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>

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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.

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 be reduced. Secondly, the nanopatterned surface of these PDMS molds needs to be aligned without any distortion on the silicon substrate. Because the PDMS nanostructures are prone to deform, it is critical to place the nanopatterned surfaces against a mirror side of the silicon substrate gently and evenly (avoid trapping air between the PDMS mold and silicon surface). The PDMS molds will be aligned as close as possible to, but not touching the neighboring molds to further minimize the unpatterned portion of the final stitch mold. Otherwise, the touched nanostructures will deform during nanoimprinting. Thirdly, the thickness of the PDMS molds may vary from batch to batch, and thus it is critical to make the thickness uniform in addition to making the thickness of each mold uniform by leveling the small Si mold perfectly before PDMS casting. Although the variation in thickness across the PDMS molds can be compensated by adjusting the thickness of the PDMS prepolymer (adhesive) layer cast on a glass slide, a thick prepolymer layer could be problematic. The prepolymer may be pulled through the interstices between the PDMS molds to the patterned surface by the capillary force, and consequently damage the nanopatterns. The thickness variation can be minimized by preparing the same amount of PDMS mixture when casting from the EBL mold. As a result, a thin PDMS prepolymer layer can be used. Alternatively, partially curing the prepolymer layer will increase its viscosity, and thus reduce its raise and eventually eliminate the possible damage of nanopatterned surfaces.
The stitch technique is limited by elastomeric nature of PDMS. Although soft lithography has been applied to replicate feature sizes as small as 2 nm32 and, in principle, can reach a resolution of less than 0.5 nm18, the nanoscale PDMS features cannot be replicated flawlessly when the aspect ratio of height to width is too high (&#62; 2) or too low (&#60; 0.2). The nanofeatures may collapse when the aspect ratio is too high, or result in insufficient relief when the PDMS stamp of a &#60; 0.2 ratio is used33. Moreover, multiple PDMS molds cannot be stitched seamlessly because of the interstices and incomplete trimming of the PDMS molds, and thus there are unpatterned and misaligned areas (in particular for continuous nanopatterns such as nanogratings). Given the small percentage of the defected area over the total surface area, the stitch technique still provides a simple and affordable way to produce a large area of nanopatterned substrates. In addition, when the stitched mold is nanoimprinted into the polymer substrate, molten polymer might flow into the interstice, resulting in an uneven surface (Figure 1a). The uneven surface makes it challenging to collect samples for cellular or molecular biological analyses. In microfluidic applications, the raise also causes an incomplete sealing when microchannels are sealed against the patterned substrate. The uneven surface problem can readily be solved by applying polymer thin film technique to minimize the raise through tuning the film thickness (Figure 1b).
Although the stitch technique needs a defined master mold for the expansion, it is simple and affordable compared with other techniques such as step-and-flash lithography and a roll-to-roll nanoimprinting lithography. The stitch technique requires only hot plates and a way of exerting compressive forces during stitching and nanoimprinting processes, but not expensive equipment. Moreover, the stitch process can be conducted in a clean environment, but not necessarily in a cleanroom.
The stitch technique is also versatile. In addition to expanding an identical nanopattern to a large area, the stitch technique can be applied for the molds consisting of micro- and/or nanoscale features of various shapes, dimensions and arrangements. To this regard, a combinatory library of micro-/nanotopographies can be built to provide high-throughput platform for investigate cell-topography interactions. This simple, affordable and versatile stitch technique can potentially be extended to create micro-/nanoscale devices with hybrid components.

A number of recent findings reveal that substrate nanotopography has pronounced influence on cell behavior, from cell adhesion, spreading and migration, to proliferation and differentiation1-6. For instance, a smaller cell size and lower proliferation rate have been observed in cells cultured on deep nanogratings, even leading to apoptosis although the cell alignment, elongation and migration were enhanced, compared with the flat controls2,7-10. Moreover, nanotopography has been shown to facilitate the differentiation of stem cells into certain lineages such as neuron2,11,12, muscle13, and bone3,4. In addition, because of increasing concerns on the toxicity of engineered nanomaterials14,15, there is a need to incorporate nanotopography into physiologically relevant in vitro models for accurate risk assessment of nanomaterials. To fulfil the biochemical and molecular biology analyses, enough cells are needed to be grown on a large area of nanopatterned substrate. However, conventional nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area.
Self-assembly including colloid lithography16 and polymer demixing17 can readily generate large-area nanostructures at low costs. Because self-assembly relies on interactions between the assembling elements such as colloidal particles and macromolecules, and possible interactions between these elements and substrate, it cannot be a stand-alone method of producing nanostructures with precise spatial positioning and arbitrary shapes18. The accompanied high density of defects is also a drawback. Precise spatial control of nanopatterns can be achieved by employing templated self-assembly, which uses top-down lithographic approaches to provide the topographical and/or chemical template to guide the bottom-up assembly of the assembling elements19-21. Alternative nanofabrication techniques such as step-and-flash lithography22 and a roll-to-roll nanoimprinting lithography23 have been developed but have limited use because of their sophisticated process or the requirement of specialized equipment. Nevertheless, a template or a master mold with defined nanoscale patterns is needed for templated or alternative nanofabrication techniques. 
Such templates and master molds are conventionally generated by using focused electron, ion, or photon beam lithography. For instance, electron beam lithography (EBL)24 and focused ion beam lithography25 can generate defined patterns with a sub-5 nm resolution. Two-photon lithography has demonstrated a feature size as small as 30 nm26. Although the focused beam lithography techniques enable generation of well-defined nanoscale structures, the capital investment and the time-consuming, costly process restrict their widespread use in academic research27. Therefore, it is highly desirable to develop enabling yet affordable techniques to produce a large area of nanopatterned surfaces with high fidelity.
We have reported a simple, cost-effective stitch technique for generating a large area of nanopatterned surface from a small well-defined mold28. This protocol provides step-by-step procedure from replication of poly(dimethylsiloxane) (PDMS) molds using an EBL-written pattern, to assembly of multiple PDMS molds into a single large mold, to generation of a master mold on polymeric such as polystyrene (PS) substrates, to production of working substrates. With the expanded nanopatterned substrates, we demonstrated nanotopographical modulation of cell spreading.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>JEOL field emission SEM</td>
			<td>JEOL</td>
			<td>JSM-7600F</td>
			<td>EBL</td>
		</tr>
		<tr>
			<td>E-beam evaporator</td>
			<td>Kurt J. Lesker</td>
			<td>Model: LAB 18 e-beam evaporator</td>
			<td>nickel deposition</td>
		</tr>
		<tr>
			<td>Trion Minilock III ICP/RIE</td>
			<td>Trion technology</td>
			<td>Model: Minilock-phantom III</td>
			<td></td>
		</tr>
		<tr>
			<td>Press machine</td>
			<td>PHI Hydraulic Press</td>
			<td>Molde: SQ-230H</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Laurell Technologies</td>
			<td>Modle: WS-400A-6NPP-LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 critical dryer</td>
			<td>Tousimis</td>
			<td>Modle: Autosamdri-815</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>University Wafer</td>
			<td>1080</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum plates</td>
			<td>McMaster-carr</td>
			<td>9057K123</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon sheets</td>
			<td>McMaster-carr</td>
			<td>8711K92</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>FALCON</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm petri dish</td>
			<td>FALCON</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Fisher Scientific</td>
			<td>12-550-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable weighing boats</td>
			<td>Fisher Scientific</td>
			<td>13-735-743</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass desiccator</td>
			<td>Fisher Scientific</td>
			<td>02-913-360</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic desiccator</td>
			<td>Bel-Art Products</td>
			<td>F42025-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Fisher Scientific</td>
			<td>1110049SH</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Ted Pella, inc.</td>
			<td>5726</td>
			<td></td>
		</tr>
		<tr>
			<td>Blade</td>
			<td>Fisher Scientific</td>
			<td>S17302</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal blocks</td>
			<td>McMaster-carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Punch</td>
			<td>Brettuns Village Leather Craft Supplies</td>
			<td>Arch punch</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(methyl methacrylate)</td>
			<td>MicroChem</td>
			<td>495 PMMA A4</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning</td>
			<td>Sylgard 184 kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene</td>
			<td>Dow Chemical</td>
			<td>Styron 685D</td>
			<td></td>
		</tr>
		<tr>
			<td>1H,1H,2H,2H-perfluorooctylmethyldichlorosilane</td>
			<td>Oakwood Chemical</td>
			<td>7142</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>MicroChem</td>
			<td>MIBK/IPA at 1: 3 ratio</td>
			<td></td>
		</tr>
		<tr>
			<td>Remover</td>
			<td>MicroChem</td>
			<td>Remover PG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Fisher Scientific</td>
			<td>T324-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified eagle medium</td>
			<td>Sigma Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microsopy Science</td>
			<td>15712-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde&#160;</td>
			<td>Fisher Chemical</td>
			<td>G151-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>ATCC CCL-185</td>
			<td></td>
		</tr>
	</tbody>
</table>

expanding nanopatterned substrates using stitch technique for nanotopographical modulation of cell behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

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 faithfully transferred to the working substrate. Nanotopography of various geometry and dimensions can be used to modulate cell behavior. As demonstrated in Figure 2 with A549, an adenocarcinomic basal epithelial cell line as model cells, the anisotropic nanogratings can elongate the cells along the nanograting direction compared with the multipolar morphology that A549 cells display on the isotropic nanopillars.
<img alt="Figure 1" src="/files/ftp_upload/54840/54840fig1.jpg" /> 
Figure 1. Generation of a large area of nanopatterned substrates using stitch technique. (a,b) Optical images of the nanopatterns transferred to PS plate and PS thin film, respectively. The arrows indicate the polymer raise in the interstices of the stitched PDMS molds. (c,d) SEM images of the nanopatterns on EBL mold and final PDMS working substrate, respectively. The scale bars are 1 &#956;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54840/54840fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54840/54840fig2.jpg" /> 
Figure 2. Nanotopographical modulated cell spreading of A549 cells. A549 cells grown on (a) nanogratings of 500 nm in line width, 500 nm in spacing and 560 nm in height and (b) nanopillars of 500 nm in diameter, 450 nm in edge-to-edge spacing and 560 nm in height, respectively. The scale bars are 10 &#956;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54840/54840fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</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.

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 Views.  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

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

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

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

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.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

A Microfluidic Technique to Probe Cell Deformability

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

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

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

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

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

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

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