2011 - Chen Et Al - Advanced Materials

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A. ChenDepartmUniversIrvine, I E-mail: Dr. D. KDepartmDivisionUniversDavis, Davis, CA 95616, USA L. FreschProf. C. DepartmUniversiIrvine, Ir J. Wang ,Stem CeLKS FacuThe UniPokfulamE-mail: r

DOI: 10

Studies of cellular responses to topographies ranging from nano- to microscales are of great importance to fundamental cell biology as well as to applications in stem cell biology and tissue engineering. [ 13 ] Leveraging traditional fabrication tech-niques originally developed for the semiconductor industry, researchers have been able to precisely control the topograph-ical features of in vitro substrata to better understand the inter-action between cells and their microenvironments. Previous studiesance, dgraphiadult sthe effcytoskeexpresscan be environthe subevant a

Whilength availabpattern

a narrow size range at either the microscale, or more recently, the nanoscale. [ 1 , 5 , 1115 ] Although such designs are helpful in studying a controlled cellular behavior, they do not represent the physiological conditions of native tissue necessary for tissue engineering. Natures ordering is dramatically different from the precisely periodic arrays produced from high precision fab-rication approaches. In vivo, the organization of the extracellular matrix (ECM) varies dramatically in its structural arrangement,

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Aaron Chen , Deborah K. Lieu , Lauren Freschauf , Valerie Lew , Himanshu Sharma , Jiaxian Wang , Diep Nguyen , Ioannis Karakikes , Roger J. Hajjar , Ajay Gopinathan , Elliot Botvinick , Charless C. Fowlkes , Ronald A. Li , * and Michelle Khine *

Shrink-Film Confi gurable Multiscale Wrinkles for Functional Alignment of Human Embryonic Stem Cells and their Cardiac Derivatives

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Prof. C. CDepartment of Computer Science 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 5785wileyonlinelibrary.comr. 2011, 23, 57855791

auf , V. Lew , D. Nguyen , Prof. E. Botvinick , C. Fowlkes, Prof. M. Khine ent of Biomedical Engineering

ty of California vine, CA 92697, USA Prof. R. A. Lill & Regenerative Medicine Consortium lty of Medicine

versity of Hong Kong , Hong Kong

[email protected]

.1002/adma.201103463

University of California Irvine, Irvine, CA 92697, USA Prof. R. A. Li, Heart, Brain Hormone & Healthy Aging Research Center Department of Medicine and Physiology LKS Faculty of Medicine University of Hong Kong, Hong Kong , H. Sharma , Prof. M. Khine ent of Chemical Engineering and Material Science

ity of California rvine, CA 92697, USA [email protected] . Lieu ent of Internal Medicine

of Cardiovascular Medicine ity of California

have demonstrated the phenomenon of contact guid-irected alignment and migration along lines of topo-

c anisotropy, using a variety of cellsfrom myocytes to tem cellswith a range of responses. [ 36 ] For example, ects of contact guidance have been shown to induce letal rearrangement, nuclear deformation, and gene ion changes in fi broblasts. [ 4 ] In addition, stem cell fate solely determined by the mechanical cues of their micro-ment in the absence of soluble factors. [ 610 ] Therefore, strates used for such studies need to be biologically rel-nd mimic the in vivo microenvironment. le it has been shown that biophysical cues of various scales affect cells differently, [ 24 ] the majority of currently le fabricated topographies have simple and repetitive s of grooves or ridges of either a homogenous size or of

contentcollagensimilar fi bers aexperiephysiolscales treadily niques.substanlimiting

To atunablecreate mnano- t

J. Wang ,CardiovaMount SNew Yor Prof. A. GSchool oUniversiMerced, texture, and fi ber bundle thickness. For example, , the main structural component in ECM, form self- brils (20100s of nm), which in turn form bundles and ross several orders of magnitude. [ 16 ] While cells in vivo ce topographies with features across a vast size range, gically comparable cellular environments with length at span several orders of magnitude have not been imulated by precision micro- or nanofabrication tech-Achieving such multiscale features typically relies on ial capital equipment and/or fabrication expertise [ 2 , 17 ] their accessibility to biological laboratories. dress these challenges, we introduce an ultrarapid,

robust, facile, and inexpensive fabrication method to ultiscaled self-similar alignment grooves ranging from micrometers as biomimetic cell culture substrates.

r. I. Karakikes , Prof. R. J. Hajjar, Prof. R. A. Li cular Research Center nai School of Medicine , NY 10029, USA opinathan Natural Sciences y of California

erced, CA 95344, USA . Fowlkes

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Commgauge)alignedareas ocult to(e-beamIn conthe plaplasmametal stiffnesshrinkneed foloxane can alssuch aalignm

OxidcreatesPE fi lmprestrebriefl y outer odistribplete fa10 minthrougwrinklthe wrihierarcscopy (which predomto 7 m(Figure

Figure 1sides an(v) to bwise. InBright fiwavelengshoulderand 15 (Pand P15 grooves ithicker athe fi rst Further wrinklesforms mkles formturing ofthere arefrom the

odity shrink fi lm, prestressed polyethylene (PE) (0.5 mil , is oxidized and subsequently shrunk uniaxially to create grooves, or wrinkles ( Figure 1 a), over relatively large f clinically relevant size (1 cm 6 cm), which is diffi -

achieve with other approaches such as electron-beam ) lithography or nanoimprint lithography (Figure 1 b).

trast, our patterned size is only limited by the size of sma chamber and is amenable to large-scale roll-to-roll systems. Instead of depositing nanometric layers of by e-beam or sputter coating in order to generate the s mismatch to create wrinkles upon the retraction of the

fi lm as previous reported, [ 18 , 19 ] we have now obviated the r a metal layer altogether. Unlike silicon, polydimethylsi-(PDMS), or glass substrates, this thermoplastic substrate

. a) Process fl ow of shrink-fi lm wrinkle formation. i) PE fi lm is treated with oxygd thermally shrunk to create unidirectional wrinkles. iii) Shrunk PE fi lm is cut i

e used in cell culture experiment. b) Preshrunk (left) and shrunk (right) fi lms. Uset shows the P5 condition rolled-up into tubes. The direction of wrinkles can eld microscopy images taken from the tubes illustrate the direction of wrinkles 2011 WILEY-VCH Verlag GmbH & Co. nelibrary.com

(AFM) nested hwith disconfi rmto the (Figure larger wplasma tskin thiclack of blayer. Th310 nm,P15, resthe rang

To evbiomim(MEFs), embryoncoated wmold foGrowth

o be easily heat-molded into various nonplanar shapes s tubes to serve as 3D scaffolds for studying endothelial ent for blood vessels (Figure 1 b). ation is achieved by oxygen plasma treatment, which a thin and relatively stiff layer at the surface of bulk . Leveraging the inherent retraction properties of the

ssed fi lm, which shrinks uniaxially by 90% by heating to 150 C, this mismatch in stiffness causes the stiff xidized layer to buckle and form predictably controllable

utions of nano- and microwrinkles ( Figure 2 a). [ 18 ] Com-brication of the topographical substrate takes less than . The properties of the aligned wrinkles can be controlled h the plasma treatment time. Interestingly, self-similar es form across various length scales with bundling of nkles apparent at longer oxidation times. The multiscale hy is revealed through various scanning electron micro-SEM) image magnifi cations. At lower magnifi cation, in the smaller wrinkles cannot be resolved, the apparent inant wavelength (major wrinkles) range shifts from 1 as plasma treatment time increases from 1 to 15 min

2 a). At higher magnifi cation, the apparent predominant ths (minor wrinkles) are 380, 100, and 200 nm with peaks at 200, 60, and 150 nm for the 1 (P1), 5 (P5), 15) min plasma treatment conditions, respectively (P1

data not shown). The formation of aligned hierarchical s due to different generations of effective layers that are nd stiffer than the previous layer. [ 20 ] Upon shrinking, generation of wrinkles and a new effective layer form. shrinking induces formation of a new generation of until the shrinking process is complete. This process ultiscale grooves as each successive generation of wrin-

with wavelengths similar to the hierarchical struc- collagen bands. [ 16 ] The data from Figure 2 a reveal that at least two generations of P5 wrinkles. As is apparent graph, and confi rmed by the atomic force microscopy

en plasma for 5 min (P5). ii) PE fi lm is constrained on opposite nto desired dimension and iv) mounted onto a glass coverslip pon heating, the PE fi lm will shrink approximately 90% length-be either parallel or perpendicular to the long axis of the tube. . Images are taken at 4 magnifi cation. KGaA, Weinheim Adv. Mater. 2011, 23, 57855791

measurements (Supporting Information Figure 1), a ierarchy is apparent at the 5 and 15 min plasma times,

tinct size populations. In addition, AFM measurements that the thin layer of Matrigel coating was conformal wrinkles and did not obscure substrate topography 2 b). The distribution of wrinkles is controllable, with rinkle sizes becoming more dominant with increased ime, in agreement with our theoretical model based on kness. [ 18 ] At 1 min, we suspect the larger wrinkles and undling is due to an insuffi ciently thick and stiff outer e average depths of wrinkles are 159 nm, 247 nm,

and 233 nm for P1, P5, P5 with Matrigel coating, and pectively (Figure 2 b). Since the wrinkles are multiscale, e of depth spans several order of magnitudes. aluate the effectiveness of contact guidance using these etic wrinkles, we align mouse embryonic fi broblasts aortic smooth muscle cells (AoSMCs), and human ic stem cells (hESCs). The wrinkles can be directly ith ECM for cell culture or, alternatively, used as a

r various tissue engineering biodegradable polymers. arrested MEFs are grown on glass coverslip, P0 (no


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to alignkles witestablishphase ofsensed over timfeeder-frtion Figpended gate) gra pluriptering don the won the

treatment), P1, P5, and P15 substrates for 24 h. Six sub-per condition are included to ensure the consistency of actin is stained to visualize cytoskeleton of MEFs. MEFs on both glass coverslip and P0 exhibit random contact e, whereas MEFs grown on the P1, P5, and P15 sub-

align within 24 h ( Figure 3 a). Moreover, more cells are on the P5 than those grown on the P1 and P15, as indi-y the orientation distribution polar plots. This is likely he range of P5 wrinkles (60 nm to 3 m), which is sim-hat of natural ECM fi brils. Therefore, the P5 substrate en for the subsequent AoSMC and hESC alignment ents. e there has been a report that hESCs align to 600 nm in the presence of differentiation media, [ 21 ] the ability

a) i) Schematic of wrinkle formation. Two generations of heirarchical wrinklesouter layer and the color represents the soft bulk polymer. ii) SEM images wlar wrinkles for P1, P5, and P15 conditions. The bundling of the wrinkles is app

of SEM images for the three different plasma conditions. Inset shows the sM magnifi cation. b) AFM indicates the bundles and the height range of the to the topography of the P5 wrinkled substrate. The scan distance of the AFMinkle depths for P0, P1, P5 with and without Matrigel coating, and P15. MM

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and maintain the pluripotent hESCs on the wrin-hout differentiation media over time is important for ed differentiation protocols that require an expansion pluripotent hESCs. [ 22 , 23 ] Despite the mechanical cues

by the hESCs, the cells remain relatively pluripotent e when cultured on the wrinkled substrate under ee pluripotent conditions (Supporting Informa-ure 2). [ 24 ] On both days 3 and 6, healthy hESCs sus-as single cells (Supporting Information Figure 2b, P1 own on both control and wrinkles express TRA-1-81, otent surface marker. Notably, forward and side scat-ata from fl ow cytometry indicate that the cells grown rinkled substrate remain healthier than those grown controls (Supporting Information Figure 2b). The

are demonstrated in the diagram. The black outline represents ith progressive zooms of 1000 and 10 000 , illustrating the arent. iii) Feature scale distribution estimated from the Fourier maller population of wrinkles at 20 000 , not resolvable at the wrinkles. The AFM image demonstrates the Matrigel coating image is 5 5 m 2 . The table summarizes the average and the

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complexthe highhowever

ase in side scattering of the same cell type over time typi- indicates a less healthy state of cells, which corresponds

increase in cellular surface granularity and internal

3 . Alignment of various types of cells on the wrinkles. a) Fluorescent microscopy imled substrates for 24 h. The polar plots reveal orientation distribution of f-actin for ce and 30 images total. 90 and 270 are the direction of wrinkles. Thinner lines indicae lapse f-actin and nuclei alignment over 72 h (bar graph, mean standard deviatio on glass coverslip, and it is an average of all controls from all time points ( n = 81 imaph, solid line) and circularity (right axis of graph, dash line) as compared to other cell = 9 images ( > 300 nuclei) per time point, and p < 0.001 and p < 0.01. For (b) anKGaA, Weinheim Adv. Mater. 2011, 23, 57855791

ity. With increasing culture time, more cells shift into side scattering region for both controls and wrinkles; , the percent of cells remaining in P1 is considerably

ages of MEFs cultured on control (glass slide), P0, P1, P5, and P15 lls grown on different substrates. Each plot is an average of 6 rep-te standard deviation. b) Subcellular hESC alignment as indicated n (s.d.), n = 9 images per time point). Control, C, represents cells ges). c) Nuclear deformation as indicated by nuclear area (left axis types including AoSMCs and MEFs. Bar graph represents mean d (c), red is f-actin (rhodamine) and blue denotes nuclei (DAPI).


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higher for cells grown on wrinkles (17.4%) than those grown on the controls (6.4%).

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demonstrate for the fi rst time the subcellular time-lapse nse to topography of feeder-free pluripotent hESCs without le differentiation factors. The alignment is assessed based actin and nuclear alignment (Figure 3 b). Time lapse ng of the cells indicate that the majority of cells align to rinkles within the fi rst 4 h of plating, with more than 40% cells stably aligning to within 15 of the wrinkle direc-As a point of comparison, to achieve roughly the same e of alignment by fl ow with endothelial cells requires shear stress for 10 h. [ 25 ] The hESC nuclei are also more d compared to a study of fi broblast nuclei on 12.5 m

microgrooved topography at 24 h culture time. [ 4 ] tably, we demonstrate for the fi rst time the deformation ripotent hESC nuclei due to topography. The deformed

i of hESCs exhibit a decreased surface area, in agree-with topography-induced direct mechanotransduction. [ 26 ] uclei of undifferentiated stem cells are more mechani-

plastic than those of differentiated cells and change as a on of differentiation. [ 5 , 27 ] We reveal the highly compliant i of hESCs through the measurements of nuclear area ircularity as compared to AoSMCs and MEFs (Figure 3 c). ESCs exhibit both a decreased average projected area,

365 to 325 m 2 , as well as a decreased average circularity , from 0.75 to 0.65, in response to the alignment ( p < ); this is in stark contrast to MEF, where nuclei circularity tered ( p < 0.001) by the wrinkles but their projected area ot affected. As apparent from the graphs, AoSMC nuclei form ( p < 0.01 for projected area and p < 0.001 for cir-ty), but to a lesser degree compared to the more plastic ferentiated hESC nuclei. derstanding how the cell perceives topographical cues and ates that information to the nucleus to commence mech-nsductive signaling could enable a strategy of controlled cell differentiation without the need for either invasive li or chemical inducement with defi ned media. [ 28 ] Impor-, because the multiscale topography inherent to this sub- is easily tuned by plasma treatment time, it is possible p the effect of local topography on subcellular responses orting Information Figure 3). This would allow, for ple, the role of each length scale in contact guidance to be ated. Because comparatively little is yet understood with

ct to the effects of hESC alignment on differentiation, it is tant and now practical to test the range of physiologically nt cues in a comprehensive format refl ective of the multi- typical of in vivo substrata. er successfully aligning a variety of cells, we next test er physiological functionality improves with alignment. best of our knowledge, the characterization of the elec-

ysiology of aligned hESC-derived cardiomyocyte (hESC-onolayer by measuring action potential (AP) propagation

an optical mapping technique is demonstrated the fi rst In the native heart tissue, alignment of cardiomyocytes butes to the anisotropic tissue structure and facilitates inated mechanical contraction and electrical propagation. lignment of cardiac muscle cells has been studied previ-using various microfabrication techniques; [ 3 , 29 , 30 ] however, l patterned substrates used in the tissue engineering do

cardiommoldednuclei, aof connNNCMsthe unpof nativment olayer. Tthroughwith orThe arrand orgFor cells8% of ttively. FremainsCM mothan thMovie 1on the the poin2.13 0reportedcontrastgation w0.20 cmtransverporting (LCV/TCLCV is olayer, wwrinklealigned of unpaanisotroand 200ANFS cOf noteon moltion expmay vermolecultheless, reprodulayer grunder tAP propand TCaligned

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ly, we demonstrated the alignment of murine neonatal yocytes (NNCMs) and hESC-CMs on PDMS substrates from metallic wrinkles by fl uorescent staining of the ctin, and cardiac troponin. [ 19 ] Furthermore, the staining xin-43, a gap junction protein, revealed that the aligned exhibited a more defi ned and consistent network than atterned cells suggesting a more natural arrangement CMs. This hypothesis is corroborated by the measure- alignment and AP propagation of hESC-CM mono-e alignment of hESC-CM monolayer is determined

f-actin stain, and the sarcomeric striation is revealed ientation perpendicular to the wrinkles ( Figure 4 a). angement of sarcomeric structures is more apparent nized for the aligned cells than the unpatterned ones. grown on the P0 and P5 substrates, 22 2% and 45 e cells align to 15 of the wrinkles on day 2, respec-rthermore, the alignment of the hESC-CM monolayer

relatively unchanged until day 7. The aligned hESC-nolayer also exhibits a more synchronized contraction unpatterned one on day 2 (Supporting Information and 2). AP propagates across the monolayer grown control (P0) substrate expands uniformly away from t of stimulation with an average conduction velocity of .11 cm s 1 , which is similar to the value we previous (Figure 4 and Supporting Information Movie 3). [ 31 ] In , the aligned monolayer exhibits an anisotropic propa-ith faster longitudinal conduction velocity (LCV, 1.82

s 1 ) parallel to the direction of wrinkles than that of the se conduction velocity (TCV, 0.95 0.08 cm s 1 ) (Sup-Information Movie 4), giving rise to an anisotropic ratio V) of 1.92 0.20 (vs. 1.00 0.05 of P0). The slower ikely related to the morphology of the hESC-CM mon-hich is a consequence of the size and depth of the . Previously, Kim et al. demonstrated that the LCV of neonatal rat ventricular myocytes is slower than that tterned cells when the ridge, groove, and depth of the pically nanofabricated substratum (ANFS) are 150, 50, nm, respectively, [ 3 ] and tuning of the dimensions of the an affect the AP propagation and conduction velocity. , the absolute conduction velocity depends critically cular properties such as connexin-encoded gap junc-ression; although microgroove-induced directionality

y well affect connexin expression and function, further ar and cellular investigations will be required. None-anisotropy seen in native heart tissue has been clearly ced. The difference in AP propagation for the mono-wn on the control and P5 substrates is more apparent e isochrone mapping (25 ms intervals). The directed

agation and nearly twofold difference between the LCV V suggests the improved functionality of hESC-CMs on the P5 substrate. lignment of various cell types including pluripotent

as well as the demonstrated functionality of aligned M are some examples of how this substrate can be rapidly and easily perform otherwise challenging bio-tudies. The ability to affect contact guidance of hESCs ucidate critical molecular pathways and lead to directed tiation into specifi c lineages. Our aligned patterned

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substrates enabled us to reproduce the anisotropy seen in the native heart and are therefore a more accurate in vitro model. Our prehESC-Ctained rare warimprovesuch a rpatible mappincell typapplicat

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Figure 4 . a) Fluorescent microscopy images of hESC-CM cultured on con-trol (P0) and P5 wrinkled substrates for 2 days. Insets represent magnifi ed regions where sarcomeric structures of hESC-CM are compared between conditions. Bar graph (mean s.d., n = 9 images per time point) repre-sents the percentage of cells aligns to 15 of the direction of wrinkles. Control is an average of both day 2 and 7 cultures. b) Characterization of electrophysiology of hESC-CM monolayers. Point stimulation (1.5 Hz, 8 V, 20 ms duration), as indicated by the white circle, was applied to the monolayers plated on P0 (control) and P5 (aligned) surfaces. The direc-tion of wrinkles is indicated by the double sided arrows, and the substrate is indicated by the white border. AP propagations at 0 ms and 400 ms are

shown, aconductithe LCV 0.95 0.1.91. p KGaA, Weinheim Adv. Mater. 2011, 23, 57855791

liminary data further show that aligned monolayers of Ms are less susceptible than unaligned controls to sus-eentry arrhythmias (data not shown). Further studies ranted and may lead to the design of safer grafts with d effi cacy for future clinical applications. Importantly, obust, easy to fabricate and confi gurable platform (com-with microtiter plates and spatiotemporal imaging/g) could enable ubiquitous alignment of any adherent e for various tissue engineering and injury repair ions.

mental Section ation of Wrinkled Substrate : Wrinkled substrates were fabricated fi lms (Cryovac D-fi lm, LD935, Sealed Air Corporation). PE fi lm, a 2.5 in. by 5.5 in. strip, was placed lengthwise onto a glass e fi lm was treated with oxygen plasma (Plasma Prep II, SPI

) for 1, 5, or 15 min. After plasma treatment, a PE piece was ed on opposite sides with binder clips (2 in. binder clips; x), and was thermally shrunk at 150 C for 3 min to generate rinkles. The PE wrinkled fi lm was trimmed and mounted to a

lass coverslip (wrinkled substrate) for cellular plating. Wrinkled s were sterilized by immersing in 70% ethanol and under UV 0 min inside a biosafety cabinet.

cterization of Wrinkles : To characterize the wrinkles, we d SEM and AFM. For the SEM, wrinkled substrates were sputter olaron SC7620) with 3 nm gold/palladium. SEM images were

on each wrinkled substrate with 1000, 5000, 10 000, and 20 000 ations, 10 kV beam, and 12 mm working distance (Hitachi FE-SEM Scanning Electron Microscope). Images were analyzed ourier transform using a MATLAB (MathWorks Inc., Natick, ) code developed in-house. AFM was conducted on a MFP-3D optical microscope (Asylum Research, Santa Barbara, CA). graphic of images of the 1, 5, and 15 min plasma treatment s were taken in tapping mode. Silicon tips with a resonant

y of about 75 kHz and force constant of 3 N m 1 were used. are used for data acquisition and analysis was IGOR Pro 6.0

trics, Portland, OR). ultures : Details of culturing AoSMC, MEF, and hESC are in the Supporting Information.

lignment : In the AoSMC, MEF, and hESC-CM alignment study, e loaded onto wrinkled substrates, coated with Matrigel (1:200 and placed inside a 24-well plate, at a density of 5 10 4 cells for both AoSMCs and MEFs, and at a density of 5 10 5 for s. End-point cell staining was performed at 24 h for both

and MEFs and at 48 h for hESC-CMs. eder-independent hESC alignment study fi rst required that the substrates be coated in Matrigel for a minimum of 24 h. The ng density was 5 10 4 cells per well. Daily exchange of hESC ed medium was required for culturing the cells. End-point cell

was performed at nine time points: 0.5, 1, 1.5, 2, 4, 6, 12, 24, . Confocal microscopy images (Laser Scanning Microscopy 710, re taken on all samples from each time point for further image

e long term culture experiment, feeder-independent hESCs tured on both control and wrinkled substrates, coated with

nd isochrone maps are spaced at 25 ms intervals. The average on velocity for the control is 2.13 0.11 cm s 1 ( n = 3), and and TCV for the aligned monolayer are 1.82 0.20 cm s 1 and 08 cm s 1 ( n = 4), respectively. The ratio of LCV to TCV is about < 0.001 and p < 0.05.


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[ 5 ] A. S. Nathan , B. M. Baker , N. L. Nerurkar , R. L. Mauck , Acta Bio-mater. 2010 , 7 , 57 .

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Matrigel and inserted in 6-well culture plates. The cell loading densities were 2.5 10 5 and 1 10 5 cells per well for 3 and 6 day culture, respectively. The medium was exchanged daily but no passaging was performpooled

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ed. Each condition had three replicates, and all replicates were and end-point fl ow cytometry was performed. iomyocyte Differentiation : Details are described in the Supporting

ation. surement of hESC-CM Electrophysiology : Optical mapping of

opagation was performed on hESC-CM monolayer by using Ultima fl uorescence imaging system (SciMedia) with a 1 cm 2 -view. The hESC-CMs were cultured on the matrigel-coated P5 te for 3 to 4 days to allow establishment of intercellular electrical tions before the imaging measurements. To visualize AP ation, the hESC-CM monolayer on P5 substrate was fi rst stained M voltage-sensitive dye di-4-ANEPPS (Invitrogen) for 5 min at emperature in Tyrodes solution (140 m M NaCl, 5 m M KCl, 1 m M 1 m M CaCl 2 , 10 m M glucose, and 10 m M HEPES at pH 7.4). The s excited by a halogen light source fi ltered by a 515 35 nm band-lter and emission was fi ltered by a 590 nm high-pass fi lter. The ere stimulated by a coaxial point stimulation electrode at 1.5 Hz, d 20 ms pulse duration. Data were collected at room temperature sampling rate of 0.2 kHz and analyzed using BV_Ana software ision, Japan). unostaining, Flow Cytometry, Image Analysis, Statistics : Details are ed in the Supporting Information.

orting Information rting Information is available from the Wiley Online Library or e author.

owledgements s the scientifi c founder of Shrink Nanotechnologies, Inc. but s no compensation nor does she have any fi nancial interest in the ny. Terms of this arrangement have been reviewed and approved Irvine in accordance with its confl ict of interest policies. work was supported in part by The Edwards Lifesciences Center anced Cardiovascular Technology Training Fellowship (A.C.), the ia Institute for Regenerative Medicine (Grant#: RN2-00921-1,

nd R.A.L.), the NIH Directors New Innovator Award Program D007283, M.K.), Defense Advanced Research Projects Agency

A) N/MEMS S&T Fundamentals Program under grant no. 1-1-4003 issued by the Space and Naval Warfare Systems Center (SPAWAR) to the Micro/nano Fluidics Fundamentals Focus (MF3) (M.K.), and Shrink Nanotechnologies Inc and the Stanford CIS M.K.). The NIH - R01 HL72857 (R.A.L.), the CC Wong Foundation ell Fund (R.A.L.) and the Research Grant Council (Project 103544, . Thanks also to Nick Sharac (Regan lab, UCI), Michelle Digman e Laboratory of Fluorescence Dynamics, and Chuck Hitzman at nford Nanocharacterization Lab.

Received: September 7, 2011 Published online: November 8, 2011

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2011 - Chen Et Al - Advanced Materials

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