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

Summary

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.

Abstract

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 (μCP) is a versatile technique to pattern such grafted PEG brushes, but manual μCP 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-μCP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal ‘click’ chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Introduction

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 substrates^1,2,3. 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 “click” chemistries can be used to facilitate directional immobilization of ligands so that they are presented in native conformations^4-6. 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 cues^6,7.

A common method for generating spatial patterns of biochemical cues entails microcontact printing (μCP) 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 substrate^8,9. However, the SAMs generated by this technique are sensitive to oxidation in long term cell culture media. Thus, μCP’d alkanethiol SAMs are often further grafted with PEG polymer brushes using surface-initiated atom transfer radical polymerization (SI-ATRP) to increase the region’s non-fouling stability¹⁰. Specifically, μCP of the alkanethiol polymerization initiator, ω-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 “click” chemistries. In this method, they use a series of μCP/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 μCP to engineer novel culture substrates is immense, it is limited by the precision and accuracy with which multiple μCP 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 μCP systems have been generated. Chakra et. al. developed a μCP 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 μm precision with no report of the accuracy achieved when performing multiple μCP steps¹². More recently, a method utilizing an integrated kinematic coupling system reported precision below 1 μm 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-μCP system capable of accurate and precise alignment of multiple μCP 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 us^6,14 and others⁷. Thus, we have merged R-μCP and sequential nucleophilic substitution reactions to develop a method for scalable manufacture of culture substrates with complex and micropatterned biochemical cues.

Protocol

1. Generating Elastomeric Stamps

1. To generate the PDMS stamp’s silicon masters, design the photomask’s feature patterns using computer-aided design software.
   1. Design the first pattern as a 20 x 20 array of annuli with 300 μm inner diameter (ID) and 600 μm OD with 1,200 μm center-to-center spacing.
   2. Design the second pattern as a 20 x 20 array of annuli with 600 μm ID and 900 μm OD with 1,200 μm center-to-center spacing.
   3. Additionally, place 1 x 1 mm² square reference marks at all four corners of each array design spaced 1,200 μm center-to-center from the corner pattern at a 45° angle.
   4. Fabricate the silicon masters for use in this experiment with 1:1 aspect ratios, correlating to a 300 μm feature depth using standard lithography techniques detailed elsewhere¹⁵ or in collaboration with a microfluidic foundry.
      NOTE: Feature depths lower than 100 μm 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.
2. 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.
3. 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 °C.
4. 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.

2. Preparing Coverslides

1. 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.
2. 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 substrate^16,17. Substrates are not required to be optically clear for fabrication; however, it facilitates microscopic analysis of fabricated substrates.
3. Rinse gold-coated coverslides with ethanol and dry under gentle nitrogen immediately before use.

3. R-μCP of Outer Annulus

1. Before R-μCP with the selectively compliant articulated robot arm (SCARA) system, calibrate the robotic tool effectors and accompanying dual camera systems using the system’s software.
   NOTE: These implements are pictured in Figure 1.

Figure 1. R-μCP System and Robotic Arm Tooling. (A) Large-scale view of R-μCP 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.

2. Manually place a PDMS stamp with 600 μm ID and 900 μm 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.
3. 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.
4. Instruct the robotic arm to etch four reference “X” 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.
5. 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)
6. Using the upward facing camera and LED illumination ring pictured in Figure 2D and E, visualize the stamp’s square reference marks and identify them with the camera software 10 times in order to determine the stamp’s average X, Y, and angular offset from the center of the robot tooling axis. (AUTOMATED PROCESS)
7. As in Figure 2A, use the downward facing camera to visualize and detect the coverslide’s etched reference marks 10 times to determine the coverslide’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’s software to precisely align the stamp’s and coverslip’s X, Y center locations and angular offsets in future R-μCP steps.
8. 3.8) Place the stamp in a bath of alkanethiol ATRP initiator, ω-mercaptoundecyl bromoisobutyrate (2 mM in ethanol) as depicted in Figure 2F. (AUTOMATED PROCESS)
9. 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’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.
10. After drying, move the stamp over the calculated center position of the gold-coated slide and lower it at 100 μm 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)
11. 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.
12. Slowly remove the stamp from the gold-coated slide and place the stamp back in the stamp nesting fixture. (AUTOMATED PROCESS)

4. SI-ATRP of PEGMEMA on Micropatterned Coverslides

1. 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.
2. 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’,2-bipyridine (3 mmol) to the Schlenk flask.
3. 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.

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.

4. 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.

5. Azide Functionalization of Micropatterned PEGMEMA Chains

1. Place the micropatterned coverslide in a 20 ml glass reaction vial.
2. Add 6 ml of N,N-dimethylformamide (DMF) containing 100 mM sodium azide to the reaction vial. Maintain this reaction at 37 °C for 24 hr.
   NOTE: Handle DMF only in a chemical fume hood. Exercise caution when weighing out sodium azide.
3. Upon completion, remove the micropatterned coverslide from the reaction vial and rinse with ethanol then water and dry under a nitrogen stream.

6. Passivation of Bromine Functionalized PEGMEMA Chains

1. Place micropatterned coverslide in 20 ml glass reaction vials.
2. Add 6 ml of dimethyl sulfoxide (DMSO) containing 100 mM ethanolamine and 300 mM triethylamine to the reaction vial. Hold this reaction at 40 °C for 24 hr.
   NOTE: Handle DMSO, ethanolamine, and triethylamine only in a chemical fume hood.
3. Upon completion remove the micropatterned coverslide from the reaction vial, rinse with ethanol then water and dry under a nitrogen stream.

7. R-μCP of Inner Annulus and SI-ATRP of PEGMEMA

1. Carry out R-μCP steps as previously described in Steps 3.2 and 3.6 – 3.12, substituting a PDMS stamp with 300 μm ID and 600 μm 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.
2. Carry out SI-ATRP steps as previously described in steps 4.1 – 4.4.

8. Acetylene Functionalization of Micropatterned PEGMEMA Chains

1. Place micropatterned coverslide in a 20 ml glass reaction vial.
2. 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.
3. Upon completion remove the micropatterned coverslide from the reaction vial and rinse with ethanol then water and dry under a nitrogen stream.

9. Copper-catalyzed “Click” Biotinylation of Acetylene Terminated PEGMEMA Chains

1. Place micropatterned coverslide in a 20 ml glass reaction vial.
2. 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 μM 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.
3. Bubble nitrogen through the reaction solution for 10 sec, seal the vial with Parafilm, and react for 24 hr at RT.
4. Upon completion remove micropatterned coverslide from the reaction vial, rinse with water and place it in a 12-well polystyrene dish.

10. Immunofluorescent Detection of Biotinylated Acetylene Groups

1. Block micropatterned coverslide in DPBS (3% Donkey Serum) for 1 hr at RT. Stain for biotinylated acetylene groups with Streptavidin-546 conjugate (2 μg/ml) in DBPS (3% Donkey Serum in PBS) for 2 hr at RT.
2. 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.

11. Copper-free “Click” Biotinylation of Azide Terminated PEGMEMA Chains

NOTE: If desired, this substrate modification step can be performed in situ during cell culture.

1. Leave micropatterned coverslide in 12-well polystyrene dish following DBPS rinses.
2. Add 2 ml of DBPS (or cell culture media) containing 20 μM DBCO-PEG₄-Biotin, and allow this reaction to continue for 24 hr at RT (or at 37 °C in an incubator).
3. Upon completion, rinse micropatterned coverslide 5 times with DPBS (or simply perform a media change).

12. Immunofluorescent Detection of Biotinylated Azide Groups

1. Stain for biotinylated azide groups with Streptavidin-488 conjugate (2 μg/ml) in 2 ml DPBS (3% Donkey Serum) for 2 hr at RT.
2. Rinse micropatterned coverslide 5 times in DPBS for 10 min with gentle agitation.
3. 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.

Results

The use of manual alignment μCP techniques to engineer culture substrates with arrays of PEG-grafted brushes functionalized with orthogonal “click” 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-μCP system is used to overcome this limitation, and its ability to accurately pattern an array of PEG brush annuli with 300 μm ID and 600 μm OD presenting terminal alkyne groups within...

Discussion

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...

Materials

List of materials used in this article

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