes de PDMS alineados e invertir rápidamente esta asamblea y transferirlo a la zona de cocción. </li><li> Aplicar una fuerza de compresión (5 kPa) usando un bloque de metal en la parte superior del conjunto para asegurar un buen contacto entre la capa de adhesivo PDMS y la parte posterior de los moldes de PDMS, y curar la capa de adhesivo PDMS a 100 ° C durante 1 hr. &lt;br /&gt; NOTA: Ajuste cuidadosamente la posición del bloque de metal para evitar la inclinación del conjunto. </li><li> Retire el bloque de metal y retire la única, del molde de PDMS cosido desde el sustrato de Si. </li></ol> 3. Generación de un molde principal en PS Sustratos Nota: El molde de PDMS cosido inmovilizada sobre un portaobjetos de vidrio se puede utilizar para generar un molde maestro en una placa de PS o una película delgada PS, de la que se pueden producir de trabajo sustratos nanoestructurada. <ol><li> Generar un molde maestro en una placa PS <ol><li> Preparar una placa PS <ol><li> Se secan los gránulos PS en un horno de vacío a 80 ° C durante dos días. </li><li> Precalentar una máquina de prensa a 230 ° C. </li><li> Montar una placa de aluminio, una lámina de politetrafluoroetileno (PTFE) y el separador de aluminio en un orden de abajo hacia arriba. </li><li> Carga de 3,5 g de gránulos de PS en el separador de aluminio con una abertura cuadrada de 3 cm (L) x 3 cm (W) x 0,3 cm (H). NOTA: El separador es de aproximadamente 0.1 cm más gruesos que los moldes de PDMS, y por lo tanto el sustrato final nanoestructurada PS es de unos 0,1 cm de espesor. </li><li> Coloque otra hoja de PTFE y luego otra placa de aluminio en el separador de aluminio. </li><li> Coloque el conjunto de la máquina de la prensa. </li><li> Precalentar los pellets PS a 230 ° C durante 30 minutos. </li><li> Aplicar una presión de compresión (0,1 MPa) sobre el conjunto durante 5 min. </li><li> Liberar la presión y vuelva a aplicar una presión de compresión de 0,5 MPa en el conjunto. </li><li> Repita el paso 3.1.1.9 con un aumento de presión de 0,5 MPa hasta que la presión deseable de 1,5 MPa se alcanza. </li><li> Apague el calentador de la máquina de la prensa y enfriarlo por debajo de 70 ° C a una presión constante de 1,5 MPa. </li><li> Tome el montaje y almacenar la placa PS en un horno de vacío a 80 ° C para evitar que la humedad vuelva a entrar en la placa PS. </li></ol></li><li> Nano-impresión del molde de PDMS cosido en la placa PS <ol><li> Coloque la placa de PS en un separador de aluminiosituado en una de 3 pulgadas oblea de Si. NOTA: Las dimensiones interiores del espaciador son los mismos que los de la placa de PS de modo que la placa de PS encaja perfectamente en el espaciador. </li><li> Calentar la placa de PS en una placa caliente a 250 ° C durante 30 min. </li><li> Ponga el molde de PDMS cosida con nanopatrones boca abajo sobre la placa PS fundido. NOTA: Un lado del molde de PDMS se pone en contacto con la superficie de la placa de PS primero y otro lado se disminuye gradualmente en contacto con la superficie de PS para evitar la formación de burbujas de aire en la interfase. Precaución: La superficie de la placa de cocción está caliente. Use thermogloves durante el proceso de nanoimpresión. </li><li> Colocar una placa de aluminio en el portaobjetos de vidrio del molde de PDMS cosido. </li><li> Aplicar una presión de compresión (12,5 kPa) mediante el uso de bloques de metal en la placa de aluminio y esperar 3 minutos. NOTA: Asegúrese de que la placa de aluminio no está inclinada. </li><li> Levante y vuelva a colocar el bloque de metal de la placa de aluminio y yoaumentar la cant presión de compresión de 25 kPa. </li><li> Repita el paso 3.1.2.6 con la presión aumentó a 50 kPa. NOTA: Este paso es eliminar el aire atrapado entre el molde de PDMS y la placa PS. </li><li> Mantener la temperatura de la zona de cocción entre 240 y 250 ° C bajo la presión constante de 50 kPa durante 15 min. </li><li> Apague la zona de cocción y enfriamiento toda la instalación. NOTA: Un ventilador puede ser usado para acelerar el proceso de enfriamiento. </li><li> Retire los bloques de metal después de que la temperatura sea inferior a 50 ° C, y con cuidado retire el molde de PDMS cosido de la placa PS. NOTA: El sustrato PS tiene las nanopatrones inversa y se puede utilizar como un molde maestro para producir PDMS de trabajo sustratos. </li></ol></li></ol></li><li> Generar un molde maestro en una película delgada PS <ol><li> Preparar una película delgada PS <ol><li> Disolver 1 g PS en 10 ml de tolueno en una campana de humos. Precaución: El tolueno puede causar la piel irritation y lesiones oculares graves, y pueden causar daños en los órganos tras exposiciones prolongadas o repetidas. Use el EPP apropiado. </li><li> Spin-capa de solución de PS 1 ml en un 2-en forma de disco a 2.500 rpm durante 1 min para formar ~ 1 de película delgada m de espesor PS. </li><li> Se evapora el tolueno de la película mediante el establecimiento de la película PS en oblea de Si en una campana de humos durante 3 días. </li><li> Recocer la película delgada PS en un horno de vacío a 80 ° C durante la noche. </li></ol></li><li> Nano-impresión del molde de PDMS en una película delgada PS <ol><li> Ponga el molde de PDMS cosida con nanotopografía boca abajo en la película delgada PS, que se fija sobre una placa caliente. </li><li> Aplicar una presión de compresión de 12 kPa en el molde de PDMS mediante el uso de bloques de metal en el lado del vidrio del molde de PDMS. </li><li> Aumentar la temperatura de la placa caliente a 180 ° C y mantener durante 15 min. Precaución: La película PS fundido puede funcionar como un lubricante. Prestar atención a evitar que los bloques de metal se deslice fuera. </li><li> Apague la zona de coccióny enfriar toda la instalación. NOTA: Un ventilador puede ser usado para acelerar el proceso de enfriamiento. </li><li> Retire los bloques de metal después de que la temperatura desciende por debajo de 50 ° C, y con cuidado retire el molde de PDMS cosido de la película PS. NOTA: La película nanoestructurada PS servirá como un molde maestro para producir sustratos PDMS de trabajo. </li></ol></li></ol></li></ol> 4. Nanotopographical modulación del comportamiento de la célula Nota: Las células epiteliales humanas se cultivan en las nanotopographies representativos que demuestren la modulación nanotopographical de difusión celular. <ol><li> Reparto de PDMS sustratos de trabajo del molde maestro generada a partir de cualquiera Paso 3.1 o 3.2 dependiendo de la aplicación. </li><li> Con un punzón de arco de acero hueco, cortar los PDMS sustratos nanoestructurada en discos para adaptarse a la configuración de una placa de múltiples pocillos específica (por ejemplo, la placa, de 24 pocillos). </li><li> Utilizar pinzas para colocar los discos de PDMS en los pocillos ofa placa de múltiples pocillos. </li><li> Esterilizar los sustratos PDMS mediante el uso de 70% de etanol y después de la exposición UV, cada uno por 30 min. </li><li> Lavar los sustratos PDMS con 1x tampón fosfato salino (PBS) tres veces. </li><li> Escudo el PDMS sustratos con proteína de la matriz extracelular (es decir, 20 mg / ml de fibronectina) durante 30 min a temperatura ambiente. </li><li> Enjuague el PDMS sustratos tres veces con PBS estéril, cada uno de 5 min. </li><li> Suspender A549 de células de cáncer de pulmón humano en un medio de Eagle modificado por Dulbecco con suero bovino fetal 10% y contar las células usando un hemocitómetro. </li><li> Placa de las células a una densidad de siembra de 2.000 células / cm 2 en los sustratos y cultura los PDMS a 37 ° C en una atmósfera humidificada que contiene 5% de CO 2 durante un día. </li><li> Se lavan las células con PBS tres veces. </li><li> Fijar las células en una mezcla de 4% de paraformaldehído y glutaraldehído al 2% en PBS durante 4 hr y deshidratar las células usando un CO 2 po críticosecador de int para la digitalización de la observación microscópica electrónica 29. Precaución: El paraformaldehído y glutaraldehído pueden causar quemaduras en la piel y lesiones oculares graves. Operar en una campana química y usar el EPP apropiado. </li></ol>

<ol>
	<li>Silva, G. A., 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=Selective+differentiation+of+neural+progenitor+cells+by+high-epitope+density+nanofibers.">Selective differentiation of neural progenitor cells by high-epitope density nanofibers.</a> Science. 303 (5662), 1352-1355 (2004).</li><li>Yim, E. K. F., Pang, S. W., 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=Synthetic+Nanostructures+Inducing+Differentiation+of+Human+Mesenchymal+Stem+Cells+into+Neuronal+Lineage.">Synthetic Nanostructures Inducing Differentiation of Human Mesenchymal Stem Cells into Neuronal Lineage.</a> Exp. Cell Res. 313 (9), 1820-1829 (2007).</li><li>Dalby, M. J., 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=The+Control+of+Human+Mesenchymal+Cell+Differentiation+Using+Nanoscale+Symmetry+and+Disorder.">The Control of Human Mesenchymal Cell Differentiation Using Nanoscale Symmetry and Disorder.</a> Nat. Mater. 6 (12), 997-1003 (2007).</li><li>Oh, 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=Stem+Cell+Fate+Dictated+Solely+by+Altered+Nanotube+Dimension.">Stem Cell Fate Dictated Solely by Altered Nanotube Dimension.</a> Proc. Natl. Acad. Sci. U. S. A. 106 (7), 2130-2135 (2009).</li><li>Brunetti, V., 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=Neurons+Sense+Nanoscale+Roughness+with+Nanometer+Sensitivity.">Neurons Sense Nanoscale Roughness with Nanometer Sensitivity.</a> Proc. Natl. Acad. Sci. U. S. A. 107 (14), 6264-6269 (2010).</li><li>McMurray, R., 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=Nanoscale+Surfaces+for+the+Long-term+Maintenance+of+Mesenchymal+Stem+Cell+Phenotype+and+Multipotency.">Nanoscale Surfaces for the Long-term Maintenance of Mesenchymal Stem Cell Phenotype and Multipotency.</a> Nat. Mater. 10 (8), 637-644 (2011).</li><li>Yim, E. K. 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=Nanopattern-induced+changes+in+morphology+and+motility+of+smooth+muscle+cells.">Nanopattern-induced changes in morphology and motility of smooth muscle cells.</a> Biomaterials. 26 (26), 5405-5413 (2005).</li><li>Gerecht, 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=The+Effect+of+Actin+Disrupting+Agents+on+Contact+Guidance+of+Human+Embryonic+Stem+Cells.">The Effect of Actin Disrupting Agents on Contact Guidance of Human Embryonic Stem Cells.</a> Biomaterials. 28 (28), 4068-4077 (2007).</li><li>Bettinger, C. J., Zhang, Z., Gerecht, S., Borenstein, J. T., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+in+vitro+Capillary+Tube+Formation+by+Substrate+Nanotopography.">Enhancement of in vitro Capillary Tube Formation by Substrate Nanotopography.</a> Adv. Mater. 20 (1), 99-103 (2008).</li><li>Thakar, R. G., Ho, F., Huang, N. F., Liepmann, D., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vascular+smooth+muscle+cells+by+micropatterning.">Regulation of vascular smooth muscle cells by micropatterning.</a> Biochem. Biophys. Res. Commun. 307 (4), 883-890 (2003).</li><li>Lee, M. R., 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=Direct+differentiation+of+human+embryonic+stem+cells+into+selective+neurons+on+nanoscale+ridge/groove+pattern+arrays.">Direct differentiation of human embryonic stem cells into selective neurons on nanoscale ridge/groove pattern arrays.</a> Biomaterials. 31 (15), 4360-4366 (2010).</li><li>Moe, A. A. 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=Microarray+with+micro-+and+nano-topographies+enables+identification+of+the+optimal+topography+for+directing+the+differentiation+of+primary+murine+neural+progenitor+cells.">Microarray with micro- and nano-topographies enables identification of the optimal topography for directing the differentiation of primary murine neural progenitor cells.</a> Small. 8 (19), 3050-3061 (2012).</li><li>Dang, J. M., 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=Myogenic+induction+of+aligned+mesenchymal+stem+cell+sheets+by+culture+on+thermally+responsive+electrospun+nanofibers.">Myogenic induction of aligned mesenchymal stem cell sheets by culture on thermally responsive electrospun nanofibers.</a> Adv. Mater. 19 (19), 2775-2779 (2007).</li><li>Dasgupta, N., 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=Thermal+co-reduction+approach+to+vary+size+of+silver+nanoparticle:+its+microbial+and+cellular+toxicology.">Thermal co-reduction approach to vary size of silver nanoparticle: its microbial and cellular toxicology.</a> Environ. Sci. Pollut. Res. 23 (5), 4149-4163 (2016).</li><li>Ranjan, 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=Microwave-irradiation-assisted+hybrid+chemical+approach+for+titanium+dioxide+nanoparticle+synthesis:+microbial+and+cytotoxicological+evaluation.">Microwave-irradiation-assisted hybrid chemical approach for titanium dioxide nanoparticle synthesis: microbial and cytotoxicological evaluation.</a> Environ. Sci. Pollut. Res. 23 (12), 12287-12302 (2016).</li><li>Deckman, H. W., Dunsmuir, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+lithography.">Natural lithography.</a> Appl. Phys. Lett. 41 (4), 377-379 (1982).</li><li>Dalby, M. J., Riehle, M. O., Johnstone, H., Affrossman, S., Curtis, A. S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Reaction+of+Endothelial+Cells+to+Polymer+Demixed+Nanotopography.">In vitro Reaction of Endothelial Cells to Polymer Demixed Nanotopography.</a> Biomaterials. 23 (14), 2945-2954 (2002).</li><li>Gates, B. D., 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=New+approaches+to+nanofabrication:+molding,+printing,+and+other+techniques.">New approaches to nanofabrication: molding, printing, and other techniques.</a> Chem. Rev. 105 (4), 1171-1196 (2005).</li><li>Yin, Y., Lu, Y., Gates, B., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Template-Assisted+Self-Assembly:+A+Practical+Route+to+Complex+Aggregates+of+Monodispersed+Colloids+with+Well-Defined+Sizes,+Shapes,+and+Structures.">Template-Assisted Self-Assembly: A Practical Route to Complex Aggregates of Monodispersed Colloids with Well-Defined Sizes, Shapes, and Structures.</a> J. Am. Chem. Soc. 123 (36), 8718-8729 (2001).</li><li>Tada, Y., 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=Directed+Self-Assembly+of+Diblock+Copolymer+Thin+Films+on+Chemically-Patterned+Substrates+for+Defect-Free+Nano-Patterning.">Directed Self-Assembly of Diblock Copolymer Thin Films on Chemically-Patterned Substrates for Defect-Free Nano-Patterning.</a> Macromolecules. 41 (23), 9267-9276 (2008).</li><li>Cheng, J. Y., Rettner, C. T., Sanders, D. P., Kim, H. C., Hinsberg, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dense+self-assembly+on+sparse+chemical+patterns:+rectifying+and+multiplying+lithographic+patterns+using+block+copolymers.">Dense self-assembly on sparse chemical patterns: rectifying and multiplying lithographic patterns using block copolymers.</a> Adv. Mater. 20 (16), 3155-3158 (2008).</li><li>Colburn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Step+and+flash+imprint+lithography:+a+new+approach+to+high-resolution+patterning.">Step and flash imprint lithography: a new approach to high-resolution patterning.</a> Proc. SPIE. 3676 ((Pt. 1, Emerging Lithographic Technologies III)), 379-389 (1999).</li><li>Ahn, S. H., Guo, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+roll-to-roll+nanoimprint+lithography+on+flexible+plastic+substrates.">High-speed roll-to-roll nanoimprint lithography on flexible plastic substrates.</a> Adv. Mater. 20 (11), 2044-2049 (2008).</li><li>Vieu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+beam+lithography:+resolution+limits+and+applications.">Electron beam lithography: resolution limits and applications.</a> Appl. Surf. Sci. 164, 111-117 (2000).</li><li>Nagase, T., Gamo, K., Kubota, T., Mashiko, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+fabrication+of+nano-gap+electrodes+by+focused+ion+beam+etching.">Direct fabrication of nano-gap electrodes by focused ion beam etching.</a> Thin Solid Films. 499 (1-2), 279-284 (2006).</li><li>Juodkazis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+lithography+of+nanorods+in+SU-8+photoresist.">Two-photon lithography of nanorods in SU-8 photoresist.</a> Nanotechnology. 16 (6), 846 (2005).</li><li>Yang, Y., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+surfacing+for+regenerative+medicine.">Nanoscale surfacing for regenerative medicine.</a> Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2 (5), 478-495 (2010).</li><li>Yang, Y., Kulangara, K., Sia, J., Wang, L., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+a+Microfluidic+Cell+Culture+Platform+Embedded+with+Nanoscale+Features.">Engineering of a Microfluidic Cell Culture Platform Embedded with Nanoscale Features.</a> Lab Chip. 11 (9), 1638-1646 (2011).</li><li>Wang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotopographical+modulation+of+cell+function+through+nuclear+deformation.">Nanotopographical modulation of cell function through nuclear deformation.</a> Acs Appl. Mater. Inter. 8 (8), 5082-5092 (2016).</li><li>Lee, J. N., Park, C., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+Compatibility+of+Poly(dimethylsiloxane)-Based+Microfluidic+Devices.">Solvent Compatibility of Poly(dimethylsiloxane)-Based Microfluidic Devices.</a> Anal. Chem. 75 (23), 6544-6554 (2003).</li><li>Yang, Y., Kulangara, K., Lam, R. T. S., Dharmawan, R., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Topographical+and+Mechanical+Property+Alterations+Induced+by+Oxygen+Plasma+Modification+on+Stem+Cell+Behavior.">Effects of Topographical and Mechanical Property Alterations Induced by Oxygen Plasma Modification on Stem Cell Behavior.</a> ACS Nano. 6 (10), 8591-8598 (2012).</li><li>Hua, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+Imprint+Lithography+with+Molecular-Scale+Resolution.">Polymer Imprint Lithography with Molecular-Scale Resolution.</a> Nano Lett. 4 (12), 2467-2471 (2004).</li><li>Delamarche, E., Schmid, H., Michel, B., Biebuyck, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+molded+polydimethylsiloxane+microstructures.">Stability of molded polydimethylsiloxane microstructures.</a> Adv. Mater. 9 (9), 741-746 (1997).</li></ol>

The authors have nothing to disclose.

Se presenta un método simple, asequible y versátil para generar una gran superficie de sustrato nanoestructurada. Para ampliar fielmente nanopatrones altamente definidos, con gran atención debe prestarse a varios pasos críticos. La primera es para recortar los múltiples moldes de PDMS. zonas sin patrón de los moldes de PDMS necesitan ser removidas. Además, las paredes laterales de los moldes se deben cortar verticalmente tan perfecta como sea posible para minimizar los huecos entre los moldes. En conjunto, la parte de las zonas sin patrón en el molde puntada final puede ser reducido. En segundo lugar, la superficie nanoestructurada de estos moldes de PDMS necesita ser alineado sin ninguna distorsión en el sustrato de silicio. Debido a que las nanoestructuras de PDMS son propensos a deformarse, es crítico para colocar las superficies nanoestructurada contra un lado espejo del substrato de silicio suavemente y de manera uniforme (evitar que quede aire atrapado entre el molde de PDMS y la superficie de silicio). Los moldes de PDMS se alinearán tan cerca como sea posible, pero sin tocar el neighboring moldes para minimizar aún más la porción del molde sin patrón de la puntada final. De lo contrario, las nanoestructuras tocados se deforman durante nanoimpresión. En tercer lugar, el grosor de los moldes de PDMS puede variar de un lote a otro, y por lo tanto es crítico para hacer el espesor uniforme, además de hacer el espesor de cada uniforme molde por la nivelación de la pequeña molde Si perfectamente antes de PDMS de colada. Aunque la variación de espesor a través de los moldes de PDMS puede ser compensada mediante el ajuste del espesor de la prepolímero (adhesivo) PDMS capa colada en un portaobjetos de vidrio, una capa de prepolímero de espesor podría ser problemático. El prepolímero se puede tirar a través de los intersticios entre los moldes de PDMS a la superficie modelada por la fuerza capilar, y por consiguiente dañar los nanopatrones. La variación de espesor puede ser minimizado mediante la preparación de la misma cantidad de mezcla de PDMS cuando la fundición del molde EBL. Como resultado, una delgada capa de PDMS prepolímero se puede utilizar. Alternativamente, el curado parcial de la prepolymecapa r aumentará su viscosidad, y por lo tanto reducir su aumento y finalmente eliminar el posible daño de las superficies nanoestructurada. La técnica de la puntada está limitada por la naturaleza elastomérica del PDMS. Aunque la litografía blanda se ha aplicado a replicar tamaños de característica tan pequeño como 2 nm 32 y, en principio, puede alcanzar una resolución de menos de 0,5 nm 18, las características de PDMS nanoescala no se pueden replicar sin problemas cuando la relación de aspecto de altura a anchura es demasiado alta (&gt; 2) o muy bajo (&lt;0,2). Los nanofeatures pueden derrumbarse cuando la relación de aspecto es demasiado alto, o resultar en un alivio insuficiente cuando se utiliza el sello de PDMS de un &lt;ratio de 0,2 33. Además, múltiples PDMS moldes no pueden ser cosidas sin problemas debido a los intersticios y el recorte incompleta de los moldes de PDMS, y por lo tanto hay zonas sin patrón y mal alineados (en particular para nanopatrones continuas como nanogratings). Dado el pequeño porcentaje de la Defectedárea encima de la superficie total, la técnica de la puntada todavía proporciona una forma sencilla y asequible para producir una amplia zona de sustratos nanoestructurada. Además, cuando el molde está cosido nanoimprinted en el sustrato de polímero, el polímero fundido podría fluir en el intersticio, lo que resulta en una superficie irregular (Figura 1a). La superficie irregular hace que sea difícil para recoger muestras para los análisis biológicos celulares o moleculares. En las aplicaciones de microfluidos, el aumento también provoca un sellado incompleto cuando microcanales están sellados contra el sustrato modelado. El problema de la superficie desigual fácilmente se puede resolver mediante la aplicación de polímero de la técnica de película delgada para reducir al mínimo el aumento a través de ajuste del espesor de la película (Figura 1b). Aunque la técnica de la puntada necesita un molde maestro definido para la expansión, es sencillo y asequible en comparación con otras técnicas como la litografía de avance y flash y una lithogra nanoimpresión de rollo a rolloPHY. La técnica requiere aumentar sólo platos calientes y una manera de ejercer fuerzas de compresión durante los procesos de costura y de nanoimpresión, pero no equipos costosos. Además, el proceso de la puntada puede llevarse a cabo en un entorno limpio, pero no necesariamente en una sala limpia. La técnica de la puntada también es versátil. Además de ampliar un nanopattern idéntica a una gran superficie, la técnica de la puntada se puede aplicar para los moldes que consisten en características micro y / o nanométrica de diversas formas, dimensiones y disposiciones. En este sentido, una biblioteca combinatoria de micro / nanotopographies puede ser construido para proporcionar una plataforma de alto rendimiento para investigar las interacciones célula-topografía. Esta técnica de puntada sencilla, asequible y versátil potencialmente puede extenderse para crear dispositivos micro / nanoescala con componentes híbridos.

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> La técnica de la puntada puede generar una gran superficie de sustratos nanoestructurada con alta fidelidad. La Figura 1a y 1b mostrar la gran área de nanopatrones transferidas desde el molde PDMS cosido a la placa de PS y PS película delgada sobre un sustrato de Si, respectivamente. La comparación entre el original molde escrito-EBL (Figura 1c) y el PDMS finales de trabajo de sustrato (Figura 1d) confirma que los nanopatrones escritas-EBL pueden transferirse fielmente al sustrato de trabajo. Nanotopografía de diversos geometría y dimensiones se puede utilizar para modular el comportamiento celular. Como se demuestra en la Figura 2 con el A549, una línea de células epiteliales basales adenocarcinomic como células modelo, los nanogratings anisotrópicas puede alargar las células a lo largo de la dirección nanograting en comparación con la morfología multipolar que las células A549 se muestran en las nanopilares isotrópicas. nt &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="Figura 1" src="/files/ftp_upload/54840/54840fig1.jpg" /> Figura 1. Generación de una gran superficie de sustratos nanoestructurada utilizando la técnica de la puntada. (A, b) las imágenes ópticas de los nanopatrones transferidos a la placa PS y PS película delgada, respectivamente. Las flechas indican el aumento de polímero en los intersticios de los moldes de PDMS cosidas. (C, d) Imágenes de SEM de las nanopatrones en molde EBL y PDMS finales de sustrato de trabajo, respectivamente. Las barras de escala son de 1 m. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54840/54840fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/54840/54840fig2.jpg" /> Figura 2. Nanotopographical modula la propagación de células de las células A549. </strong&gt; A549 células cultivadas en (a) nanogratings de 500 nm de ancho de línea, de 500 nm de separación y 560 nm de altura y (b) nanopilares de 500 nm de diámetro, 450 nm en el espaciado de borde a borde y 560 nm de altura , respectivamente. Las barras de escala son de 10 micras. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54840/54840fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

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

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

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

La expansión de nanoestructurada Los sustratos usando la técnica de la puntada para Nanotopographical modulación del comportamiento de la célula

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

6.5K vistas.  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.

Video: 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 las células madre pluripotentes para el sondeo Organización Espacial de la diferenciación Parcas

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

Investigación

Biología del Desarrollo

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 Fuerza Litografía Cardiaca de Ingeniería de Tejidos

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

Bioingeniería

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.

Microfabricación de los patrones de oro Nanoporous de Estudios de interacción célula-materiales

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.

Patrones celular en fotolitográficamente Definido Parylene-C: SiO<sub&gt; 2</sub&gt; Sustratos

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.

El estiramiento células micropatterned en una membrana de PDMS

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.

Micropatrón simple de células únicas sin litografía con plantillas de corte láser

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.

Un método versátil de Patterning proteínas y 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.

Fabricación de Nanopattern gradiente térmico Nanoimprinting técnica y proyección de la respuesta de células formadoras de colonias endoteliales 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.

Control de la geometría celular a través de micropatterning asistido por láser infrarrojo

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.

Generación de patrones para microscopía de tracción de micropatrones

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