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.
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.
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 “click” 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 (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 substrate8,9. 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 us6,14 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.
1. Generating Elastomeric Stamps
2. Preparing Coverslides
3. R-μCP of Outer Annulus
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.
4. SI-ATRP of PEGMEMA on Micropatterned Coverslides
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.
5. Azide Functionalization of Micropatterned PEGMEMA Chains
6. Passivation of Bromine Functionalized PEGMEMA Chains
7. R-μCP of Inner Annulus and SI-ATRP of PEGMEMA
8. Acetylene Functionalization of Micropatterned PEGMEMA Chains
9. Copper-catalyzed “Click” Biotinylation of Acetylene Terminated PEGMEMA Chains
10. Immunofluorescent Detection of Biotinylated Acetylene Groups
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.
12. Immunofluorescent Detection of Biotinylated Azide Groups
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 a separate array of PEG brush annuli with 600 μm ID and 900 μm OD presenting terminal azide groups is demonstrated14. Following the reaction of alkyne presenting PEG brushes with Azide-PEG3-Biotin and the reaction of azide presenting PEG brushes with DBCO-PEG4-Biotin, the substrate was immunostained with fluorescent probes and imaged using a confocal microscope (Figure 4). Analysis of these images using a custom MATLAB program calculated that the two PEG brush arrays were aligned with sub-10 μm accuracy, i.e., X ~ 6.4 μm, Y ~ 1.7 μm, and θ ~ 0.02°, which is at the manufacturer’s cited limit of our SCARA model (i.e., ~10 μm) and indicative of the R-μCP system’s prior performance14. Thus, we believe that the use of higher-end SCARAs in this system would provide even lower, sub-micron resolution. These results demonstrate the versatile substrate engineering capabilities enabled by the combined use of the R-μCP system and sequential nucleophilic substitution reactions. The minimal cross-reactivity evidenced by fluorescent labeling of the orthogonal surface chemistries serves to illustrate the potential of this system for precise immobilization of biochemical cues to generate complex culture substrates for tissue engineering applications.
Figure 2. Schematic of R-μCP Process. (A) Downward facing camera visualizing immobilized gold-coated slide, (B) etching reference marks on gold-coated coverslide, (C) removing PDMS stamp from stamp nesting fixture, (D–E) visualizing PDMS stamp reference marks with upward facing camera, (F) placing PDMS stamp in alkanethiol initiator bath, (G) drying alkanethiol initiator solvent over nitrogen streams, and (H) stamping alkanethiol coated PDMS stamp on gold-coated coverslide.
Figure 4. Immunostained Images of MicroPatterned Slide. R-μCP micropatterned coverslide orthogonally functionalized to present terminal (A) azide and (B) alkyne groups, biotinylated using copper-free and copper-mediated click chemistry, and detected with Streptavidin-488 and -546 respectively. (C) Overlay of both fluorescent channels. All scale bars 500 μm.
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 orientations on substrates. While simulating all the endogenous factors of a cell’s niche in vitro is unreasonable, the R-μCP system described here allows for immobilization of multiple regions of orthogonal bioactive chemistries with microscale control of spatial orientation.
The utility of these substrates increases further when considering that while PEG brush arrays bound with immobilized ligands can elicit biospecific interactions, unbound PEG brushes remain resistant to protein adsorption and thus can serve to control cell adhesion and migration. Although passive, micropatterned adsorption of extracellular matrix (ECM) proteins or polymer-PEG conjugates is a simpler method for controlling cell adhesion and migration, these technique only provide transient control since adsorption is a reversible process11. Also, molecules adsorb to surfaces in unpredictable orientations and at random concentrations thereby limiting the fidelity and control of biospecific interactions. Moreover, compounds typically used to create non-fouling regions, such as bovine serum albumin or Pluronic F127, do not possess designated reactive sites for further functionalization limiting post-adsorption in situ modifications. SAMs of alkanethiols can be generated with such reactive groups for additional surface modification, but they too have limited durability in tissue culture conditions due to the lack of oxidative shielding afforded by grafted PEG brushes10,11. Conversely, the merger of our R-μCP platforms with PEG-brush grafting and sequential nucleophilic substitution reactions provides the ability to engineer substrates with durable, microscale cytophobic regions that can be modified a priori or in situ with bio-orthogonal functionalities. This will permit greater control of biospecific interactions that can regulate in vitro tissue morphology and growth.
A key strength of R-μCP compared to other systems for aligning sequential microcontact printings is the ability to remove the substrate from the system between repeated stampings while still maintaining high alignment accuracy12,13. This enables greater diversity in the types of surface chemistries that can be applied, and the duration of surface modification reactions can be varied to tune the densities of subsequently immobilized ligands6. Thus, R-μCP can be used to establish focal concentration gradients in the biochemical cues that mediate biospecific interactions14. With the capacity to precisely control both the location and degree of biospecific interactions, the R-μCP system offers a unique method for investigating the roles of bioactive ligands in tissue morphogenesis and establishes a robust system for generating culture substrates simulating the presentation of biological cues in the in vivo niche.
A critical aspect of cellular niches, specifically within development, is spatiotemporal modulation of signaling factors18. Though soluble biochemical cues can be added to culture media in a temporally defined manner, there is limited spatial control over which cells encounter these cues. With the R-μCP system described here, it is possible to construct micropatterned culture substrates with terminally functionalized PEG brushes presenting azide functional groups that have no effect on cell fate in culture. While azide groups are capable of undergoing copper-catalyzed “click” reactions with alkyne presenting molecules, this cannot be performed in the presence of cells in culture given the toxicity of copper. However, high-strain molecules like dibenzocyclooctyne (DBCO) undergo a highly selective and biocompatible copper-free azide-alkyne cycloaddition reaction19,20. Through conjugation of DBCO-containing linkers, micropatterned culture substrates can be modified in situ with new biological ligands21. With the ability to render cytophobic regions cytophilic or add different biochemical cues for use in multi-step signaling procedures, culture substrates generated with this method have novel adaptive capabilities and thus provide a more robust system for instructing tissue morphogenesis in vitro.
Despite the immense potential and utility of the R-μCP platform, it does have certain drawbacks. The use of numerous SI-ATRP steps to generate PEG-grafted substrates greatly increases the fabrication time compared to methods involving inkjet printing or microdroplet deposition of ECM proteins. While the precision of inkjet printing techniques rivals that of the current R-μCP system, the adsorption of ECM proteins is reversible thereby providing less control of cell-protein interactions. Also, substrates generated via inkjet printing techniques cannot be removed during fabrication, thereby impeding the use of diverse substrate modification chemistries that permit, for example, spatially restricted in situ substrate modifications22. Due to these limitations inkjet techniques are better suited for rapid generation of culture substrates primarily concerned with short-duration, static arrangements of biomolecules and cell types23 whereas the R-μCP system is much better suited toward generating multi-faceted substrates designed to exhibit long-term, dynamic control over both spatial and temporal cellular interactions in 2D with diverse biological ligands.
Microcontact printing enables rapid deposition of nano-to-microscale ‘ink’ features over large surface areas, but there has always been significant heterogeneity in the concentration of deposited ‘ink’ substances. This is also a current limitation of the R-μCP platform as can be observed in Figure 4. We hypothesize that the heterogeneity in fluorescent modification of the PEG brushes is due to the robot’s application of an uneven normal force over the entire PDMS stamp, which is also likely not perfectly flat. This results in non-uniform contact pressure between all regions of the stamp and the substrate thereby causing uneven deposition of the alkanethiol initiator and subsequent thickness of the grafted PEG brush. In order to manufacture PEG-grafted substrates with even deposition of alkanethiols and thereby surface functionality, the stamping tool design will need to be optimized to apply a distributed and uniform normal force over the entire PDMS stamp. This will facilitate uniform contact with the gold-coated slides across the entire interface independent of stamp imperfections. Also, in Figure 4B, one can observe slight cross-contamination of acetylene groups on the azide terminated PEG brush. While the surface density of immobilized ligands due to cross-contamination is minimal compared to that on the intended PEG-brush, this can be reduced to near zero by increasing the duration of the ethanolamine passivation reaction (see Section 6) as demonstrated previously6.
The primary application of the R-μCP system described here is for manufacturing complex tissue culture substrates that could be used to exert spatial and temporal control of cell fate. However, the high precision and accuracy of the R-μCP platform makes it an attractive method for other applications as well. The ability to pattern cytophilic areas with differential ligand chemistries followed by in situ modification of previously inert, cytophobic regions allows for co-culture of multiple cell lines with tight control over their spatial orientation. This coupled with the inherent high-throughput nature of micropatterning present an alternative to current methods for high-throughput screening of both single cells and multi-cell combinations24. While the R-μCP system has great potential in the realm of biology, it could also be applied to the field of microelectromechanical systems (MEMS). In MEMS fabrication, it is desirable to transfer MEMS components with high precision and accuracy for mass production. With novel kinetic stamping techniques, the R-μCP system described here could be adapted to effectively print components of silicon or gallium nitride on silicon wafers for use in generating MEMS25. Thus, the R-μCP platform could be used for a wide range of potential applications.
In conclusion, use of the R-μCP system to generate PEG-grafted culture substrates orthogonally functionalized using sequential nucleophilic substitution reactions presents not only an ideal platform for potentially controlling tissue morphology and growth in vitro, but an excellent system for investigating the roles of multiple bioactive ligands on cell fate. The ability to pattern multiple biochemical cues in distinct and highly ordered patterns establishes the groundwork for building culture substrates capable of instructing the formation of tissue structures with multiple cell types organized at the micron scale. This, coupled with the ability to modify micropatterned substrates in situ, could allow for unparalleled control of tissue morphogenesis and cell fate in culture. The patterning techniques described here provide a versatile system for manufacturing culture substrates that could one day facilitate rational and reproducible production of organotypic tissue structures in vitro.
The authors have nothing to disclose.
Funding for this work, GTK, TK, and JDM were provided by the Wisconsin Institute for Discovery and the Wisconsin Alumni Research Foundation.
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. |
24mm 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 | |
20mL 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. |