Name: fabricating complex culture substrates using robotic microcontact printing (r-µcp) and sequential nucleophilic substitution
Uploaded: 2014-10-31T15:00:00.000+00:00
Duration: 8 min 23 s
Description: Watch this Scientific Journal Video about Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R-Ã‚ÂµCP and Sequential Nucleophilic Substitution at JoVE.com

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><tbody><tr><td>SCARA&amp;nbsp;</td><td>Epson</td><td>LS3-401ST</td><td>Higher end models with increased precision are available if desired.&amp;nbsp;</td></tr><tr><td>(Tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane</td><td>Gelest</td><td>SIT8174.0</td><td>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><td>Sylgard 184 Silicone Elastomer Kit</td><td>Ellsworth Adhesive Co</td><td>NC9020938</td><td>Thouroughly degass solutions via vacuum exposure before use. Alternative kits such as Kit 182 are acceptable.</td></tr><tr><td>24 mm x 50 mm #1 Cover Glass Slides</td><td>Fisher Scientific</td><td>48393106</td><td>These can be purchased from a number of suppliers with varying dimensions to suit need.</td></tr><tr><td>CHA-600 Telemark Electron Beam Evaporator</td><td>Telemark</td><td>SEC-600-RAP</td><td>Requries specialized training.</td></tr><tr><td>EPSON LS3 SCARA</td><td>EPSON</td><td>LS3-401ST</td><td></td></tr><tr><td>&amp;omega;-mertcaptoundecyl bromoisobutyrate</td><td>Prochimia</td><td>FT 015-m11-0.2</td><td>Store at -20 &amp;deg;C. Other ATRP initiators may be used as this R-&amp;mu;CP platform is applicable to all micropatterning modalities.&amp;nbsp;</td></tr><tr><td>Schlenk Tube Flask 50 ml</td><td>Synthware</td><td>60003-078</td><td>Requires rubber stoppers with diaphram.</td></tr><tr><td>Poly(ethylene glycol) methyl ether methacrylate</td><td>Sigma Aldrich</td><td>447943</td><td>Shipped containing MEHQ and BHT free readical inhibitors.</td></tr><tr><td>Methanol (Certified ACS)</td><td>Fisher Scientific</td><td>A412-4</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>Copper(II) Bromide</td><td>Sigma Aldrich</td><td>437867</td><td>CAUTION, limit exposure with surgical mask.</td></tr><tr><td>2,2&#039;-Bipyridine</td><td>Sigma Aldrich</td><td>D216305</td><td>CAUTION, limit exposure with surgical mask.</td></tr><tr><td>Sodium L-Ascorbate</td><td>Sigma Aldrich</td><td>A4034</td><td></td></tr><tr><td>20 ml Borosilicate Glass Scintillation Vials</td><td>Fisher Scientific</td><td>03-340-4E</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma Aldrich</td><td>S2002</td><td>CAUTION, limit exposure with surgical mask.</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-dimethyformamide</td><td>Sigma Aldrich</td><td>227056</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>Ethanolamine</td><td>Sigma Aldrich</td><td>398136</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>Triethylamine</td><td>Sigma Aldrich</td><td>T0886</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>Dimethylsulfoxide</td><td>Sigma Aldrich</td><td>276855</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>Propargylamine</td><td>Sigma Aldrich</td><td>P50900</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>200 Proof Ethanol</td><td>University of Wisconsin Material Distribution Services</td><td>2292</td><td>CAUTION, only handle in chemical fume hood.</td></tr><tr><td>Azide-PEG3-Biotin</td><td>ClickChemistryTools</td><td>AZ104-100</td><td>Solubilized in DMF</td></tr><tr><td>Copper(II) Sulfate</td><td>Sigma Aldrich</td><td>C1297</td><td>CAUTION, limit exposure with surgical mask.</td></tr><tr><td>Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td><td>Sigma Aldrich</td><td>678937</td><td></td></tr><tr><td>L-Ascorbic Acid</td><td>Sigma Aldrich</td><td>A7506</td><td></td></tr><tr><td>Phosphate Buffer Saline</td><td>Invitrogen</td><td>14190144</td><td></td></tr><tr><td>Donkey Serum</td><td>Sigma Aldrich</td><td>D9663</td><td>Donkey serum contaminated items are considered bio-hazardous material and should be disposed of accordingly. Various other compounds (&lt;em&gt;e.g.&lt;/em&gt; BSA) are available and serve this purpose.</td></tr><tr><td>12-Well Polystyrene Plate</td><td>Thermo Scientifit - NUNC</td><td>07-200-81</td><td>Plates can be purchased form a number of suppliers with varying dimensions.</td></tr><tr><td>DBCO-PEG4-Biotin</td><td>Clickchemistytools</td><td>A105P4-10</td><td>Solubilized in DMF</td></tr><tr><td>Streptavidin, Alexa Fluor 488 Conjugate</td><td>Life Technologies</td><td>S-11223</td><td>Solubilized in PBS</td></tr><tr><td>Streptavidin, Alexa Fluor 546 conjugate</td><td>Life Technologies</td><td>S-11225</td><td>Solubilized in PBS</td></tr><tr><td>Nikon A1-R Confocal Microscope</td><td>Nikon</td><td>Nikon Eclipse Ti, A1R</td><td>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-Ã‚ÂµCP and Sequential Nucleophilic Substitution at JoVE.com

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

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

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- µCP and Sequential Nucleophilic Substitution

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

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 ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

Biology

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

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

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

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

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

Applications of 3D-Printing for Micro-Scale Neuronal Cell Culture Devices

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

Author Spotlight: Modular Neuronal Networks for Analyzing Brain Functions

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-µCP) and Sequential Nucleophilic Substitution

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