Name: Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior (Video) | JoVE Journal
Uploaded: 2016-12-08T14:00:00.000+00:00
Duration: 9 min 6 s
Description: Learn to produce large-area nanopatterned substrates using a simple stitch technique with PDMS molds. This cost-effective method enables nanotopographical studies of cell phenotype and focal adhesion protein expression.

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5 minutes

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. Replication of PDMS Molds from an EBL Mold
<ol>
	<li>Fabricate silicon mold29
	<ol>
		<li>Spin coat 200 &#956;l polymethyl methacrylate (PMMA) solution on a 1&#160;&#215; 1 cm silicon (Si) substrate at 2,500 rpm for 1 min to form a thin film.</li>
		<li>Bake the PMMA film on the Si substrate at 180 &#176;C for 2 min.</li>
		<li>Write the designed nanopattern on the PMMA film by using a focused electron beam at an area dose of 300 &#181;C/cm2.</li>
		<li>Develop the PMMA nanopattern in developer for 80 sec.</li>
		<li>Deposit the PMMA nanopattern with a nickel layer of 50 nm in thickness using an E-beam evaporator at an output voltage of 10 kV, emission current of 0.5 mA and a deposition rate of 0.5 &#197;/sec.</li>
		<li>Lift off the PMMA part in 20 ml remover at 80 &#176;C for 20 min.</li>
		<li>Reactive ion etch (RIE) the nanopattern into the Si substrate to get a Si mold of the desired depth. 
		&#8203;NOTE: Gas mixture of tetrafluoromethane (CF4)/oxygen (O2) (90%/10%) at an inductively coupled plasma (ICP) power of 400 W and RIE power of 150 W is used to etch Si substrate to a depth of 560 nm.</li>
	</ol>
	</li>
	<li>Silanize Si mold
	<ol>
		<li>Put a glass coverslip and the Si mold in a 100 mm PS Petri dish and transfer them in a glass desiccator located in a fume hood.</li>
		<li>Drop 10 &#181;l 1H,1H,2H,2H-Perfluorooctyltrichlorosilane on the coverslip. 
		&#8203;Caution: 1H,1H,2H,2H-Perfluorooctyltrichlorosilane may cause skin corrosion and serious eye damage. Wear appropriate personal protective equipment (PPE).</li>
		<li>Cover the Petri dish partially.</li>
		<li>Keep the desiccator under vacuum for 5 hr in a fume hood to complete the silanization of the Si mold.</li>
	</ol>
	</li>
	<li>Prepare PDMS prepolymer
	<ol>
		<li>Weigh 10 g PDMS resin and 1.05 g curing agent in a disposable weighing boat.</li>
		<li>Mix the PDMS prepolymer thoroughly by using a plastic spoon.</li>
		<li>Degas the PDMS prepolymer in a plastic desiccator under vacuum for about 20 min till a clear mixture is observed.</li>
	</ol>
	</li>
	<li>Replicate PDMS molds
	<ol>
		<li>Put the silanized Si mold in a 60 mm Petri dish.</li>
		<li>Pour the PDMS prepolymer on the Si mold in the Petri dish.</li>
		<li>Place the Petri dish in a plastic desiccator and degas for about 10 min till all bubbles disappear.</li>
		<li>Transfer the Petri dish to a hotplate and cure the PDMS prepolymer at 70 &#176;C for 4 hr.</li>
		<li>Peel off the PDMS mold from the Si mold carefully by using tweezers. 
		&#8203;NOTE: PDMS molds can be stored at ambient conditions for up to one week. After curing, there are some uncrosslinked PDMS resin molecules and residual curing agent in the PDMS molds30. The low molecular weight molecules will gradually diffuse out and accumulate at the surface over time. This affects the topographical and mechanical properties of the PDMS surface31. The diffusion is not significant within one week.</li>
	</ol>
	</li>
</ol>
2. Stitching of PDMS Molds into a Large, Single Mold
<ol>
	<li>Prepare multiple PDMS molds by repeating Step 1.4. 
	NOTE: Weigh same amount of PDMS mixture each time to obtain PDMS molds of same thickness.</li>
	<li>Determine the orientation of anisotropic PDMS nanopatterns such as nanogratings under an optical microscope and mark it on the backside of the PDMS molds with a marker pen. 
	NOTE: It is not necessary to mark the orientation of isotropic nanotopography such as nanopillars.</li>
	<li>Clean a Si substrate with ethanol in a fume hood and dry it with compressed air.</li>
	<li>Trim off the unpatterned areas of each PDMS mold with a blade. 
	&#8203;NOTE: For the PDMS molds that will be placed at the periphery of the stitched mold, only the unpatterned areas in contact with others should be trimmed off.</li>
	<li>Place the trimmed PDMS mold with the nanopattern face-down on the mirror side of the Si substrate and then align other molds close to but not touching the surrounding mold(s).</li>
	<li>Prepare a PDMS adhesive layer
	<ol>
		<li>Cast 1 g degassed PDMS prepolymer (PDMS resin and curing agent ratio: 10:1.05) on a clean glass slide (7.5 cm&#160;&#215; 2.5 cm) to form a 0.5 mm thick layer.</li>
		<li>Bake the PDMS layer at 100 &#176;C on a hotplate for 3-5 min. Use a needle to touch the layer and ensure that the layer is partially but not completely cured. 
		NOTE: Partially cured PDMS cannot flow like uncured PDMS prepolymer, but it is sticky compared with cured PDMS.</li>
	</ol>
	</li>
	<li>Place the PDMS layer on the backside of aligned PDMS molds and quickly invert this assembly and transfer it to the hotplate.</li>
	<li>Apply a compressive force (5 kPa) using a metal block on the top of assembly to ensure a good contact between the PDMS adhesive layer and the backside of PDMS molds, and cure the PDMS adhesive layer at 100 &#176;C for 1 hr. 
	NOTE: Carefully adjust the position of the metal block to avoid the tilt of the assembly.</li>
	<li>Remove the metal block and peel off the single, stitched PDMS mold from the Si substrate.</li>
</ol>
3. Generation of a Master Mold on PS Substrates
Note: The stitched PDMS mold immobilized on a glass slide can be used to generate a master mold on a PS plate or a PS thin film, from which working nanopatterned substrates can be produced.
<ol>
	<li>Generate a master mold on a PS plate
	<ol>
		<li>Prepare a PS plate
		<ol>
			<li>Dry the PS pellets in a vacuum oven at 80 &#176;C for two days.</li>
			<li>Preheat a press machine at 230 &#176;C.</li>
			<li>Assemble an aluminum plate, a polytetrafluoroethylene (PTFE) sheet and the aluminum spacer in an order from bottom to top.</li>
			<li>Load 3.5 g PS pellets in the aluminum spacer with a square opening of 3 cm (L) &#215; 3 cm (W) &#215; 0.3 cm (H). 
			&#8203;NOTE: The spacer is approximately 0.1 cm thicker than the PDMS molds, and thus the final nanopatterned PS substrate is about 0.1 cm thick.</li>
			<li>Place another PTFE sheet and then another aluminum plate on the aluminum spacer.</li>
			<li>Place the assembly in the press machine.</li>
			<li>Preheat the PS pellets at 230 &#176;C for 30 min.</li>
			<li>Apply a compressive pressure (0.1 MPa) on the assembly for 5 min.</li>
			<li>Release the pressure and then reapply a compressive pressure of 0.5 MPa on the assembly.</li>
			<li>Repeat Step 3.1.1.9 with a pressure increase of 0.5 MPa till the desirable pressure of 1.5 MPa is reached.</li>
			<li>Turn off the heater of the press machine and cool it down below 70 &#176;C at a constant pressure of 1.5 MPa.</li>
			<li>Take the assembly out and store the PS plate in a vacuum oven at 80 &#176;C to prevent moisture from re-entering the PS plate.</li>
		</ol>
		</li>
		<li>Nanoimprint the stitched PDMS mold into PS plate
		<ol>
			<li>Place the PS plate in an aluminum spacer set on a 3-inch Si wafer. 
			&#8203;NOTE: The inner dimensions of the spacer are the same as those of the PS plate so the PS plate fits right in the spacer.</li>
			<li>Heat the PS plate on a hotplate at 250 &#176;C for 30 min.</li>
			<li>Put the stitched PDMS mold with nanopatterns face-down on the molten PS plate. 
			NOTE: One side of the PDMS mold is put in touch with the surface of the PS plate first and another side is lowered gradually in contact with the PS surface to avoid the formation of air bubbles at the interface. 
			&#8203;Caution: The surface of the hotplate is hot. Wear thermogloves during nanoimprinting process.</li>
			<li>Place an aluminum plate on the glass slide of the stitched PDMS mold.</li>
			<li>Apply a compressive pressure (12.5 kPa) by using metal blocks on the aluminum plate and wait for 3 min. 
			&#8203;NOTE: Make sure that the aluminum plate is not tilted.</li>
			<li>Lift and replace the metal block from the aluminum plate and increase the compressive pressure to 25 kPa.</li>
			<li>Repeat Step 3.1.2.6 with the pressure increased to 50 kPa. 
			&#8203;NOTE: This step is to remove the air trapped between the PDMS mold and PS plate.</li>
			<li>Maintain the temperature of the hotplate between 240 and 250 &#176;C under the consistent pressure of 50 kPa for 15 min.</li>
			<li>Turn off the hotplate and cool down the whole setup. 
			&#8203;NOTE: A fan can be used to expedite the cooling process.</li>
			<li>Remove the metal blocks after the temperature is below 50 &#176;C, and carefully peel off the stitched PDMS mold from the PS plate. 
			&#8203;NOTE: The PS substrate has the reverse nanopatterns and can be used as a master mold to produce working PDMS substrates.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Generate a master mold on a PS thin film
	<ol>
		<li>Prepare a PS thin film
		<ol>
			<li>Dissolve 1 g PS in 10 ml toluene in a fume hood. 
			&#8203;Caution: Toluene can cause skin irritation and serious eye damage, and may cause damage to organs through prolonged or repeated exposure. Wear appropriate PPE.</li>
			<li>Spin-coat 1 ml PS solution on a 2-in wafer at 2,500 rpm for 1 min to form ~1 &#956;m thick PS thin film.</li>
			<li>Evaporate toluene from the film by setting the PS film on Si wafer in a fume hood for 3 days.</li>
			<li>Anneal the PS thin film in a vacuum oven at 80 &#176;C overnight.</li>
		</ol>
		</li>
		<li>Nanoimprint the PDMS mold on a PS thin film
		<ol>
			<li>Put the stitched PDMS mold with nanotopography face-down on the PS thin film, which is set on a hotplate.</li>
			<li>Apply a compressive pressure of 12 kPa on the PDMS mold by using metal blocks on the glass side of the PDMS mold.</li>
			<li>Increase the temperature of the hotplate to 180 &#176;C and maintain it for 15 min. 
			&#8203;Caution: The molten PS film can function as a lubricant. Pay attention to prevent the metal blocks from sliding off.</li>
			<li>Turn off the hotplate and cool down the whole setup. 
			&#8203;NOTE: A fan can be used to expedite the cooling process.</li>
			<li>Remove the metal blocks after the temperature drops below 50 &#176;C, and carefully peel off the stitched PDMS mold from the PS film. 
			&#8203;NOTE: The nanopatterned PS film will serve as a master mold to produce working PDMS substrates.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Nanotopographical Modulation of Cell Behavior
Note: Human epithelial cells are cultured on the representative nanotopographies to demonstrate nanotopographical modulation of cell spreading.
<ol>
	<li>Cast PDMS working substrates from the master mold generated from either Step 3.1 or 3.2 depending on the application.</li>
	<li>Using a hollow steel arch punch, cut the nanopatterned PDMS substrates into discs to fit the configuration of a specific multi-well plate (e.g., 24-well plate).</li>
	<li>Use tweezers to place the PDMS discs into the wells of a multi-well plate.</li>
	<li>Sterilize the PDMS substrates by using 70% ethanol and then UV exposure, each for 30 min.</li>
	<li>Wash the PDMS substrates with 1x sterile phosphate buffered saline (PBS) three times.</li>
	<li>Coat the PDMS substrates with extracellular matrix protein (i.e., 20 &#181;g/ml fibronectin) for 30 min at room temperature.</li>
	<li>Rinse the PDMS substrates three times with sterile PBS, each for 5 min.</li>
	<li>Suspend human lung cancer cell A549 in Dulbecco&#39;s modified eagle medium with 10% fetal bovine serum and count the cells using a hemocytometer.</li>
	<li>Plate the cells at a seeding density of 2,000 cells/cm2 on the PDMS substrates and culture them at 37 &#176;C in a humidified atmosphere containing 5% CO2 for one day.</li>
	<li>Wash the cells with PBS three times.</li>
	<li>Fix the cells in a mixture of 4% paraformaldehyde and 2% glutaraldehyde in PBS for 4 hr and dehydrate cells using a CO2 critical point dryer for scanning electronic microscopic observation29. 
	Caution: Paraformaldehyde and glutaraldehyde may cause severe skin burns and eye damage. Operate in a chemical hood and wear appropriate PPE.</li>
</ol>

<ol><li>Silva, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+differentiation+of+neural+progenitor+cells+by+high-epitope+density+nanofibers%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Synthetic+Nanostructures+Inducing+Differentiation+of+Human+Mesenchymal+Stem+Cells+into+Neuronal+Lineage%5BTitle%5D)+AND+%22Exp.+Cell+Res%22%5BJournal%5D)">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/pubmed?term=((The+Control+of+Human+Mesenchymal+Cell+Differentiation+Using+Nanoscale+Symmetry+and+Disorder%5BTitle%5D)+AND+%22Nat.+Mater%22%5BJournal%5D)">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/pubmed?term=((Stem+Cell+Fate+Dictated+Solely+by+Altered+Nanotube+Dimension%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">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/pubmed?term=((Neurons+Sense+Nanoscale+Roughness+with+Nanometer+Sensitivity%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">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/pubmed?term=((Nanoscale+Surfaces+for+the+Long-term+Maintenance+of+Mesenchymal+Stem+Cell+Phenotype+and+Multipotency%5BTitle%5D)+AND+%22Nat.+Mater%22%5BJournal%5D)">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/pubmed?term=((Nanopattern-induced+changes+in+morphology+and+motility+of+smooth+muscle+cells%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((The+Effect+of+Actin+Disrupting+Agents+on+Contact+Guidance+of+Human+Embryonic+Stem+Cells%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Enhancement+of+in+vitro+Capillary+Tube+Formation+by+Substrate+Nanotopography%5BTitle%5D)+AND+%22Adv.+Mater%22%5BJournal%5D)">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/pubmed?term=((Regulation+of+vascular+smooth+muscle+cells+by+micropatterning%5BTitle%5D)+AND+%22Biochem.+Biophys.+Res.+Commun%22%5BJournal%5D)">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/pubmed?term=((Direct+differentiation+of+human+embryonic+stem+cells+into+selective+neurons+on+nanoscale+ridge%2Fgroove+pattern+arrays%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Microarray+with+micro-+and+nano-topographies+enables+identification+of+the+optimal+topography+for+directing+the+differentiation+of+primary+murine+neural+progenitor+cells%5BTitle%5D)+AND+%22Small%22%5BJournal%5D)">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/pubmed?term=((Myogenic+induction+of+aligned+mesenchymal+stem+cell+sheets+by+culture+on+thermally+responsive+electrospun+nanofibers%5BTitle%5D)+AND+%22Adv.+Mater%22%5BJournal%5D)">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/pubmed?term=((Thermal+co-reduction+approach+to+vary+size+of+silver+nanoparticle%3A+its+microbial+and+cellular+toxicology%5BTitle%5D)+AND+%22Environ.+Sci.+Pollut.+Res%22%5BJournal%5D)">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/pubmed?term=((Microwave-irradiation-assisted+hybrid+chemical+approach+for+titanium+dioxide+nanoparticle+synthesis%3A+microbial+and+cytotoxicological+evaluation%5BTitle%5D)+AND+%22Environ.+Sci.+Pollut.+Res%22%5BJournal%5D)">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/pubmed?term=((Natural+lithography%5BTitle%5D)+AND+%22Appl.+Phys.+Lett%22%5BJournal%5D)">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/pubmed?term=((In+vitro+Reaction+of+Endothelial+Cells+to+Polymer+Demixed+Nanotopography%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((New+approaches+to+nanofabrication%3A+molding%2C+printing%2C+and+other+techniques%5BTitle%5D)+AND+%22Chem.+Rev%22%5BJournal%5D)">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/pubmed?term=((Template-Assisted+Self-Assembly%3A+A+Practical+Route+to+Complex+Aggregates+of+Monodispersed+Colloids+with+Well-Defined+Sizes%2C+Shapes%2C+and+Structures%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">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/pubmed?term=((Directed+Self-Assembly+of+Diblock+Copolymer+Thin+Films+on+Chemically-Patterned+Substrates+for+Defect-Free+Nano-Patterning%5BTitle%5D)+AND+%22Macromolecules%22%5BJournal%5D)">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/pubmed?term=((Dense+self-assembly+on+sparse+chemical+patterns%3A+rectifying+and+multiplying+lithographic+patterns+using+block+copolymers%5BTitle%5D)+AND+%22Adv.+Mater%22%5BJournal%5D)">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/pubmed?term=((Step+and+flash+imprint+lithography%3A+a+new+approach+to+high-resolution+patterning%5BTitle%5D)+AND+%22Proc.+SPIE%22%5BJournal%5D)">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/pubmed?term=((High-speed+roll-to-roll+nanoimprint+lithography+on+flexible+plastic+substrates%5BTitle%5D)+AND+%22Adv.+Mater%22%5BJournal%5D)">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/pubmed?term=((Electron+beam+lithography%3A+resolution+limits+and+applications%5BTitle%5D)+AND+%22Appl.+Surf.+Sci%22%5BJournal%5D)">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/pubmed?term=((Direct+fabrication+of+nano-gap+electrodes+by+focused+ion+beam+etching%5BTitle%5D)+AND+%22Thin+Solid+Films%22%5BJournal%5D)">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/pubmed?term=((Two-photon+lithography+of+nanorods+in+SU-8+photoresist%5BTitle%5D)+AND+%22Nanotechnology%22%5BJournal%5D)">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/pubmed?term=((Nanoscale+surfacing+for+regenerative+medicine%5BTitle%5D)+AND+%22Wiley+Interdiscip.+Rev.+Nanomed.+Nanobiotechnol%22%5BJournal%5D)">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/pubmed?term=((Engineering+of+a+Microfluidic+Cell+Culture+Platform+Embedded+with+Nanoscale+Features%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">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/pubmed?term=((Nanotopographical+modulation+of+cell+function+through+nuclear+deformation%5BTitle%5D)+AND+%22Acs+Appl.+Mater.+Inter%22%5BJournal%5D)">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/pubmed?term=((Solvent+Compatibility+of+Poly%28dimethylsiloxane%29-Based+Microfluidic+Devices%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">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/pubmed?term=((Effects+of+Topographical+and+Mechanical+Property+Alterations+Induced+by+Oxygen+Plasma+Modification+on+Stem+Cell+Behavior%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">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/pubmed?term=((Polymer+Imprint+Lithography+with+Molecular-Scale+Resolution%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">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/pubmed?term=((Stability+of+molded+polydimethylsiloxane+microstructures%5BTitle%5D)+AND+%22Adv.+Mater%22%5BJournal%5D)">Stability of molded polydimethylsiloxane microstructures.</a> Adv. Mater. 9 (9), 741-746 (1997).</li>
</ol>

The authors have nothing to disclose.

We present a simple, affordable, yet versatile method to generate a large area of nanopatterned substrate. To faithfully expand highly defined nanopatterns, great attention should be paid to several critical steps. The first one is to trim the multiple PDMS molds. Unpatterned areas of the PDMS molds need to be removed. Additionally, the sidewalls of the molds should be cut vertically as perfect as possible to minimize the gaps between the molds. Collectively, the portion of unpatterned areas in the final stitch mold can ...

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

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.

The stitch technique can generate a large area of nanopatterned substrates with high fidelity. Figure 1a and 1b display the large area of nanopatterns transferred from the stitched PDMS mold to PS plate and PS thin film on a Si substrate, respectively. The comparison between the original EBL-written mold (Figure 1c) and the final PDMS working substrate (Figure 1d) confirms that the EBL-written nanopatterns can be faithful...

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

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

The overall goal of this procedure is to produce a large area of nanopatterned substrate from small nanopatterned molds by using a simple and affordable yet versatile stitch technique. This method can help study substrate nanotopography modulation of cell phenotype and function at cellular and molecular levels, such as expression of focal adhesion proteins. The main advantage of this technique is that it is simple and cost effective.To begin, prepare the silanized silicon mold in the PDMS prepolymer, as described in the text protocol. Put the silanized silicon mold in a 60 millimeter Petri dish. Pour the PDMS prepolymer on the silicon mold in the Petri dish.Place the Petri dish in a plastic desiccator and degas for about 10 minutes, until all bubbles disappear. Transfer the Petri dish to a hot plate and cure the PDMS prepolymer at 70 degrees Celsius for four hours. Carefully peel off the PDMS mold from the silicon mold by using tweezers.Determine the orientation of anisotropic PDMS nanopatterns, such as nanogratings, under an optical microscope, and mark it on the backside of the PDMS molds with a marker pen. Clean the silicon substrate with ethanol in a fume hood, and then dry it with compressed air. Trim off the unpatterned areas of each PDMS mold with a blade.The PDMS molds should be aligned as close as possible, but not touching the surrounding mold, to minimize the unpatterned area and to avoid touching to the nanopattern deformation when the compressor force is applied. Place the trimmed PDMS mold with the nanopattern face-down on the mirror side of the silicon substrate, and then align other molds close to but not touching the surrounding molds. It is critical to try to cure the PDMS at the basal layer because uncured PDMS may flow through the gap between the multiple molds and damage the nanopatterns.However, the completely cured PDMS cannot bond the multiple molds together. To prepare a PDMS adhesive layer, cast one gram of degassed PDMS prepolymer on a clean glass slide to form a 0.5 millimeter thick layer. Bake the PDMS layer at 100 degrees Celsius on a hot plate for three to five minutes.Use a needle to touch the layer and ensure that the layer is partially but not completely cured. Place the PDMS layer on the backside of the aligned PDMS molds, quickly invert this assembly, and transfer it to the hot plate. Apply a compressive force using a metal block on top of the assembly to ensure good contact between the PDMS adhesive layer and the backside of the PDMS molds.Cure the PDMS adhesive layer at 100 degrees Celsius for one hour. Turn off the hot plate, remove the metal block, and peel off the single-stiched PDMS mold from the silicon substrate. To nanoimprint the stitched PDMS mold into the PS plate, place the PS plate in an aluminum spacer set on a three inch silicon wafer.Heat the PS plate on a hot plate at 250 degrees Celsius for 30 minutes. Then, place the stitched PDMS mold, with nanopatterns face-down, on the molten PS plate. Next, place an aluminum plate on the glass slide of the stitched PDMS mold.Apply a compressive pressure by using metal blocks on the aluminum plate, and wait for three minutes. Lift and replace the metal block from the aluminum plate, and increase the compressive pressure to 25 kilopascals. Then, repeat this step with the pressure increased to 50 kilopascals.Maintain the temperature of the hot plate at between 240 and 250 degrees Celsius under consistent pressure of 50 kilopascals for 15 minutes. Turn off the hot plate, and cool down the whole setup. Remove the metal blocks after the temperature is below 50 degrees Celsius, and carefully peel off the stitched PDMS mold from the PS plate.To nanoimprint the PDMS mold on a PS thin film, place the stitched PDMS mold with nanotopography face-down on the PS thin film, which is set on a hot plate. Apply compressive pressure of 12 kilopascals on the PDMS mold by using metal blocks on the glass slide of the PDMS mold. Increase the temperature of the hot plate to 180 degrees Celsius, and maintain it for 15 minutes.Turn off the hot plate, and cool down the whole setup. Remove the metal blocks after the temperature drops below 50 degrees Celsius, and carefully peel off the stitched PDMS mold from the PS film. Using a hollow steel arch punch, cut the nanopatterned PDMS substrates into discs to fit the configuration of a specific multi-well plate.Then, use tweezers to place the PDMS discs into the wells of the multi-well plate. Sterilize the PDMS substrates by using 70%ethanol for 30 minutes. Afterwards, aspirate the ethanol, and follow this by UV exposure for 30 minutes.Wash the PDMS substrates with 1X sterile phosphate-buffered saline three times. Next, coat the PDMS substrates with extracellular matrix protein for 30 minutes at room temperature. Rinse the PDMS substrates three times with sterile PBS, each for five minutes.Suspend human A549 lung cancer cells in Dulbecco's Modified Eagle Medium with 10%fetal bovine serum, and count the cells using a hemocytometer. Plate the cells at a seeding density of 2, 000 cells per square centimeter on the PDMS substrates. Culture the cells at 37 degrees Celsius in a humidified atmosphere containing 5%carbon dioxide for one day, and observe them afterwards.The generated nanopattern on the PS plate and the thin film are shown here. The arrows indicate the polymer raise in the interstices of the stitched PDMS molds. These SEM images demonstrate that PDMS-working substrates are faithfully transferred from the electron beam lithography-written nanopattern by applying the stitch technique.Representative SEM images of A549 cells grown on nanogratings and nanopillars are shown here. The cells show aligned cell morphology on the nanogratings, while the cells spread randomly on the nanopillars. After watching this video, you should have a good understanding of how to generate a large area of nanopatterned substrate by stitching multiple, small defined nanopatterned molds.Don't forget that working with toluene and paraformaldehyde can be extremely hazardous, and always wear appropriate personal protection equipment while performing the procedures.

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Research

JoVE Journal

Bioengineering

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement.  Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

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.

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

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.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Stretching Micropatterned Cells on a PDMS Membrane

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

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

A Versatile Method of Patterning Proteins and Cells

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.

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

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.

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

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 of Cell Geometry through Infrared Laser Assisted Micropatterning

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.

Pattern Generation for Micropattern Traction Microscopy

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

Learn to produce large-area nanopatterned substrates using a simple stitch technique with PDMS molds. This cost-effective method enables nanotopographical studies of cell phenotype and focal adhesion protein expression.

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

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