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-uniformity hotplate.</li>
<li>Photopattern the substrate using either a direct-write photolithography system or a mask aligner system and appropriate mask. Masks can be purchased from a number of commercial sources. Additionally, transparencies printed with UV absorbent ink, taped to an optical flat, may be used to produce patterns with large features.</li>
<li>Develop the patterned wafer in 1:2 400K developer:semi-conductor grade deionized water for 1 m 45 s with gentle agitation. Rinse thoroughly with semi-conductor grade deionized water and dry with a stream of nitrogen (N2) gas. Pattern development can be checked using a microscope with a UV filter. If the pattern is not fully developed, the wafer can be returned to the developing solution for additional time.</li>
</ol>
Note: For best results, photopatterning should be carried out in a cleanroom environment.
2. Preparation of PDMS Stamp (Figure 2)
<ol>
<li>Prepare a 10:1 by weight resin:hardener mixture of Sylgard 182 (PDMS) and completely cover the master (photopatterned wafer) with the mixture in a disposable petri dish.</li>
<li>De-gas the PDMS-covered master in a vacuum dessicator until no bubbles are visible and allow the stamp to cure in an oven at 65&deg;C for 1.5 h. Prior to curing, it is important make sure the master is at the bottom of the dish as it may rise to the surface during the de-gassing step.</li>
<li>Cut the PDMS stamp out of the master and trim to the appropriate size. Store the stamp in a covered container (feature side up) to protect it from dust and debris.</li>
</ol>
3. Preparation of the Patterned Gold Substrate (Figure 3)
<ol>
<li>Prepare 25 mm no. 1 round glass coverslips for metal deposition by treating them with oxygen plasma for 10 m. Rinse twice with 18.2 M&Omega; deionized water followed by twice with ethanol, drying with a stream of N2 gas between each step.</li>
<li>Using a multi-pocket electron-beam deposition system, deposit 50 &Aring; titanium followed by 150 &Aring; gold. Do not vent the evaporator between deposition of the titanium layer and gold layer. Alternatively, gold-coated coverslips may be purchased from a variety of suppliers, however, it is important that the metal layers are prepared by electron-beam evaporation and not thermal evaporation. If purchased from an outside source, gold substrates may be cleaned by plasma oxidation or "piranha" (7:3 Conc. H2SO4:30% H2O2)11 prior to use.</li>
</ol>
Note: "Piranha" solution is explosive in the presence of organic compounds.
<ol start="3">
<li>Prepare the stamping solution, 10 mM hexadecanethiol in absolute ethanol, and the backfilling solution, 1 mM glycol-terminated thiol in absolute ethanol.</li>
<li>Rinse the stamp with ethanol and dry thoroughly with N2 gas. Apply stamping solution to the PDMS stamp dropwise until fully coated. Dry the stamp thoroughly with N2 gas. Proceed to 5a or 5b as appropriate based on the features of the PDMS stamp.</li>
<li>
<ol class="lalpha">
<li>Gently press the stamp onto the gold substrate and allow the monolayer to form for 15 s.</li>
<li>Place the gold substrate into a petri dish containing 18.2 M&Omega; deionized water, ensuring the substrate is submerged. Gently press the stamp onto the gold substrate and allow the monolayer to form for 15 s.</li>
</ol></li>
<li>Rinse the stamped substrate twice with ethanol, drying with N2 gas after each rinse and place the substrate in a petri dish.</li>
<li>Cover the substrate with backfilling solution and seal the dish with parafilm to prevent evaporation.</li>
<li>Allow the background monolayer to form in the dark for 12-14 h.</li>
<li>Remove the patterned coverslip from the backfilling solution and rinse twice with ethanol, drying with N2 gas after each rinse.</li>
</ol>
4. Applying Protein and Cells to the Patterned Substrate
<ol>
<li>Place the patterned coverslip in a small petri dish or cell culture chamber and cover with 500 &mu;L to 1 mL of Dulbecco's Phosphate Buffered Saline (DPBS). The DPBS must completely cover the substrate during protein incubation to ensure even protein coverage.</li>
<li>Add a concentrated solution of protein to the DPBS and mix the solution by pipeting several times. Incubate the protein mixture with the substrate at 37&deg;C for 1 h. Final concentrations for laminin and fibronectin are typically 12 &mu;g/mL and 20 &mu;g/mL, respectively. Protein can be labeled with an amine reactive fluorescent dye to allow for easy pattern visualization. However, labeled protein should be mixed 1:1 with unlabeled protein as protein labeling can interfere with biological activity.</li>
<li>After incubation, rinse the substrate thoroughly with DPBS (4-5x) to remove unbound protein, taking care not to dry the substrate or bring it through the air-water interface. After the first three rinses, add approximately 500 &mu;L of complete cell growth media to maintain a wet substrate.</li>
<li>Replace the growth media used to rinse the substrate with fresh media.</li>
<li>Dissociate and count cells for plating onto the substrate. Either dissociated primary cells, such as hippocampal neurons, or immortalized cell lines, such as CHO-K1 cells, can be utilized.</li>
<li>Plate dissociated cells onto the substrate. Typically 30 to 200 cells/mm2 are used.</li>
</ol>
5. Representative Results:
<img src="/files/ftp_upload/3164/3164fig1.jpg" alt="Figure 1"/> Figure 1.General schematic for the photolithographic preparation of a patterned master. In this process, a silicon wafer is cleaned with acetone, coated with photoresist, exposed to the pattern of interest, and the pattern is developed.
<img src="/files/ftp_upload/3164/3164fig2.jpg" alt="Figure 2"/> Figure 2. General schematic for PDMS stamp preparation. In this process, the patterned master is covered with Sylgard (10:1 resin:hardener), de-gassed in a vacuum desiccator, cured in an oven at 60&deg;C, and cut to size.
<img src="/files/ftp_upload/3164/3164fig3.jpg" alt="Figure 3"/> Figure 3. General schematic for substrate patterning. In this process, glass substrates are coated with titanium (50&Aring;) and gold (150&Aring;) using an electron beam evaporator, patterned by microcontact printing hexadecanethiol using a PDMS stamp, backfilled with glycol terminated alkane thiols, and coated with fluorescently-labeled protein.
<img src="/files/ftp_upload/3164/3164fig4.jpg" alt="Figure 4"/> Figure 4. Patterned master (A) and PDMS stamp (B) prepared using the described methods. Scale bars are 100&mu;m.
<img src="/files/ftp_upload/3164/3164fig5.jpg" alt="Figure 5"/> Figure 5. Patterned SAMs visualized with AlexaFluor 647-labeled fibronectin (A) and CHO-K1 cell confinement (B). Scale bars are 100 &mu;m.
<img src="/files/ftp_upload/3164/3164fig6.jpg" alt="Figure 6"/> Figure 6. Patterned laminin seeded with E18 mouse hippocampal neurons at 4 days in vitro. AlexaFluor 350-conjugated anti-laminin antibody is used for pattern visualization (A) and E18 mouse hippocampal neurons are stained with MitoTracker Red 580 (B). Scale bars are 100 &mu;m.
<img src="/files/ftp_upload/3164/3164fig7.jpg" alt="Figure 7"/> Figure 7. Potential pitfalls in patterned substrate preparation visualized by AlexaFluor 647-conjugated fibronectin adsorption. (A) Insufficent mixing leads to uneven protein adsorption. (B) Uneven application of pressure during stamping leads to partial patten transfer. (C) Excessive pressure during stamping can lead to stamp collapse. (D) Exposure of patterned surface to the air water interface during rinsing can result in background protein adsorption. Scale bars are 100 &mu;m.
<img src="/files/ftp_upload/3164/3164fig8.jpg" alt="Figure 8"/> Figure 8.Submerged patterning can produce patterns with small features that are difficult to print by conventional microcontact printing in air. Images (A) and (B) show different regions of the same pattern, printed with the same PDMS stamp in air (A) or deionized water (B). 10&mu;m-wide support lines which surround the pattern (added to help prevent stamp collapse) are seen in (A), however, the smaller dot features as shown in (B) are not seen. This demonstrates that printing in air works well for larger features, but printing in water may be necessary for patterns with smaller features. Scale bars are 20 &mu;m.
<table border="1">
 <tbody>
 <tr>
 <td>Cycle</td>
 <td>Acceleration Rate (rpm/s)</td>
 <td>Final Speed (rpm)</td>
 <td>Time (s)</td>
 </tr>
 <tr>
 <td>1</td>
 <td>500</td>
 <td>1000</td>
 <td>5</td>
 </tr>
 <tr>
 <td>2</td>
 <td>3800</td>
 <td>3800</td>
 <td>30</td>
 </tr>
 </tbody>
</table>
Table 1. Two-cycle spin program used to create a 4.5 &mu;m thick coating of AZ9245 on a silicon wafer.
To prepare PDMS stamps for the formation of patterned substrates, a master in photoresist is first fabricated (Figures 1 and 4A). The master is the inverse of the stamp and is created using either a direct-write lithography system or a mask aligner. When a positive photoresist, such as AZ9245, is used for master production, the resist-coated wafer is exposed to light with the same pattern that will appear on the final substrate. While it is not always possible, it has been reported that the ideal aspect ratio (feature size to resist thickness) for PDMS stamp masters is 1:2.13 We have found that aspect ratios of 1:40 are possible, depending on the nature of the pattern. AZ9245 coated silicon wafers under the conditions described here give photoresist with a nominal thickness of 4.5 &mu;m. We have found that this thickness of AZ9245 can be used to produce PDMS masters with features ranging from &gt;100 &mu;m to 2 &mu;m.
PDMS stamps are cast from Sylgard 182 (or Sylgard 184) using the master fabricated from photoresist (Figure 2). Photoresist masters can be used multiple times to create many copies of the same stamp. After hardening the PDMS, stamps are removed from the master using a razor blade and the resulting stamp can be visualized under a microscope by placing stamp feature side down on a glass coverslip (Figure 4B)
Proper stamping results in a sharp, clear protein pattern that can be visualized by application of fluorescently labeled protein (Figures 3 and 5). Alternatively, immunohistochemistry may be used to visualize the protein pattern after cell fixation (Figure 6). Cell growth is well confined to the protein pattern for both immortalized cell lines and primary cells (Figures 5 and 6).
While this technique is easily mastered, several common problems may arise. The application of protein without sufficient mixing of the concentrated protein solution in the DPBS can lead to uneven protein patterns (Figure 7A). Improper stamping can lead to partial pattern transfer or stamp collapse (Figure 7B-C). In addition, exposing the patterned substrate containing adsorbed protein to air can disrupt the monolayer causing decreased resistance in the background (Figure 7D). Patterns comprised of very small features (&lt; 5 &mu;m) and high aspect ratios often require the use of submerged microcontact printing. In this procedure (3.5b) water is used as a barrier to prevent hexadecanethiol from depositing onto the substrate outside of the pattern (Figure 8).14

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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>

No conflicts of interest declared.

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 by the photoresist manufacturer). The most difficult masters to produce combine both large and small features.
Fabrication of a PDMS stamp from a master is straightforward using the procedure described in section 2. However, care must be taken to completely mix the two components of Sylgard 182. Failure to achieve complete mixing of the components will result in an inability to harden the stamp and likely destruction of the master. Additionally, the master may shatter when separating it from the PDMS stamp if excess force is used or twisting occurs during separation. If the master does break in the process of removing the stamp, it may still be possible to use the stamp after cleaning with ethanol. However, this stamp may produce patterns with defect sites.
While microcontact printing of SAMs using a PDMS stamp is versatile and may be useful in many applications, a number of crucial steps must be followed to achieve successful protein and cell confinement. To obtain good protein and cell confinement, gold substrates must be prepared by electron-beam deposition, since thermal evaporation leads to rough gold surfaces that do not support proper monolayer formation. Moreover, we have found that how the glass substrate is prepared for titanium and gold deposition also affects the quality of the gold substrate and monolayer stability. While glass substrates can be cleaned for deposition using a wide variety of techniques, including boiling methanol, "piranha",11 and "buzzard" solution,12 in our hands coverslips prepared by plasma oxidation are far superior to coverslips cleaned using other methods. We have observed that solvent, acid, and base-based cleaning methods give monolayers with decreased temporal stability and an increased number of defect sites.
The exact stamping technique required to produce optimal protein and cellular patterns varies based on the size and shape of the features to be stamped and aspect ratio of the stamp. Typically, the exact stamping technique for each new stamp must be optimized and stamping technique improves after several trials. Applying too much pressure during stamping will lead to stamp collapse, which is prone to happen with stamps containing larger aspect ratios (Figure 7C). Alternatively, applying too little pressure results in partial pattern transfer (Figure 7B). It is also possible to take advantage of stamping technique to slightly alter the features of the pattern. For example, we have found that features can be slightly increased in size by stamping for longer periods of time and others have reported that the dimensions of certain features can be reduced using submerged microcontact printing techniques.15 Often air bubbles become trapped beneath the stamp when performing submerged stamping; however, this technique may give the best results when small features in conjunction with large aspect ratios are desired.
There are many advantages to using microcontact printing in conjunction with self-assembled monolayer chemistry. When directly printing proteins onto substrates, conformational changes have been shown to occur16 attributable to the step where proteins are dried during the stamping process. This drying step is thought to lead to protein aggregation and denaturation, which in turn affects protein function. In the SAM system, proteins adsorb to the substrate from a buffered aqueous solution, identical to how substrates are typically prepared for cell culture. In addition, microcontact printing of proteins does not achieve cell confinement for extended periods of time, due to the lack of protein resistant background molecules.17 In contrast, the use of a glycol background monolayer provides for excellent confinement.5 In cases where the use of a gold substrate is either not possible or undesirable, trichlorosilane monomers can be utilized instead to allow for protein and cell confinement.9While there are methods for the confinement of proteins and cells that do not rely on microcontact printing, these methods tend to be more labor intensive and are not amenable to producing the large numbers of identical substrates typically required for biological studies.18,19

<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 ...

creating-two-dimensional-patterned-substrates-for-protein-cell

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.5K Views.  Washington University in St. Louis. 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.

Video: Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

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 ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

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.

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 ...

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 ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Summary

Explore More Videos

Chapters in this video

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

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

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

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

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Summary

Explore More Videos

Chapters in this video

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

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

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

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

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates