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In vitro generation of differentiated cardiac myofibers on

micropatterned laminin surfaces

Todd C. McDevitt,1 John C. Angello,2 Marsha L. Whitney,1,3 Hans Reinecke,3 Stephen D. Hauschka,2

Charles E. Murry,3 Patrick S. Stayton1

1Department of Bioengineering, University of Washington, Seattle, Washington 981952Department of Biochemistry, University of Washington, Seattle, Washington 981953Department of Pathology, University of Washington, Seattle, Washington 98195

Received 17 April 2001; revised 3 July 2001; accepted 19 July 2001

Abstract: Cardiac muscle fibers consist of highly alignedcardiomyocytes containing myofibrils oriented parallel tothe fiber axis, and successive cardiomyocytes are intercon-nected at their ends through specialized junctional com-plexes (intercalated disks). Cell culture studies of cardiacmyofibrils and intercalated disks are complicated by the factthat cardiomyocytes become extremely flattened and exhibitdisorganized myofibrils and diffuse intercellular junctionswith neighboring cells. In this study we sought to direct theorganization of cultured cardiomyocytes to more closely re-semble that found in vivo. Lanes of laminin 550 m widewere microcontact-printed onto nonadhesive (BSA-coated)surfaces. Adherent cardiomyocytes responded to the spatialconstraints by forming elongated, rod-shaped cells whosemyofibrils aligned parallel to the laminin lanes. Patternedcardiomyocytes displayed a striking, bipolar localization of

the junction molecules N-cadherin and connexin43 that ul-trastructurally resembled intercalated disks. When lamininlanes were widely spaced, each lane of cardiomyocytes beatindependently, but with narrow-spacing cells bridged be-tween lanes, yielding aligned fields of synchronously beat-ing cardiomyocytes. Similar cardiomyocyte patterns wereachieved on the biodegradable polymer PLGA, suggestingthat patterned cardiomyocytes could be used in myocardialtissue engineering. Such highly patterned cultures could beused in cell biology and physiology studies, which requireaccurate reproduction of native myocardial architecture. 2002 Wiley Periodicals, Inc. J Biomed Mater Res 60: 472479, 2002; DOI 10.1008/jbm.1292

Key words: cardiomyocyte; tissue engineering; cell arrays;intercalated disks; micropatterning

INTRODUCTION

Spatially defined adhesive cues play important rolesduring biological development and later in directingtissue organization and repair in mature tissues. Re-cent advances in microfabrication have provided newapproaches to control the spatial organization of pro-teins on surfaces, in ways that mimic naturally occur-

ring spatial cues.1,2 Microfabrication techniques arethus providing important new avenues for investigat-ing fundamental biological questions, including stud-ies designed to define the relationships between cellshape and function.35 A variety of cell types, includ-ing macrophages and neural and bone cells, have beenpatterned on microfabricated surfaces.69 The abilityto spatially organize these cells into complex and dif-

ferentiated structures is also providing new opportu-nities for developing better sensing, drug screening,and tissue engineering technologies.1012

Cardiomyocytes in native myocardial tissue are or-ganized into parallel cardiac muscle fibers with intra-cellular contractile myofibrils oriented parallel to thelong axis of each cell and junctional complexes be-tween abutting cells concentrated at the ends of eachcardiomyocyte. This highly oriented cytoarchitectureis critical for the proper electromechanical coupling ofcardiomyocytes to stimulate the transmission of di-rected contraction over long distances. In contrast, cul-tured cardiomyocytes typically spread to form an epi-

Correspondence to: P. S. Stayton; e-mail: stayton@u.washington.edu; or C. E. Murry; e-mail: murry@u.washington.edu; or S. D. Hauschka; e-mail: haus@u.washington.edu

Contract grant sponsor: National Science FoundationContract grant sponsor: University of Washington Engi-

neered Biomaterials Engineering Research Center; contractgrant number: EEC-9529161

Contract grant sponsor: National Institutes of Health; con-tract grant numbers: HL64387-01 (to P.S., S.H., C.E.M.),HL61553 (to C.E.M.)

2002 Wiley Periodicals, Inc.

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thelioid sheet, with disorganized myofibrils and dif-fuse intercellular junctions, bearing little similarity tonormal myocardial morphology.

Previous attempts to align cardiomyocytes in vitrohave used etching or photolithographic techniques togenerate linear surface features, with subsequent ad-

sorption of serum protein mixtures to support di-rected cell adhesion.1315 These important studieshave shown that conduction velocities and action po-tentials were faster in the oriented strands of cardio-myocytes and, in fact, were similar to adult mousemyocardium.

Here we used microcontact printing of laminin toestablish an in vitro system in which spatially definedcues from the substrate guided cardiomyocyte align-ment and the development of normal cytoarchitecture.Microcontact printing is a simple, versatile method todirectly pattern adhesive proteins on a wide variety ofsurfaces, including common polystyrene dishes. Theprinted protein patterns provide high resolution tostudy and control how cardiomyocytes respond tospatial adhesion cues. We show that neonatal cardio-myocytes cultured on laminin lanes form rod-shapedcells with highly aligned myofibrils and bipolar inter-calated disks. Such micropatterned cells form synchro-nously beating myofibers that resemble those in nativemyocardium. This route to organizing cardiomyocytesinto more natural structures should provide new op-portunities for studying their cell biology and physi-ology and may also be of use for cell array-basedscreening and tissue engineering applications.

MATERIALS AND METHODS

Micropatterning of extracellular matrix proteins

Laminin patterning was performed using microcontactprinting techniques, similar to methods previously de-scribed.1618 Silicon wafers were patterned with photoresist(AZ1512; Clariant Corporation) by standard photolithogra-phy using a photomask purchased from Photosciences.Polydimethylsiloxane stamps (Sylgard 184; Dow) were cast

against the patterned silicon wafers and cured overnight at65C. Stamps were cut to 12 cm2 and coated with laminin-1(Becton Dickinson, derived from Engelbreth-Holm-Swarmmouse tumor) at 45 g/mL in PBS, pH 7.4, for 3045 min atroom temperature and then rinsed and dried under nitro-gen. Stamps were placed laminin-side down for 510 min atroom temperature onto 35-mm polystyrene dishes (Falcon)that had been pre incubated with 1% BSA in PBS overnightat 4C, rinsed, and then dried under nitrogen immediatelybefore printing. Protein patterned dishes were stored in ster-ile PBS before cell plating. Thin PLGA membranes (85:15composition) spin-coated onto glass coverslips were pro-vided by Dr. Jonathan Mansbridge of Advanced Tissue Sci-ences, Inc. PLGA-coated coverslips were patterned as de-

scribed above and secured with double-sided Scotch tape to35- or 60-mm polystyrene dishes. Laminin lane pattern sta- bility was assessed using laminin conjugated to OregonGreen 488 (Molecular Probes).

Cell culture

Cardiomyocytes were freshly isolated from the ventriclesof 1- to 2-day-old rat pups and cultured at 37C, 5% CO2 aspreviously described.19,20 Culture media consisted of a 3:1mixture of DMEM:M199 supplemented with 10% horse se-rum, 5% fetal bovine serum, L-glutamine, HEPES (17 mM),and penicillin-streptomycin. After isolation, the cells wereplated onto the patterned 35-mm polystyrene dishes andallowed to attach overnight (1517 h). The plates were rinsedwith Dulbeccos phosphate-buffered saline (DPBS, pH 7.4;Sigma) to remove nonadherent cells and then refed withculture media containing 1 M cytosine arabinofuranoside

(ara-C; Sigma) to prevent fibroblast overgrowth. Thereafter,cultures were refed with ara-C containing media every 23days.

Immunostaining

Cardiomyocyte cultures were fixed for 23 min with 3%paraformaldehyde (PFA) in PBS, pH 7.4, 5 mM EGTA, 0.2%Triton X-100 at room temperature and then fixed with 3%PFA in PBS for 30 min. The samples were blocked with 2%rabbit serum in PBS for 1 h at room temperature or over-night at 4C; all subsequent antibodies and stains were di-luted in the same blocking buffer. Samples were treated withprimary antibodies to sarcomeric myosin heavy chain(MF20), connexin43 (Chemicon) or pan-cadherin (Sigma) asdescribed.20 Primary antibodies were incubated for 6090min at room temperature, followed by a secondary rabbitanti-mouse FITC-conjugated antibody (1:20; DAKO) at roomtemperature for 6090 min or overnight at 4C. Lastly, thecells were counterstained with BODIPY phalloidin 558/568(1:100; Molecular Probes) to detect actin filaments and DAPI(1:500; Sigma) to detect nuclei, mounted with Vectashieldmedia (Vector), coverslipped, and stored in the dark at 4Cbefore microscopy. Cardiac tissue from adult rats was em-bedded in OCT (Miles Scientific) and cryosectioned at 5 m.Sections were dried overnight, fixed, and immunostainedfor connexin43 and N-cadherin as described for the culturedcells.

Microscopy

Fluorescent images were captured with a Nikon EclipseE800 microscope equipped with a Photometrix SenSys digi-tal camera. Phase contrast imaging of live cultures was per-formed using a Nikon Eclipse TE200 microscope within aplexiglass enclosure heated to 37C. Still images were cap-tured by a Hamamatsu C4742-98 digital camera, and video

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microscopy was recorded using a Hamamatsu C2400 CCDcamera. Time-lapse microscopy was performed with a Ni-kon Diaphot microscope equipped with a video camera (Se-ries 65; Dage-MTI, Inc.) and a time-lapse recorder (ModelTLC 2015R; GYYR Products). Time-lapse cultures on 35-mmplates were enclosed in a T25 flask and gassed with 5% CO2to equilibrate the atmosphere.

For electron microscopy, cultures were fixed with Karnov-sky solution in 0.1% cacodylate buffer, processed through agraded alcohol series and propylene oxide, and embeddedin LR/White plastic (Polyscience, Warrington, PA) in thetissue culture dishes. Random areas were cut out and thin-sectioned en face. Rat heart samples were fixed and pro-cessed with the same solutions. Cell culture and tissue thinsections were poststained with uranyl acetate and lead cit-rate and examined with a JEOL electron microscope(JEM-1200EXII).

RESULTS

Cardiomyocyte patterning

Spatially defined laminin patterns on a nonadhesivebackground were constructed by microcontact print-ing onto a BSA monolayer applied to polystyrenedishes (Fig. 1, insets). Protein patterns were stable inaqueous buffer or in serum-containing media at 37Cin the absence of cells for at least 4 weeks (longest timetested). Rat neonatal cardiomyocytes took 26 h to at-tach and spread on the laminin lanes as assessed by

time-lapse video microscopy, and the cells displayedvery little motility along the lanes thereafter. Becausemost neonatal cardiomyocytes are nonproliferative,cell coverage of the laminin lanes depended on the

initial seeding density and subsequent cell spreading.At seeding densities of 250,000400,000 cells/35-mmdish, there were many gaps between cells along indi-vidual lanes at 1824 h; by the next day the patternedlanes became almost completely filled due to addi-tional cell spreading. After 48 h, nearly all of the car-

diomyocytes had formed cell-cell junctions with adja-cent cells in the same lane and were contracting.

Although cardiac fibroblasts constituted

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accommodate 1 or 2 adjacent cells, and 45- to 50-mlanes contained up to 4 adjacent cells.

The dimensions of typical adult rat ventricular cellsin vivo are 1530 m in diameter by 100130 mlong.21,22 Ventricular cardiomyocytes in the develop-ing rat heart have cross-sectional diameters of about 6m at birth, and this dimension increases to

14 m

by 60 days23; cardiomyocyte length undergoes a com-parable relative increase during this period. These di-mensions generate an aspect ratio (AR), defined as themajor axis divided by the minor axis, of 37. To de-termine how the shapes of neonatal rat cardiomyo-cytes were affected by culture on laminin lanes, ARswere calculated for isolated cells 3 days after seedingon different lane widths. The mean AR decreased aslane width increased: AR = 9.2 3.8, 4.9 1.5, and 3.0 1.4 on 5-, 15-, and 30-m lanes, respectively. Indi-vidual cardiomyocytes on unpatterned laminin or onlaminin lanes >30 m wide were more highly spread,with an average AR of 1.8 0.7. Although we wereunable to obtain accurate measurements of cellheights, it was evident that cardiomyocytes grown on5- to 15-m laminin lanes had a much more three-dimensional cell topology compared with thosegrown on unpatterned laminin. However, fewer cellsdeveloped end-to-end contact with adjacent cells on5-m lanes, whereas most of the cells on 10- to 20-mlane widths made bipolar contacts, and most of thelane surfaces were covered. Although lane coveragewas largely dependent on the cell plating density,these data suggested 5-m lanes were too narrow to

support optimal cell adhesion and junction formation.

Myofibril alignment

The mechanical work of cardiomyocytes in hearttissue requires myofibril alignment parallel to the longaxis of cardiac muscle fibers. Because the myofibrils incardiomyocytes grown on unpatterned surfaces arerandomly aligned, it was of interest to determinewhether their orientation would be influenced bygrowth on laminin patterns. Immunostaining with a

myosin heavy chain antibody (data not shown) as wellas electron microscopic analysis indicated that myofi-

bril orientation was strongly aligned by culture onlaminin lanes. Patterned cells had a high density ofparallel myofibrils, whereas the myofibrils in cellsgrown on unpatterned laminin were in disarray andoften branched at acute angles (Fig. 2). Like normalmyocardium, sarcomeres in patterned cultures wereoften in register across an entire cell width, and theaverage widths of myofibrils closely resembled thosein the neonatal rat heart. In addition, the myofibrils on

both sides of junctions between patterned cardiomyo-

cytes were oriented in the same direction, whereas inunpatterned cultures, myofibrils often occurred at ran-dom orientations relative to those in the adjoining cell(Fig. 2). The elongated shape of mitochondria andtheir locations between myofibrils in patterned cellswere also more similar to that observed in native myo-

cardial tissue. These results demonstrate that cardio-myocytes respond to the imposed adhesive cues byorganizing normal myofibril structures over long dis-tances in a manner very similar to that found in vivo.

Formation of intercalated disks

Cardiomyocytes in heart tissue connect to the abut-ting cells within cardiac muscle fibers by intercalateddisk cell-cell junctions containing N-cadherin and con-nexin43. These proteins play key roles in adherens andelectrochemical gap junctions. If cardiomyocytes onlaminin lanes exhibited localized concentrations of N-cadherin and connexin43 and cytoarchitecture resem-

bling intercalated disks, this could potentiate thetransmission of cell-to-cell linear electrochemical sig-nals, as occurs in vivo.

When cardiomyocytes were cultured on lane widthssimilar to adult cellular diameters (i.e. 1520 m), theyresponded by forming precisely aligned and bipolarcell-cell junctions. Electron microscopy showed thatthese junctions resembled normal intercalated disksfound in vivo (Fig. 2) and that they contained bothdesmosomes and intermediate (adherens) junctions.

Expression of N-cadherin was visible by immuno-staining after 1 day in vitro and increased in intensityover the next 48 h. N-cadherin was concentrated at the

bipolar cell junctions in discrete bands that resembledintercalated disks (Fig. 3). In contrast, on wider lami-nin patterns of 3050 m, which accommodated 24cells per lane-width, some N-cadherin staining wasobserved along both the short and the long axes ofadjacent cardiomyocytes. The gap junctional proteinconnexin43 was also observed predominantly at the

bipolar cell junctions (Fig. 3), and its localization ap-peared more punctate than the concentrated bands ofN-cadherin. In cardiomyocytes grown on unpatterned

laminin, a similar time course of N-cadherin and con-nexin43 appearance was observed, but the stainingwas not distributed in the bipolar fashion found innative tissue. Instead, it occurred circumferentiallyaround the cell perimeter, wherever there was contact

between cells (Fig. 3).

Contractile activity

Contraction of individual cells was first detectedabout 24 h after plating, and by 48 h, after the forma-

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tion of intercalated disks, entire lanes of cardiomyo-cytes were contracting in synchrony. Contraction ratesof the patterned cardiomyocytes reached maximal lev-els of 150 beats/min after 34 days. No significant

differences in contraction rates were observed for car-diomyocytes cultured on different adhesive lanewidths. However, the beat synchrony between adja-cent lanes was due to the ability of cells to extendbridges between adjacent lanes, which was depen-dent on the distance between lane patterns (Fig. 4).Many cell bridges across lanes were observed with10-m separation distances, leading to a high degreeof contraction synchrony between adjacent lanes (Fig.4). Significantly fewer bridges were observed as thespacing was increased (i.e., 20- and 40-m separationdistances) and 80 m spacing essentially inhibited car-

diomyocyte bridging between lanes, thus adjacentlanes beat asynchronously.

Organization on PLGA surfaces

To determine whether microcontact printing andcardiomyocyte patterning could be performed on bio-degradable synthetic polymer surfaces, such as thosecommonly used in tissue engineering scaffolds, lami-nin lanes were printed onto thin PLGA films. Analysisof laminin persistence beneath cell lanes (Fig. 5, inset)showed a lower fluorescent intensity and a pitted ap-pearance after 5 days, consistent with the degradationof PLGA in the aqueous culture media. The alignment,

Figure 2. Transmission electron microscopy characterization of myofibril structure and organization (top row) and celljunction morphologies (bottom row). Unpatterned cultures (left panels) and patterned cultures (30-m lane 20-m spacing,middle panels) after 4 days, are compared to rat neonatal cardiac tissue (right panels). Myofibrils (Mf), mitochondria (mito),nuclei (Nu), and capillaries (Cap) are identified. Arrowheads: the sarcolemmal boundary of individual cardiomyocytes.Arrows: the sites of intercellular junctions containing intermediate junctions and desmosomes (bottom row). Myofibril andcell junction assembly in patterned cultures is comparable to native tissue, whereas unpatterned cultures exhibit no suchorganization.

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cytoarchitecture and contraction of cardiomyocytepatterns on PLGA were comparable to those on poly-styrene for at least a week (longest time assessed),suggesting that PLGA could be used as a transientscaffold for patterning cardiomyocytes.

DISCUSSION

These studies indicate that microcontact printingcan be used to create patterns of extracellular matrixproteins that organize cardiomyocytes into fibers thatresemble those found in native tissue. Although mul-ticellular strands of cardiomyocytes have been orga-nized on photolithographically patterned chemicalsurfaces, microcontact printing of matrix proteins isless technically demanding than photolithographyand is compatible with many substrate materials. Mi-

crocontact printing should thus provide a convenientmethod for studying extracellular matrix-cell interac-tions as well as developmental and physiologicalquestions pertaining to the mechanisms of myofibril,sarcoplasmic reticulum, and intercalated disk forma-tion, and the electrochemical and mechanical couplingof cardiomyocytes.

We have shown that neonatal rat cardiomyocytesform highly organized arrays in response to spatiallycontrolled adhesive cues. The cardiomyocytes assumerod-like geometries and develop highly aligned myo-fibrils with normal diameters and bipolar cell junc-tions with intercalated disk connections that includespatially localized N-cadherin and connexin43. The re-sulting cardiomyocyte organization closely resemblesthat found in native tissue. In addition, by controllingthe distances between laminin lanes, cardiomyocytesin adjacent lanes can be engineered to contract inde-pendently or in synchrony.

Figure 3. Immunofluorescent staining for electrical and mechanical components of intercalated disks. Unpatterned culturesafter 7 days (left), 20 20 m patterned cultures after 4 days (middle), and sections from adult rat heart (right) wereimmunostained for either N-cadherin (top row, green) or connexin43 (bottom row, green), actin filaments were counter-stained with phalloidin (red), and nuclei were stained with DAPI (blue). N-cadherin and connexin43 localization betweenadjacent cells in patterned cultures resembled that found in the intercalated disks of mature cardiac tissue, whereas unpat-terned cardiomyocytes exhibited circumferential staining. Cell nuclei of nonmyocytes appear between the cardiac myofibersof the native tissue.

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A recent study by Thomas et al.15 reported on theelectrophysiological properties of neonatal mouse car-diomyocytes grown in strands 3586 m in diameter,guided by photolithographic patterning of coverslipsthat directed subsequent serum protein adsorption.They found that conduction velocities and action po-tentials were faster and closer to adult mouse myocar-dium in cardiomyocytes grown in strands versus ran-domly oriented cultures. These physiological mea-surements complement our structural and molecularobservations and provide further evidence that spatialorganization can direct cardiomyocyte cytoarchitec-

ture to resemble that observed in vivo.In addition to their usefulness for studies of cardiac

cell biology and physiology, patterned cardiomyocytecultures should be well suited to array technologiesfor screening and diagnostic applications that require

better reproduction of myocardial architecture andsynchronized contraction. Also, because the micro-patterning technique can be readily applied to biode-gradable polymeric substrates such as PLGA, mi-cropatterning strategies could be used for controllingthe development of oriented muscle for cardiac tissueengineering applications. These strategies comple-ment those of other investigators who have incorpo-

Figure 4. Synchrony of cardiomyocyte contraction in patterned cultures. Videomicroscopy was performed to record thecontraction of live cardiomyocyte cultures after 2 days, and the rates of individual lanes of beating cells were quantified.Representative phase images are shown for cardiomyocytes on either (A) 30 80- or (B) 30 10-m patterns. The contractionrates within individual lanes were plotted. Adjacent, widely spaced lanes of cardiomyocytes beat asynchronously (A),whereas narrowly spaced lanes of the same width exhibited a high degree of synchronous contraction (B), because of cellbridging between lanes. Some of the cellular bridges are identified with arrows.

Figure 5. Cardiomyocyte patterning on a biodegradabletissue engineering scaffold. Cardiomyocytes were culturedfor 5 days on thin PLGA membranes with 20 20 m pat-terns of laminin conjugated to Oregon Green 488. Cultureswere then fixed and stained with phalloidin (red) and DAPI(blue) to permit fluorescent microscopy analysis. The greaternumber of fibroblasts bridging between lanes is due to ahigher percentage of these cells in this particular cardiomyo-cyte preparation. Laminin lanes underlying the cells werewell retained on the PLGA surface during this time course(inset).

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rated cardiomyocytes into three-dimensional syn-thetic and natural polymer matrices.2426 The resultingorganization of cardiomyocytes into highly aligned ar-rays with natural cytoarchitecture and cell junctionscould greatly improve engineered tissue function.

The authors thank Mr. Ron Hanson for performing thecardiomyocyte isolations, Ms. Veronica Poppa for extensiveassistance with immunostaining and electron microscopy,and Dr. Kip Hauch for his microscopy expertise. The authorsalso gratefully acknowledge the Washington TechnologyCenter Microfabrication Laboratory.

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