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: tokano@abmes.twmu.ac.jp

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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