Name: creating two-dimensional patterned substrates for protein and cell confinement
Uploaded: 2011-09-06T13:00:00
Description: Watch this Scientific Journal Video about Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement at JoVE.com

We would like to acknowledge the entire Maurer group at Washington University whose collective knowledge has made this protocol possible. Funding for this work is provided by the National Institute of Mental Health (1R01MH085495).

1. Preparation of the Patterned Master (Figure 1)
<ol>
<li>Center the silicon wafer on the spin-coater and rinse the wafer with acetone during the initial step of the two-cycle spin program in Table 1. The acetone will evaporate during the second step of the spin program leaving a clean, dry wafer.</li>
<li>Apply approximately 1 mL AZ9245 photoresist/in (in diameter) to the wafer and spin-coat using the conditions described in Table 1.</li>
<li>Soft-bake the photoresist-coated wafer at 110&deg;C for 2 m using a high-u...

<ol>
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R., Kuck, V., Katz, H., Amundson, K., Ewing, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper-like+electronic+displays:+Large-area+rubber-stamped+plastic+sheets+of+electronics+and+microencapsulated+electrophoretic+inks.">Paper-like electronic displays: Large-area rubber-stamped plastic sheets of electronics and microencapsulated electrophoretic inks.</a> Proc Natl Acad Sci U S A. 98, 4835-4840 (2001).</li><li>Yanker, D., Maurer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+printing+of+trichlorosilanes+on+glass+for+selective+protein+adsorption+and+cell+growth.">Direct printing of trichlorosilanes on glass for selective protein adsorption and cell growth.</a> Molecular Biosystems. 4, 502-504 (2008).</li><li>Johnson, D., Maurer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recycling+and+reusing+patterned+self-assembled+monolayers+for+cell+culture.">Recycling and reusing patterned self-assembled monolayers for cell culture.</a> Chemical Communications. , 520-522 (2011).</li><li>Herne, T., Tarlov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+DNA+probes+immobilized+on+gold+surfaces.">Characterization of DNA probes immobilized on gold surfaces.</a> J. Am. Chem. Soc. , 8916-8920 (1997).</li><li>Hanson, E., Schwartz, J., Nickel, B., Koch, N., Danisman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bonding+self-assembled,+compact+organophosphonate+monolayers+to+the+native+oxide+surface+of+silicon.">Bonding self-assembled, compact organophosphonate monolayers to the native oxide surface of silicon.</a> J. Am. Chem. Soc. , 16074-16080 (2003).</li><li>Johannes, M., Cole, D., Clark, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscope+based+nanofabrication+of+master+pattern+molds+for+use+in+soft+lithography.">Atomic force microscope based nanofabrication of master pattern molds for use in soft lithography.</a> Applied Physics Letters. , (2007).</li><li>Bessueille, F., Pla-Roca, M., Mills, C. A., Martinez, E., Samitier, J., Errachid, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submerged+microcontact+printing+(S&#x03BC;CP):+An+unconventional+printing+technique+of+thiols+using+high+aspect+ratio,+elastomeric+stamps.">Submerged microcontact printing (S&#x03BC;CP): An unconventional printing technique of thiols using high aspect ratio, elastomeric stamps.</a> Langmuir. , 12060-12063 (2005).</li><li>Xia, Y., Whitesides, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extending+microcontact+printing+as+a+microlithographic+technique.">Extending microcontact printing as a microlithographic technique.</a> Langmuir. , 2059-2067 (1997).</li><li>Biasco, A., Pisignano, D., Krebs, B., Pompa, P. P., Persano, L., Cingolani, R., Rinaldi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformation+of+microcontact-printed+proteins+by+atomic+force+miroscopy+molecular+sizing.">Conformation of microcontact-printed proteins by atomic force miroscopy molecular sizing.</a> Langmuir. , 5154-5158 (2005).</li><li>Shen, K., Qi, J., Kam, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+printing+of+proteins+for+cell+biology.">Microcontact printing of proteins for cell biology.</a> J Vis Exp. (22), e1065-e1065 (2008).</li><li>Piner, R., Zhu, J., Xu, F., Hong, S., Mirkin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Dip-pen"+nanolithography.">"Dip-pen" nanolithography.</a> Science. 283, 661-663 (1999).</li><li>Ryan, D., Parviz, B. A., Linder, V., Semetey, V., Sia, S. K., Su, J., Mrksich, M., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+multiple+aligned+self-assembled+monolayers+using+light.">Patterning multiple aligned self-assembled monolayers using light.</a> Langmuir. , 9080-9088 (2004).</li></ol>

A number of issues can arise in the lithographic production of the master used for PDMS stamp creation. Underexposure of the resist-coated wafer results in hazy and indistinct patterns and overexposure of the resist-coated wafer results in enlarged or missing features. In general, masters with large feature sizes (&gt;10 &mu;m) are relatively easy to pattern and develop, while masters with smaller features can require extensive optimization of photopatterning and development parameters (beyond the parameters recommended ...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>Wafer Reclaim Services</td>
			<td>&nbsp;</td>
			<td>2 inch</td>
		</tr>
		<tr>
			<td>Spin coater/hot plate		</td>
			<td>Brewer Science</td>
			<td>Cee 200CB Spin-Bake System</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>AZ9245 Photoresist		</td>
			<td>Mays Chemical Company</td>
			<td>105880034-1160</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Direct-write photolithography system		</td>
			<td>Microtech s.r.l.</td>
			<td>LW325 LaserWriter System</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mask Aligner		</td>
			<td>HTG</td>
			<td>3HR</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>AZ 400K Developer		</td>
			<td>Mays Chemical Company</td>
			<td>105880018-1160</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sylgard 182 Silicone Elastomer Kit	</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>25 mm no. 1 round glass coverslips</td>
			<td>VWR</td>
			<td>16004-310</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plasma Oxidizer		</td>
			<td>Diener</td>
			<td>Femto</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Titanium pieces	Kamis 		</td>
			<td>Incorporated</td>
			<td>&nbsp;</td>
			<td> 99.95% pure</td>
		</tr>
		<tr>
			<td>Gold pellets			 </td>
			<td>Kamis Incorporated</td>
			<td>&nbsp;</td>
			<td>99.999% pure</td>
		</tr>
		<tr>
			<td>Electron-beam evaporator</td>
			<td>Kurt J. Lesker	</td>
			<td>PVD 75 Thin Film Deposition System</td>
			<td>with electron-beam accessory</td>
		</tr>
		<tr>
			<td>Hexadecanethiol		</td>
			<td>Alfa Aesar</td>
			<td>A11362</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1-mercaptoundec-11-yl)tetra(ethyleneglycol)		</td>
			<td>Sigma Aldrich</td>
			<td>674508</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol			 </td>
			<td>Pharmco-aaper</td>
			<td>111000200</td>
			<td>200 proof, absolute</td>
		</tr>
		<tr>
			<td>Parafilm	</td>
			<td>VWR	</td>
			<td>52858-000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DPBS			 </td>
			<td>VWR</td>
			<td>4500-434</td>
			<td>Without calcium and magnesium</td>
		</tr>
		<tr>
			<td>Mouse Laminin I	</td>
			<td>VWR	</td>
			<td>95036-762</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Human Plasma Fibronectin		</td>
			<td>Invitrogen</td>
			<td>33016-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>AlexaFluor&reg; 647 carboxylic acid, succinimidyl ester		</td>
			<td>Invitrogen</td>
			<td>A-20006</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MitoTracker Red 580		</td>
			<td>Invitrogen</td>
			<td>M22425</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>AlexaFluor&reg; 350 carboxylic acid, succinimidyl ester	</td>
			<td>Invitrogen</td>
			<td>	A-10168</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-laminin antibody</td>
			<td>Fisher Scientific</td>
			<td>AB2034MI</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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

Watch this Scientific Journal Video about Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement at JoVE.com

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

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 ...

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