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Adv. Mate
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,
,
ficnohs t
d,
o
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
Dsik
ft M
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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wileyonli
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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plasma strates data. F-grown guidancstrates alignedcated bdue to
tilar to tis chosexperim
Whilgrooves
Figure 2 .the stiff self-simitransformlower SEconformrange wr
2011 WILEY-VCH Verlag GmbH & Co. Kr. 2011, 23, 57855791
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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N5787GaA, Weinheim wileyonlinelibrary.com
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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wileyo
increcallyto an
Figurewrinklicatesby timgrownof gras.d., n 2011 WILEY-VCH Verlag
GmbH & Co. nlinelibrary.com
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%).
Weresposolubon f-imagithe wof thetion. degre1.5 Paaligne2 m
Noof plunuclement The ncally functinucleand cThe hfrom
index0.001are alare ndo deculariundif
Untranslanotrastem stimutantlystrateto
ma(Suppexamelucidrespeimporrelevascales
AftwhethTo thetrophCM) musingtime. contricoordThe aously
typica
not exhibit the relevant physiological multiscale topographies.
Previous
e
efh
a
hu
e
l
s
e
oh
a
2011 WILEY-VCH Verlag GmbH & Co. Kter. 2011, 23,
57855791
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
The hESCs hESC-Cused tological scould eldifferen5789GaA,
Weinheim
MM
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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
Experi Fabric
from PE cut into plate. ThSuppliesconstrainOffi ceMaaligned w12
mm gsubstratelight for 3
Charaperformecoated (Pobtainedmagnifi cS-4700-2by fast FMA,
USAinverted The toposubstratefrequencThe softw(Waveme
Cell Cdescribed
Cell Acells werdilution)per well hESC-CMAoSMCs
The fewrinkled cell loadiconditionstaining and 72 hZeiss)
weanalysis.
For thwere cul
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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Adv. Ma
[ 5 ] A. S. Nathan , B. M. Baker , N. L. Nerurkar , R. L. Mauck
, Acta Bio-mater. 2010 , 7 , 57 .
[ 6 ] F.
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
CardInform
MeaAP prMiCamfi eld-ofsubstraconnecpropagwith 10room tMgCl 2
,dye wapass fi cells w8 V, anwith a (BrainV
Immdescrib
Supp Suppofrom th
Ackn M.K. ireceivecompaby UC
Thisfor AdvCalifornM.K. a(1DP2O(DARPN6600Pacifi c Centergrant
(Stem CR.A.L.)and ththe Sta
[ 1 ] C. Sc
[ 2 ] M C.
[ 3 ] D MU
[ 4 ] M Ex 2011 WILEY-VCH Verlag GmbH & Co.ter. 2011, 23,
57855791
198 [ 7 ] A.
677 [ 8 ] B. M
200 [ 9 ] S.
G. E. T
[ 10 ] S. F. E. T
[ 11 ] E. Kma
[ 12 ] C. 200
[ 13 ] N. G.
[ 14 ] M. [ 15 ] X.
P. L [ 16 ] G.
73 , [ 17 ] G.
R. L [ 18 ] C.
S. G 447
[ 19 ] J. I. R. 201
[ 20 ] K. EMa
[ 21 ] S. G.
[ 22 ] M. J. A S. P S. 101
[ 23 ] K. R V. M Pro
[ 24 ] C. M.
[ 25 ] A. D 201
[ 26 ] M. C. D
[ 27 ] J. D Pro
[ 28 ] L. R. O
[ 29 ] H. 28 ,
[ 30 ] H. 200
[ 31 ] T. X G.
[ 32 ] L. Y M. L. J
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
S. Chen , M. Mrksich , S. Huang , G. M. Whitesides , D. E.
Ingber , ience 1997 , 276 , 1425 . . J. Dalby , N. Gadegaard , R.
Tare , A. Andar , M. O. Riehle , P. Herzyk , D. Wilkinson , R. O.
Oreffo , Nat. Mater. 2007 , 6 , 997 . . H. Kim , E. A. Lipke , P.
Kim , R. Cheong , S. Thompson , . Delannoy , K. Y. Suh , L. Tung ,
A. Levchenko , Proc. Natl. Acad. Sci. SA 2010 , 107 , 565 . . J.
Dalby , M. O. Riehle , S. J. Yarwood , C. D. Wilkinson , A. S.
Curtis , p. Cell Res. 2003 , 284 , 274 . 5791 KGaA, Weinheim
MUN
ICATIO
N
wileyonlinelibrary.com
M. Watt , P. W. Jordan , C. H. ONeill , Proc. Natl. Acad. Sci.
USA 8 , 85 , 5576 .
J. Engler , S. Sen , H. L. Sweeney , D. E. Discher , Cell 2006 ,
126 , . urtuza , J. W. Nichol , A. Khademhosseini , Tissue Eng.
Part B Rev.
9 , 15 , 443 . Pagliari , A. C. Vilela-Silva , G. Forte , F.
Pagliari , C. Mandoli , Vozzi , S. Pietronave , M. Prat , S.
Licoccia , A. Ahluwalia , raversa , M. Minieri , P. Di Nardo , Adv.
Mater. 2011 , 23 , 514 . Soliman , S. Pagliari , A. Rinaldi , G.
Forte , R. Fiaccavento , Pagliari , O. Franzese , M. Minieri , P.
Di Nardo , S. Licoccia , raversa , Acta Biomater. 2010 , 6 , 1227 .
. Yim , E. M. Darling , K. Kulangara , F. Guilak , K. W. Leong ,
Bio-
terials 2010 , 31 , 1299 . J. Bettinger , R. Langer , J. T.
Borenstein , Angew. Chem. Int. Ed. 9 , 48 , 5406 . Bowden , S.
Brittain , A. G. Evans , J. W. Hutchinson ,
M. Whitesides , Nature 1998 , 393 , 146 . T. Lam , W. C. Clem ,
S. Takayama , Biomaterials 2008 , 29 , 1705 . Jiang , S. Takayama ,
X. Qian , E. Ostuni , H. Wu , N. Bowden , eDuc , D. E. Ingber , G.
M. Whitesides , Langmuir 2002 , 18 , 3273 .
D. Pins , D. L. Christiansen , R. Patel , F. H. Silver ,
Biophys. J. 1997 , 2164 . C. Engelmayr , Jr. , M. Cheng , C. J.
Bettinger , J. T. Borenstein , anger , L. E. Freed , Nat. Mater.
2008 , 7 , 1003 .
C. Fu , A. Grimes , M. Long , C. G. L. Ferri , B. D. Rich , S.
Ghosh , hosh , L.P. Lee , A. Gopinathan , M. Khine , Adv. Mater.
2009 , 21 ,
2 . Luna , J. Ciriza , M. E. Garcia-Ojeda , M. Kong , A. Herren
, D. Lieu , A. Li , C. C. Fowlkes , M. Khine , K. E. McCloskey ,
Tissue Eng. C 1 , 17 , 579 . fi menko , J. Finlay , M. E. Callow ,
J. A. Callow , J. Genzer , ACS Appl. ter. Interfaces 2009 , 1 ,
1031 . Gerecht , C. J. Bettinger , Z. Zhang , J. T. Borenstein
,
Vunjak-Novakovic , R. Langer , Biomaterials 2007 , 28 , 4068 .
A. Lafl amme , K. Y. Chen , A. V. Naumova , V. Muskheli ,
. Fugate , S. K. Dupras , H. Reinecke , C. Xu , M. Hassanipour ,
olice , C. OSullivan , L. Collins , Y. Chen , E. Minami , E. A.
Gill ,
Ueno , C. Yuan , J. Gold , C. E. Murry , Nat. Biotechnol. 2007 ,
25 , 5 . . Stevens , K. L. Kreutziger , S. K. Dupras , F. S. Korte
, M. Regnier , uskheli , M. B. Nourse , K. Bendixen , H. Reinecke ,
C. E. Murry ,
c. Natl. Acad. Sci. USA 2009 , 106 , 16568 . Xu , M. S. Inokuma
, J. Denham , K. Golds , P. Kundu , J. D. Gold , K. Carpenter ,
Nat. Biotechnol. 2001 , 19 , 971 . . van der Meer , A. A. Poot , J.
Feijen , I. Vermes , Biomicrofl uidics
0 , 4 , 11103 . J. Dalby , N. Gadegaard , P. Herzyk , D.
Sutherland , H. Agheli , . Wilkinson , A. S. Curtis , J. Cell
Biochem. 2007 , 102 , 1234 .
. Pajerowski , K. N. Dahl , F. L. Zhong , P. J. Sammak , D. E.
Discher , c. Natl. Acad. Sci. USA 2007 , 104 , 15619 . E. McNamara
, R. J. McMurray , M. J. P. Biggs , F. Kantawong ,
. C. Oreffo , M. J. Dalby , J. Tissue Eng . 2010, 2010 , 1 . T.
Au , I. Cheng , M. F. Chowdhury , M. Radisic , Biomaterials 2007 ,
4277 . T. Heidi Au , B. Cui , Z. E. Chu , T. Veres , M. Radisic ,
Lab Chip 9 , 9 , 564 . ue , H. C. Cho , F. G. Akar , S. Y. Tsang ,
S. P. Jones , E. Marban ,
F. Tomaselli , R. A. Li , Circulation 2005 , 111 , 11 . ang , M.
H. Soonpaa , E. D. Adler , T. K. Roepke , S. J. Kattman , Kennedy ,
E. Henckaerts , K. Bonham , G. W. Abbott , R. M. Linden , . Field ,
G. M. Keller , Nature 2008 , 453 , 524 .