Funding for this work, GTK, TK, and JDM were provided by the Wisconsin Institute for Discovery and the Wisconsin Alumni Research Foundation.

1. Generating Elastomeric Stamps
<ol style="margin-left: 40px;">
	<li>To generate the PDMS stamp&#8217;s silicon masters, design the photomask&#8217;s feature patterns using computer-aided design software.
	<ol>
		<li>Design the first pattern as a 20 x 20 array of annuli with 300 &#956;m inner diameter (ID) and 600 &#956;m OD with 1,200 &#956;m center-to-center spacing.</li>
		<li>Design the second pattern as a 20 x 20 array of annuli with 600 &#956;m ID and 900 &#956;m OD with 1,200 &#956;m center-to-center spacing.</li>
		<li>Additionally, place 1 x 1 mm2 square reference marks at all four corners of each array design spaced 1,200 &#956;m center-to-center from the corner pattern at a 45&#176; angle.</li>
		<li>Fabricate the silicon masters for use in this experiment with 1:1 aspect ratios, correlating to a 300 &#956;m feature depth using standard lithography techniques detailed elsewhere15 or in collaboration with a microfluidic foundry. 
		NOTE: Feature depths lower than 100 &#956;m can lead to abnormal deformation of stamps prior to contact with substrate surfaces. 
		NOTE: This protocol begins with having already obtained silicon masters with the described patterns of photoresist, which requires specialized equipment and clean rooms to create. It is best to consult with a fabricator or core facility to create these patterned masters.</li>
	</ol>
	</li>
	<li>Silanize silicon masters O/N by incubation with (tridecafluoro-1,1,2,2-tetrahydroocty) trichlorosilane vapor. 
	NOTE: Silane vapor is highly toxic and should only be handled in a chemical fume hood.</li>
	<li>Create inverse replicas of the silicon masters by curing a 10:1 ratio of PDMS pre-polymer and PDMS curing agent on top of the silanized silicon masters in a petri dish O/N at 60 &#176;C.</li>
	<li>Remove the PDMS stamps from the silicon masters, and bond the stamps to acrylonitrile butadiene styrene (ABS) backings or any other rigid materials using glue. 
	NOTE: The material of choice need not be transparent or biocompatible. However, it must provide a flat surface for interface with the robotic tooling and higher rigidity than PDMS.</li>
</ol>
2. Preparing Coverslides
<ol style="margin-left: 40px;">
	<li>Rinse microscope coverslides (24 mm x 50 mm #1) in toluene, methanol and sonicate in acetone for 1 min before rinsing with ethanol and drying under a gentle nitrogen stream. 
	NOTE: Handle toluene and acetone only in a chemical fume hood.</li>
	<li>Coat coverslides with ~3.5 nm titanium (Ti) followed by 18.0 nm gold (Au) using a focused electron-beam evaporator. 
	NOTE: This procedure is compatible with a variety of gold-coated substrates including silicon and polystyrene. While it is possible to generate these on-site, gold-coated substrates are also available via commercial suppliers. 
	NOTE: Ti layers should be at least 3 nm thick, while Au layers on substrates should be at least 5 nm thick and can range all the way to 1mm in thickness depending on the ideal optical properties of the final substrate16,17. Substrates are not required to be optically clear for fabrication; however, it facilitates microscopic analysis of fabricated substrates.</li>
	<li>Rinse gold-coated coverslides with ethanol and dry under gentle nitrogen immediately before use.</li>
</ol>
3. R-&#956;CP of Outer Annulus
<ol style="margin-left: 40px;">
	<li>Before R-&#956;CP with the selectively compliant articulated robot arm (SCARA) system, calibrate the robotic tool effectors and accompanying dual camera systems using the system&#8217;s software. 
	NOTE: These implements are pictured in Figure 1.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/52186/52186fig1highres.jpg" /> 
Figure 1. R-&#956;CP System and Robotic Arm Tooling. (A) Large-scale view of R-&#956;CP system with all tooling and fixtures, (a) vacuum chuck, (b) reagent bath, (c) downward facing camera, (d) stamp nesting fixture, (e) robotic tool. (B) Robotic arm tooling depicting (f) diamond-tipped etching tool and (g) pneumatic suction tool holding a (h) ABS-backed PDMS stamp.
<ol start="2" style="margin-left: 40px;">
	<li>Manually place a PDMS stamp with 600 &#956;m ID and 900 &#956;m OD protruding annuli face down in the stamp nesting fixture. Manually place a freshly cleaned gold-coated coverslide on top of the vacuum chuck, displayed in Figure 1A, and immobilize it using the connected lab vacuum.</li>
	<li>Using the robot controls, align the downward facing camera over the center point of the gold-coated coverslide, as in Figure 2A. 
	NOTE: This point can initially be defined by visual inspection and does not require great accuracy, as the PDMS stamp is much larger than the gold-coated slide.</li>
	<li>Instruct the robotic arm to etch four reference &#8220;X&#8221; marks at the vertices of a square with dimensions 3.8 mm by 3.8 mm centered on the gold-coated coverslide using the diamond-tipped etching tool attached to the robotic arm. (Depicted in Figure 2B; AUTOMATED PROCESS) 
	NOTE: This ensures all four reference marks are within one visual frame of the downward facing camera.</li>
	<li>Using the robots pneumatic suction tooling, pick-up and hold the PDMS stamp 1 mm above the stamp nesting fixture as in Figure 2C. (AUTOMATED PROCESS)</li>
	<li>Using the upward facing camera and LED illumination ring pictured in Figure 2D and E, visualize the stamp&#8217;s square reference marks and identify them with the camera software 10 times in order to determine the stamp&#8217;s average X, Y, and angular offset from the center of the robot tooling axis. (AUTOMATED PROCESS)</li>
	<li>As in Figure 2A, use the downward facing camera to visualize and detect the coverslide&#8217;s etched reference marks 10 times to determine the coverslide&#8217;s average X, Y center locations and angular offset from the center of the robot tooling axis. (AUTOMATED PROCESS). 
	NOTE: An internal computation is performed by the robotic system&#8217;s software to precisely align the stamp&#8217;s and coverslip&#8217;s X, Y center locations and angular offsets in future R-&#956;CP steps.</li>
	<li>3.8) Place the stamp in a bath of alkanethiol ATRP initiator, &#969;-mercaptoundecyl bromoisobutyrate (2 mM in ethanol) as depicted in Figure 2F. (AUTOMATED PROCESS)</li>
	<li>Remove the stamp from the alkanethiol solution and place it over nitrogen stream pressurized to 0.48 bar (5 psi) to evaporate the ethanol as in Figure 2G. After 1 min increase the nitrogen stream&#8217;s pressure is to 1.03 bar (15 psi) to ensure uniform dryness. (AUTOMATED PROCESS) 
	NOTE: Incomplete drying will result in total or partial loss of pattern fidelity.</li>
	<li>After drying, move the stamp over the calculated center position of the gold-coated slide and lower it at 100 &#956;m increments while monitoring Z-axis motor force as depicted in Figure 2H. 
	NOTE: This is communicated as torque experienced by the Z-axis motor and displayed in the robot guidance software. (AUTOMATED PROCESS)</li>
	<li>Stop lowering the stamp once the predetermined pressure of 79.2 kPa has been achieved, and maintain contact with the gold-coated slide for 15 sec. (AUTOMATED PROCESS) 
	NOTE: The pressure values here are optimized for the current stamp design and feature height. If the stamp design is changed, specifically for high aspect ratio stamps, tune these accordingly by a trial and error process.</li>
	<li>Slowly remove the stamp from the gold-coated slide and place the stamp back in the stamp nesting fixture. (AUTOMATED PROCESS)</li>
</ol>
4. SI-ATRP of PEGMEMA on Micropatterned Coverslides
<ol style="margin-left: 40px;">
	<li>Release vacuum pressure holding the micropatterned coverslide, and transfer it to a 50 ml Schlenk flask. Seal and degas the Schlenk flask using a vacuum pump.</li>
	<li>Add 5.5 ml of ATRP reaction mixture containing the macromonomer PEGMEMA (208.75 mmol), water (34.4 ml), methanol (43.8 ml), copper(II) bromide (1 mmol), and 2&#8217;,2-bipyridine (3 mmol) to the Schlenk flask.</li>
	<li>Add 0.5 ml of L-sodium ascorbate (454.3 mM) in water to initiate the reaction, and allow it to continue for 16 hr at RT under inert gas. 
	NOTE: Following addition of L-sodium ascorbate, reaction color will shift from light green to dark brown as shown in Figure 3A-B. 
	NOTE: The reaction can continue beyond 16 hr, but it will not significantly increase the length of the PEG brushes.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/52186/52186fig3highres.jpg" /> 
Figure 3. Initiation of SI-ATRP. (A) Initiation of reaction and (B) subsequent color change following addition of L-sodium ascorbate. (C) Microscope image of micropatterned coverslide surface following SI-ATRP procedure. Scale bar 1 mm.
<ol start="4" style="margin-left: 40px;">
	<li>Remove the micropatterned coverslide from the Schenk flask and rinse with ethanol, water, and ethanol and dry under a gentle nitrogen stream. 
	NOTE: Following SI-ATRP, surface modifications should be visible to the eye and can be imaged and analyzed under a microscope as in Figure 3C. 
	NOTE: This is the simplest method to test the precision of the trial as it obviates the need to immunostain the substrates.</li>
</ol>
5. Azide Functionalization of Micropatterned PEGMEMA Chains
<ol style="margin-left: 40px;">
	<li>Place the micropatterned coverslide in a 20 ml glass reaction vial.</li>
	<li>Add 6 ml of N,N-dimethylformamide (DMF) containing 100 mM sodium azide to the reaction vial. Maintain this reaction at 37 &#176;C for 24 hr. 
	NOTE: Handle DMF only in a chemical fume hood. Exercise caution when weighing out sodium azide.</li>
	<li>Upon completion, remove the micropatterned coverslide from the reaction vial and rinse with ethanol then water and dry under a nitrogen stream.</li>
</ol>
6. Passivation of Bromine Functionalized PEGMEMA Chains
<ol style="margin-left: 40px;">
	<li>Place micropatterned coverslide in 20 ml glass reaction vials.</li>
	<li>Add 6 ml of dimethyl sulfoxide (DMSO) containing 100 mM ethanolamine and 300 mM triethylamine to the reaction vial. Hold this reaction at 40 &#176;C for 24 hr. 
	NOTE: Handle DMSO, ethanolamine, and triethylamine only in a chemical fume hood.</li>
	<li>Upon completion remove the micropatterned coverslide from the reaction vial, rinse with ethanol then water and dry under a nitrogen stream.</li>
</ol>
7. R-&#956;CP of Inner Annulus and SI-ATRP of PEGMEMA
<ol style="margin-left: 40px;">
	<li>Carry out R-&#956;CP steps as previously described in Steps 3.2 and 3.6 &#8211; 3.12, substituting a PDMS stamp with 300 &#956;m ID and 600 &#956;m OD protruding annuli, such that the annuli with smaller features are placed inside the previously micropatterned annuli. 
	NOTE: The pressure threshold for this stamp is 132.0 kPa. As previously mentioned, these pressure settings are optimized for the stamp features being used in this experiment.</li>
	<li>Carry out SI-ATRP steps as previously described in steps 4.1 &#8211; 4.4.</li>
</ol>
8. Acetylene Functionalization of Micropatterned PEGMEMA Chains
<ol style="margin-left: 40px;">
	<li>Place micropatterned coverslide in a 20 ml glass reaction vial.</li>
	<li>Add 6 ml of DMSO containing 100 mM propargylamine to the reaction vial. Maintain this reaction at RT for 24 hr. 
	NOTE: Handle DMSO and propargylamine only in a chemical fume hood.</li>
	<li>Upon completion remove the micropatterned coverslide from the reaction vial and rinse with ethanol then water and dry under a nitrogen stream.</li>
</ol>
9. Copper-catalyzed &#8220;Click&#8221; Biotinylation of Acetylene Terminated PEGMEMA Chains
<ol style="margin-left: 40px;">
	<li>Place micropatterned coverslide in a 20 ml glass reaction vial.</li>
	<li>Add 6 ml of copper sulfate (15 mM) / Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, 30 mM) (1:1 v/v water/DMF) containing 562 &#956;M azide-PEG4-Biotin conjugate to the reaction vial. Add 1.2 ml of L-ascorbic acid (0.15 mM) in water to the mixture to initialize the reaction.</li>
	<li>Bubble nitrogen through the reaction solution for 10 sec, seal the vial with Parafilm, and react for 24 hr at RT.</li>
	<li>Upon completion remove micropatterned coverslide from the reaction vial, rinse with water and place it in a 12-well polystyrene dish.</li>
</ol>
10. Immunofluorescent Detection of Biotinylated Acetylene Groups
<ol style="margin-left: 40px;">
	<li>Block micropatterned coverslide in DPBS (3% Donkey Serum) for 1 hr at RT. Stain for biotinylated acetylene groups with Streptavidin-546 conjugate (2 &#956;g/ml) in DBPS (3% Donkey Serum in PBS) for 2 hr at RT.</li>
	<li>Rinse micropatterned coverslide 5 times with DPBS for 10 min with gentle agitation. 
	NOTE: Figure 4B depicts an image of the slide after this step is completed.</li>
</ol>
11. Copper-free &#8220;Click&#8221; Biotinylation of Azide Terminated PEGMEMA Chains
NOTE: If desired, this substrate modification step can be performed in situ during cell culture.
<ol style="margin-left: 40px;">
	<li>Leave micropatterned coverslide in 12-well polystyrene dish following DBPS rinses.</li>
	<li>Add 2 ml of DBPS (or cell culture media) containing 20 &#956;M DBCO-PEG4-Biotin, and allow this reaction to continue for 24 hr at RT&#160;(or at 37 &#176;C in an incubator).</li>
	<li>Upon completion, rinse micropatterned coverslide 5 times with DPBS (or simply perform a media change).</li>
</ol>
12. Immunofluorescent Detection of Biotinylated Azide Groups
<ol style="margin-left: 40px;">
	<li>Stain for biotinylated azide groups with Streptavidin-488 conjugate (2 &#956;g/ml) in 2 ml DPBS (3% Donkey Serum) for 2 hr at RT.</li>
	<li>Rinse micropatterned coverslide 5 times in DPBS for 10 min with gentle agitation.</li>
	<li>Image micropatterned coverslide using confocal fluorescence microscope. Figure 4A-C depicts images of the slide after this step is completed. 
	NOTE: When using substrates for tissue culture assays, skip sections 10 and 12 of this protocol. After the desired degree of functionalization, sterilize the engineered coverslide by rinsing both sides with 100% ethanol and drying under a nitrogen stream. Transfer the slide to a sterile biological safety cabinet and rinse the slide with PBS five times before proceeding with cell seeding and culture.</li>
</ol>

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The authors declare that they have no competing financial interest.

Ideal substrates for tissue engineering would be bioinspired and thereby recapitulate the spatial distribution of critical bioactive ligands found within the native tissues. They would also possess dynamic properties that enable temporal adjustments of the ligands and the spatial patterns in which they are presented to permit directed tissue morphogenesis and spatially restricted induction of cell fate. Fabrication of such substrates requires the immobilization of multiple biochemical cues in complex and highly ordered o...

The ability of PEG-grafted surfaces to display covalently bound biochemical ligands while simultaneously maintaining inherent non-fouling properties make them an ideal choice for engineering custom microscale environments on culture substrates1,2,3. The biospecific interactions mediated by ligand conjugated PEG brushes enables reductionistic analysis of the effects of biochemical cues found within complex in vivo tissue microenvironments on individual cell phenotypes. Furthermore, bio-orthogonal &#8220;click&#8221; chemistries can be used to facilitate directional immobilization of ligands so that they are presented in native conformations4-6. Thus, microscale spatial patterning of PEG brushes is a versatile tool to create designer in vitro niches to investigate cell signaling induced by immobilized biochemical cues6,7.
A common method for generating spatial patterns of biochemical cues entails microcontact printing (&#956;CP) gold-coated substrates with patterns of PEG conjugated alkanethiols. Then, the micropatterned self-assembled monolayers (SAMs) of PEG-ylated alkanethiols restricts physical adsorption of biochemical molecules, e.g., proteins, only to non-patterned regions of the substrate8,9. However, the SAMs generated by this technique are sensitive to oxidation in long term cell culture media. Thus, &#956;CP&#8217;d alkanethiol SAMs are often further grafted with PEG polymer brushes using surface-initiated atom transfer radical polymerization (SI-ATRP) to increase the region&#8217;s non-fouling stability10. Specifically, &#956;CP of the alkanethiol polymerization initiator, &#969;-meraptoundecyl bromoisobutyrate, on gold-coated surfaces followed by SI-ATRP of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) monomers generates surfaces with micropatterned long-term, stable, and non-fouling PEG brushes. Moreover, these are capable of being further modified to present diverse chemical moieties11.
Taking advantage of this property, Sha et. al. developed a method to engineer culture substrates with multicomponent PEGMEMA brushes presenting orthogonal &#8220;click&#8221; chemistries. In this method, they use a series of &#956;CP/SI-ATRP steps interspersed with sequential sodium azide, ethanolamine, and propargylamine nucleophilic substitutions to create culture substrates presenting microscale patterns of multiple immobilized ligands6. While the potential of using such chemistries in conjunction with manual &#956;CP to engineer novel culture substrates is immense, it is limited by the precision and accuracy with which multiple &#956;CP steps can be aligned on a single substrate. A high level of precision and accuracy would be required to reproducibly manufacture complex in vitro niches using these versatile techniques.
To address this limitation, several automated and semi-automated &#956;CP systems have been generated. Chakra et. al. developed a &#956;CP system in which custom stamps are placed on a rail system and brought into conformal contact with gold-coated slides using a computer-controlled pneumatic actuator. However, this method requires the precise fabrication of custom stamp designs and reports a 10 &#956;m precision with no report of the accuracy achieved when performing multiple &#956;CP steps12. More recently, a method utilizing an integrated kinematic coupling system reported precision below 1 &#956;m using a single pattern, but were unable to accurately align multiple patterns due to a lack of precise control of stamp features from mold to mold13. Additionally, both of the previous methods require the substrate to remain fixed between patterning steps, thereby significantly limiting the diversity of surface modification chemistries that can be utilized. Here, we describe an automated R-&#956;CP system capable of accurate and precise alignment of multiple &#956;CP steps while allowing maximal flexibility in stamp design and fabrication. Furthermore, the patterned substrates can be repeatedly removed from the system between stampings, thereby permitting the use of diverse substrate modification chemistries, including sequential nucleophilic substitutions. Substrates engineered using such chemistries have been used for cell culture previously by both us6,14 and others7. Thus, we have merged R-&#956;CP and sequential nucleophilic substitution reactions to develop a method for scalable manufacture of culture substrates with complex and micropatterned biochemical cues.

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Name</td>
			<td style="width:185px;">Company</td>
			<td style="width:185px;">Catalog Number</td>
			<td style="width:185px;">Comments</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">SCARA&#160;</td>
			<td style="width:185px;">Epson</td>
			<td style="width:185px;">LS3-401ST</td>
			<td style="width:185px;">Higher end models with increased precision are available if desired.&#160;</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:185px;">(TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL)TRICHLOROSILANE</td>
			<td style="width:185px;">Gelest</td>
			<td style="width:185px;">SIT8174.0</td>
			<td style="width:185px;">CAUTION, Should only be handled in a chemical fume hood. When silanizing wafers no one should enter the hood until all silane has been evaporated.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:185px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:185px;">Ellsworth Adhesive Co</td>
			<td style="width:185px;">NC9020938</td>
			<td style="width:185px;">Thouroughly degass solutions via vacuum exposure before use. Alternative kits such as Kit 182 are acceptable.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:185px;">24mm X 50 mm #1 Cover Glass Slides</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">48393106</td>
			<td style="width:185px;">These can be purchased from a number of suppliers with varying dimensions to suit need.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">CHA-600 Telemark Electron Beam Evaporator</td>
			<td style="width:185px;">Telemark</td>
			<td style="width:185px;">SEC-600-RAP</td>
			<td style="width:185px;">Requries specialized training.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">EPSON LS3 SCARA</td>
			<td style="width:185px;">EPSON</td>
			<td style="width:185px;">LS3-401ST</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:185px;">&#969;-mertcaptoundecyl bromoisobutyrate</td>
			<td style="width:185px;">Prochimia</td>
			<td style="width:185px;">FT 015-m11-0.2</td>
			<td style="width:185px;">Store at -20&#176;C. Other ATRP initiators may be used as this R-&#956;CP platform is applicable to all micropatterning modalities.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Schlenk Tube Flask 50 mL</td>
			<td style="width:185px;">Synthware</td>
			<td style="width:185px;">60003-078</td>
			<td style="width:185px;">Requires rubber stoppers with diaphram.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">Poly(ethylene glycol) methyl ether methacrylate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">447943</td>
			<td style="width:185px;">Shipped containing MEHQ and BHT free readical inhibitors.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Methanol (Certified ACS)</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">A412-4</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Copper(II) Bromide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">437867</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">2&#39;,2-Bipyridine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">D216305</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Sodium L-Ascorbate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">A4034</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">20mL Borosilicate Glass Scintillation Vials</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">03-340-4E</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Sodium Azide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">S2002</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">N,N-dimethyformamide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">227056</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Ethanolamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">398136</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Triethylamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">T0886</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Dimethylsulfoxide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">276855</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Propargylamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">P50900</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">200 Proof Ethanol</td>
			<td style="width:185px;">University of Wisconsin Material Distribution Services</td>
			<td style="width:185px;">2292</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Azide-PEG3-Biotin</td>
			<td style="width:185px;">ClickChemistryTools</td>
			<td style="width:185px;">AZ104-100</td>
			<td style="width:185px;">Solubilized in DMF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Copper(II) Sulfate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">C1297</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">678937</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">L-Ascorbic Acid</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">A7506</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Phosphate Buffer Saline</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:185px;">14190144</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:185px;">Donkey Serum</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">D9663</td>
			<td style="width:185px;">Donkey serum contaminated items are considered bio-hazardous material and should be disposed of accordingly. Various other compounds (e.g. BSA) are available and serve this purpose.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">12-Well Polystyrene Plate</td>
			<td style="width:185px;">Thermo Scientifit - NUNC</td>
			<td style="width:185px;">07-200-81</td>
			<td style="width:185px;">Plates can be purchased form a number of suppliers with varying dimensions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">DBCO-PEG4-Biotin</td>
			<td style="width:185px;">Clickchemistytools</td>
			<td style="width:185px;">A105P4-10</td>
			<td style="width:185px;">Solubilized in DMF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Streptavidin, Alexa Fluor 488 Conjugate</td>
			<td style="width:185px;">Life Technologies</td>
			<td style="width:185px;">S-11223</td>
			<td style="width:185px;">Solubilized in PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Streptavidin, Alexa Fluor 546 conjugate</td>
			<td style="width:185px;">Life Technologies</td>
			<td style="width:185px;">S-11225</td>
			<td style="width:185px;">Solubilized in PBS</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:185px;">Nikon A1-R Confocal Microscope</td>
			<td style="width:185px;">Nikon</td>
			<td style="width:185px;">Nikon Eclipse Ti, A1R</td>
			<td style="width:185px;">An epifluorescent microscope is sufficient to image functionalized micropatterned substrates.</td>
		</tr>
	</tbody>
</table>

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.

The use of manual alignment &#956;CP techniques to engineer culture substrates with arrays of PEG-grafted brushes functionalized with orthogonal &#8220;click&#8221; chemistries has been demonstrated in previous work6. However, this offers minimal control of pattern orientation and often results in overlap of functionalized areas. Here, a novel R-&#956;CP system is used to overcome this limitation, and its ability to accurately pattern an array of PEG brush annuli with 300 &#956;m ID and 600 &#956;m OD presenti...

Watch this Scientific Journal Video about Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R-ÂµCP and Sequential Nucleophilic Substitution at JoVE.com

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &amp;#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.

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 ...

fabricating-complex-culture-substrates-using-robotic-microcontact

Research

JoVE Journal

Bioengineering

10.4K Views.  University of Wisconsin, Madison. 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. 

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

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 ...

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

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 ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Four-Dimensional CT Analysis Using Sequential 3D-3D Registration

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data remains difficult because segmentation in all volumetric frames is time-consuming. We aimed to analyze joint kinematics using a sequential 3D-3D registration technique to provide the kinematics of the moving bone with respect to the fixed bone semiautomatically using 4DCT DICOM data and existing software. Surface data of the source bones are reconstructed from 3DCT. The trimmed surface data are respectively matched with surface data from the first frame in 4DCT. These trimmed surfaces are sequentially matched until the last frame. These processes provide positional information for target bones in all frames of the 4DCT. Once the coordinate systems of the target bones are decided, translation and rotation angles between any two bones can be calculated. This 4DCT analysis offers advantages in kinematic analyses of complex structures such as carpal or tarsal bones. However, fast or large-scale motions cannot be traced because of motion artifacts.

We analyzed joint kinematics from four-dimensional computed tomography data. The sequential 3D-3D registration method semiautomatically provides the kinematics of the moving bone with respect to the subject bone from four-dimensional computed tomography data.

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...