Photolithographic Patterning of C2C12 Myotubes using Vitronectin as GrowthSubstrate in Serum-Free Medium

Peter Molnar,* Weishi Wang, Anupama Natarajan, John W. Rumsey, and James J. Hickman

Nanoscience Technology Center, University of Central Florida, 12424 Research Parkway, Suite 400, Orlando, Florida 32826

The C2C12 cell line is frequently used as a model of skeletal muscle differentiation. In ourserum-free defined culture system, differentiation of C2C12 cells into myotubes required surface-bound signals such as substrate-adsorbed vitronectin or laminin. On the basis of this substraterequirement of myotube formation, we developed a photolithography-based method to patternC2C12 myotubes, where myotubes formed exclusively on vitronectin surface patterns. We havedetermined that the optimal line width to form single myotubes is approximately 30µm. Toillustrate a possible application of this method, we patterned myotubes on the top of commercialsubstrate-embedded microelectrodes. In contrast to previous experiments where cell patterningwas achieved by selective attachment of the cells to patterned surfaces in a medium that containedall of the factors necessary for differentiation, this study illustrates that surface patterning of asignaling molecule, which is essential for skeletal muscle differentiation in a defined system,can result in the formation of aligned myotubes on the patterns. This technique is being developedfor applications in cell biology, tissue engineering, and robotics.

Introduction

Potential applications for artificial skeletal muscle includestissue replacement, physiological and pharmacological studies,disease models, and robotics (1-5). Although skeletal muscleengineering has shown significant success in the past decade,there are still several challenges, such as low contractile forceand lack of spatial control of myotube formation and attachment,hindering the development of functional artificial skeletal muscle(1, 2, 6-8).

The goal of our investigation was to develop a method forthe integration of skeletal muscle with silicon structures usingthe standard lithographic manufacturing steps for applicationsusing MicroElectroMechanical Systems (MEMS) or othermicrodevices. As a model system, we utilized the C2C12 mouseskeletal muscle cell line because it has been frequently usedfor the study of myoblast differentiation, neuromuscular junctionformation, or muscle pharmacology (9-15). The control of theattachment of the myoblasts and their subsequent myotubeformation was accomplished by adapting a photolithography-based method that has previously been applied to pattern neuronsand other cell types (16, 17). Besides photolithography, severalother techniques (micro contact printing, micro fluidic pattern-ing, inkjet printing, etc.) have already been established to patterncells (18-28).

One of the common problems in patterning cells when usingchemical cues on a culture surface has been the necessity ofutilizing serum-free medium, because adsorbed proteins fromserum may obscure the surface patterns (17, 29). C2C12 cellshave already been shown to form myotubes under serum-freeconditions (12, 30-32). Synthetic surfaces, for example,N-(2-aminoethyl)(3- aminopropyl) trimethoxysilane (EDA) and tride-cafluoro-1,1,2,2-tetrahydrooctyl-1-dimethylchlorosilane (13F),have also been used for the study of the attachment, morphology,

and proliferation of C2C12 cells (36), but myotube formationhas not been reported on these surfaces. In the course of ourstudy we have observed that in our serum-free media formula-tion C2C12 cells also required specific surface-bound signalsto form myotubes. It was found that adsorbed Vitronectinpromoted myotube formation. Vitronectin has been an importantserum component and was among the first proteins to adsorbonto hydrophilic surfaces upon exposure to serum (33).Vit-ronectin has been reported to play a role in myotube attachmentand differentiation (34, 35). The combination of a surfacedependent cue for myotube formation and a serum-free mediumthat did not promote myotube formation by itself opened thepossibility for selective control of C2C12 myotube formationon surface patterns.

Materials and Methods

Surface Modification. Glass coverslips were cleaned usingHCl/methanol (1:1), soaked in concentrated H2SO4 for 30 min,and then rinsed in double deionized H2O. Coverslips were thenboiled in deionized water, rinsed with acetone, and oven dried.The trimethoxysilylpropyldiethylenetriamine (DETA, UnitedChemical Technologies) film was formed by the reaction ofthe cleaned surfaces with a 0.1% (v/v) mixture of the organosi-lane in toluene. The DETA coverslips were heated to just belowthe boiling point of toluene, rinsed with toluene, reheated tojust below the boiling temperature, and then oven dried. Surfaceswere characterized by contact angle and X-ray photoelectronspectroscopy methods (29). In some experiments DETA-coatedcoverslips were incubated in (1) cell culture medium+ 10%fetal bovine serum, (2) 10µg/mL (in water) vitronectin(Invitrogen), or (3) 10µg/mL (in water) laminin (Invitrogen)for 1 h at 37°C.

Surface Patterning.Quartz photomasks were designed usingthe CleWin layout editor (WieWeb, Hengelo, The Netherlands)and fabricated through a commercial vendor (BandwidthFoundry Pty Ltd., Australia). The surface of the protein-coated

* To whom correspondence should be addressed. Ph: (407) 625 5700.Fax: (407) 882 2819. Email: pmolnar@mail.ucf.edu.

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10.1021/bp060302q CCC: $37.00 © 2007 American Chemical Society and American Institute of Chemical EngineersPublished on Web 01/10/2007

coverslips that was not covered by the photomask was ablatedusing a 193 nm Ar/F LPX200i laser beam (Lambda Physik, Ft.Lauderdale, FL) combined with a beam homogenizer (Microlas,Ft. Lauderdale, FL; energy density 50 mJ/cm2) to create thepatterns.

C2C12 Culture. C2C12 cell line was obtained from ATCC.Cell stock was grown in T-75 flasks in DMEM (HyClone,SH30243.01)+ 10% fetal bovine serum at 5% CO2 and 37°C.After confluence, the cells were dissociated in cell dissociationsolution (HyClone, HyQTase) by tituration in a 5 mL pipet.After centrifugation (500g, 5 min), the C2C12 cells werereplated in differentiation medium (DMEM+ 1% B27 supple-ment, Invitrogen) on the glass coverslips.

All cell culture experiments were repeated 3 times with 3coverslips in each of the experiments.

Immunohistochemistry. Coverslips were fixed in-20 °Cmethanol for 5 min and followed by permeabilization in PBS+ 1% BSA+ 0.05% saponin (permeabilization solution) for 5min. The nonspecific binding sites were blocked using perme-abilization solution+ 5% donkey serum (blocking solution) for

30 min at room temperature. C2C12 myotube coverslips werethen incubated in anti-embryonic, myosin heavy chain, primaryantibody (F1.652, Developmental Hybridoma Studies Bank) anddiluted 1:4 in a blocking solution overnight at 4°C. Thecoverslips were then incubated in secondary antibody (MolecularProbes A-21202), diluted 1:200 in a blocking solution for 2 hat room temperature.

Results and Discussion

Surface-Bound Signals Required for Myotube Formation.Our first observation was that, in contrast to the published results(12, 30-32), C2C12 myoblasts did not differentiate intomyotubes when replated in our serum-free medium formulation.However, we observed that the myotube formation occurredonly when, instead of replating the cells at confluency in theserum-free medium, we just replaced the serum-containingmedium with the serum-free formulation. On the basis of thisobservation, we repeated our initial experiments, but beforereplating we incubated the culture surface (which was eithercell-culture quality plastic or DETA-coated coverslips) with the

Figure 1. Substrate dependence of myotube formation of the C2C12 myoblasts. Cells were cultured on the surfaces for 10 days in serum-freemedium. (A) Cell-culture quality plastic. (B) Plastic coated with serum proteins. (C) DETA coverslips coated with serum proteins. (D) DETAcoverslips. (E) DETA coverslip coated with laminin. (F) DETA coverslip coated with vitronectin. Phase contrast, scale bar) 200 µm.

Figure 2. Line-width dependence of myotube formation. (A) Mask design with 50, 30, 20, 10, 5, 2, and 1µm lines. (B) Vitronectin-coatedcoverslips were patterned using this mask, and the C2C12 cells were then plated on the patterns. After 5 days single myotubes were observed onthe 30µm lines. Thicker lines resulted in the formation of multiple myotubes, whereas myotubes did not form on the thinner lines. (C) Myotubes,plated on 30µm lines, were visualized by immunostaining for the myosin heavy chain. Scale bar) 200 µm.

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serum-containing medium for 1 h and followed that with rinsingthe coverslips in serum-free medium 3 times. This short surfacetreatment resulted in myotube formation under serum-freeconditions using our medium formulation. Our conclusion wasthat factors that were adsorbed from the serum-containingmedium to the culture substrate were necessary for C2C12myoblast differentiation. To support the observation, Fauxcheauet al. (2004) had reported that vitronectin (adsorbed to the culturesurface from serum) was the main attachment protein mediatingthe initial adhesion of the cells in the presence of serum (33).Therefore, we repeated our experiments with C2C12 cells usingcell-culture quality plastic and DETA-coated coverslips incu-bated with 10% serum, 10µg/mL vitronectin, or 10µg/mLlaminin (a commonly used cell-culture substrate interacting withintegrin receptors) for 1 h. Although initial cell attachment wasthe same on all surfaces, myotube formation was not observedon the untreated surfaces; see Figure 1. Moreover, by day 5 invitro C2C12 cells were detaching from these surfaces. Myotubeformation was started at day 3 for the vitronectin-treated surface,day 5 for serum, and about day 7 for laminin. Myotubeformation was strongest on the vitronectin-coated surface. Theeffect of vitronectin on myotube formation was concentration-dependent (0.01-10 µg/mL range, data not shown). Bothvitronectin and laminin are known substrates of integrinreceptors. They have been shown to cause muscle differentiationin Drosophila embryo cells and follow alternate intermediatedifferentiation steps without affecting the final outcome (35).

Control of Myotube Formation through Patterning ofVitronectin on the Surface. In our serum-free conditions thedifferentiation of the C2C12 cells was very sensitive to thesurface coatings. Myotubes were formed only on those surfaceareas where vitronectin was present. On the basis of thisobservation, we adopted a photolithography-based technique(laser ablation,37) in order to create vitronectin patterns onthe culture surface and used these patterns to subsequentlycontrol the myotube formation. Using patterns of different linewidths, we determined that below a 10µm line width myotubesdid not form, at a 30µm width single myotubes formed, andabove a 30µm width multiple myotubes formed on the lines(Figure 2). This observation was important because thisdependence of myotube formation on the pattern dimensionsmakes it possible to separate different cell types, for example,neurons and myotubes, just by pattern geometry (neurons growrelatively well on 5µm lines) in future co-culture experiments.

To demonstrate the control of myotube formation utilizingsurface patterns on a recording platform, we registered thesepatterns with an array of substrate-embedded microelectrodes(Figure 3). These microelectrodes could be useful for selective

stimulation of single myotubes in robotics, MEMS, or drugscreening applications.

Conclusion

We have shown that myotube formation by C2C12 myoblastsis highly dependent on the culture surface under serum-freeconditions. In contrast to previous experiments where surfacepatternes were created to guide only the initial attachment ofthe cells in a medium that contained all of the factors necessaryfor differentiation, in this study patterning was achieved by thecombination of a surface dependent cue for myotube formationand a serum-free medium that did not promote myotubeformation by itself. This study is an example for the develop-ment of an active surface that determines the behavior of abiological system through surface-immobilized and patternedsignaling molecules and could inspire a wide range of applica-tions of this technique. In our laboratory this method is beingfurther developed for applications in cell biology, tissueengineering and robotics.

Acknowledgment

This work was supported by the NIH Career DevelopmentAward K01 EB003465-03.

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Figure 3. Patterning of the C2C12 myotubes on the surface of the substrate embedded microelectrodes. The pattern consisted of 30µm thickparallel lines connected with 2µm thick lines at the electrodes. Myotubes formed almost exclusively on the 30µm lines, but some myoblasts (notdifferentiating into myotubes) were attaching to the 2µm lines. In rare cases myotubes formed on the 2µm lines, which could be the result of anerror in the patterning process. Phase contrast, Day 5. (A) Scale bar) 200 µm. (B) Scale bar) 100 µm.

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Received October 2, 2006. Accepted December 7, 2006.

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