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TRANSCRIPT
Manuscript for Reactive and Functional Polymers
Thermoresponsive thin hydrogel-grafted surfaces for
biomedical applications
Jun Kobayashi and Teruo Okano*
Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical
University (TWIns), 8-1 Kawada-cho, Shinjuku, Tokyo 162-8666, Japan
* To whom correspondence should be addressed.
Tel: +81-3-5367-9945 Extn. 6201, Fax: +81-3-3359-6046
E-mail: [email protected]
Keywords
thermoresponsive surface, thermoresponsive thin hydrogel,
poly(N-isopropylacrylamide), thermoresponsive chromatography, tissue engineering,
cell sheet engineering
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Abstract
Thin poly(N-isopropylacrylamide) (PIPAAm) hydrogels were introduced onto
biomaterials surfaces for accelerating the kinetics of the swelling and shrinking for
PIPAAm hydrogels in response to temperature changes. Thin PIPAAm hydrogels on
the biomaterials surfaces exhibit rapid and reversible phase transitions and act as
switching sequences to regulate the interaction between the surfaces and biological
materials by external temperature-induced changes. By utilizing the
temperature-dependent changes of thin PIPAAm hydrogels grafted onto surfaces, our
laboratory has pursued unique approaches for developing useful biomedical materials
as new types of chromatographic matrices and cell culture surfaces.
Aqueous thermoresponsive chromatography systems using PIPAAm-grafted
stationary phases enable us to separate biomolecules with high biological activity.
Additionally, thermoresponsive cell culture surfaces allow for the recovery of confluent
cell monolayers, which have been clinically applied to ophthalmological treatments,
dilated cardiomyopathy, esophageal ulcerations, periodontal disease, and cartilage
injury. Furthermore, next-generation thermoresponsive cell culture surfaces for
large-scale cell cultivation and the capture of specific cells have been considered a
key technology for expanding small quantities of stem cells and isolating the resulting
differentiated cells for therapeutic use.
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1. Introduction
Stimuli-responsive polymeric hydrogels exhibit phase transitions in volume in
response to external stimuli such as solvent components [1], pH [2], light [3], chemical
species [4], and temperature [5]. In biomedical applications, much attention has
been paid to thermoresponsive cross-linked poly(N-isopropylacrylamide) (PIPAAm)
hydrogels, exhibiting temperature-dependent changes in volume in aqueous media.
PIPAAm hydrogels have been investigated as drug delivery carriers for
stimuli-responsive drug delivery systems [6] and as biomimetic actuators [7].
PIPAAm exhibits a reversible temperature-dependent phase transition in aqueous
solutions at the transition temperature of approximately 32 °C, which is called the
lower critical solution temperature (LCST) [5, 8]. In the case of macroscopic PIPAAm
hydrogels, however, a sudden increase in temperature such as immersion in hot
water above the LCST induced the formation of densely shrunken layer on the
outermost of PIPAAm hydrogels, called skin layers (Figure 1A) [9]. The skin layer
hampers the water penetration from inside the hydrogels to the outer environments,
resulting in a very slow kinetics of the shrinking.
For accelerating the kinetics of the swelling and shrinking of PIPAAm
hydrogels, our laboratory has conducted several strategies to avoid skin layer
formation by tailoring molecular architectures at the molecular level such as the
introduction of free mobile PIPAAm chains [10] or hydrophilic moieties [11] into the
PIPAAm hydrogel networks. By contrast, the downscaling of PIPAAm hydrogels was
also expected to exhibit the rapid response times. In general, the relaxation time of
the swelling and shrinking of conventional hydrogels is proportional to the square of
the characteristic hydrogel length [12]. Typically, the relaxation time of phase
transition is 107 s for a spherical hydrogel of 1-cm diameter, but only 10-1 s for that of
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1-μm diameter, assuming that the collective diffusion coefficient of the hydrogels is on
the order of 10-7 cm2/s [13].
When thin thermoresponsive hydrogel layers are grafted onto material
surfaces, the grafted hydrogels show rapid and reversible temperature-dependent
soluble/insoluble changes due to the hydration/dehydration of N-isopropylacrylamide
side chains, resulting in hydrophilic/hydrophobic surface property changes [14].
Thus, the interaction between the thermoresponsive surfaces grafted with thin
PIPAAm hydrogel and biological structures can be rapidly regulated by external
temperature changes (Figure 1B). In addition, the thermoresponsive property of thin
PIPAAm hydrogel layers is used to switch its functions as a temperature-dependent
charge density (Figure 1C) and affinity interaction (Figure 1D).
Our laboratory has pursued unique approaches to utilizing thermoresponsive
surfaces in useful biomedical applications. Thermoresponsive surfaces have been
applied to new types of chromatographic matrices and cell culture surfaces.
Thermoresponsive chromatographic matrices have been used to separate biologically
active biomolecules in solely aqueous media [15]. Aqueous chromatographic
systems using PIPAAm-grafted surfaces facilitate control over the retention time of
biomolecules toward grafted PIPAAm as stationary phases by changing the external
column temperature. Moreover, PIPAAm-grafted cell culture surfaces have exhibited
temperature-dependent changes in cell detachment/attachment across the LCST [16,
17]. Confluently cultured cells on PIPAAm-grafted cell culture surfaces can be
harvested as monolayers, called “cell sheets,” by lowering the culture temperature
from 37°C to 20°C without the use of digestive enzymes or chelating agents. Our
unique approach, referred to as “cell sheet engineering,” has been used to construct
transplantable tissues composed exclusively of cells and three-dimensionally layered
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tissues [18].
This study provides brief overviews of biomedical applications using
thermoresponsive surfaces for bioseparation and cell-sheet-based tissue engineering.
Moreover, the design of the next generation of thermoresponsive surfaces is also
reviewed.
2. Thermoresponsive surfaces for bioseparation
2.1. Thermoresponsive chromatography
Chromatographic techniques are widely used for separation of biomolecules
using columns, which are selected according to the size, hydrophobicity, ionicity,
affinity of the target biomolecules [19]. Reversed phase chromatography (RPC), in
which the interaction (partitioning) between the stationary phase and solutes is
controlled by changing the polarity of the mobile phase, is commonly used as an
effective separation tool, particularly in pharmaceutics and biochemistry [20]. RPC,
however, has limited applications because solute bioactivity (particularly for proteins
and peptides) is frequently compromised by the use of organic solvent mobile phases.
A novel aqueous chromatographic system that regulates hydrophobic and/or
ionic interactions (partitioning) with solutes through external temperature changes
was achieved by grafting thermoresponsive PIPAAm chains onto the surface of the
device as the stationary phase (Figure 1B, C). Furthermore, temperature-induced
conformational changes in the PIPAAm stationary phase will regulate the affinity of
interactions between the functionalized surface and target biomolecules based on
masking and forced-release effects (Figure 1D). This system has the advantage of
preserving analyte bioactivity and has a low environmental impact (e.g., no
organic-mobile disposal issue). It is important to note that the retention of
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biomolecules is readily modulated simply by changing the external column
temperature.
2.2. Thermoresponsive hydrophobic interaction chromatography
Our group collaborated with Kanazawa's group proposed the effectiveness
and characteristics of thermoresponsive hydrophobic interaction chromatography in
solely aqueous media [21]. The separation of hydrophobic steroids with different
hydrophobicities was achieved on the surface of PIPAAm-grafted silica beads
measuring 5 µm in diameter that were used as the high performance liquid
chromatography (HPLC) packing material. While overlapping steroid
chromatograms were observed at lower temperatures, the retention times of the
steroids increased with temperatures above the LCST of PIPAAm, leading to highly
resolved chromatograms. The force driving steroids toward PIPAAm-grafted
stationary phases is mainly due to hydrophobic interactions (Figure 1B). The
retention of hydrophobic solutes was enhanced by increasing the hydrophobicity of
the thermoresponsive stationary phase, into which a hydrophobic co-monomer,
n-butyl methacrylate (BMA), was introduced through copolymerization [22].
Additionally, the hydrophobic interaction between the grafted surfaces and solutes
was amplified on the surface of thin PIPAAm hydrogel layers due to the enhanced
partitioning of hydrophobic solutes toward the cross-linked PIPAAm layers (Figure 1B)
[14]. Recently, PIPAAm brushes on the surface of column matrices were prepared
by surface-initiated atom transfer radical polymerization (SI-ATRP), which facilitates
the variation of the density and/or molecular weight of PIPAAm brushes by changing
the density of the ATRP initiator and the ATRP duration [23, 24].
The use of polymeric beads as a base column matrix is useful because silica
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bead matrices are unstable under alkaline conditions due to the hydrolysis of silica.
Recently, porous cross-linked polystyrene [25, 26] and hydroxylated cross-linked
polymethacrylate beads [27] were used as column matrices to improve stability under
an all-aqueous mobile phase. A PIPAAm-grafted polymeric bead column helped
enhance the retention of basic peptides, bradykinins, by increasing the pH level of the
mobile phases due to the increasing deprotonation of basic amino acids in bradykinin;
this led to an increase in the hydrophobicity of the peptides [26].
2.3. Thermoresponsive ionic interaction chromatography
Most bioactive molecules, such as drugs, peptides, and proteins, contain ionic
moieties on their surfaces. Thus, the effective retention and separation of ionic
biomolecules can be achieved by introducing the opposite ionic moiety into
thermoresponsive stationary phases.
An anionic poly(IPAAm-co-acrylic acid-co-tert-butylacrylamide)
(poly(IPAAm-co-AAc-co-tBAAm)) thin hydrogel on the surface of silica beads was
used as the stationary phase to separate positively charged biomolecules in an
aqueous mobile phase [28-30]. With increasing column temperature, the collapse
and dehydration of thermoresponsive thin hydrogels on the surface prevented
carboxyl groups within the polymer network from accessing the hydrophilic
environment in the mobile phase, leading to an increase in the apparent pKa [28, 29]
and eventually the reduction of surface charge densities (Figure 1C). The separation
of catecholamines [28, 29] and basic bioactive peptides (angiotensin subtypes I, II,
and III) [30] was achieved by using thermoresponsive aqueous chromatography on
anionic poly(IPAAm-co-AAc-co-tBAAm) thin hydrogels as the stationary phases. The
retention behavior of angiotensins can be dramatically modulated by applying
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stepwise column temperature gradients, leading to a reduction in the duration of
separating operations [30]. In addition, cationic
poly(IPAAm-co-N,N-dimethylaminopropylacrylamide-co-BMA)
(poly(IPAAm-co-DMAPAAm-co-BMA)) thin hydrogels were also applied to the column
matrix to separate adenosine nucleotides (AMP, ADP, and ATP) [31] and
oligonucleotides [32] in the aqueous mobile phase.
In contrast, with the aqueous mobile phase possessing the ionic strength of a
buffer, the adsorption/desorption behavior of proteins on the ionic thermoresponsive
surfaces was different from that of small biomolecules. Albumin was strongly
adsorbed on the cationic thermoresponsive copolymer-grated stationary phases due
to both enhanced electrostatic interaction with the cationic copolymer at low buffer salt
concentrations at pH 7.0 and increased hydrophobic interaction with the dehydrated
copolymer at high temperatures [33]. The enhancement in the electrostatic
interaction was due to an increase in the Gibbs-Donnan effect at lower ionic strengths.
Another plasma protein, γ-globulin, was not able to adhere to the surfaces at all the
temperatures because γ-globulin is almost neutral at pH 7.0. With decreasing
temperatures, captured albumin was eluted from the column because the expansion
of the cationic copolymer chains reduced the extent of hydrophobic interaction and
hampered albumin’s access to cationic moieties by steric hindrance. Additionally, on
anionic thermoresponsive copolymer-grated stationary phases, lysozymes adhered
strongly at higher column temperatures [34], exhibiting a retention behavior similar to
that of the cationic thermoresponsive column. Purified lysozyme was isolated from
whole egg white through the anionic thermoresponsive column by using stepwise
temperature gradients.
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2.4. Thermoresponsive affinity chromatography
By utilizing temperature-induced conformational changes in PIPAAm
molecules, a novel concept of affinity regulation based on masking and forced-release
effects was proposed (Figure 1D). PIPAAm chains and Cibacron Blue (CB)
molecules, which have an affinity toward albumin, were independently immobilized
onto column matrix surfaces [35]. Albumin may easily access immobilized CB
ligands in the collapsed state of PIPAAm chains above the LCST, but it is hampered
by the steric hindrance of the expanded PIPAAm chains below the LCST (i.e.,
masking effect). Through chromatographic analyses using PIPAA/CB coimmobilized
beads as a column matrix, the binding capacity of albumin below the LCST of PIPAAm
was shown to be significantly more reduced than that above the LCST. Furthermore,
the captured albumin can be released from the matrix surface by lowering the
temperature to below the LCST of PIPAAm (i.e., forced-release effect) while
maintaining the properties of the mobile phase, e.g., pH and ionic strength.
By utilizing thermoresponsive affinity chromatography, the selective recovery
of human albumin from human serum was achieved. By using the same concept, a
thermoresponsive affinity chromatographic matrix, on which both Ricinus communis
agglutinin (RCA120) lectin and lactose were co-immobilized within PIPAAm molecules,
facilitated the temperature-regulated elution of the glycoprotein asialotransferrin [36].
Recently, the targets to be separated by using thermoresponsive surfaces
have been extended to mammalian cells. Development of novel technologies for
purifying specific cells without any labeling and modification is required for implanting
the purified cells safely. Separation system using thermoresponsive surfaces would
be useful because of the noninvasive recovery of the target cells. Currently, our
laboratory has focused on the design of thermoresponsive polymer-grafted surfaces
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to efficiently separate cells and the development of separation system such as
chromatographic and microfluidic platforms.
3. Thermoresponsive surfaces for cell culture
3.1. Thermoresponsive cell culture dish
In the conventional cell culture method, cells proliferate and grow on the
surface of a tissue culture polystyrene (TCPS) dish. Cultured cells are harvested by
treating with trypsin to digest extracellular matrices (ECMs) and by chelating Ca2+ ions
to disrupt cell-cell junctions, followed by subculturing the cells onto another TCPS dish.
These procedures degrade plasma membrane proteins and deposited ECMs, leading
to a reduction in cell viability [17].
Over 20 years ago, our group proposed a new concept for cell culturing using
thermoresponsive cell culture surfaces, on which cross-linked thin PIPAAm hydrogels
are covalently grafted, allowing cultured cells to be recovered by lowering the
temperature below the LCST [16, 17, 37]. In contrast with the conventional cell
culture method, the detachment of cells from the surface of a PIPAAm-grafted
thermoresponsive cell culture dish involves a unique process. At 37°C, various
types of cells adhere to and proliferate on the thermoresponsive cell culture surface,
which is hydrophobic due to the dehydration of the grafted PIPAAm above the LCST.
When the culture temperature is decreased to 20°C, the PIPAAm-grafted surface
becomes hydrophilic, resulting in the detachment of cultured cells. Cross-linked
PIPAAm-grafted cell culture surfaces were prepared by irradiating a uniformly spread
IPAAm monomer solution on TCPS dishes with an electron beam [16, 17]. Using this
technology, a thermoresponsive TCPS dish called UpCellTM has been marketed
globally by Thermo Fisher Scientific under the NuncTM brand.
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3.2. Cell-sheet-based tissue engineering
Tissue engineering, a concept proposed by Langer and Vacanti [38], has been
widely accepted as a promising methodology for the in vitro reconstruction of
three-dimensional tissues and the in vivo regeneration of damaged tissues and
organs. In typical tissue engineering, the cells are seeded in or on biodegradable
materials, which serve as temporary scaffolds and promote the reorganization of the
cells to form a functional tissue. However, this approach has fundamental limitations,
such as host inflammatory responses to the implanted scaffolds during their
degradation [39].
By contrast to conventional approaches, our group has developed a novel
method to prepare a cell sheet using a thermoresponsive cell culture surface and to
build three-dimensional tissues in vitro by stacking cell sheets [18]. A unique
approach called “cell sheet engineering” has been used to construct ideal
transplantable tissues composed exclusively of cells. Confluently cultured cells on
the surface of hydrophobic PIPAAm-grafted TCPS at 37°C could be detached as a
single cell sheet when the culture temperature was decreased to 20°C [40].
Detached cell sheets preserved the membrane proteins and ECMs beneath the cell
sheet and cell-cell junctions because the cell sheets were recovered without the use
of any enzymatic proteolysis treatment. Fluorescent labeling reveals that the
recovered cell sheet retains typical ECMs such as fibronectin [40] and type IV
collagen [41] beneath the cell sheets. ECM proteins such as fibronectin and type IV
collagen are known to be major components of the basement membrane that
underlies the epithelium, facilitating the transplantation of the cell sheets on the
surface of the body. Furthermore, layering cell sheets facilitates the formation of
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thick tissues that exhibit spontaneous pulsation of the layered cardiac cell sheets;
such tissues are easily visible to the naked eye [42].
3.3. Cell-sheet-based clinical applications
The clinical parameters for the application of cell sheet engineering
technology in regenerative treatments have been successfully created. The current
status of clinical applications using cell-sheet-based regenerative treatment is
summarized in Table 1.
Our first clinical research study involved the treatment of patients with burn
injuries or scars using cultured epidermal keratinocytes [43]. A successful clinical
study was conducted on the treatment of patients with unilateral or bilateral total
corneal stem-cell deficiencies resulting from alkali burns or Stevens-Johnson
syndrome [44]. An autologous oral mucosal epithelial cell sheet was transplanted on
the ocular surface of patients with total corneal stem-cell deficiencies. In France, a
clinical trial involving the treatment of a bilateral limbal stem-cell deficiency was
conducted by the transplantation of autologous oral mucosal epithelial cell sheets [45].
Twenty-five cases with a one-year follow-up revealed that the treatment was safe and
effective; only two patients experienced serious adverse events classified as not
product-related. Based on these results, an application for marketing authorization
has been submitted to the European Medicines Agency (EMA).
For the treatment of dilated cardiomyopathy (DCM) or ischemic heart disease,
clinical research has begun, using autologous skeletal myoblast sheets. In the case
of a 56-year-old male suffering from idiopathic DCM, autologous skeletal myoblast
sheet transplantation on the surface of his dilated heart improved the patient’s clinical
condition: the patient showed no arrhythmia, discontinued the use of a left ventricular
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assist device (LVAD), and avoided cardiac transplantation [46]. Cytokines secreted
from transplanted myoblast sheets were considered to induce the reduction of fibrosis,
angiogenesis, and the recruitment of hematopoietic stem cells; however, myoblasts
cannot differentiate into cardiomyocytes. Now, a Japanese medical device company,
Terumo Corporation, is planning to begin a clinical trial to treat patients suffering from
ischemic heart disease by transplanting autologous skeletal myoblast sheets.
Other clinical studies using cell sheet engineering have also been carried out
for the treatment of esophageal ulcerations, periodontal disease, and cartilage injury.
To treat esophageal ulcerations after the removal of a superficial neoplasm and to
prevent stenosis, autologous mucosal epithelial cell sheets were transplanted [47].
Periodontal treatment using periodontal ligament-derived cell sheets has been applied
to regenerate damaged periodontal support structures [48]. Chondrocyte sheet
transplantation onto the knee joints has been applied in treatments to repair and
regenerate articular cartilage [49]. Preclinical investigations using cell sheet
engineering to create an air leak sealant for the lungs [50] and generate liver tissue
[51] and pancreatic islets [52] have been conducted in animal models and
summarized in other reviews [18, 53].
4. Next-generation thermoresponsive cell culture dishes
Successful cell-sheet-based therapies have been developed for the treatment
of the superficial layer of tissues and organs due to the ease of transplantation. Our
attention has recently shifted to address some challenging issues in tissue
engineering. One of these issues concerns the expansion of a few cells (e.g., stem
cells) to a large number of cells and their differentiation into desired cells.
Furthermore, the expanded cells should be purified to clinically safe grades. To
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overcome these challenges, our laboratory has designed the next generation of
thermoresponsive cell culture surfaces.
4.1. Thermoresponsive microcarriers for large-scale cultivation
To produce a wide variety of recombinant proteins, including antibodies,
vaccines, and enzymes, the large-scale cultivation of mammalian cells has been used
in biotechnological and clinical applications. Recently, large-scale culture systems
have been applied to the expansion of undifferentiated cells, such as human
mesenchymal stem cells (hMSCs) and human embryonic stem cells (hESCs) on
microcarriers (MCs) [54] and to embryoid body (EB) formation [54, 55]. Cell culture
systems using MCs represent a promising way of expanding these cells to obtain
sufficient quantities for the practical applications of tissue engineering and cell therapy.
In the MC culture system, cells grow as a monolayer on the MC surface and are
harvested by digesting the ECMs using trypsin during the subculturing process.
However, repeated trypsin treatments may damage the cell membrane, resulting in a
reduction in cell viability.
Recently, our laboratory developed a novel technology for large-scale cell
cultivation in which the cultured cells can be harvested simply by lowering the
temperature of the surface of PIPAAm-immobilized MCs [56, 57]. PIPAAm was
grafted onto MCs by using SI-ATRP on the surface of chloromethylated polystyrene
beads. By using the thermoresponsive MCs, Chinese hamster ovary (CHO-K1) cells
were expanded by a factor of approximately 50 in a 7-day stirred suspension culture
at 37°C. Furthermore, the cultured cells were harvested by lowering the temperature
to 20°C; a harvest efficiency of 76.1±16.3% was attained by optimizing both the
amount of grafted PIPAAm and the bead diameter.
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The introduction of quaternary amino monomer (3-acrylamidopropyl
trimethylammonium chloride; APTAC) into the grafted PIPAAm chains on the bead
surfaces improved both the efficiency of cell detachment from the bead surfaces and
cell proliferation [58]. Repulsive electrostatic interactions among positively charged
PIPAAm copolymer-grafted bead surfaces facilitated the dispersion of MCs in the cell
culture medium at 37°C, resulting in enhanced cell proliferation relative to a system
featuring nonionic PIPAAm-grafted beads. In addition, the efficiency of cell
detachment from the surface of the positively charged MCs after reducing the
temperature to 20°C was also increased, presumably due to the enhanced hydration
caused by introducing positively charged APTAC moieties. Thus, large-scale cell
cultivation systems using thermoresponsive MCs have significant potential in
expanding cell quantities for therapeutics without any proteolytic treatments.
4.2. Affinity regulation between cells and surface-immobilized ligands on
thermoresponsive cell culture surfaces
Expanded human pluripotent stem cells such as hESCs and human-induced
pluripotent stem cells (hiPSCs) in an undifferentiated state can differentiate into many
types of cells, including cardiac cells. To obtain differentiated cells with high
efficiency, expansion/differentiation methods, including the use of suspension cultures
of EBs in bioreactors [55] and utilizing synthetic compounds to promote the
differentiation of stem cells into desired cells [59], have been thoroughly investigated.
Moreover, the obtained cells should be highly purified and contain no undifferentiated
cells to satisfy clinical safety requirements. Currently, the most widely used methods
for separating and purifying specific cells are flow cytometry and the
magnetic-activated cell sorter system (MACS) technique. However, these methods
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have several disadvantages, such as the requirement of a labeling procedure using a
fluorescent dye-conjugated antibody. Thus, the development of labeling-free
procedures for separating and purifying target cells with high efficiency is required to
develop regenerative therapies that use stem cells as a source.
To regulate specific cell adhesion, our laboratory developed
antibody-immobilized thermoresponsive cell culture surfaces. Anti-human CD90
antibody-immobilized thermoresponsive cell culture surfaces were prepared as a
model to examine the temperature-dependent regulation of the affinity of interactions
between CD90-positive cells and immobilized antibodies [60]. Thymic carcinoma
cells (Ty-82) and neonatal normal human dermal fibroblasts (NHDFs), which express
CD90 on their cellular membranes, were used as models. Both cells selectively
adhered to anti-human CD90 antibody-immobilized thermoresponsive surfaces.
Moreover, the cells were detached from the surfaces by lowering the temperature to
20°C and by applying additional forces, such as those induced by pipetting and
contraction forces caused by shrinking the cell sheets. These results indicate that
hydrated PIPAAm chains are able to diminish the affinity of interactions between
immobilized CD90 antibodies and cell surfaces with a decrease in temperature.
A separation system using antibody-immobilized thermoresponsive surfaces
would be useful for the noninvasive purification of specific cells because the
recovered cells are free from any labeling agent such as antibodies. Although the
system using a cell culture dish is unsuitable for large-scale cell separation, this
problem will be overcome by incorporating, for example, a chromatographic system
and microfluidic channels. Moreover, more precise separation and purification of
cells can be achieved by controlling the flow of shear stress within the column or
microfluidic channels.
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5. Conclusions
This review introduced thermoresponsive thin hydrogel-grafted surfaces for
chromatographic stationary phases, cell culture surfaces, and the application of these
surfaces in cell-sheet-based tissue engineering. Aqueous thermoresponsive
chromatography systems utilizing PIPAAm-grafted surfaces as stationary phases
facilitate the separation of biomolecules with high biological activity. Furthermore,
thermoresponsive cell culture surfaces allow for the recovery of confluent cell
monolayers as contiguous living cell sheets. The biological activity of biomolecules
and cells recovered from thermoresponsive PIPAAm-grafted surfaces can be
preserved because the phase transition of grafted PIPAAm in aqueous media occurs
at the LCST of 32°C, which is close to the natural temperature of the human body.
We have already applied cell sheet engineering in clinical settings for the treatment of
ophthalmological disease, dilated cardiomyopathy, esophageal ulcerations,
periodontal disease, and cartilage injury. The development of next-generation
thermoresponsive cell culture surfaces for large-scale cell cultivation and the capture
of specific cells have the potential to expand small quantities of stem cells and to
isolate the differentiated cells for therapeutic use.
6. Acknowledgements
Part of this work was financially supported by the Global COE Program,
Multidisciplinary Education and Research Center for Regenerative Medicine
(MERCREM) from the Ministry of Education, Culture, Sports, Science and Technology
(MEXT) of Japan, Creation of Innovation Centers for Advanced Interdisciplinary
Research Areas Program in the Project for Developing Innovation Systems “Cell
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Sheet Tissue Engineering Center (CSTEC)” from MEXT of Japan and by a
Grant-in-Aid for Scientific Research (Grant No. 23106009) on Innovative Areas "Bio
Assembler" (Area No. 2305) from MEXT of Japan.
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Table 1. Clinical applications using cell-sheet-based therapies.
Tissue/organ Target illness Cell sheets Implementation site
(country) Current status
Corneal limbal-derived cell sheet
Osaka University (Japan) Started in 2003 [44] Corneal epithelium
Limbal stem-cell deficiency
Oral mucosal epithelial cell sheet
Les Hospices Civils de Lyon (France) Collaborator: Cellseed, Inc. (Japan)
Finished European clinical trial (25 cases with one-year follow-up [45]), and applied to the European Medicines Agency (EMA) for marketing authorization
Osaka University (Japan) Started in May 2007 [46]Myocardium Severe cardiac disease (e.g., ischemic heart disease, dilated cardiomyopathy)
Skeletal myoblast sheet
Terumo Corporation (Japan) Collaborator: Cellseed, Inc. (Japan)
Japanese clinical trial in preparation
Tokyo Women’s Medical University (Japan)
Started in April 2008 [47]Esophagus Prevention of stenosis after endoscopic submucosal dissection of esophageal cancer
Oral mucosal epithelial cell sheet
Karolinska Institute (Sweden) Approved by ethical committee, and in preparation
Periodontal ligament
Periodontal disease
Periodontal ligament-derived cell sheet
Tokyo Women’s Medical University (Japan)
Started in October 2011
Cartilage Knee cartilage injury
Cartilage cell sheet Tokai University (Japan) Started in November 2011
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A) Macroscopic PIPAAmhydrogel
B) Thin PIPAAmhydrogel
C) Thin ionic PIPAAmhydrogel
D) Thin PIPAAmhydrogel having ligands
Rapid Slow
Hydrophobic biomolecules
Cationic biomolecules
Target proteins
Downscaling
Enhancedhydrophobic interactionEnhancedpartitioning into thinPIPAAmhydrogel
Decreasedcharge densityEnhancedhydrophobic interaction
DecreasedstreichindranceIncreasedaffinity interaction
20 °C 37 °C37 °C
Temp.increase
Temp.increase
Skin layer
Temp.decrease
Rapid
Temp.increase
Temp.decrease
Rapid
Temp.increase
Temp.decrease
Rapid
Figure 1. Schematics showing the kinetics of the shrinking for (A) macroscopic
PIPAAm hydrogel and (B-D) thin PIPAAm hydroglel-grafted surfaces when the
temperature is increased from 20 to 37 °C.
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