Technical Note

Novel transparent nano- to micro-heterogeneous substrates for in-situ cellmigration study

Irene Y. Tsai,1 J. Angelo Green,2 Masahiro Kimura,3 Bruce Jacobson,2 Thomas P. Russell11Polymer Science and Engineering Department, University of Massachusetts, Amherst, Massachusetts 010032Program in Molecular and Cellular Biology and Department of Biochemistry and Molecular Biology,University of Massachusetts at Amherst, Massachusetts 010033Films and Film Products Research Laboratories, Toray Industries, Inc., 1-1, Sonoyama 1-chome, Otsu,Shiga 520-8558, Japan

Received 29 June 2005; revised 10 May 2006; accepted 23 May 2006Published online 21 November 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30935

Abstract: Transparent substrates having heterogeneitiesranging from nanometer to micrometer lateral length scalewere fabricated to study cell migration. The surfaces weregenerated using thin films of block copolymers and homo-polymer blends on ultra smooth transparent polyethyleneterephthalate films. Results show that the lateral size scaleof the surface heterogeneities affects fibroblast (NIH-3T3)adhesion, spreading and motility. More specifically, fibro-blasts migrate faster on micron-sized than on nanometer-sized heterogeneities. Cell movements and morphology onthe micron patterned surfaces resemble cells cultured in a

3D environment. These surfaces, therefore, can potentiallybe utilized as models to study cell behavior in physiologi-cally relevant conditions which can add to our fundamen-tal understanding of cell-substrate interactions and facili-tate development of surfaces for medical devices. � 2006Wiley Periodicals, Inc. J Biomed Mater Res 80A: 509–512,2007

Key words: heterogeneous surfaces; topography; fibro-blasts; cell migration; lateral length scales

INTRODUCTION

Understanding cell migration is important in bio-technological applications including cellular trans-plantation, tissue engineering, and the making ofbiochips,1,2 and, as a consequence, material design pa-rameters that control cell migration are under intenseinvestigation.3–5 Here, we describe a method to fabri-cate transparent substrate with patterned topographicfeatures, nanometer to micros in size, suitable forin situmicroscopy investigations of cell motility. Under-standing the role of the lateral size of the features is a

key in the design of materials for medical devices anddeveloping in vitromodel systems.

Cells live in the extracellular matrix, an environ-ment consisting of proteins, polysaccharides, pro-teoglycans, and other macromolecules that formhierarchical structures over multiple length scales.Collagen fibrils, for example, with a 66 nm structuralrepeating period form macroscopic aggregates. Theseassembled hierarchical structures are evident in tis-sues as cables of the tendons, as regular layers in thebones, and as matrix of the cornea.6 Studying theinteractions between the cells and the hierarchicalstructures are central in understanding cell adhesionand migration.7 Many experiments have shown thatcell adhesion can be influenced by patterning thesurface on the nanometer and micrometer lengthscales.8–10 However, studies of cell migration onnanoscopic features have been limited, since mostpatterning techniques rely on conventional photoli-thography to generate the patterns. Yet, developingrobust routes to fabricate topographic features onany length scales and methods to determine optimallength scales for a given application is a key to un-derstanding cell–substrate interactions. Here, we de-

Correspondence to: T. P. Russell; e-mail: russell@mail.pse.umass.eduContract grant sponsor: National Institutes of Health;

contract grant numbers: GM-29127, T32-GM08515, 5 T32HL007284-27Contract grant sponsor: The U.S. Department of Energy

and The National Science Foundation (Material ResearchScience and Engineering Center)

' 2006 Wiley Periodicals, Inc.

scribe the use of homopolymer blends and diblockcopolymers on ultra-smooth polyethylene terephtha-late (PET) film to fabricate a transparent substrate tovisualize cell motility. Thin films of homopolymerblends macroscopically phase separate with featuresizes typically on the micron length scale, whilediblock copolymers microphase separate into well-defined arrays of domains tens of nanometers insize.11,12 Thus, the size scale can be tuned continu-ously from the nanoscopic to the macroscopic byusing mixtures of blends with copolymers where therelative concentrations of the components are varied.Our previous studies demonstrated that cell adhe-sion was affected by size scale of topographic fea-tures on a surface.13 Here, we extend these studiesonto ultra-smooth transparent PET films to investi-gate cell migration. This will have tremendous impli-cations on optimizing scaffolds for tissue engineeringor surfaces for microfluidic devices, biochips, ormedical applications.

Studies have shown that cell migration is influ-enced by both surface chemistry and topography.14

Cells will migrate along topographic features whenplated onto a chemically uniform surface, a phenom-enon known as contact guidance15 that is crucial inembryonic morphogenesis and wound healing. Cellsalso respond to chemical signals when they areseeded onto adhesive and nonadhesive patterns.16 Inthis study, cell movements were recorded on PETsurfaces coated with topographic features of oxi-dized polystyrene (PS) films and surfaces coatedwith random copolymers of PS and poly(methylmethacrylate), P(S-r-MMA), where the lateral size ofthe heterogeneities was varied from the nanometerto micrometer length scale. We have already shownthat the cell spreading area and degree of actin stressfiber formation increased as the lateral length scalebetween the oxidized PS surfaces decreased. Cellspreading area analysis revealed preferential interac-tion of the cells with the oxidized PS as opposed tothe surface modified with P(S-r-MMA); and, manyfilopodia and lamellipodia were found to interactwith the oxidized PS surfaces. This report extendsthese previous studies to cell migration.

EXPERIMENTAL

Surface preparation

Substrates with heterogeneities varying in lateral lengthscale from nanometers to micrometers were prepared byusing thin films of mixtures of homopolymers with diblockcopolymers. The relative concentrations of homopolymersand block copolymers were changed to control feature size.Asymmetric diblock copolymers of PS and PMMA denotedP(S-b-MMA) with a molecular weight of 73,000 and a poly-

dispersity 1.04 were prepared by standard anionic polymer-ization methods. The volume fraction of PS in the PS-b-MMA was 0.7 and, consequently, the equilibrium morphol-ogy of the copolymer consists of 20 nm diameter cylindricaldomains of PMMA in a PS matrix with an average separa-tion distance of 30 nm. PS (MW ¼ 52,000) and PMMA(MW ¼ 29,000) with narrow molecular weight distributionswere purchased from Polymer Laboratories and used with-out further purification. Four separate surfaces havingdifferent concentrations of PS-b-MMA mixed with PS andPMMA were used. One percent solutions of the blends intoluene were spin coated onto an ultra-smooth PET surfaceto which a hydroxy-terminated random copolymer of styreneand methyl methacrylate, P(S-r-MMA), having a styrene frac-tion of 0.58, was anchored. With the anchored P(S-r-MMA),the interfacial interactions of the substrate with P(S-b-MMA),PS, and PMMA are balanced.

The thin film mixtures were annealed at 1708C, reactiveion etched with oxygen to remove the top *6 nm of thefilm, and then exposed to ultraviolet radiation for 35 minto crosslink the PS and degrade the PMMA. The films weresubsequently washed with acetic acid to remove PMMA,rinsed with water, and dried. The PS remaining on thesubstrate is oxidized under these conditions. A schematicdiagram of the surface preparation is shown in Figure 3.The AFM images of the heterogeneous surfaces were usedto determine the average length scale of the surface pat-terns using autocorrelation.

Tissue culture and cell migration assays

NIH-3T3 cells were from American Type Culture Collec-tion (Rockville, MD). Cells were maintained as subconflu-ent monolayers in DMEM supplemented with 10% (vol/

Figure 1. AFM images of (A) micron and (B) nano pat-terns and their corresponding optical images of time-lapsecell migration at time ¼ 0 min.

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vol) calf serum (Atlanta Biologicals, Atlanta, GA), 100 mg/mL dihydrosteptomycin, and 60 U/mL penicillin (Sigma,St. Louis, MO), in a humidified 378C incubator with 5%CO2. Tissue culture plates and flasks were from VWR(West Chester, PA).

Cells were detached from culture vessels with 0.01%trypsin-EDTA, washed with Hank’s Balanced Salt Solution,and resuspended in fresh DMEM without phenol red(Sigma) in 10% serum. Cells were plated on the patternedsurfaces, which were placed in a 150 � 25 mm2 tissue cul-ture plate and allowed to spread for 3 h in a 378C/5% CO2

incubator. The cells were then transferred to a concealedmicroscope chamber maintained at 378C with 5% CO2.Time-lapse phase images were collected at 10 min intervalsover a period of 600 min, using a Nikon Eclipse TE-300inverted microscopes and a CCD100 video camera andScion Corporation LG3-01 frame grabber.

RESULTS AND DISCUSSION

Transparent ultra-smooth PET films (Mictron,Toray Industries) were used as a substrate. Hydroxy-ter-minated random copolymer of P(S-r-MMA) was end-grafted onto the PET film by coating a thin film ofP(S-r-MMA) from toluene followed by annealing at1708C for 2 days. Subsequently, the films were washedwith toluene, leaving a 6 nm layer of P(S-r-MMA)on the PET surface. The fraction of styrene in theP(S-r-MMA) was 0.58, which corresponds to a com-position where interaction of the P(S-r-MMA) withPS and poly(methyl methacrylate) (PMMA) are bal-anced. This was done to orient the microdomain

morphology normal to the surface. One percent solu-tions of homopolymer blends and mixtures of theblends with diblock copolymers were spin coatedonto the modified PET film. Subsequently, a oxygenreactive ion etch was used to remove a layer of PS atthe surface, and then the sample was exposed to UVand washed with acetic acid to remove the PMMA.This leaves an oxidized film of PS on the surfacewith topographic features that are characteristic ofthe morphology of the thin polymer mixture or co-polymer film.

Fibroblasts (NIH 3T3) on surface with nanoscopicfeatures showed distinctly different cellular morphol-ogy, cytoskeletal organization, and migration speedfrom those cultured on surfaces having micron-sizedfeatures. The majority of cells on micron patternedsubstrates exhibit more polarized morphology thancells on the nano patterns. Many more filopodia, thinprojections from the cell, were visible on the micron-sized patterned surfaces than on the nanopatternedsurface (Fig. 1). On the other hand, fibroblasts on thenano features have more lamellipodia or fan-shapedmorphology. These phenotypes are consistent with theobservations seen previously reported by us for celladhesion. Cells are larger and form more stress fiberson nano patterns than on micron-sized features of thesame surface chemistry. Furthermore, the cells on themicron features showed similar characteristics as thosereported for cells in 3D matrices,17 and therefore, themicron-sized topographic surfaces may be a goodmodel for the study of cell migration or cell division inmore physiologically relevant conditions.

Quantitative cell migration studies on the heteroge-neous surfaces with heterogeneities on the nanometerto micrometer length-scale shows a dramatic differ-ence in cell speed. As shown in Figure 2, there is a dra-matic change in cell speed when the feature size is*60 nm when compared with cells migrating on*110 nm features. This is consistent with the resultsfrom cell adhesion kinetics of Massia and Hubbell,where it was demonstrated that the surface thresholdspacings for focal contact and stress fiber formationoccurs at 140 nm.18 Maheshwari et al.,19 on the otherhand, found significant changes in focal adhesion andstress fiber formation for substrate features of *60 nmin size. This difference may arise from the differencesin cell type, rigidity of the substrate, and the level ofaffinity between the cell and the surface. Our result isconsistent with the observation that cells that migratefaster form fewer stress fibers, and have less spreadingarea. Although no significant difference in cell speedwas detected for cells on surfaces with features rang-ing from *110 nm to the microns, other changes incell signaling, genotype, or matrix remodeling mayoccur. Further studies in these areas would offer in-triguing insights to better design materials for biomed-ical applications. It should also be noted that cell

Figure 2. Average cell migration speed on patterned sub-strates of characteristic length scales of 2 mm to 60 nm andflat oxidized PS. Error bars indicate standard error of themean (SEM) of three experiments. *Statistically significantdeviations (p ¼ 0.05 using ANOVA) between 60 and 110nm. **Statistically significant deviations (p ¼ 0.05 usingANOVA) between 2 mm and flat oxidized PS.

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speeds on the nano patterned and flat oxidized PS sur-faces were not significantly different from each other,which is in keeping with our previous results on celladhesion.

SUMMARY

In summary, a transparent smooth PET substratemodified with heterogeneities ranging from thenanometer to micrometer length scale were preparedon smooth PET films using thin films of blockcopolymers and their mixtures with homopolymers.These substrates were used for in situ cell migrationstudies. Results show that fibroblast (NIH-3T3) adhe-sion, spreading, and motility are influenced by sizescale of the surface heterogeneities. Fibroblasts mi-grate faster on micron-sized than on nanometer-sizedheterogeneities. Moreover, cell movement on the micronpatterned surfaces are reminiscent to the cells culturedin a 3D environment, opening the potential use ofthe surfaces as models to study cell behavior inphysiological relevant conditions which may provideimportant advances to our fundamental understand-

ing of cell–substrate interactions and developmentfor materials of medical devices.

We thank Toray for supplying the ultra smooth PET film.

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Figure 3. Schematic diagram of the fabrication of trans-parent nano- to microheterogeneous substrates usingdiblock copolymers and homopolymer blends on ultra-smooth PET film.

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