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Light-Responsive Hierarchically Structured Liquid Crystal Polymer Networks for Harnessing Cell Adhesion and Migration


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Extracellular microenvironment is highly dynamic where spatiotemporal regulation of cell-instructive cues such as matrix topography tightly regulates cellular behavior. Recapitulating dynamic changes in stimuli-responsive materials has become an important strategy in regenerative medicine to generate biomaterials which closely mimic the natural microenvironment. Here, light responsive liquid crystal polymer networks are used for their adaptive and programmable nature to form hybrid surfaces presenting micrometer scale topographical cues and changes in nanoscale roughness at the same time to direct cell migration. This study shows that the cell speed and migration patterns are strongly dependent on the height of the (light-responsive) micrometer scale topographies and differences in surface nanoroughness. Furthermore, switching cell migration patterns upon in situ temporal changes in surface nanoroughness, points out the ability to dynamically control cell behavior on these surfaces. Finally, the possibility is shown to form photoswitchable topographies, appealing for future studies where topographies can be rendered reversible on demand.
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1606407 (1 of 8) © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Light-Responsive Hierarchically Structured Liquid Crystal
Polymer Networks for Harnessing Cell Adhesion
and Migration
Gülistan Koçer, Jeroen ter Schiphorst, Matthew Hendrikx, Hailu G. Kassa,
Philippe Leclère, Albertus P. H. J. Schenning,* and Pascal Jonkheijm*
DOI: 10.1002/adma.201606407
The extracellular matrix (ECM) continuously submits to cells
tightly regulated spatiotemporal changes in chemical, physical,
and mechanical cues. These dynamic changes in signals to cells
eventually prompt changes in cell and tissue behavior.[1] In regen-
erative medicine, various strategies have been used to engineer
materials to present ECM inspired cues to direct (stem) cell fate
and tissue function.[1b,2] Notwithstanding these achievements,
Extracellular microenvironment is highly dynamic where spatiotemporal
regulation of cell-instructive cues such as matrix topography tightly regulates
cellular behavior. Recapitulating dynamic changes in stimuli-responsive mate-
rials has become an important strategy in regenerative medicine to generate
biomaterials which closely mimic the natural microenvironment. Here, light
responsive liquid crystal polymer networks are used for their adaptive and
programmable nature to form hybrid surfaces presenting micrometer scale
topographical cues and changes in nanoscale roughness at the same time
to direct cell migration. This study shows that the cell speed and migra-
tion patterns are strongly dependent on the height of the (light-responsive)
micrometer scale topographies and differences in surface nanoroughness.
Furthermore, switching cell migration patterns upon in situ temporal changes
in surface nanoroughness, points out the ability to dynamically control cell
behavior on these surfaces. Finally, the possibility is shown to form photo-
switchable topographies, appealing for future studies where topographies can
be rendered reversible on demand.
G. Koçer, Prof. P. Jonkheijm
Bioinspired Molecular Engineering Laboratory
MIRA Institute for Biomedical Technology and Technical Medicine
and Molecular Nanofabrication Group
MESA+ Institute for Nanotechnology
Department of Science and Technology
University of Twente
7500 AE, Enschede, The Netherlands
J. ter Schiphorst, M. Hendrikx, Dr. P. Leclère, Prof. A. P. H. J. Schenning
Functional Organic Materials and Devices
Department of Chemical Engineering and Chemistry
Eindhoven University of Technology
5612 AE, Eindhoven, The Netherlands
J. ter Schiphorst, M. Hendrikx, Dr. P. Leclère, Prof. A. P. H. J. Schenning
Institute for Complex Molecular Systems
Eindhoven University of Technology
P.O. Box 513, 5600 MB, Eindhoven, The Netherlands
Dr. H. G. Kassa, Dr. P. Leclère
University of Mons (UMONS)
Laboratory for Chemistry of Novel Materials
Center for Innovation and Research in Materials and Polymers
Research Institute for Materials Science and Engineering
Place du Parc, 20 B-7000 Mons, Belgium
The ORCID identification number(s) for the author(s) of this article
can be found under
the majority of biomaterials that present
these cues are static, with predefined
properties that cannot submit a changing
environment to cells at any specific point
in time. Therefore, designing new bio-
materials with the ability to change proper-
ties in time is important as such dynamic
materials would mimic more closely the
natural microenvironment of cells.[1] This
can be achieved by incorporating stimuli-
responsive elements into materials using,
for example, thermosensitivity.[1a,3] Control
over size (from the nano to micrometer
scale) and arrangement of topographical
features as one of the matrix cues is known
to have great influence on cell adhesion,
spreading, migration and differentiation,
and tissue organization.[2a,4] Therefore,
recapitulating topographical changes of a
natural matrix in situ (i.e., in presence of
cells) is highly interesting for the genera-
tion of new biomaterials. Several studies
on dynamic (i.e., in situ at a specific point in time changed,
therefore temporal changes, either irreversibly or reversibly)
topographies have used strain-responsive buckling of plasma
oxidized polymethylsiloxane thin films[5] or thermally activated
shape-memory polymer (i.e.,
-polycaprolactone)[6] and strain-
responsive liquid crystal elastomers[7] in dynamic control of
(stem) cell alignment, spreading, and differentiation.
Adv. Mater. 2017, 1606407
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Nevertheless, in these systems, careful selection of the stim-
ulus considering in situ experiments as well as the versatility of
surface modifications in terms of feature generation is essen-
tial. Generally, light offers flexibility and remote control, nonin-
vasiveness and physiological compatibility, i.e., when tuned in
correct doses and wavelength, to generate changes in surface
or bulk properties of biomaterials.[1a,8] In fact, light has been
extensively used to pattern and control the presentation of cell
adhesive ligands in vitro[8,9] and in vivo[10] to tune chemical
and physical properties of cell encapsulated hydrogels,[1a,3a] to
control tissue assembly and to release patterned cells from sur-
faces.[11] The conceivable benefits of employing light responsive
systems are recognized and encourage utilization to advance
dynamic surface topographies across larger areas.[12] Yet, there
are only a few examples where light has been used to form (re)
configurable topographies in polymer systems consisting of
photoswitchable molecules in order to control cell spreading
and alignment while challenges regarding in situ studies still
remain.[12,13] In a very recent example, Netti and co-workers
elegantly showed in situ spatiotemporal control of topography
to direct cell alignment on azopolymers.[14] However, so far no
study has documented if in situ light-induced changes (i.e.,
temporal changes) in surface structure effects cell migration,
which is one of the essential processes in tissue remodeling
and wound healing as well as in colonization of biomaterials
by desired cell types for successful implantation.[4f,15] Here, we
employ the adaptive and programmable nature of light respon-
sive liquid crystal polymer network (LCN) coatings[16] to achieve
new spatially arranged patterned biointerfaces for in situ tem-
poral control of surface properties to guide cell behavior. These
azobenzene based LCN systems can operate in solvent free
environments and present a versatile way to change surface
topography both in a reversible and irreversible fashion.[16,17]
We show that these LCNs are biocompatible and can be used as
bioinstructive responsive materials to control cell adhesion and
migration. Furthermore, we exploit these materials to induce
in situ temporal changes (permanent in this case) in surface
structural properties (i.e., roughness) to control cell migration.
The preparation of the responsive LCN coatings (with com-
ponents given Scheme 1) is given in Figure 1A (mesogenic
monomers, Table S1, in the Supporting
Information). A mixture of (meth)acrylate
functionalized azobenzene and liquid crys-
talline monomers were used to create a so-
called chiral nematic phase that is subse-
quently aligned in plane by shear forces and
then photopolymerized (see details in the
Supporting Information). Mask illumination
of the films lead to local trans to cis isomeri-
zation of azobenzene molecules conjugated
in the network resulting in a local formation
of protrusions (volume generation) in the
illuminated areas yielding (microscale) topo-
graphical cues for cells (Figure S1A (Sup-
porting Information) and Figure 1B–D).[17]
These surface topographies can be rendered
permanent by modifying the starting mix-
ture composition.[17a] Cell adhesion and
migration were studied either on surfaces
with predefined (fixed) microscale topographical cues or on
surfaces where at a specific point in time the surface structure
was changed in situ. By using a mask containing a hexagonal
pattern, hexagonally arranged pillars (HP) were formed. The
height of the HPs was determined for cellular studies in the
micrometer scale by conveniently varying the illumination
dose (intensity). A series of surfaces were prepared with pil-
lars ranging in height from 0.2 to 1.6 µm, having a diameter of
20 µm and spaced 20 µm from each other as was verified using
optical profilometry (Figure 1B,C, HP-0.2 µm, HP-0.3, HP-0.5,
HP-1.2, and HP-1.6 µm). Other patterns were achieved using
other masks, such as circular patterns (CP) of, e.g., 15 µm in
width and separation and with a height of 0.3 µm (CP-0.3),
demonstrating the versatility of this contactless method to fabri-
cate topographical polymeric biointerfaces (Figure 1D). As con-
trol surfaces to the films with microscale topographies, “flat”
(with respect to microscale) LCN surfaces were used that were
or were not entirely illuminated (Flat_i or Flat_ni). Upon illu-
mination the morphology of the film alternates, which is veri-
fied by a change in the absorbance properties of the polymer
films (Figure S1B, Supporting Information). Atomic force
microscopy (AFM) measurements on Flat_ni and Flat_i sur-
faces revealed an increase in surface roughness on the illumi-
nated areas changing from 9.9 ± 0.9 to 18.2 ± 0.4 nm showing
that the nanoscale topography was changed upon illumination
(Figures S1C,D and S2A,B, Supporting Information). It should
be noted that the formation of pillars further increases the
nanoroughness (vide infra). The surfaces were hydrophobic
irrespective of illumination with contact angles of 88° ± 1° and
101° ± 4° for Flat_ni and Flat_i, respectively. In order to facili-
tate protein adsorption and hence cell adhesion on these sur-
faces, all the LCN films were incubated and coated with serum
proteins overnight before performing the cell experiments.
Use of established cell lines such as NIH3T3 fibroblasts is
informative in revealing adhesion related cellular processes.[4b,14]
Adhered NIH3T3 fibroblast cells and their supported growth
on all the fabricated LCN surfaces (Figure 1E–M and Videos
S1–S9 and Figure S3 (Supporting Information)) validated the
biocompatibility of these LCN films similar to polystyrene,
which is commonly used for cell cultures. From time-lapse
Adv. Mater. 2017, 1606407
Scheme 1. Molecular structures of the materials that were used for preparing the light-respon-
sive liquid crystal polymer (Compounds are 1: Light responsive molecule, 2: Chiral dopant, 3–5:
Nematic hosts, 6: Photoinitiator, 7: Chain stopper/inhibitor).
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imaging experiments performed on the films that were incu-
bated with fibroblasts, single cell migration behavior and the
mean cell migration speed (total distance covered per time of
measurements) were determined (Figures 2 and 3). The initial
mean cell migration speed of 0.85 µm min1 observed on flat
LCN films significantly dropped to 0.32 µm min1 when such
films were illuminated entirely, while qualitatively no changes
in cell morphology were observed (Figures 2H and 3A–C and
Videos S1 and S2 (Supporting Information)). Furthermore, cell
migration patterns were derived from the time-lapse images and
plotted as a change in cell migration speed in intervals of 5 min
(Figure 3G,H). These plots not only confirm that cells became
Adv. Mater. 2017, 1606407
Figure 1. Encoding topography in films using light in LCNs to harness cell behavior. A) Schematic representation of the method to fabricate HP
topographies in LCN films by using a hexagonally patterned mask and B) 3D representation of a HP surface. C) Height profiles of the HP surfaces.
D) 3D representation of the CP surface. E–H) Phase contrast images of NIH3T3 cells after 1 d on HP-1.6, HP-1.2, HP-0.5, HP-0.2 µm, respectively.
I) SEM image of cells on a HP surface (with 15 µm diameter and separation, and 0.15 µm height) after 12 h. J) Phase contrast image of cells on CP-0.3
surface after 1 d. K,L) SEM images of cells on CP-0.3 surface after 12 h and M) phase contrast image of cells after 1 d on tissue culture polystyrene as
control surface (scale bar: 50 µm for (E–H, J, M); 10 µm for (I), and 100 µm for (K, L)).
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more static on illuminated substrates but also reveal changes in
migration speed that occurred when cells were observed over
the course of a few hours. These changes in migration speed
occurred similarly frequent, yet of a markedly lower magni-
tude upon illumination (Figure 3G,H). These observations
can be related to the increase in nanoscale surface roughness
upon illumination, as was measured using AFM (9.9 ± 0.9
versus 18.2 ± 0.4 nm, for Flat_ni versus Flat_i, respectively,
Figure S1C,D, Supporting Information). It is known that cells
are able sense nanoscale features down to 8 nm and distinguish
the size of the features by their nanopodial extensions.[4b,18] Yet,
studies also show that in general, cell attachment is influenced
by changes in mechanical properties.[19] To gain more insight in
the nanoscale topographical and mechanical properties of the
Adv. Mater. 2017, 1606407
Figure 2. Characterization of single cell migration behavior on LCN surfaces. A) AFM morphology and height image of a 0.75 µm pillar and its sur-
rounding. B) Derjaguin–Müller–Toporov (DMT) effective elastic modulus map obtained from one ImAFM scan of the 0.75 µm HP (given in Figure A)
(The color contrasts and the scale bars depict the value of the stiffness on the sample surface in GPa. The scan size is 40 × 40 µm2). Snapshots from
live cell imaging for representative cells C,D) residing on the pillars and E) stationary and in contact with the pillars on the flat surfaces on HP-0.3,
HP-1.6, HP-1.2 topographies, respectively (black arrow: a representative stationary cell). F,G) Representative cells that are motile over the pillars and
on the flat areas on HP-1.2 and HP-1.6 µm surfaces, respectively (scale bar: 50 µm). H) Single cell migration speed on different LCN surfaces. The
black circles are nonclassified cell categories, while the blue circles (open and closed) show category 1 and 2, respectively (ni: not illuminated, red
line: the mean cell speed, mean ± standard deviation, **p < 0.01 and *p < 0.05). I) Percentage of the cells on HP surfaces representing the whole cell
population counted on the surfaces in both categories.
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pillars and surrounding areas we imaged a hierarchically struc-
tured HP-0.75 µm film using intermodulation atomic force
microscopy (ImAFM) (Figure 2A,B and Figure S2 and Table S2
(Supporting Information)). Height and roughness on top of
the pillars and in their surrounding areas were determined and
compared to illuminated flat surfaces. Illumination of flat films
resulted in a 20 nm increase in nanoscale roughness (Table S2,
Supporting Information). The surfaces of illuminated pillars
were found to also be rougher (55 ± 3.3 nm) compared with
the nonilluminated surrounding areas (10 ± 1.4 nm). These
results verified that we have fabricated hybrid interfaces pre-
senting light-induced topographical patterns with significantly
increased nanoscale roughness. ImAFM inspections also reveal
simultaneously nanoscale mechanical properties of the sur-
face of the pillars and their surrounding areas (Figure 2B and
Figure S2C,D (Supporting Information)). Figure 2B is a typical
effective elastic modulus map showing an average effective
elastic modulus (0.5–0.75 GPa) of the surfaces of illuminated
Adv. Mater. 2017, 1606407
Figure 3. Characterization of surface topology and pillar height dependent switch in cell migration behavior. A) Average speed of cells on both
flat surfaces and cells on HP-0.3 µm surface in Category 1. Phase contrast images of cells after 1 d on B) flat not illuminated, C) flat illuminated,
and D,E) HP-0.3 µm surface (scale bar: 50 µm). F) Time-lapse imaging for a cell that represents Category 1 on HP-0.3 µm surface (scale bar: 50 µm).
G–J) Cell migration patterns as change in single cell speed at every 5 min for four representative cells on different LCN surfaces.
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pillars, which was found lower than on nonilluminated
surrounding areas (1.2 GPa) (see the Supporting Information
for details). Even though the elastic modulus has decreased
upon illumination, we believe that both illuminated and nonil-
luminated areas present stiff matrices to cells to a similar
extent due to their high elastic modulus (in GPa) compared to
elastic natural matrix (elastic modulus ranging from a few hun-
dred Pa to a few hundred kPa).[19,20] Forces generated by cells
are in the range of 1–5 nN µm2 and cells would need to pull
against and deform the underlying substrate to be able to sense
and respond to stiffness.[19a,20,21] Therefore, our hierarchical
interfaces allow us to relate the effect of nanoscale roughness
and micrometer scale topographical cues independently of the
stiffness of the surfaces.
The derived mean cell migration speeds on LCN films with the
HP topographical cues revealed that surfaces with 0.3 µm high
pillars (HP-0.3 µm) induced a significantly lower mean cell speed
of 0.23 µm min1 when compared to the HP-0.5, HP-1.2, and
HP-1.6 surfaces, which had a mean cell speed of 0.87, 0.81,
and 0.87 µm min1, respectively (Figure 2H and Videos S4–S7
(Supporting Information)). Interestingly, the latter topogra-
phies induced cell speeds in the same range as observed on the
nonilluminated flat LCN films, while the cell speed observed on
the HP-0.3 topography is similar to that on the illuminated flat
LCN films. The mean cell speed of 0.52 µm min1 observed on
HP-0.2 topographies represents an intermediate case (vide infra).
As a control surface for cell adhesion and migration, a speed of
0.55 µm min1 was observed on classical tissue culture polystyrene
surfaces, which lies in between the values observed for both flat
LCN surfaces (Figure S4 and Video S9, Supporting Information).
To closer inspect how the HP topographies were harnessing
the spatiotemporal adhesion and organization of cells on these
surfaces during migration, the live cell imaging data were ana-
lyzed in more detail (see the Experimental Section). From the
analysis, two modes of cell behavior were identified; cells in cat-
egory 1 were mainly stationary with mean cell speeds as low
as 0.15 µm min1 and these cells were mainly in contact with
the pillars while having minimal contact with the areas sur-
rounding the pillars (Figure 2C,E); cells in category 2 were gen-
erally mobile with mean cell speeds as high as 1.13 µm min1
and these cells were having minimal contact with pillars and
were mainly moving across areas surrounding the pillars
(Figure 2F,G). Much to our surprise, the analysis shows that the
majority (84%) of the cells on HP-0.3 µm belong to static, cat-
egory 1 type cells (Figure 2H,I), while the majority (70%–87%)
of cells on films with higher pillars (HP-0.5, 1.2, and 1.6 µm)
fall to mobile, category 2 type cells. These category 2 cells
migrate with no significant differences on the HP-0.5, 1.2, and
1.6 topographies (Figure 2H), yet they do migrate significantly
faster when compared to the category 1 cells on HP-0.3 topog-
raphies. It is known that higher cell speed can be related to the
persistence in directional movement.[4f,22] However, trajectory
analysis of cells in category 2 on all HP surfaces did not reveal
any direct correlation of cell speed with persistence and direc-
tionality (Figure S5, Supporting Information). As the average
speeds of the two categories correspond with the speed of the
illuminated and nonilluminated flat surfaces, we tentatively
assign the remarkable, static versus mobile appearance of cells
to the extent that cells were in contact with a rougher surface
existing on illuminated parts of the films versus their contact
with a smoother surface existing on nonilluminated parts of the
films. The cell migration behavior that was imaged on HP-0.2
topographies could not be categorized due to a lack of resolving
the topography during imaging (black circles in Figure 2H).
Yet, cells on HP-0.2 µm were spread over both pillars and
nonilluminated areas indicative of cells that fall into category 2
(Figure 1H and Video S3 (Supporting Information)).
Most notable are the consequences for cell migration
behavior when switching from flat surfaces to a HP-0.3 topog-
raphy. Category 1 cells on HP-0.3 topographies were adherent
and residing only on the pillars, mainly elongating over the pil-
lars for extended periods of up to 5 h (Figure 3D–F) in agree-
ment with the observed contact of cellular protrusions on the
rougher pillars (Figure 1I). In contrast, cells on the flat LCN
surfaces, irrespective of illumination, were spread, but not as
elongated, which is typical for cell morphologies on 2D sur-
faces (Figures 3B,C and 1M). This clearly points toward topog-
raphy guided cell morphology, which is known to influence cell
motility.[2a,4d–f] Intriguingly, even though cells were residing
on illuminated parts of the topographic films, presumably due
to increased roughness, the mean cell migration speed of the
category 1 type cells on HP-0.3 significantly dropped further
to 0.15 µm min1 when comparing to the cell migration speed
of 0.32 µm min1 on flat, illuminated films (Figure 3A). The
migration pattern plots for HP-0.3 topographies reveal that
this category of cells became even more static with lower and
less frequent changes observed in cell speed (Figure 3H,I). In
strong contrast, cells on HP-1.2 topographies yielded a dramatic
shift in the cell migration pattern to a highly dynamic behavior
with increasing frequencies and magnitudes of changes in cell
migration speed over time (Figure 3I,J and Figure S6 (Sup-
porting Information)).
Next to the HP topographic patterned films, single cell migra-
tion speed and cell behavior were also imaged and analyzed on
the circular patterns of comparable heights and spacing as the
HP patterns (Video S8, Supporting Information). This anal-
ysis similarly revealed the existence of two distinct cell popu-
lations with cells aligning on the patterns of 0.3 µm in height
and 15 µm spacing. These results show that cell adhesion and
migration was guided on a different surface geometry indicating
that the observations are generic (Figure 1J–L and Figure S7
(Supporting Information)). These results demonstrate also the
versatility of employing light responsive LCN films as new
biointerfaces to direct cell movement and migration speed.
The changes in cell migration patterns are clearly associated
with synergistic cell contact guidance by the micrometer scale
topographies and the nanoscale roughness both of which are
consequences of the light-induced isomerization of the azoben-
zene moieties in the bulk of the film material.[2a,4b] Switching
cell migration behavior by switching the surface structure have
important implications regarding in situ dynamic experiments
where cell migration could be differentially harnessed to guide
cells to specific locations on the surface.
To explore the possibility to guide cell migration and col-
lect these migrating cells in local spots, the following surface
topographic pattern was designed. Films were prepared with
flat, nonilluminated areas surrounded by HP-1.1 topographies
(Figure 4A,B and Figure S8A (Supporting Information)). Our
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observations on such mixed surface-structured films revealed
that the cell migration speed was similar on both areas of the
surface and in agreement with the speeds and trends observed
on either of the individual, nonmixed surface-structured sur-
faces (Figure 2H and Figure S8B and Video S10 (Supporting
Information)). However, much to our surprise, the majority of
the cells (82%, 9 out of 11 cells that were monitored for 4 h)
starting from the pillar area, entered the flat area and remained
in this area with a spread morphology (Figure 4B). This result
shows the possibility to spatially control the localization of the
cell population through cell migration on these surfaces, which
has important implications in colonization of cells on biomate-
rials to enhance their performance.[15]
With these results in hand, consequences of in situ temporal
(i.e., changes occur in time in the presence of cells) light-induced
permanent surface structural changes on the cell behavior
were studied representing a first step in exploring the poten-
tial of these materials in dynamically controlling the surface
topography. For this purpose, cells were seeded on flat, nonil-
luminated surfaces and before illumination of the substrate the
typical response of cells on these surfaces was
observed (Figures 3G and 4D). For this type
of experiments, a critical consideration is the
biocompatibility of the illumination dose.[1a]
By illuminating the film from the bottom,
light penetration measurements using an
intensity detector (details are given in the
Supporting Information) showed no UV pen-
etration through the samples to the cells indi-
cating that UV light was absorbed by the film.
According to this measurement, the films
were exposed to light of
= 390–440 nm for
10 min (35 mW cm2), which was sufficient
to induce the structural changes in rough-
ness. According to AFM measurements on
this sample an increase in surface roughness
from 9.0 ± 1.2 to 11.1 ± 2 nm was observed
upon illumination for 10 min (Figure S9,
Supporting Information), where a change in
cell migration can be expected.[4b,f,18,23]
Upon illumination, cells were monitored
for 12 h before measuring the cell migra-
tion speed, to observe and confirm their
viability after illumination. Live cell imaging
data revealed that 87% of the cells remained
viable within this time (Videos S11 and S12,
Supporting Information). Changes in cell
migration speed occurred less frequent and
to a smaller extent with a decrease in mean
cell speed from 1.21 to 0.54 µm min1 after
illumination showing a shift toward a more
static behavior (Figures 3H and 4E and
Figure S10 (Supporting Information)). These
observations are in accordance with the ex
situ experiments (Figure 3A) where speed
decreased with increased roughness.
In conclusion, consequences for cell
migration on light-induced topographi-
cally patterned polymer network films were
studied as function of micrometer scale topographical height
and pattern and nanoscale surface roughness on similarly stiff
surfaces for cells. Cell motility patterns could be switched from
static to highly mobile by increasing the height of the pillars or
from dynamic to moderately static by increasing the nanoscale
roughness or to highly static on a selective topographic pattern.
In situ temporal experiments further demonstrated the effect of
nanoscale changes. Preliminary experiments on patterned LCN
surfaces showed that it is also possible to form photoswitchable
(reversible) topographies on demand (Figures S11 and S12, Sup-
porting Information) at the nanoscale which is another impor-
tant step for in situ control of cell behavior. These results demon-
strate a new approach in engineering bioinstructive light respon-
sive surface structures. Future experiments aim at in situ revers-
ible studies as well as manipulation of microscale topographies
where light responsive elements that are switchable at longer
wavelengths can be used to match the volume generation prop-
erties with cytocompatible illumination doses.[24] The freedom
that is offered in this system will enable us to generate mate-
rials that are more closely recapitulating the dynamic natural
Figure 4. Spatial and temporal switches in cell behavior on the same LCN surface. A) 3D
representation of the mixed surface with a Flat_ni region surrounded by HP-1.1 topographies.
B) Phase contrast image of NIH3T3 cells on LCN surfaces presenting a flat not illuminated area
surrounded by 1.1 µm high HPs after 3 d of culture. C) Live cell imaging for a representative cell
going from the pillar area to the flat area (scale bar: 50 µm). D,E) In situ switch in cell migration
patterns on flat LCN surfaces as change in single cell speed at every 5 min.
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Adv. Mater. 2017, 1606407
microenvironment not just giving better insights in funda-
mental biological processes but will also enable us to enhance
biomaterial performance for regenerative medicine applications.
Experimental Section
Preparation and Characterization of Liquid Crystal Polymer Network
Surfaces: Detailed information on preparation of light responsive
LCN materials is given in the Supporting Information. Material
characterization was performed with UV–vis spectroscopy, AFM, optical
profilometry, and contact angle measurements. Details of surface
characterization are also given in the Supporting Information.
Characterization of Cell Behavior: NIH 3T3 (ATTC CRL-1658) cells
were seeded at a density of 5000 cells cm2 on the surfaces and time
lapse images were taken every 5 min using Cytomate live cell imaging
equipment (Eindhoven, The Netherlands). Cell trajectories and cell
migration speed were derived using the MTrackJ plugin of ImageJ
(NIH) by following individual cells. In situ switching experiments were
performed at room temperature for 10 min, after 1 d of cell adhesion,
in live cell imaging medium. Live cell imaging was performed as
explained above to observe cell migration before and after illumination
of the substrates. Cell adhesion and organization on the surfaces were
analyzed with phase contrast and scanning electron microscopy (SEM).
See the Supporting Information for details.
Statistics: Data are presented as mean ± standard deviation and cell
migration data were analyzed using Kruskal–Wallis one way-ANOVA
for statistical significance. Multiple comparison tests were performed
with Mann–Whitney test by adjusting
level downward according to
Bonferroni correction comparing all groups of interest.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
P.J. and G.K. thank NWO (VIDI Grant No. 723.012.106) for funding.
J.t.S. and A.P.H.J.S. thank EU FP7 for funding (NAPES Project
No. 604241). M.H. thanks NWO (TOP PUNT Grant No. 10018944) for
funding. P.L. thanks FEDER (BELSPO-PAI VII/5), P.L. and H.G.K. thank
FRS-FNRS PDR (ECOSTOFLEX). Danqing Liu, Sisi Tang, Erik F.G.A.
Homburg, Marc P.F.H.L. van Maris, Dick J. Broer, Jenny Brinkmann,
Koen Nickmans, and Jeffrey N. Murphy are acknowledged for helpful
discussions and assistance during measurements.
Conflict of Interest
The authors declare no conflict of interest.
biointerfaces, cell–biomaterial interactions, liquid crystals, stimuli-
responsive topographies
Received: November 26, 2016
Revised: March 20, 2017
Published online:
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... It is worth emphasizing that these inputs should involve multiple and typically local components. So programmable materials differ fundamentally from the more traditional responsive materials that only react to a global and ambient stimulus (such as heat-responsive shape memory alloys [3,4] or light-responsive liquid crystal polymers [5][6][7]). Moreover, users should be able to reset and re-design the input set for different output properties. ...
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This study investigates the programming of elastic wave propagation bandgaps in a kirigami material by intentionally sequencing its constitutive mechanical bits. Such sequencing exploits the multi-stable nature of the stretched kirigami, allowing each mechanical bit to switch between two stable equilibria with different external shapes (aka. "(0)" and "(1)" states). Therefore, by designing the sequence of (0) and (1) bits, one can fundamentally change the underlying periodicity and thus program the phononic bandgap frequencies. To this end, this study develops an algorithm to identify the unique periodicities generated by assembling "$n$-bit strings" consisting of $n$ mechanical bits. Based on a simplified geometry of these $n$-bit strings, this study also formulates a theory to uncover the rich mapping between input sequencing and output bandgaps. The theoretical prediction and experiment results confirm that the (0) and (1) bit sequencing is a versatile approach to programming the phonic bandgap frequencies. Moreover, one can additionally fine-tune the bandgaps by adjusting the global stretch. Overall, the results of this study elucidate new strategies for programming the dynamic responses of architected material systems.
... In general, stimuli should be mild and biocompatible to avoid cell dysfunctions [176]. Light responsive AZO-based liquid crystal polymeric coatings can be utilized to form surfaces presenting micrometer scale topographical cues and changes in nanoscale roughness due to volume generation arising from trans-to-cis isomerization of AZO molecules upon UV irradiation to direct cell migration [177]. Polymeric coatings containing SP may exhibit water repellency and cell adhesion with adjustable surface polarity by photo-isomerization [178]. ...
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Continuous interests in stimuli-responsive macromolecules significantly impacted new developments in polymeric coatings. Responsiveness to bacterial attacks, ice or fog formation, anti-fouling properties, autonomous self-cleaning and self-healing, or drug delivery systems, are just a few examples of modern functions of macromolecules and other components utilized in polymeric coatings. These autonomous responses to various external stimuli combined with the suitable protection and appearance are particularly attractive functions. This review outlines recent advances in the development of novel stimulus-responsive polymeric coatings in the context of current and future trends. A combination of stimuli-responsiveness, protection and durability, appearance, and other “smart” functions make polymeric coatings particularly attractive as integral components of future engineered systems. This review consists of four sections, (1) stimulus-responsive protection, (2) stimulus-responsive appearance, (3) smart functions, and (4) future trends and opportunities. The purpose of this monograph is not to list all stimuli and responsiveness utilized in polymeric coatings, but address favorable and unpropitious, in our view, scientific advances and technological opportunities in the development of a new generations of “smart” coatings that still maintain traditional functions of protection and appearance.
... 71,72 We fabricated lightresponsive changeable surface topographies on films of liquid crystal polymer networks and explored these topographies for controlling cell adhesion, polarization, and migration (Fig. 1C). 73 Switching cell migration patterns upon in situ temporal topographical changes, points out the ability to control dynamically cell behavior on these biointerfaces. ...
Synthetically designed biomaterials strive to recapitulate and mimic the complex environment of natural systems. Using natural materials as a guide, the ability to create high performance biomaterials that control cell fate, and support the next generation of cell and tissue-based therapeutics, is starting to emerge. Supramolecular chemistry takes inspiration from the wealth of non-covalent interactions found in natural materials that are inherently complex, and using the skills of synthetic and polymer chemistry, recreates simple systems to imitate their features. Within the past decade, supramolecular biomaterials have shown utility in tissue engineering and the progress predicts a bright future. On this 30th anniversary of the Netherlands Biomaterials and Tissue Engineering society, we will briefly recount the state of supramolecular biomaterials in the Dutch academic and industrial research and development context. This review will provide the background, recent advances, industrial successes and challenges, as well as future directions of the field, as we see it. Throughout this work, we notice the intricate interplay between simplicity and complexity in creating more advanced solutions. We hope that the interplay and juxtaposition between these two forces can propel the field forward.
Dynamic interplay between extracellular matrix with anisotropic structure and biological cells has directed the scaffold path of development toward the utilization of actuation-responsive biomaterials. Among stimuli-responsive polymers, liquid crystalline elastomers (LCEs), owing to attachment of highly anisotropic mesogenic units to the amorphous elastomeric chains, are able to reveal reversible orientational order. Dynamic order–disorder phase transition and stimuli-responsiveness are the main factors to consider them as rationally smart mimickers of biological cell topology. This Review explains briefly the structures and synthesis methods of LCEs as well as their materiobiology with an emphasis on biochemical and biomechanical features of cells from molecular view. In this regard, some indispensable factors such as quasi-liquid crystalline behavior of cells, degree of orientational order in different cells and liquid crystalline induced topographical signals as growth enhancing agent for cells are investigated. Moreover, the interactions of cell-liquid crystalline substrate and the performance of LCE scaffolds on various cell lines response are given as an overview.
Programming shape changes in soft materials requires precise control of the directionality and magnitude of their mechanical response. Among ordered soft materials, liquid crystal elastomers (LCEs) exhibit remarkable and programmable shape shifting when their molecular order changes. In this work, we synthesized, remotely programmed, and modeled reversible and complex morphing in monolithic LCE kirigami encoded with predesigned topological patterns in its microstructure. We obtained a rich variety of out-of-plane shape transformations, including auxetic structures and undulating morphologies, by combining different topological microstructures and kirigami geometries. The spatiotemporal shape-shifting behaviors are well recapitulated by elastodynamics simulations, revealing that the complex shape changes arise from integrating the custom-cut geometry with local director profiles defined by topological defects inscribed in the material. Different functionalities, such as a bioinspired fluttering butterfly, a flower bud, dual-rotation light mills, and dual-mode locomotion, are further realized. Our proposed LCE kirigami with topological patterns opens opportunities for the future development of multifunctional devices for soft robotics, flexible electronics, and biomedicine.
Accurately obtaining information on the heterogeneity of CTCs at the single-cell level is a very challenging task that may facilitate cancer pathogenesis research and personalized therapy. However, commonly used multicellular population capture and release assays tend to lose effective information on heterogeneity and cannot accurately assess molecular-level studies and drug resistance assessment of CTCs in different stages of tumor metastasis. Herein, we designed a near-infrared (NIR) light-responsive microfluidic chip for biocompatible single-cell manipulation and study the heterogeneity of CTCs by a combination of the lateral flow microarray (LFM) chip and photothermal response system. First, immunomagnetic labeling and a gradient magnetic field were combined to distribute CTCs in different regions of the chip according to the content of surface markers. Subsequently, the LFM chip achieves high single-cell capture efficiency and purity (even as low as 5 CTCs per milliliter of blood) under the influence of lateral fluid and magnetic fields. Due to the rapid dissolution of the gelatin capture structure at 37 °C and the photothermal properties of gold nanorods, the captured single CTC cell can be recovered in large quantities at physiological temperature or released individually at a specific point by NIR. The multifunctional NIR-responsive LFM chip demonstrates excellent performance in capture and site release of CTCs with high viability, which provides a robust and versatile means for CTCs heterogeneity study at the single-cell level.
Soft materials comprising polyethyleneimine (PEI), that integrate low pH-stimulated higher-order assemblies (fibres and sheets) with light responsiveness, have been shown. Excitation wavelength light-driven interactions enable the formation of bead-necklace-type structures...
Photoresponsive soft materials are everywhere in the nature, from human’s retina tissues to plants, and have been the inspiration for engineers in the development of modern biomedical materials. Light as an external stimulus is particularly attractive because it is relatively cheap, noninvasive to superficial biological tissues, can be delivered contactless and offers high spatiotemporal control. In the biomedical field, soft materials that respond to long wavelength or that incorporate a photon upconversion mechanism are desired to overcome the limited UV-visible light penetration into biological tissues. Upon light exposure, photosensitive soft materials respond through mechanisms of isomerization, crosslinking or cleavage, hyperthermia, photoreactions, electrical current generation, among others. In this review, we discuss the most recent applications of photosensitive soft materials in the modulation of cellular behavior, for tissue engineering and regenerative medicine, in drug delivery and for phototherapies.
Deformations triggered by body heat are desirable in the context of shape-morphing applications because, under the majority of circumstances, the human body maintains a higher temperature than that of its surroundings. However, at present, this bioenergy-triggered action is primarily limited to soft polymeric networks. Thus, herein, the programming of body temperature-triggered deformations into rigid azobenzene-containing liquid crystalline polymers (azo-LCPs) with a glass-transition temperature of 100 °C is demonstrated. To achieve this, a mechano-assisted photo-programming strategy is used to create a metastable state with room-temperature stable residual stress, which is induced by the isomerization of azobenzene. The programmed rigid azo-LCP can undergo large-amplitude body temperature-triggered shape changes within minutes and can be regenerated without any performance degradation. By changing the programming photomasks and irradiation conditions employed, various 2D to 3D shape-morphing architectures, including folded clips, inch-worm structures, spiral structures, and snap-through motions are achieved. When programmed with polarized light, the proposed strategy results in domain-selective activation, generating designed characteristics in multi-domain azo-LCPs. The reported strategy is therefore expected to broaden the applications of azo-LCPs in the fields of biomedical and flexible microelectronic devices.
Tissue engineering is an alternative medical approach to reconstructing neuronal injuries. Topographical features direct neuronal responses, such as cell adhesion, proliferation, morphological changes, alignment, and gene expression, which are controlled by the size, shape, and pattern of the adherent surface. Several studies have reported different aspects of neuronal behavior on aligned micro/nanopatterns and pillars. In this review, we focus on the recent progress in the development of topographical patterns for neuronal outgrowth, guidance, and differentiation. We discuss the neuronal responses to various patterns, such as grooves, fibers, and pillar patterns. These topographical features induce neuronal alignment and enhance their differentiation. Furthermore, we review the underlying mechanisms linked to the effects of micro- and nanopatterns.
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High aspect ratio pillared topographies provide a large number of mechanical cues that cells can sense and react to. High aspect ratio pillars have been employed effectively to promote stem cell differentiation and to probe cellular tractions. Yet, the full potential of these topographies for mechanobiology remains insufficiently characterized. Here, the response of progenitor neural stem cells to dense high aspect ratio polymer pillars in the nano- and microscale is investigated. Thermal nanoimprinting is utilized to fabricate with high precision well-defined pillars with high density and aspect ratio. Studies on cell viability, morphology, cell spreading, and migration are performed comparatively to a control flat substrate. The traction forces exerted by the cells on the pillar structures are probed quantitatively by a combined focused ion beam scanning electron microscopy (FIB-SEM) technique. The cell responses observed are distinctive for each dimension, following the trend that an increase in aspect ratio and feature size from nano- to micronscale results in more confined cell morphology with large cytoplasmic penetrations and nuclear deformation. Accordingly, cells seeded on the micrometer scale topography show reduced mobility, a persistent quasi-directional migration, high traction forces, and a lower rate of proliferation. Cells on the nanotopography show higher rate of proliferation, a large cell spread, high mobility with random migration altogether with lower traction forces.
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Photoactivated generation of disorder in a liquid crystal network produces free volume that leads to the controlled formation of dynamic corrugations at its surface. The liquid crystal order amplifies the deformation of copolymerized azobenzene, which takes place on molecular length scales, to a micrometre-sized macroscopic phenomenon based on changes in density. We postulate a new mechanism in which continuous oscillating dynamics of the trans-to-cis isomerization of the azobenzene overrules the net conversion, which is currently considered as the origin. This is supported by a significant local density decrease when both the trans and cis isomers are triggered simultaneously, either by dual-wavelength excitation or by the addition of a fluorescent agent converting part of the light to the cis-actuating wavelengths. This new insight provides a general guideline to boost free volume generation leading not only to larger macroscopic deformations but also to controllable and faster non-equilibrium dynamics.
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Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have recently been realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell-adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials.
Cell response to exogenous cues is the result of a complex integration of multiple biochemical/biophysical signals, which might occur simultaneously and might be characterized by specific spatial and temporal patterns. Among these signals, surface topography plays an important role in affecting cell func-tions and fate. However, the current understanding of the interplay between cells and topography relies on static environment. Here the intrinsic light-responsive properties of azopolymers and the versatility of laser-based confocal microscope technique is exploited, aiming to induce spatio-temporal dynamic topographic changes in situ during cell culture. Diverse patterns can be designed on cell-populated azopolymer films with high control on time, space, and on-off signal modification. The technique proposed in this study enables the development of synthetic platforms that finely control cell orientation and migration both in time and space. The results may pave the way to unravel complex processes involved in cell-topography interactions, thus allowing to define the spatio-temporal features that most effectively influence cell functions.
The extracellular matrix (ECM) is a dynamic environment that constantly provides physical and chemical cues to embedded cells. Much progress has been made in engineering hydrogels that can mimic the ECM, but hydrogel properties are, in general, static. To recapitulate the dynamic nature of the ECM, many reversible chemistries have been incorporated into hydrogels to regulate cell spreading, biochemical ligand presentation and matrix mechanics. For example, emerging trends include the use of molecular photoswitches or biomolecule hybridization to control polymer chain conformation, thereby enabling the modulation of the hydrogel between two states on demand. In addition, many non-covalent, dynamic chemical bonds have found increasing use as hydrogel crosslinkers or tethers for cell signalling molecules. These reversible chemistries will provide greater temporal control of adhered cell behaviour, and they allow for more advanced in vitro models and tissue-engineering scaffolds to direct cell fate.
Liquid crystals are the basis of a pervasive technology of the modern era. Yet, as the display market becomes commoditized, researchers in industry, government and academia are increasingly examining liquid crystalline materials in a variety of polymeric forms and discovering their fascinating and useful properties. In this Review, we detail the historical development of liquid crystalline polymeric materials, with emphasis on the thermally and photogenerated macroscale mechanical responses-such as bending, twisting and buckling-and on local-feature development (primarily related to topographical control). Within this framework, we elucidate the benefits of liquid crystallinity and contrast them with other stimuli-induced mechanical responses reported for other materials. We end with an outlook of existing challenges and near-term application opportunities.
In this work, the development of a photoresponsive platform for the presentation of bioactive ligands to study receptor-ligand interactions has been described. For this purpose, supramolecular host-guest chemistry and supported lipid bilayers (SLBs) have been combined in a microfluidic device. Quartz crystal microbalance with dissipation monitoring (QCM-D) studies on methyl viologen (MV)-functionalized oligo ethylene glycol-based self-assembled monolayers, gel and liquid-state SLBs have been compared for their nonfouling properties in the case of ConA and bacteria. In combination with bacterial adhesion test, negligible nonspecific bacterial adhesion is observed only in the case of methyl-viologen-modified liquid-state SLBs. Therefore, liquid-state SLBs have been identified as most suitable for studying specific cell interactions when MV is incorporated as a guest on the surface. The photoswitchable supramolecular ternary complex is formed by assembling cucurbit[8]uril (CB[8]) and an azobenzene-mannose conjugate (Azo-Man) onto MV-functionalized liquid-state SLBs and the assembly process has been characterized using QCM-D and fluorescence techniques. Mannose has been found to enable binding of E. coli via cell-surface receptors on the nonfouling supramolecular SLBs. Optical switching of the azobenzene moiety allows us to "erase" the bioactive surface after bacterial binding, providing the potential to develop reusable sensors. Localized photorelease of bacterial cells has also been shown indicating the possibility of optically guiding cellular growth, migration, and intercellular interactions.
Recently, there has been a great deal of interest in using the photoisomerization of azobenzene compounds to control specific biological targets in vivo. These azo compounds can be used as research tools or, in principle, could act as optically controlled drugs. Such "photopharmaceuticals" offer the prospect of targeted drug action and an unprecedented degree of temporal control. A key feature of azo compounds designed to photoswitch in vivo is the wavelength of light required to cause the photoisomerization. To pass through tissue such as the human hand, wavelengths in the red, far-red, or ideally near infrared region are required. This Account describes our attempts to produce such azo compounds. Introducing electron-donating or push/pull substituents at the para positions delocalizes the azobenzene chromophore and leads to long wavelength absorption but usually also lowers the thermal barrier to interconversion of the isomers. Fast thermal relaxation means it is difficult to produce a large steady state fraction of the cis isomer. Thus, specifically activating or inhibiting a biological process with the cis isomer would require an impractically bright light source. We have found that introducing substituents at all four ortho positions leads to azo compounds with a number of unusual properties that are useful for in vivo photoswitching. When the para substituents are amide groups, these tetra-ortho substituted azo compounds show unusually slow thermal relaxation rates and enhanced separation of n-π* transitions of cis and trans isomers compared to analogues without ortho substituents. When para positions are substituted with amino groups, ortho methoxy groups greatly stabilize the azonium form of the compounds, in which the azo group is protonated. Azonium ions absorb strongly in the red region of the spectrum and can reach into the near-IR. These azonium ions can exhibit robust cis-trans isomerization in aqueous solutions at neutral pH. By varying the nature of ortho substituents, together with the number and nature of meta and para substituents, long wavelength switching, stability to photobleaching, stability to hydrolysis, and stability to reduction by thiols can all be crafted into a photoswitch. Some of these newly developed photoswitches can be used in whole blood and show promise for effective use in vivo. It is hoped they can be combined with appropriate bioactive targets to realize the potential of photopharmacology.
Topography of material surfaces is known to influence cell behavior at different levels: from adhesion up to differentiation. Different micro- and nano-patterning techniques have been employed in order to create patterned surfaces to investigate various aspects of cell behavior, most notably cellular mechanotransduction. Nevertheless, conventional techniques, once implemented on a specific substrate, fail in allowing dynamic changes of the topographic features. Here we investigated NIH-3T3 cell response to reversible topographic signals encoded on light responsive azopolymer films. Switchable patterns were fabricated by means of a well established holographic set-up. Surface relief gratings (SRGs) were realized with Lloyd’s mirror system and erased with circular polarized or incoherent light. Cell cytoskeleton organization and focal adhesions assembly proved to be very sensitive to the underlying topographic signal. Thereafter, pattern reversibility was tested in air and wet environment by using temperature or light as triggers. Additionally, pattern modification was dynamically performed on substrates with living cells. This study paves the way towards an in-situ and real-time investigation of the material-cytoskeleton crosstalk owing to the intrinsic proprieties of azopolymers.
Responsive, biocompatible substrates are of interest for directing the maturation and function of cells in vitro during cell culture. This can potentially provide cells and tissues with desirable properties for regenerative therapies. Here, we demonstrate a straightforward and scalable approach to attach, align, and dynamically load cardiomyocytes on responsive liquid crystal elastomer (LCE) substrates. Monodomain LCEs exhibit reversible shape changes in response to cyclic heating, and when immersed in an aqueous medium on top of resistive heaters, shape changes are fast, reversible, and produce minimal temperature changes in the surroundings. We systematically characterized the strain response of LCEs in water and demonstrated the attachment and alignment of neonatal rat ventricular myocytes on LCE substrates. Cardiomyocytes attached to both static and stimulated LCE substrates, and under cyclic stimulation, cardiomyocytes aligned along the primary direction of strain. This work demonstrates the potential of LCEs as stimuli-responsive substrates for dynamic cell culture.