Engineering cellematerial interfaces for long-term expansion of human
pluripotent stem cells
Chien-Wen Changa,1,2, Yongsung Hwanga,2, Dave Brafmanb,2, Thomas Hagana, Catherine Phungb,
aDepartment of Bioengineering, University of California, San Diego, 9500 Gilman Drive, MC 0412, La Jolla, CA 92093, USA
bDepartment of Cellular and Molecular Medicine, University of California, San Diego, CA 92093, USA
a r t i c l e i n f o
Received 25 August 2012
Accepted 8 October 2012
Available online 3 November 2012
Human pluripotent stem cells
Embryonic stem cells
Synthetic heparin mimics
a b s t r a c t
Cost-effective and scalable synthetic matrices that support long-term expansion of human pluripotent
stem cells (hPSCs) have many applications, ranging from drug screening platforms to regenerative
medicine. Here, we report the development of a hydrogel-based matrix containing synthetic heparin-
mimicking moieties that supports the long-term expansion of hPSCs (?20 passages) in a chemically
defined medium. HPSCs expanded on this synthetic matrix maintained their characteristic morphology,
colony forming ability, karyotypic stability, and differentiation potential. We also used the synthetic
matrix as a platform to investigate the effects of various physicochemical properties of the extracellular
environment on the adhesion, growth, and self-renewal of hPSCs. The observed cellular responses can be
explained in terms of matrix interface-mediated binding of extracellular matrix proteins, growth factors,
and other cell-secreted factors, which create an instructive microenvironment to support self-renewal of
hPSCs. These synthetic matrices, which comprise of “off-the-shelf” components and are easy to
synthesize, provide an ideal tool to elucidate the molecular mechanisms that control stem cell fate.
? 2012 Elsevier Ltd. All rights reserved.
Since the isolation of human embryonic stem cells (hESCs),
there has been a tremendous interest in developing defined, scal-
able invitro culture conditions that can support their growth. These
efforts have led to the development of multiple defined growth
media, but these still require either feeder layers such as mouse
embryonic fibroblasts (MEFs) or biologically derived matrices such
as Matrigel for maintenance of pluripotency and self-renewal of
hPSCs [1e6]. Development of chemically defined matrices is
a challenging task because the myriad of physicochemical signals
that MEFs and Matrigel provide. Within these limitations, recent
advances in the field of biomaterials have led to identification of
substratesdboth naturally derived and syntheticdfor the self-
renewal of hPSCs [7e16]. High-throughput screening technolo-
gies have contributed significantly toward the development of
these chemically defined, synthetic materials [10,17].
Accumulating evidence suggests that heparin molecules play
a key role in maintaining self-renewal of hPSCs [4,12,18]. Studies by
Levenstein et al. showed the role of MEF-secreted heparan sulfate
proteoglycans on self-renewal of hESCs . To harness the bene-
ficial effects of heparin moieties on the self-renewal of hPSCs, Klim
et al. have developed synthetic matrices that display heparin-
binding peptides to support long-term self-renewal of hPSCs .
The role of heparin moieties in self-renewal of hPSCs is not
surprising given that heparin molecules can bind to soluble bFGF
molecules and modulate their bioactivity [19e21]; bFGF is a crucial
biomolecule required for maintenance of self-renewal of hPSCs
in vitro. Additionally, heparin molecules have been shown to
protect bFGF from denaturation and proteolytic degradation,
thereby increasing its longevity and function [21,22].
Recently we have shown that synthetic heparin mimics such as
poly(sodium 4-styrenesulfonate) (PSS) can bind to soluble bFGF
and regulate FGF signaling akin to heparin molecules . Based on
these findings along with the known role of bFGF molecules on
in vitro self-renewal of hPSCs, we developed synthetic hydrogels
containing PSS moieties to support long-term culture of hPSCs
while maintaining their pluripotency. Employing hydrogel-based
synthetic matrices, we further elucidated the role of physico-
chemical cues of the matrix on self-renewal of hPSCs. Such easy to
* Corresponding author. Fax: þ1 858 534 5722.
E-mail address: email@example.com (S. Varghese).
1Current address: Department of Biomedical Engineering and Environmental
Sciences, National Tsing Hua University, Taiwan.
2C.C, Y.H, and D.B contributed equally to this work.
Contents lists available at SciVerse ScienceDirect
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0142-9612/$ e see front matter ? 2012 Elsevier Ltd. All rights reserved.
Biomaterials 34 (2013) 912e921
synthesize and cost-effective synthetic matrices would not only
accelerate the translational potential of hPSCs, but also provide
a platform to decipher the interplay between various physico-
chemical cues on self-renewal of hPSCs. Additionally, these
matrices would help to identify the myriad of molecular and
signaling pathways that dictate stem cell fate and commitment.
2. Materials and methods
N-acryloyl amino acid (AA) monomers, such as N-acryloyl 2-glycine (A2AGA), N-
acryloyl 4-aminobutyric acid (A4ABA), N-acryloyl 6-aminocaproic acid (A6ACA), and
N-acryloyl 8-aminocaprylic acid (A8ACA), were synthesized from glycine (Fisher
Scientific, Inc.), 4-aminobutyric acid, 6-aminocaproic acid, and 8-aminocaprylic acid
(Acros Organics Inc.), respectively, as described elsewhere . Sodium 4-
vinylbenzenesulfonate (SS), 3-sulfopropyl acrylate potassium salt (SPA), and [2-
(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (MEDSAH)
were purchased from Aldrich. Acrylamide (Am) was purchased from Invitrogen and
N,N0-methylenebisacrylamide (BisAm), ammonium persulfate (APS) and N,N,N0,N0-
tetramethylethylenediamine (TEMED) were obtained from Sigma. The monomers
used in this study are summarized in Supplementary Table S1.
2.2. Hydrogel synthesis
The hydrogels containing varying functional groups and hydrophilicity were
synthesized through copolymerization of acrylamide with monomers containing
either carboxylate or sulfonate groups. The PSS-based hydrogels (PAm6-co-PSS2,
PAm6-co-PSS1, PAm6-co-PSS0.5) were synthesized by copolymerizing acrylamide
(Am, 7.5 mmol) with sodium 4-vinylbenzenesulfonate (SS, 2.5 mmol) at 6:2, 6:1, and
6:0.5 mole ratios. The monomers were dissolved in deionized (DI) water, and
polymerized in Bio-Rad 1 mm spacer glass plates at room temperature using 0.26,
0.19, and 0.10 mmol of BisAm as a crosslinker and 1.3% w/v of APS/TEMED (redox
initiator/accelerator). Hydrogels containing SPA and MEDSAH moieties (PAm6-co-
PSPA2, PAm6-co-PMEDSAH2) were synthesized by copolymerizing Am (7.5 mmol)
with SPA (2.5 mmol) or MEDSAH (2.5 mmol) at a mole ratio of 6:2. The precursors
were dissolved in DI water and polymerized using 0.26 mmol of BisAm and 1.3% w/v
of APS/TEMED. Lastly, hydrogels with carboxyl groups were synthesized by
copolymerizing Am (7.5 mmol) with AA monomers (2.5 mmol) at a mole ratio of 6:2
as described elsewhere . Briefly, the monomers were dissolved in 1 M NaOH and
polymerized using 0.26 mmol of BisAm and 1.3% w/v of APS/TEMED. The compo-
sitions and nomenclature of the hydrogels are summarized in Supplementary
Table S2. The hydrogels were sterilized with 70% ethanol and washed with fresh
phosphate buffered saline (PBS) solution for 72 h. The rinsed hydrogels were incu-
bated in culture media (high glucose DMEM with 2 mM L-glutamine and 50 units/ml
penicillin/streptomycin) containing 10% fetal bovine serum (premium select) over-
night before plating the cells.
2.3. Surface roughness
Surface roughness of hydrogels was evaluated using a Multimode AFM equipped
with a Nanoscope IIIA controller from Veeco Instruments (Santa Barbara, CA) run by
Nanoscope software v5.30 as previously reported . AFM images were acquired in
contact mode at forces of w4 nN with an “E” scanner (maximum scan area
12 ? 12 mm2) using Si3N4 cantilevers (Veeco) with 0.06 N/m nominal spring
constants. Hydrogels were prepared as described above. Upon synthesis, hydrogels
were washed in PBS for 36 h to leach out unreacted reactants and to reach equi-
librium swelling. For a given scan area, the reported roughness value is the average
root mean square (RMS) roughness obtained from two different spots of triplicate
samples. Using the nanoscope software, data analysis was carried out where a flat-
tening order 3 was applied to all images to correct for tilt and bow before roughness
2.4. Elastic modulus
Equilibrium swollen hydrogels in PBS were used for compression measurements
. The measurements were performed using Bose ElectroForce 3200 Test
Instrument (Bose, Minnesota, USA). Samples were compressed by two parallel plates
at a maximum loading of 225 N with a crosshead speed of 0.1 mm/min. The elastic
moduli were calculated from the linear region of the stressestrain curve (0e5%
strain). All measurements were carried out as quadruplicates for each set of
2.5. Water contact angle
The water contact angle of the hydrogels was determined by a sessile drop
method at room temperature using a contact angle meter (CAM100, KSV
Instruments Ltd.) . A 5ml droplet of water was placed on the surface of hydrogels.
All samples were prepared as triplicates and results were shown as a mean value
with standard deviation.
Thelentiviral construct that was used togeneratethe Oct4-GFPreporterlinewas
kindly provided by Dr. Alexey Terskikh. The reporter line was generated as described
single clones were isolated and screened for stable GFP expression levels.
2.7. Culture of hPSCs
HUES9, HUES9-Oct4-GFP, HUES6, and hiPSC were expanded in defined medium
(StemPro; DMEM/F-12 supplemented with StemPro supplement, 2% bovine serum
albumin (BSA), 55 mM 2-mercaptoethanol, and 1% Gluta-MAX) or in MEF-
conditioned medium. The MEF-conditioned medium was collected after culturing
MEF for 24 h using growth medium (Knockout DMEM supplemented with 10%
Knockout Serum Replacement,10% humanplasmonate (Talecris Biotherapeutics),1%
non-essential amino acids, 1% penicillin/streptomycin, 1% Gluta-MAX, and 55 mM 2-
mercaptoethanol) as described elsewhere . The hPSCs were cultured on mitot-
ically inactivated MEF at an initial seeding density of 104cells/cm2in MEF-
conditioned medium prior to their culture on Matrigel or synthetic matrices. The
hPSCs were manually passaged as small clumps of 30e40 mm size after 6 days of
culture onto different matrices (Matrigel and synthetic matrices) by using a splitting
ratio of 1:4. All the sequential passages were carried out similarly by passaging the
cells manually. The hPSCs on PAm6-co-PSS2hydrogels were passaged after 10e12
days depending upon the colony size and morphology. All cultures were supple-
mented with fresh medium containing 30 ng/ml of bFGF (Life Technologies) daily.
2.8. Population doubling time
Population doubling time (PDT) of HUES9 cells grown on Matrigel, MEF, and
PAm6-co-PSS2hydrogel was calculated using the equation below :
ðT2 ? T1Þ
3:32*ðlog N2 ? log N1Þ
where T1 and T2 represents days 3 and 5, respectively; N1 and N2 are the number of
cells at T1 and T2, respectively. The number of cells at each time point was counted
using TC10? Automated Cell Counter.
For PDT measurements, HUES9 cells were cultured as single cells by enzymat-
ically splitting the cells using Accutase. The cell count was carried out after 3 and 5
days of culture to calculate the population doubling time.
Immunofluorescent staining was performed using the following primary anti-
bodies: OCT4 (1:200; Santa Cruz), NANOG (1:200; Santa Cruz), SOX17 (1:200; R & D
systems), SMA (1:500; R & D systems), and NESTIN (1:50; BD Biosciences). The
following secondary antibodies were used: goat anti-rabbit Alexa 647 (1:400; Life
Technologies), donkey anti-mouse Alexa 546 (1:250; Life Technologies), and donkey
anti-goat Alexa 546 (1:250; Life Technologies). For immunofluorescent staining,
cells were fixed in 4% PFA for 5 min at 4?C, followed by 10 min at room temperature.
Immediately before staining, the cells were permeabilized with 0.2% (v/v) Triton X-
100 and blocked with 1% (w/v) BSA and 3% (w/v) nonfat dry milk for 30 min. Cells
were stained with primary antibodies diluted in 1% BSA overnight at 4?C, washed 3
times with TBS, and incubated with secondary antibodies for 1 h at 37?C. The nuclei
were stained with Hoechst 33342 (2 mg/ml; Life Technologies) for 5 min at room
temperature. Imaging was performed using an automated confocal microscope
(Olympus Fluoview 1000 with motorized stage and incubation chamber).
2.10. RNA isolation and quantitative PCR
RNA isolation was carried out by using TRIzol (Invitrogen), and treated with
DNase I (Invitrogen). Reverse transcription was performed by using qScript cDNA
Supermix (Quanta Biosciences). Quantitative PCR was carried out by using TaqMan
probes (Applied Biosystems) and TaqMan Fast Universal PCR Master Mix (Applied
Biosystems) on a 7900HT Real-Time PCR machine (Applied Biosystems). Taqman
gene expression assay primers (Applied Biosystems) listed in Supplementary
Table S3 were used. Gene expression was normalized to 18S rRNA levels. Delta Ct
values were calculated as Ctarget
. All experiments were performed with three
2.11. FACS analysis
HPSCs were dissociated with Accutase. The cells were re-suspended in buffer
(2% FBS/0.09% sodium azide/DPBS; BD Biosciences) and stained directly with Alexa
647 conjugated Tra-1-81 (Biolegend) or Alexa Fluor 647 mouse IgM,K isotype
C.-W. Chang et al. / Biomaterials 34 (2013) 912e921
control. Cells were stained for 30 min on ice, washed, and re-suspended in buffer.
Samples were analyzed by using BD Biosystems FACSCanto.
2.12. Embryoid body formation
The hPSCs cultured on PAm6-co-PSS2were Accutased for 2e3 min and re-
suspended in growth medium without the supplementation of bFGF, plated onto
ultralowattachment plates,and cultured in 37?C/5% CO2incubator for 8 daytoform
embryoid body (EBD).
2.13. In vitro differentiation
All media components used were procured from Life Technologies unless indi-
cated otherwise. For endoderm differentiation, hPSCs were cultured on Matrigel in
MEF-conditioned medium supplemented with 30 ng/ml FGF2 until confluency. The
medium was then changed to RPMI medium supplemented with 1% (v/v) Gluta-
MAX and 100 ng/ml recombinant human Activin A (R&D Systems). Cells were
cultured for 3 days, with FBS concentrations at 0% for the first day and 0.2% for the
second and third days. Cultures were supplemented with 30 ng/ml purified mouse
Wnt3a on the first day.
To initiate ectoderm differentiation, hPSCs were cultured on Matrigel in MEF-
conditioned medium supplemented with 30 ng/ml FGF2. Cells were then Accu-
tased for 5 min and re-suspended in neural progenitor cell medium (10% FBS,1% N2,
1% B27, DMEM/F-12), 5 mM ROCK inhibitor (Y-27632, Stemgent), 50 ng/ml
recombinant mouse Noggin (R&D Systems), 0.5 mM dorsomorphin (Tocris Biosci-
ence). Roughly, 7.5 ? 105cells suspended in neural progenitor cell medium were
added to each well of several 6 well ultra low attachment plates. The plates were
then placed on an orbital shaker at 95 rpm in a 37?C/5% CO2incubator for overnight.
The formed spherical clusters were then cultured in neural progenitor cell medium
supplemented with 50 ng/ml recombinant mouse Noggin and 0.5mM Dorsomorphin,
but no FBS. The medium was subsequently changed every other day. After 5 days in
suspension culture, the EBs were then transferred toa 10 cm dish coated (3 ? 6 wells
per 10 cm dish) with growth factor-reduced Matrigel (1:25 in KnockOut DMEM; BD
Biosciences). The cells adhered onto the Matrigel-coated dishes were then cultured
in neural progenitor cell medium supplemented with 50 ng/ml recombinant mouse
Noggin and 0.5 mM Dorsomorphin. After 7 days of attachment, rosette-forming EBs
were collected by manual dissection. Isolated rosettes were incubated in Accutase
for 15 min in a 37?C, 5% CO2tissue culture incubator. The rosettes were then plated
onto poly-L-ornithine (PLO; 10 mg/ml; Sigma) and mouse laminin (Ln; 5 mg/ml)
coated plates in neural progenitor cell expansion medium [(1% N2,1% B27, DMEM/F-
12) supplemented with 30 ng/ml FGF2 and 30 ng/ml EGF (R & D systems)]. For
mesoderm induction, hPSCs were cultured on Matrigel in DMEM supplemented
with 20% FBS and 1% penicillin/streptomycin for 21 days.
2.14. Karyotype analysis
To monitor genomic integrity, cells grown on PAm6-co-PSS2 hydrogel with
at passage 16 and 20 using standard protocols for G-banding (Cell Line Genetics).
2.15. PCR array analysis for various extracellular matrix proteins, integrins, and
Briefly, RNA was isolated from cells using TRIzol (Invitrogen), and treated with
DNase I (Invitrogen). Reverse transcription was performed by using RT2First Strand
Kit (SABioscience, Cat# 330401) and 200 ng of cDNA was processed for quantitative
real-time PCR for 84 genes involved in extracellular matrix proteins and adhesion
molecules by using PCR array kit (RT2Profiler? PCR Arrays Extracellular matrix and
adhesion molecules, PAHS-013A-2, SABioscience) using an ABI Prism 7700 Sequence
Detection System (Applied Biosystems). PCR products were quantified by measuring
SYBR Green fluorescent dye incorporation with ROX dye reference.
2.16. Protein adsorption
The amount of various proteins adsorbed onto PAm6-co-A2AGA2and PAm6-co-
PSS2hydrogels was quantified by a modified Bradford protein assay using Bio-Rad
Protein Assay kit (Cat# 500-0006) as previously reported . Circular hydrogels
having 6 mm diameter were prepared and placed onto 96-well plate. These
hydrogels were incubated with 200 ml of bovine serum albumin (Sigma, Cat#
A8412), vitronectin (Sigma, Cat# V8379-50UG), collagen type I (BD Biosciences, Cat#
354231), collagen type IV (Sigma, Cat# C5533), laminin (Sigma, L6274), and fibro-
nectin (Gibco, Cat# 33016-015) solutions of varying concentrations (0, 2.5, 5,10, and
15 mg/ml) in PBS for 15 h at 4?C. 30 ml of each supernatant solution was mixed with
200 ml of Bradford dye reagent solution, which was prepared according to manu-
facturer’s protocol.100 ml of the above solution was transferred to a flat-bottom 96-
well plate to measure their absorbance at 595 nm by using a Multimode Detector
(Beckman Coulter, DTX 880). Three biological replicates were used for the
measurements. The adsorption was calculated from a standard curve generated for
the corresponding proteins of known concentrations.
2.17. Enzyme-linked immunosorbent assay (ELISA)
The amount of bFGFadsorbed by PAm6-co-A2AGA2and PAm6-co-PSS2hydrogels
was carried out by bFGF ELISA kit (RayBiotech, Inc., cat# ELH-bFGF-001) following
the manufacturer’s protocol. Similar to the protein adsorption assay, circular
hydrogels measuring 6 mm in diameter were prepared and placed onto a 96-well
plate. These hydrogels were incubated with 250 ml of bFGF (30 ng/ml) at 37?C for
approximately 12 h 100 ml of the each supernatant solution was transferred to
a bFGF microplate (96-wells coated with anti-human bFGF) and incubated overnight
at 4?C, followed by incubation with a biotinylated antibody and streptavidin solu-
tion. After washing, 100 ml of a TMB substrate solution was added to the wells and
samples were incubated for 30 min. Finally, 50 ml of the stop solution was added to
samples and their absorbance at 450 nm was measured by using a Multimode
Detector (Beckman Coulter, DTX 880). Three biological replicates were used for the
measurements. The adsorption was calculated from a standard curve generated by
bFGF standards provided by the manufacturer.
3.1. Design and characterization of synthetic matrices
We synthesized a series of copolymer hydrogels with varying
elastic modulus, functional group, and hydrophilicity by copoly-
merizing acrylamide (Am) with monomers containing either
a sulfonate or a carboxylate functional group as described in
Supplementary Table S1, Table S2 and Fig. S1. Together, these
hydrogels with varying physicochemical properties could provide
information on the effect of chemistry, functional group, rigidity,
and hydrophilicity of the matrix on supporting self-renewal of
hPSCs in vitro. The copolymer hydrogels are referred to as PAmx-co-
PBy, where PAm and PB represent the polymer components of the
hydrogel, and x and y denote the mole ratio of the two monomers
used in hydrogel synthesis (see Supplementary Table S2 fordetails).
For instance, the hydrogels synthesized by copolymerizing acryl-
amide (Am) and sodium 4-vinylbenzenesulfonate (SS) at a mole
ratio of 6:2 is denoted as PAm6-co-PSS2. Fig.1a shows the schematic
of the synthetic matrices containing PAm and PSS moieties (e.g.
PAmx-co-PSSy). The elastic modulus and hydrophilicity of these
hydrogels are listed in Supplementary Table S2. For example, the
343.7 ? 5.1 kPa, and a hydrophilicity of 23.0 ? 2.0?.
3.2. PSS-based hydrogels support self-renewal and long-term
expansion of hPSCs
Our initial observation was that the HUES9 cells cultured on
PAm6-co-PSS2hydrogels adhered ontothe underlying hydrogel and
formed bright and compact colonies. However, differences in cell
adhesion were observed between PAm6-co-PSS2 hydrogels and
their control counterpartsdMEF- and Matrigel-supported cultures.
Observations after 24 h of cell seeding indicated that the number of
cells adhered onto both MEF and Matrigel are significantly higher
compared to PAm6-co-PSS2hydrogels. Despite these differences,
the cells adhered onto PAm6-co-PSS2hydrogels, proliferated and
formed compact colonies similar to those observed on Matrigel-
and MEF-supported cultures (Fig. 1bed). The population doubling
time of HUES9 cells on PAm6-co-PSS2hydrogels was found to be
w38 h, while that on MEFs and Matrigel was found to be w23 h
(Fig.1e). The estimated population doubling time on PAm6-co-PSS2
hydrogels is likelyan overestimategiven that the cellecell adhesion
of cells grown on these hydrogels were significantly higher
compared to those on Matrigel- and MEF-supported cultures;
strong cellecell adhesion limits the uniform dissociation of hESC
colonies into single cells.
Although the aforementioned findings show that HUES9 cells
can adhere and grow on PAm6-co-PSS2hydrogels, it is vital to test
whether the developed matrix can support the long-term
C.-W. Chang et al. / Biomaterials 34 (2013) 912e921
expansion of hPSCs without compromising their pluripotency and
karyotypic stability. The PAm6-co-PSS2hydrogels indeed supported
adhesion and long-term growth of HUES9 cells both in MEF-
conditioned medium and chemically defined StemPro medium
(Supplementary Fig. S2a, Fig. 2). HUES9 cells expanded on PAm6-
co-PSS2hydrogels with frequent splitting for over 20 passages (>8
months) using StemPro medium exhibited characteristic stem cell
morphology and tight colony formation (Fig. 2a). The expanded
HUES9 cells were positive for OCT4 and NANOG. The real-time PCR
(qPCR) results indicate that hPSCs exhibited similar gene expres-
sion levels of OCT4 and NANOG compared to those cultured on
Matrigel (Fig. 2b). The pluripotency of HUES9 cells expanded on
PAm6-co-PSS2hydrogels was further confirmed by FACS analysis,
which revealed a similar percentage of pluripotent cells between
those cultured on PAm6-co-PSS2and Matrigel, as evidenced by the
population of OCT4 and TRA-1-81 positive cells (Fig. 2c).
One of the unique characteristics of pluripotent stem cells is
their ability to form embryoid bodies (EBs) in suspension culture
and differentiate into all three germ layers. The HUES9 cells grown
on PAm6-co-PSS2hydrogels formed EBs (Supplementary Fig. S3).
The cells expanded on PAm6-co-PSS2matrices were also differen-
tiated into mesoderm, ectoderm, and endoderm, further confirm-
ing that the cells grown extensively on these hydrogels maintained
their ability to differentiate into multiple germ layers (Fig. 3a,b).
Additionally, the cells cultured on PAm6-co-PSS2 maintained
a normal karyotype (Fig. 3c and Supplementary Fig. S2b). Together,
these findings demonstrate the potential of PAm6-co-PSS2 to
support long-term culture of undifferentiated hPSCs while main-
taining their pluripotency.
To determine whether the PAm6-co-PSS2-assisted self-renewal
of HUES9 cells is applicable to other hPSCs, we have investigated
the potential of PAm6-co-PSS2to support the growth of HUES6 and
human induced pluripotent stem cells (hiPSCs) in vitro. Similar to
HUES9 cells, HUES6 and hiPSCs cultured and passaged over
multiple times on PAm6-co-PSS2hydrogels in StemPro medium
displayed characteristic hPSC morphology, bright colony formation,
and OCT4 and NANOG expression comparable to Matrigel (Fig. 4a,b
and Supplementary Fig. S4a,b).
Fig. 1. PSS-based hydrogels support growth of HUES9 cells in vitro. (a) Schematic of PSS-based hydrogel(s) synthesized by copolymerizing acrylamide with sodium 4-
vinylbenzenesulfonate with bisacrylamide as a crosslinking agent. Representative phase contrast images of HUES9 colonies on (b) PAm6-co-PSS2hydrogel, (c) Matrigel, and (d)
mouse embryonic fibroblasts (MEFs) after 7 days in culture in StemPro medium. (e) Population doubling of HUES9 cells over five days of bed. Scale bar: 200 mm. Values are shows as
mean ? SD. ***p < 0.001.
C.-W. Chang et al. / Biomaterials 34 (2013) 912e921
3.3. Effect of matrix rigidity on hPSCs
Having established the unique ability of PAm6-co-PSS2hydro-
gels containing PSS moieties to support the growth of hPSCs in vitro
while maintaining their pluripotency, we next determined the
effect of matrix rigidity on hPSCs. To this end, we synthesized
PAm6-co-PSS2hydrogels with different bulk moduli (w54, w138,
Supplementary Table S2). Note that the changes in modulus also
introduce subtle changes in matrix hydrophilicity as rigidity affects
swelling, which in turn affects the surface density of sulfonate
functional groups accessible at the interface. As seen from Fig. 5a,
an increased cell adhesion and colony formationwas observed with
increasing rigidity of PAm6-co-PSS2hydrogels. Hydrogels having
a compressive modulus of w344 kPa supported adhesion, colony
Supplementary Fig. S5 demonstrates the growth of HUES9 cells on
these PAm6-co-PSS2 hydrogels having higher elastic modulus
(w344 kPa). PAm6-co-PSS2 hydrogels with low bulk rigidity
(w54 kPa) supported minimal cell adhesion while those having
a rigidity of w138 kPa exhibited moderate cell adhesion, but the
attached cells underwent spontaneous differentiation.
3.4. Effects of chemical functional group(s) and matrix
hydrophilicity on hPSCs
We next investigated the effect of hydrogel composition on
adhesion and growth of hPSCs by varying the amount of PSS
content within the hydrogels (mole ratio of Am:SS 6:0.5, 6:1, 6:2).
Significant differences in adhesion and colony formation of HUES9-
Oct4-GFP cells were observed amongst the hydrogels; specifically
a monotonic dependence with the PSS content was observed
(Fig. 5b, Supplementary Fig. S6a). No cell adhesionwas observed on
hydrogels containing lower amounts of PSS moieties (PAm6-co-
PSS0.5). While an increase in PSS content in the hydrogel (PAm6-co-
PSS1) supported cell adhesion, they failed to support the colony
formation of adhered cells. A further increase in PSS content, as in
PAm6-co-PSS2, supported both adhesion and colony formation of
HUES9-Oct4-GFP cells (Fig. 5b, Supplementary Fig. S6a). Note that
varying the hydrogel composition also introduced subtle changes
to their hydrophilicity and bulk rigidity (Supplementary Table S2).
Given the importance of functional group and matrix hydro-
philicity on cell adhesion, we also evaluated cellular responses of
HUES9 cells on different hydrogels with varying hydrophilicity.
These hydrogels were created by reacting Am with different
monomers (SS, SPA and MEDSAH) terminating with sulfonate
functional group at a mole ratio of 6:2 (Am:comonomer). These
hydrogels have similar elastic moduli and functional groups but
varying matrix hydrophilicities (Supplementary Table S2). Similar
to PAm6-co-PSS2hydrogels, significant cell adhesion was observed
initially on PAm6-co-PSPA2 hydrogels, while minimal to no cell
adhesion was observed on PAm6-co-PMEDSAH2(Fig. 5c). However,
unlike PAm6-co-PSS2hydrogels, cells on PAm6-co-PSPA2did not
grow to form bright compact colonies (Fig. 5c).
These results clearly indicate the effect of multiple physical and
chemical cues of the underlying matrix on hPSC response. In an
effort to delineate the effect of various material properties from
that of the functional group, we synthesized copolymer hydrogels
bearing carboxylate groups (PAm6-co-PA2AGA2) having similar
elastic modulus, hydrophilicity, and topography to that of PAm6-co-
Fig. 2. PSS-based synthetic matrices supported long-term maintenance of HUES9 in StemPro medium. (a) HUES9 cells grown on PAm6-co-PSS2hydrogels in StemPro medium for 20
passages stained positive for NANOG (Red) and OCT4 (Green). The nuclei are stained blue with Hoescht 33342. The inset shows higher magnification images. Scale bar: 200 mm
(main images) and 100 mm (inset images). (b) Quantitative PCR of HUES9 cells grown on PAm6-co-PSS2hydrogels showed similar expression level of OCT4 and NANOG to that on
Matrigel. (c) Representative FACS profiles of HUES9 cells grown on PAm6-co-PSS2hydrogel and Matrigel, which again shows PAm6-co-PSS2hydrogels can support in vitro self-
renewal of HUES9 cells similar to Matrigel. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
C.-W. Chang et al. / Biomaterials 34 (2013) 912e921
PSS2(Supplementary Table S2 and Fig. S7). Unlike PAm6-co-PSS2,
the HUES9 cells on PAm6-co-PA2AGA2hydrogels exhibited minimal
to no cell adhesion (Supplementary Fig. S6b,c). We also examined
the effect of carboxyl functional groups on hPSCs by employing
different PAm6-co-PB2hydrogels having carboxyl functional groups
but varying hydrophilicity (Supplementary Table S2) . Similar
to PAm6-co-PA2AGA2, no cell adhesion was observed on hydrogels
with carboxyl functional groups (data not shown). These findings
demonstrate the importance of sulfonate groups on the observed
PAm6-co-PSS2-mediated cell response.
3.5. Cell-matrix interface on adhesion and growth of hPSCs
As the interface of the hydrogels was not functionalized with
proteins or peptides and a short incubation of the hydrogels in
serum medium prior to cell seeding was needed for initial cell
adhesion, we examined the adsorption of various extracellular
matrix proteins (ECM) onto the hydrogel surfaces. It is well known
that matrix interfacial properties (hydrophilicity, functional group,
surface roughness, rigidity, etc.) affect protein adsorption and
conformation, thereby influencing cell adhesion [23,26,27]. We
examined protein adsorption on PAm6-co-PSS2 hydrogels and
compared it to PAm6-co-PA2AGA2. We chose these two hydrogels
based on our observation that despite having similarhydrophilicity,
surface roughness, and rigidity, PAm6-co-PSS2hydrogels support
hPSCs while PAm6-co-PA2AGA2hydrogels do not. We also exam-
ined the adsorption of bFGF on either hydrogel. While both the
hydrogels supported adsorption of ECM proteins and bFGF, PAm6-
co-PSS2hydrogels was found to adsorb slightly higher amounts of
certain proteins such as BSA and VN compared to PAm6-co-
Cell surface adhesion molecules such as integrins play an
important role in the adhesion of hPSCs to the underlying ECM, and
also in the regulation of their self-renewal [28e30]. Similarly, ECM
Fig. 3. In vitro differentiation of HUES9 cells passaged 20 times using PAm6-co-PSS2hydrogels. (a) Immunofluorescence staining shows differentiation of these cells into endoderm
(SOX17), mesoderm (SMA), and ectoderm (Nestin) lineages. Scale bar ¼ 100 mm. (b) Quantitative PCR results for differentiated HUES9 cells shows expression of ectoderm (CXCR4,
FOXA2, SOX17), mesoderm (SMA, ACTC1), and ectoderm markers (Nestin, SOX1, SOX2). (c) Karyotype analysis of HUES9 cells grown on PAm6-co-PSS2hydrogel shows a normal
euploid karyotype. Values are shown as mean ? SD. **p < 0.01 and ***p < 0.001.
C.-W. Chang et al. / Biomaterials 34 (2013) 912e921
components secreted by the hPSCs and the feeder cells have also
been shown to play an important role in maintaining the pluripo-
tency of hPSCs [18,31]. To this end, we determined the endogenous
expression levels of various cell surface adhesion molecules and
ECM components of HUES9 cells cultured on PAm6-co-PSS2and
PAm6-co-PSPA2, and compared the results against those of MEF-
and Matrigel-supported culture under identical conditions. The
PAm6-co-PSPA2hydrogel was chosen as it supported initial adhe-
sion of HUES9 cells similar to PAm6-co-PSS2, but failed to support
their growth and colony formation. As seen from Fig. 6c, the
underlying matrix had a significant effect on the gene expression
profile of the cells. In short, the cells on PAm6-co-PSS2hydrogels
exhibited higher expression levels of various integrins and ECM
proteins that are known to be relevant to self-renewal of hPSCs.
Specifically, HUES9 cells cultured on PAm6-co-PSS2 expressed
higher levels of fibronectin, laminin, collagen, and vitronectin, as
well as integrins a1, a2, a8, and aV. Many of these ECM proteins and
integrins have been implicated to play an important role in self-
renewal of hPSCs [10,11,28,30,32]. Additionally, cells on PAm6-co-
PSS2hydrogels expressed higher levels of MMP family of proteins
indicating the potential role of ECM remodeling.
HPSCs such as hESCs and iPSCs grow best when cultured on
feeder cells such as MEFs or Matrigel [33,34]. Emerging evidence
shows that biomaterial-based matrices can also support in vitro
expansion of hPSCs without compromising their phenotypic and
differentiation potential [8,10,14,15]. In this study, we demonstrate
the potential of synthetic hydrogels containing heparin-mimicking
PSS moieties in supporting the in vitro growth and self-renewal of
hPSCs. The synthetic matrix, PAm6-co-PSS2, supported the growth
and expansion of multiple hPSC lines (HUES9, HUES6, and hiPSCs)
through multiple passages (?20 passages) while maintaining their
Our results indicate that the presence of sulfonate groups alone
is not sufficient to support self-renewal of hPSCs, but a combination
of physical cues such as hydrophilicity and elastic modulus is
required, thus exemplifying the delicate balance of insoluble and
soluble cues of the niche on various cellular responses of hPSCs.
Previous studies by Villa-Diaz et al. have shown that PMEDSAH-
coated dishes having a water contact angle of w17?supported
self-renewal of hESCs in StemPro medium . However, PAm6-co-
Fig. 4. PAm6-co-PSS2hydrogel supported in vitro growth of HUES6 and human induced pluripotent stem cells (hiPSCs) grown over multiple passages in StemPro medium. Immu-
nofluorescence staining of (a) HUES6 and (b) hiPSCs after passages 7 and 9, respectively. Red for NANOG and Green for OCT4, while nuclei are stained blue with Hoescht 33342. Scale
bar: 200 mm (main images) and 100 mm (inset images). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
C.-W. Chang et al. / Biomaterials 34 (2013) 912e921
PMEDSAH2 hydrogels failed to support hPSCs adhesion and
growth; this could be attributed to the differences in chemical
composition and/or the hydrophilicity of the matrix. Indeed,
functional groups and hydrophilicity have been shown to playa key
role in modulating the adsorption and conformation of proteins,
and the sequestration of growth factors [10,23,35]. These subtle
changes of the cell-matrix interface can have a significant effect on
mediating the initial adhesion of hPSCs onto the matrix .
Despite having the same hydrophilicity, elastic modulus, and
surface roughness, PAm6-co-PSS2and PAm6-co-PA2AGA2hydrogels
elicited different cellular responses. These differences in cellular
responses could be attributed to changes in ECM proteins that are
adsorbed ontothe hydrogel interface, with significant alterations in
the extent of BSA and VN adsorbed between the surfaces. Previous
studies have shown that the adsorption of BSA and VN onto
hydrogel surfaces can foster adhesion and self-renewal of hPSCs
[10,15]. It is also possible that besides the amount of proteins at the
interface, the conformation of proteins plays a role in mediating cell
adhesion [36e40]. Another possibility is the differences in matrix-
bFGF binding strength, which could lead to changes in bFGF
Together, our results suggest that the adhesive interface of the
PAm6-co-PSS2 matrices, mediated through protein adsorption,
supports initial adhesion of hPSCs, which in turn facilitate both cell-
matrix and cellecell interactions to allow colony formation of the
adhered cells. While the adsorbed proteins support initial adhesion
of seeded cells, it is likely that the cell-secreted ECM proteins are
the ones that support long-term maintenance and growth of these
cells, as shown by the transcription profile. The reciprocal inter-
actions of cells with their surrounding ECM play an important role
in their fate determination as ECM components can induce various
intracellular signals to drive self-renewal vs. differentiation deci-
sions. Recent studies have indicated the importance of a combina-
tion of integrins and ECM proteins in maintaining stemness of
pluripotent cells [28,30]. For instance, a recent study by Meng et al.,
demonstrated the superior effect of matrices comprising of several
peptides over that of single peptide on supporting self-renewal of
hESCs . A similar finding was also reported by Brafman et al.,
which showed the beneficial effect of a combination of ECM
proteins on supporting self-renewal of hESCs . In addition to
ECM proteins and integrins, the cells cultured on PAm6-co-PSS2
hydrogels exhibit higher levels of MMP proteins indicating poten-
tial ECM remodeling in hPSC self-renewal. This result is consistent
with a recent study, which demonstrated the role of ECM remod-
eling and endogenous cell-secreted factors on self-renewal of
mouse embryonic stem cells (mESCs) . Any perturbations to the
ESC-secreted signaling resulted in the mESCs exiting their self-
renewal state, thus demonstrating the importance of autocrine
factors on self-renewal of pluripotent stem cells.
In conclusion, this study demonstrates that synthetic hydrogels
having a combination of physicochemical properties support
adhesion and growth of hPSCs by activating cellular processes and
Fig. 5. Effect of matrix rigidity, chemistry, and hydrophilicity on hPSCs. (a) Representative phase contrast images of adhered HUES9-Oct4-GFP cells on PSS-based hydrogels with
varying bulk rigidity (top) and their corresponding fluorescence images (bottom). (b) Images of HUES9-Oct4-GFP cells on PSS-based hydrogels with varying mole ratio of acrylamide
to sodium 4-vinylbenzene sulfonate (Am:SS; 6:0.5, 6:1, and 6:2); PAm6-co-PSS0.5, PAm6-co-PSS1, PAm6-co-PSS2(top) (c) Representative phase contrast images of hPSCs grown on
hydrogels containing different chemistries and hydrophilicities while maintaining identical sulfate functional groups and matrix rigidities. Scale bar: 200 mm.
C.-W. Chang et al. / Biomaterials 34 (2013) 912e921
harnessing autocrine factors that are conducive for self-renewal of
hPSCs. The hydrogel-based synthetic matrices introduced here
support adhesion of hPSCs and their long-term growth without
compromising their pluripotency and karyotypic stability. Such
tunable synthetic matrices also serve as platforms to elucidate the
roles of different biophysical and biochemical cues in cell-matrix
and cellecell interactions.
Conflict of interest
The authors declare no conflict of interest.
C.W. and S.V. conceptualized the study. C.W, Y.H, D.B., and S.V,
designed the experiments, and analyzed the data. C.W., Y.H., D.B.,
T.H., and C.P. performed the experiments. C.W., Y.H., D.B., and S.V.
contributed to the data interpretation, discussion, and writing the
We acknowledge Profs. S. Chien and G. Arya for valuable
discussions. We also thank the financial support from California
Institute of Regenerative Medicine (RN2-00945 and RT2-01889).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
Fig. 6. Characterization of cellematerial interface. (a) Quantification of various extracellular matrix proteins adsorbed onto PAm6-co-PSS2hydrogel (blue) and PAm6-co-PA2AGA2
hydrogel (red). BSA; bovine serum albumin, VN; vitronectin, Col1; collagen type I, Col4; collagen type IV, LN; laminin, FN; fibronectin. (b) bFGF adsorption onto PAm6-co-PSS2(blue)
and PAm6-co-PA2AGA2(red) hydrogel(s). Values are shown as mean ? SD. *p < 0.05 and **p < 0.01, (c) Hierarchical cluster analysis of transcription profile of HUES9 cells cultured
on MEFs, PAm6-co-PSS2, and PAm6-co-PSPA2. Expression levels are normalized to that of Matrigel. The notations *, **, #, and ## indicates 2e5 times, 5e10 times, 10e15 times, and
>15 times of relative fold inductions, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
C.-W. Chang et al. / Biomaterials 34 (2013) 912e921
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