Hydrophobized Thermoresponsive Copolymer Brushes for Cell

Sep 4, 2013 - Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University (TWIns), 2-2 Wakamatsuc...
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Hydrophobized Thermoresponsive Copolymer Brushes for Cell Separation by Multistep Temperature Change Kenichi Nagase,† Yuri Hatakeyama,†,‡ Tatsuya Shimizu,† Katsuhisa Matsuura,† Masayuki Yamato,† Naoya Takeda,‡ and Teruo Okano*,† †

Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University (TWIns), 8-1 Kawadacho, Shinjuku, Tokyo 162-8666, Japan ‡ Department of Life Science and Medical Bioscience, School of Advanced Science and Engineering, Waseda University (TWIns), 2-2 Wakamatsucho, Shinjuku, Tokyo 162-8480, Japan S Supporting Information *

ABSTRACT: For preparing a thermally modulated biointerface that separates cells without the modification of cell surfaces for regenerative medicine and tissue engineering, poly(N-isopropylacrylamide-co-butyl methacrylate) (P(IPAAm-co-BMA), thermoresponsive hydrophobic copolymer brushes with various BMA composition were formed on glass substrate through a surfaceinitiated atom transfer radical polymerization (ATRP). Characterization of the prepared surface was performed by X-ray photoelectron spectroscopy (XPS), attenuated total reflection Fourier transform infrared spectroscopy (ATR/FT-IR), and gelpermeation chromatography (GPC) measurement. Prepared copolymer brush surfaces were characterized by observing the adhesion (37 °C) and detachment (20 or 10 °C) of four types of human cells: human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts (NHDFs), human aortic smooth muscle cells (SMCs), and human skeletal muscle myoblast cells (HSMMs). HUVECs and NHDFs exhibited their effective detachment temperature at 20 and 10 °C, respectively. Using cells’ intrinsic temperature sensitivity for detachment from the copolymer brush, a mixture of green fluorescent protein (GFP)-expressing HUVECs (GFP-HUVECs) and NHDFs was separated.

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INTRODUCTION

proliferate on PIPAAm modified cell culture substrate, because PIPAAm is hydrophobic due to its dehydration. Then, with reducing temperature to 20 °C, the cultured cells detach themselves as a contiguous cell sheet from the surface, due to the hydration and swelling of grafted PIPAAm on the culture surfaces. Since fabricated cell sheets on PIPAAm grafted cell culture substrate completely preserve their cell−cell junctions and cell surface proteins as well as the extracellular matrix (ECM),7 the sheets adhere tightly onto host tissues without suture or cell loss after transplantation.8 Additionally, threedimensional tissues with a high cell density can be fabricated by layering the cell sheets, because tissues can be fabricated by cells only without using scaffolds. Therefore, cell sheets have been

Recently, regenerative medicine that reproduces the lost functions of the tissue and organs has been becoming one of promising therapy for patients in medical fields. Especially, cellbased regenerative medicine has been progressing rapidly, and a number of clinical trials have already started. Cell therapy with direct injection shows enormous potential for recovering the functions of the tissues and organs.1 Also, tissue engineering using biodegradable scaffolds has been widely used for constructing the tissues,2 and some of the bioengineered tissues were implanted to patients successfully.3 In contrast, our laboratory has developed a novel tissue engineering approach without any scaffolds, which is called “cell sheet technology”.4,5 In this approach, thermo-responsive cell culture dishes, prepared by modifying poly(N-isopropylacrylamide) (PIPAAm)6 on tissue culture polystyrene, are used for preparing an artificial tissue consisting of monolayer cells. At 37 °C, cells adhere and © 2013 American Chemical Society

Received: May 9, 2013 Revised: August 31, 2013 Published: September 4, 2013 3423

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Figure 1. Scheme of the preparation of the poly(N-isopropylacrylamide (IPAAm)-co-butyl methacrylate (BMA)) brush grafted glass surface through surface-initiated ATRP as an intelligent biointerface for cell separation.

used for the various types of tissue engineering9−11 and regenerative medicines.12−15 Particularly, in several types of tissues, clinical trials using cell sheets have already started.16−18 In this way, the fabrication of transplantable tissue in vitro is a key concept in current regenerative medicine. For fabricating transplantable tissues in vitro, effective cell separation methods that can provide an adequate purity, yield, and function after separation have been desired, because the purity of cells or individual cell contents in cocultured cells is important for fabricating functional tissues.19,20 To date, the various types of cell separation methods have been developed such as field-flow fractionation (FFF),21,22 affinity adsorption,23,24 and flow sorting.25,26 Especially, fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS) are widely used as precise cell separation methods.25−27 However, these cell separation methods require the modification of cell surfaces with fluorescent antibody or magnetic particles, leading to a serious problem upon the transplantation of separated cells to human body. Therefore, a cell separation method that requires no modification on the surface of cell is preferable for using separated cells for transplantation. With investigating new cell separation tools, our laboratory paid attention to PIPAAm modified surfaces as a cell-separating material, because various types of cells are found to exhibit their specific cell adhesion and detachment properties on PIPAAm grafted surfaces. Using cells’ intrinsic adhesive and detachment properties, targeted cells are expected to be collected by an external temperature change. In addition, recovered cells have already been proved to be safe for transplantation, because cell sheets fabricated on a PIPAAm modified surface have already been used for clinical applications without any problems.16−18 Additionally, a surface-initiated atom transfer radical polymerization (ATRP), one of the effective living radical polymerizations, was used for preparing a cell-separating surface. Several modification methods for PIPAAm on substrate have been established, such as electron-beam radiation,4,28 radical polymerization,29 reversible addition−fragmentation chain transfer radical (RAFT) polymerization,30 ATRP,31 and polymer casting.32 Among them, ATRP is expected to be a good candidate method for preparing cell-separating surfaces, because ATRP provides a densely packed PIPAAm brush structure (more than 0.1 chains/nm2),33 leading to a reduction in the amount of

undetachable cells and protein on substrate.34 Also, brush length can be precisely controlled by changing feed monomer concentration or polymerization period in ATRP.33,35 Our laboratory has already investigated PIPAAm brush surfaces as cell-separating materials for separating lymphocyte subpopulations and cells for fabricating cardiovascular tissues.36,37 A certain level of separations of these cells was performed by using various cells’ intrinsic adhesion and detachment properties.36,37 For further improving the cell separation efficiency, the copolymerization of PIPAAm brush is speculated to be an effective approach, because thermoresponsive copolymer brushes having hydrophobic or ionic groups have successfully separated peptides or proteins in our previous attempts.38−40 Especially, the copolymerization of hydrophobic monomer, such as n-butyl methacrylate (BMA), increases the hydrophobic properties of grafted copolymer,41 which is speculated to give an effective cell adhesion. In the present study, hydrophobized thermoresponsive copolymer brushes, made by copolymerizing BMA into PIPAAm, were prepared by a surface-initiated ATRP on glass substrates. Temperature-dependent adhesion and detachment properties of human cells were observed for investigating a possibility to allow the prepared surface to become a cellseparating material.

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EXPERIMENTAL SECTION

Materials. N-Isopropylacrylamide (IPAAm) was kindly provided by Kohjin (Tokyo, Japan) and recrystallized from n-hexane. Butyl methacrylate (BMA), obtained from Wako Pure Chemicals (Osaka), was purified by distillation at 35 °C at a vacuum of 3 mmHg. CuCl and αchloro-p-xylene were purchased from Wako Pure Chemicals. Tris(2aminoethyl)amine (TREN) was purchased from Acros Organics (Pittsburgh, PA, USA). Formaldehyde, formic acid, and sodium hydroxide were purchased from Wako Pure Chemicals. Tris(2-N,Ndimethylaminoethyl)amine (Me6TREN) was synthesized from TREN, according to the previous reports.42 Glass coverslips (24 × 50 mm, 0.2 mm in thickness) were purchased from Matsunami Glass (Osaka). Ethylenediamine-N,N,N′,N′-tetraacetic acid disodium salt dehydrate (EDTA·2Na) were purchased from Wako Pure Chemicals. ((Chloromethyl)phenylethyl) trimethoxysilane (mixed m, p isomers) as an ATRP initiator was obtained from Gelest (Morrisville, PA). 2Propanol (HPLC grade), methanol, acetone, and toluene (dehydrate) were purchased from Wako Pure Chemicals. Tissue culture polystyrene dishes (TCPS) (Falcon 3002) were obtained from BD Bioscience 3424

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Table 1. Elemental Analyses of Poly(N-isopropylacrylamide-co-butyl methacrylate) (P(IPAAm-co-BMA)) Brush Grafted Glass Surfaces by an X-ray Photoelectron Spectroscope (XPS) at a Take-off Angle of 15° atom (%) codea initiator modified glass IPB-0 IPB-1 IPB-2 IPB-5 calcd of IPAAmb calcd of BMAc

BMA feed ratio (mol %)

C

N

O

Si

Cl

N/C ratio

0 1 2 5

24.4 66.8 65.8 68.3 69.2

0.1 10.3 9.57 9.37 8.05

48.7 16.0 17.1 15.1 15.7

26.0 6.40 7.23 7.04 6.76

0.81 0.46 0.37 0.23 0.24

0 0.154 0.146 0.137 0.116

75.0 80.0

12.5

12.5 20.0

0.17 0

a

All sample surfaces were abbreviated as IPB-X, where X is the feed composition of BMA (Table 1). bTheoretical atomic composition of Nisopropylacrylamide monomer. cTheoretical atomic composition of butyl methacrylate monomer.

(Billerica, MA). Cells and cell culture media were obtained from Takara Bio (Shiga). Green fluorescent protein (GFP)-expressing human umbilical vein endothelial cells (GFP-HUVEC) was obtained from Angio-Proteomie (Boston, MA). Water used in this study was Milli-Q water prepared by an ultrapure water purification system (Synthesis A10) (Millipore, Billerica, MA) unless otherwise mentioned. Preparation of ATRP Initiator Immobilized Cover Glass Slips. Glass coverslips with a silane layer comprising of 2-(m/p-chloromethylphenyl) ethyltrimethoxysilane, an ATRP initiator, was prepared as shown in the first step in Figure 1. Glass coverslips were cleaned by oxygen plasma irradiation (the irradiation intensity: 400 W, oxygen pressure: 0.1 mmHg) for 180 s in a plasma dry cleaner (PX-1000) (March Plasma Systems, Concord, CA). Immediately after the plasma oxidation, these glass coverslips were placed in a separable flask (1000 mL), which was humidified at 60% relative humidity for 2 h. Toluene solution of ATRP initiator (46.3 mmol/L in 700 mL) was poured into the separable flask, of which solution was stirred for 18 h at room temperature. The ATRP initiator immobilized glass coverslips were rinsed with toluene and acetone and dried in a vacuum oven at 110 °C. Surface Modification of Glass Surface with Thermoresponsive Copolymer by ATRP. P(IPAAm-co-BMA) grafted surfaces with various BMA composition were prepared by modulating the feed monomer of IPAAm in surface initiated-ATRP as shown in the second step in Figure 1. The typical preparation procedure was as follows: the total monomer concentration was set to be 875 mmol/L with the following monomer composition in feed; IPAAm (44.1 g, 389 mmol) and BMA (0.56 g, 3.94 mmol) (the monomer composition: BMA 1 mol %) were dissolved in 450 mL of 2-propanol. The feed composition of BMA was varied to be 0, 1, 2, or 5 mol %. The solution was deoxygenated by argon gas bubbling for 30 min. CuCl (295 mg, 3 mmol) and Me6TREN (765 mg, 3.3 mmol) were added under an argon atmosphere, and the solution was stirred for 20 min to obtain a CuCl/Me6TREN catalyst system. Both monomer solution in a flask and the silanemodified coverslips in a 500 mL separable flask were placed separately into a glovebox, which was purged with dry argon gas by repeated vacuum and argon flush (three times). The monomer solution was then poured into the flask containing the glass coverslips, followed by adding α-chloro-p-xylene (39.48 μL, 0.3 mmol) to the reaction solution. The ATRP reaction proceeded for 16 h at 25 °C under continuous stirring on a magnetic stirrer (AMG-S) (ASH, Chiba). P(IPAAm-co-BMA) grafted glass coverslips were washed with acetone, methanol, 50 mmol/L EDTA solution, and finally water, and the modified coverslips were dried in a high vacuum oven at 50 °C for 5 h. The reaction solution after polymerization was dialyzed against Milli-Q water using dialysis membrane [Spectra/Por standard regenerated cellulose dialysis membrane, molecular weight cut off (MWCO): 1000] (Spectrum Laboratories, Rancho Dominguez, CA) for 1 week with daily water changed, and the polymer was recovered by freeze-drying. Numberaverage molecular weights and polydispersity index (PDI) values of the copolymers were determined by a GPC system (sequentially connected columns: TSKgel SuperAW2500, TSKgel SuperAW3000, and TSKgel SuperAW4000) (Tosoh, Tokyo) controlled with GPC-8020 model II

ver. 5.0 (Tosoh). A calibration curve was obtained using poly(ethylene glycol) standards. The flow rate was 1.0 mL/min. The mobile phase was DMF containing 50 mmol/L LiCl, and the column temperature was controlled at 45 °C using an equipped column oven. The elution profiles were monitored by an equipped refractometer. BMA content in the copolymers was determined by 1H NMR (UNITYINOVA 400 MHz spectrometer) (Varian, Palo Alto, CA) using N,N-dimethylformamided7 as solvent. XPS Analysis of Initiator-and Copolymer-Modified Surfaces. Elemental analysis was performed for both ATRP-initiator modified and P(IPAAm-co-BMA) brush grafted glass coverslips by an X-ray photoelectron spectroscope (XPS) (K-Alpha, Thermo Fisher Scientific, Waltham, MA). Excitation X-rays were produced from a monochromatic Al Kα1,2 source and a takeoff angle of 15°. Wide scans were recorded to analyze all existing elements on the surface, and high resolution narrow scan analysis was performed for the peak deconvolution of carbon C1s signals. All binding energies were referenced to a C1s hydrocarbon peak at 285.0 eV. Amount of P(IPAAm-co-BMA) on Glass Substrates. Amount of grafted P(IPAAm-co-BMA) was determined by an attenuated total reflection Fourier transform infrared spectroscope (ATR/FT-IR) (Nicolet 6700) (Thermo Fisher Scientific) using germanium as an ATR crystal. Glass as the base substrate showed a strong absorption arising from Si−O at 1000 cm−1. Absorption of amide carbonyl derived from P(IPAAm-co-BMA) copolymer appeared in the region of 1650 cm−1. The peak intensity ratio of I1650/I1000 was used to determine the amount of P(IPAAm-co-BMA) grafted surface using a calibration curves prepared from a series of known amounts of P(IPAAm-co-BMA) copolymer casts on unmodified glass surfaces. (The calibration curves were shown in Supporting Information Figure S1.) Prepared glass coverslips were abbreviated as IPB-X where X represents the feed composition of BMA in the ATRP procedure. Phase Transition of P(IPAAm-co-BMA) in Cell Culture Medium. Phase transitions of P(IPAAm-co-BMA) in cell culture media were observed through their optical transmittance changes. Solutions of P(IPAAm-co-BMA) with various molecular weights were prepared using four different cell culture media: endothelial cell growth medium (EGM), fibroblast cell growth medium (FGM), smooth muscle cell growth medium (SmGM), and skeletal muscle cell growth medium (SkGM), and water at a concentration of 10 mg/mL. Optical transmittance changes of the polymer solutions were monitored at 600 nm with a UV−vis spectrometer (V-660, JASCO, Tokyo). The sample cuvette was thermostatted with a Peltier-effect cell holder (ETC717, JASCO) with a heating rate of 0.10 °C/min. The lower critical solution temperature (LCST) of copolymer was defined as the temperature at 90% transmittance of solution. Cell Culture and Cell Adhesion Behavior. Four types of cells, utilized for fabricating cell sheets on tissue, were used for investigating cell adhesion and detachment properties from the prepared surfaces. Human umbilical vein endothelial cells (HUVECs),43,44 normal human dermal fibroblasts (NHDFs),44−46 human aortic smooth muscle cells (SMCs), 47 −49 and human skeletal muscle myoblast cells 3425

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Table 2. Characterization of P(IPAAm-co-BMA) Brush Modified Glass Substrate IPAAm/BMAb (molar ratio) a

in feed

in copolymer

amount of copolymerc) (μg/cm2)

Mnd)

Mw/Mnd)

grafted density (chains/nm2)

IPB-0 IPB-1 IPB-2 IPB-5

100/0 99/1 98/2 95/5

100/0 98.9/1.1 95.4/4.6 93.6/6.4

0.948 ± 0.175 1.30 ± 0.162 1.81 ± 0.410 1.64 ± 0.272

11200 13200 14100 14400

1.40 1.31 1.36 1.52

0.51 0.59 0.77 0.66

code

a

All sample surfaces were abbreviated as IPB-X, where X is the feed composition of BMA (Table 1). bDetermined by 1H NMR measurement. Determined by ATR/FT-IR measurement. d)Determined by GPC using DMF containing 50 mmol/L LiCl. (HSMMs)17,50,51 were cultured on conventional TCPS dishes (Becton, Dickinson and Company, Franklin Lakes) with endothelial cell medium (EGM), fibroblast cell medium (FGM), smooth muscle cell medium (SmGM), and skeletal muscle myoblast cell medium (SkGM), respectively. Cells were recovered from conventional TCPS dishes by treating with 0.1% trypsin containing 1.1 mmol/L EDTA in phosphatebuffered saline (PBS). Recovered single cells were seeded on the prepared P(IPAAm-co-BMA) brush surfaces (coverslips) at 3.3 × 104 cells/mL for observing their adhesion and detachment. Cell suspensions were put on P(IPAAm-co-BMA) brush surfaces of cover glass slips in conventional culture dishes, which were incubated at 37 °C in a humidified atmosphere of 5% CO2 for 24 h, then transferred to another incubator set at 20 or 10 °C, and again incubated for 4 h for observing both cell adhesion and detachment. Cell morphology was also photographed at predetermined time by a phase-contrast microscope (ECLIPSE TE2000-U) (Nikon, Tokyo) equipped with a digital camera (OXM1200C) (Nikon, Tokyo). GFP-HUVEC was photographed by a fluorescence microscope (ECLIPSE TE2000-U) equipped with a digital camera (AxioCam HRc, Carl ZEISS, Oberkochen, Germany) for observing the adhesion of GFP-HUVEC. Adhering cells were counted randomly on the microphotographs in multiple areas. Percent cell adhesion was then calculated as the mean of three measurements with SD.

c)

Characterization of P(IPAAm-co-BMA) brush grafted surfaces was summarized in Table 2. The mole fraction of BMA in the copolymer was larger than that in the feed composition, probably due to the higher reactivity ratio of BMA, compared to that of IPAAm in ATRP procedure using a CuCl/Me6TREN catalyst system with 2-propanol as a solvent.38 Amounts of grafted P(IPAAm-co-BMA) on glass surfaces, determined by ATR/FTIR, were slightly increased with increasing BMA composition. Similarly, the molecular weight of prepared P(IPAAm-co-BMA) in reaction solution increased with increasing feed BMA composition. These were also attributed to a higher reactivity of BMA compared to that of IPAAm. The polydispersity index of prepared P(IPAAm-co-BMA) copolymer in reaction solution was slightly larger than that of a polymer prepared by polymerization only in solution without grafting substrate, as previously reported.38 This was attributed to a relatively smaller CuII complex in ATRP reaction. In our previous works for grafting PIPAAm on substrate through a surface-initiated ATRP, our laboratory has used CuCl2 for controlling polymerization in previous works,31,35 because the addition of CuCl2 increases the CuII complex in reaction solution, leading to a controlled polymerization.53 Similarly, previous reports regarding grafting copolymer onto substrate via surface-initiated ATRP have indicated that the addition of unbounded-initiator (free initiator) into ATRP reaction solution at the same time on grafting polymer onto substrate promotes to control polymerization, because the unbounded initiator increases the CuII complex, resulting in the control of polymerization.53 In the present study, although the polymerization was controlled by some extent in the present ATRP, an increase in free initiator or additional CuCl2 was required for more controlled polymerization. The estimated graft density exhibited a relatively higher value (>0.1 chains/nm2), indicating that the ATRP reaction formed densely packed P(IPAAm-coBMA) copolymer brush on glass substrates.54 The dense copolymer brush structure was speculated to suppress an undetachable cell adhesion and other extracellular proteins adsorption on the glass substrate. Also, for comparing the parameters of prepared copolymer brush surfaces with theoretical investigation in previous reports,55,56 the estimated brush thickness, polymerization degree, and area per chain were summarized in Table S1 in the Supporting Information. The graft densities and chain lengths of the copolymers were slightly different from a theoretical investigation regarding thermo-responsive cell culture substrates prepared by RAFT polymerization,55 because the copolymer brush in this study prepared through surface-initiated ATRP has a higher graft density than polymer brushes prepared by other polymerization methods.30 Therefore, a relatively shorter brush length than that of the theoretical prediction was speculated to be suitable for thermally modulated cell adhesion and detachment.

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RESULTS AND DISCUSSION Characterization of P(IPAAm-co-BMA) Brush Grafted Glass Surfaces. For investigating the elemental composition of the prepared surface, XPS measurement was performed. Table 1 summarizes the elemental compositions of the prepared surfaces, and the peak deconvolutions of XPS carbon C1s peaks of the ATRP-initiator-modified surface and P(IPAAm-co-BMA) grafted glass surfaces were shown in Figure S2 in Supporting Information. All samples were abbreviated as IPB-X where X represent the feed composition of BMA in ATRP procedure. For example, P(IPAAm-co-BMA) brush grafted glass coverslip prepared by ATRP using 1 mol % BMA feed composition was named IPB-1. In the spectrum of P(IPAAm-co-BMA) grafted glass surfaces (Figure S2B−E), an additional peak was observed at higher binding energy region, corresponding to the CO bond of IPAAm and BMA, while there was no peak in the spectrum of ATRP-initiator-modified glass slide (Figure S2A). These results indicated that P(IPAAm-co-BMA) was successfully grafted on glass surfaces through a surface-initiated ATRP. Additionally, nitrogen content decreased, and carbon content increased with increasing the BMA feed monomer composition. This indicated that the composition of BMA in grafted copolymer increased with increasing BMA feed composition, because BMA monomer contains no nitrogen and has a larger carbon content than that of IPAAm monomer. In addition, the chlorine composition also decreased after ATRP. This was attributed to a poor photoelectron accessibility to the structure during XPS analysis, because chlorine was buried in copolymer layer or lost during the termination reaction of radical coupling.52 3426

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Figure 2. Phase transition profiles of poly(N-isopropylacrylamide (IPAAm)-co-butyl methacrylate (BMA)). (A) Phase transition profiles in Milli-Q water, (B) in endothelial cell growth medium (EGM), (C) in fibroblast cell growth medium (FGM), (D) in smooth muscle cell growth medium (SmGM), and (E) in skeletal muscle cell growth medium (SkGM). The closed diamonds represent IPB-0, PIPAAm homopolymer; the open circles, IPB-1; the closed triangles, IPB-2; and the closed circles, IPB-5. All sample surfaces were abbreviated as IPB-X, where X is the feed composition of BMA (Table 1).

Table 3. Phase Transition Temperature of P(IPAAm-co-BMA) in Various Cell Culture Media IPAAm/BMAb (molar ratio) a

in feed

IPB-0 IPB-1 IPB-2 IPB-5

100/0 99/1 98/2 95/5

code

phase transition temperature (°C)c

in copolymer

Mnb

water

EGMd

FGM

SmGM

SkGM

100/0 98.9/1.1 95.4/4.6 93.6/6.4

11200 13200 14100 14400

32.7 24.0 19.2 16.6

29.9 22.1 18.0 15.4

30.2 22.4 19.1 15.0

30.1 22.4 17.8 14.7

30.0 22.6 19.6 14.8

a All sample surfaces were abbreviated as IPB-X, where X is the feed composition of BMA (Table 1). bDetermined by GPC using DMF containing 50 mmol/L LiCl. cDefined as temperature at 90% transmittance. dEGM represents endothelial cell medium; FGM, fibroblast cell medium; SmGM, smooth muscle cell medium; and SkGM, skeletal muscle myoblast cell medium.

and detachment behavior of four different types of cells. Figures 3−6 show the cell morphologies on prepared P(IPAAm-coBMA) brush-grafted surfaces and cell-adhesion and detachment profiles on the prepared surfaces. Figure 7 shows cell adhesion and detachment profiles on each prepared P(IPAAm-co-BMA) brushes for comparison. Four types of human cells were adhered on and detached from prepared copolymer brush surfaces at 37 and 20 °C, respectively. On the comparison of individual cell adhesion and detachment profiles in Figure 7, NHDFs exhibited a relatively higher adhesion ratio than those of other cell types. Also, on detachment profiles, HSMMs retain their adhesion on copolymer brushes, while other cell types detached themselves promptly. These differences in cell adhesion and detachment profiles would be applicable to the separation of cells, as suggested our previous report.37 However, differences in these adhesion and detachment profiles were relatively small, leading to a low separation efficiency. On the comparison of each cell adhesion with changing hydrophobic moiety in copolymer brushes, cell adhesion was enhanced with increasing BMA composition. Especially, IPB-5 exhibited a cell adhesion character similar to those of tissue culture polystyrene (TCPS), because the increased hydrophobicity of the copolymer brush was speculated to enhance cell adhesion. Our previous reports regarding thermoresponsive

For investigating copolymer’s hydrophobicity and lower critical solution temperature, the phase transition behavior of P(IPAAm-co-BMA) copolymer with various BMA composition was observed in Milli-Q water. In addition, the phase transition temperatures in various cell culture media were measured, because the phase transition behavior of PIPAAm and its copolymer is known to be influenced by ion concentration57 Figure 2A−E shows the phase transition behavior of P(IPAAmco-BMA) copolymer in Milli-Q water and cell culture media. Table 3 summarized the phase transition temperature of P(IPAAm-co-BMA) in Milli-Q water and cell culture media. The phase transition temperature decreased with increasing BMA composition, because the incorporation of BMA increases the hydrophobicity of the random copolymer,41,58 and the increased hydrophobicity enhances the aggregation of copolymers. Therefore, the incorporation of BMA monomer in the copolymer decreased the copolymers’ LCST. Also, a smaller decrease in phase transition temperatures was found compared to that in Milli-Q water. This was attributed to a salting-out effect57 induced by salt in cell culture media. Cells Adhesion on and Detachment from Intelligent Interfaces. Prepared P(IPAAm-co-BMA) brush surfaces with various BMA composition were evaluated as a thermoresponsive cell separating interface by observing the adhesion 3427

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Figure 3. Human umbilical vein endothelial cells (HUVECs) morphology on P(IPAAm-co-BMA) copolymer brushes with various BMA composition (A) and HUVEC adhesion on and detachment from these brushes (B). Cell morphologies at 37 and 20 °C were observed after 24 and 4 h incubations, respectively. The closed squares (■) represent IPB-0; the open triangles (△), IPB-1; the closed diamonds (◆), IPB-2; the open circles (○), IPB-5; the closed circles (●), tissue culture polystyrene (TCPS). All sample surfaces were abbreviated as IPB-X, where X is the feed composition of BMA (Table 1).

Figure 4. Normal human dermal fibroblasts (NHDFs) morphology on P(IPAAm-co-BMA) copolymer brushes with various BMA composition (A) and NHDF adhesion on and detachment from these brushes (B). Cell morphologies at 37 and 20 °C were observed after 24 and 4 h incubations, respectively. The closed squares (■) represent IPB-0; the open triangles (△), IPB-1; the closed diamonds (◆), IPB-2; the open circles (○), IPB-5; the closed circles (●), tissue culture polystyrene (TCPS). All sample surfaces were abbreviated as IPB-X, where X is the feed composition of BMA (Table 1).

copolymer having hydrophobic moiety has indicated that hydrophobized thermoresponsive copolymer exhibits a stronger interaction with small molecular drugs, peptides, and proteins38,41,59 than those between PIPAAm homopolymer and them. In this study, hydrophobized thermoresponsive copolymer brush was speculated to adsorb cellular proteins, resulting in a strong cell adhesion character. Actually, fibronectin (FN) adsorption on prepared P(IPAAm-co-BMA) brush grafted surfaces was observed by using rhodamine-labeled fibronectin, although all data were unable to be measured, because of the insufficient amount of the prepared copolymer brush modified surfaces for protein adsorption experiment. The result indicated that fibronectin had a tendency to be adsorbed on hydrophobized thermoresponsive copolymer brush modified surfaces compared to PIPAAm homopolymer brush surfaces (details of the experiment and result are shown in Figure S3 and Table S2 in the Supporting Information). Therefore, hydrophobized thermoresponsive surface adsorbed cellular proteins, leading to sufficient cell adhesion at 37 °C. On the contrary, cell detachment from hydrophobized IPB-5 surface was insufficient compared to those on other surfaces. This was probably due to its relatively lower phase transition temperature of IPB-5. Unlike the phase transition temperatures of other PIPAAm surfaces, that of IPB-5 surface was lower than 20 °C, and cell detachment from IPB-5 at 20 °C was insufficient because of the insufficient hydration of grafted copolymer. Therefore, cell detachment behavior from IPB-5 at 10 °C was investigated. Figure 8A−D shows the comparison of cell detachment profile from IPB-5 surface between 10 and 20 °C. Effective detachment of HUVECs was observed at 20 °C compared to 10 °C. On the contrary, the effective detachment of NHDFs was also observed at 10 °C compared to 20 °C. On SMC and HSMM detachment, similar cell detachment profiles were observed between 10 and 20 °C.

These were attributed to the balance between cell intrinsic metabolic activity and the hydration of grafted copolymer brush. Our research institute has reported that cell detachment from thermoresponsive-polymer-modified surfaces is strongly affected by not only the hydration of PIPAAm but also the cellular metabolism for detachment.60 Therefore, each cell has a specific adequate temperature for detachment from thermoresponsive polymer-modified surfaces. Actually, for investigating the effect of cell metabolic activity on detachment from the copolymer brush surfaces, cell detachment behavior at 20 °C was observed with adding sodium azide in cell culture medium for inhibiting cytochrome C oxidase in mitochondria and suppressing ATP generation. By the disruption of ATP dependent cellular activities in the cell, the detachment of HUVECs from copolymer brush surfaces were suppressed, indicating that cellular metabolic activity is essential for detachment from the copolymer brush (Figure S4 in the Supporting Information). Additionally, SMC is known to have a tendency to detach from normal temperature responsive culture dish at 20 °C. On the other hand, HSMM has a tendency to stay on the dish at 20 °C. For observing a clear detachment from the thermoresponsive copolymer brush, this study incubated SMC and HSMM at both 20 and 10 °C. Probably, due to their intrinsic adhesive characters, no difference in their temperature dependent detachment characters were observed at both 20 and 10 °C, indicating that SMC and HSMM seemed to have a poor temperature sensitivity. In the case of copolymer brush surface, IPB-5 copolymer brush tended to be more hydrated at 10 °C than at 20 °C. However, the 3428

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Figure 5. Human aortic smooth muscle cells (SMCs) morphology on P(IPAAm-co-BMA) copolymer brushes with various BMA composition (A) and SMC adhesion on and detachment from these brushes (B). Cell morphologies at 37 and 20 °C were observed after 24 and 4 h incubations, respectively. The closed squares (■) represent IPB-0; the open triangles (△), IPB-1; the closed diamonds (◆), IPB-2; the open circles (○), IPB-5; the closed circles (●), tissue culture polystyrene (TCPS). All sample surfaces were abbreviated as IPB-X, where X is the feed composition of BMA (Table 1).

Figure 6. Human skeletal muscle myoblast cells (HSMMs) morphology on P(IPAAm-co-BMA) copolymer brushes with various BMA composition (A) and HSMM adhesion on and detachment from these brushes (B). Cell morphologies at 37 and 20 °C were observed after 24 and 4 h incubations, respectively. The closed squares (■) represent IPB-0; the open triangles (△), IPB-1; the closed diamonds (◆), IPB-2; the open circles (○), IPB-5; the closed circles (●), tissue culture polystyrene (TCPS). All sample surfaces were abbreviated as IPB-X, where X is the feed composition of BMA (Table 1).

HUVEC cellular metabolism rate upon the detachment at 10 °C was probably lower than that at 20 °C, suggested by our report regarding similar endothelial cells metabolism.60 Therefore, HUVEC detachment at 20 °C was enhanced compared to that at 10 °C. On the contrary, in the case of NHDFs, the hydration of copolymer provided a more significant effect on the detachment than NHDF metabolism. These results indicated that HUVECs and NHDFs had a suitable temperature for detachment at 20 and 10 °C, respectively. In addition, the activities of HUVECs and NHDFs after incubation at 20 and 10 °C were investigated by alamar blue assay. The result indicated that incubation at a lower temperature scarcely affected the activity of cells (Table S3 in the Supporting Information). Therefore, the difference in cell sensitivity for detachment temperature would be useful for cell separation. These cell adhesion and detachment experiment results indicated that (1) cell adhesion was enhanced on IPB-5 brush obtained by grafting P(IPAAm-co-BMA) copolymer with 5 mol % of BMA feed composition and (2) specific suitable temperatures for cell detachment were observed on HUVECs and NHDFs at 20 and 10 °C, respectively. Using these properties, the separation of HUVECs and NHDFs was attempted, because the coculture of HUVEC and NHDF is known to be necessary for obtaining the various types of tissues having an requiring angiogenetic ability,61,62 and the cell separation is important for preparing cell mixture having various cell composition ratios. GFP-expressing HUVEC (GFPHUVEC) was used in this separation for distinguishing

individual cell adhesion and detachment properties. Mixed cell suspensions consisting of GFP-HUVEC and NHDF were seeded on IPB-5 with endothelial cells growth medium as culture medium. Various other culture media were investigated, such as Dulbecco’s modified Eagle’s medium (DMEM) and a mixture of EGM and FGM. However, effective cell adhesion on the surface was unable to be performed using these cell culture medium, probably due to the insufficient cellular adhesion factor in these cells culture medium. Therefore, EGM was found to be an appropriate cell culture medium for GFP-HUVECs and NHDFs separation. Figure 9 shows GFP-HUVEC and NHDF adhesion on and detachment from IPB-5, and the cells morphologies on IPB-5. Cell detachment was performed at 20 and 10 °C. Both of HUVECs and NHDFs were able to adhere on IPB-5 at 37 °C. Upon detachment at 20 °C, both HUVECs and NHDFs were promptly detached with a similar detachment behavior. Upon detachment at 10 °C, NHDFs were promptly detached themselves from IPB-5 surfaces with a higher recovery rate, while HUVECs was unable to detach in initial incubation period until 30 min, and more than half of adhered HUVECs were remained at IPB-5 surface. These results indicated that, in a cocultured system, NHDFs were selectively detached from IPB-5 surface at 10 °C, and HUVECs were detached from IPB-5 surfaces at 20 °C. Therefore, these cells were speculated to be separated by changing temperature stepwise from 37 to 10 °C and 20 °C. 3429

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Figure 7. Cells adhesion and detachment profiles on (A) IPB-0, (B) IPB-1, (C) IPB-2, and (D) IPB-5 for comparing cell detachment profiles on each copolymer brush surfaces. The open circles (○) represent HUVECs; the closed triangles (▲), NHDFs; the open diamonds (◇), SMCs; and the closed squares (■), HSMMs. All sample surfaces were abbreviated as IPB-X, where X is the feed composition of BMA (Table 1).

Figure 8. Cells detachment profiles from IPB-5 brush at 10 or 20 °C. (A) HUVECs, (B) NHDFs, (C) SMCs, and (D) HSMMs. The open circles represent incubation for cells recovery at 20 °C. The closed triangles represent incubation at 10 °C.

recovering HUVECs. In incubation period at 10 °C, NHDFs promptly detached while HUVECs adhered on IPB-5 surfaces. Then, incubation period at 20 °C, adhered HUVECs were recovered from the IPB-5 surface. Therefore, the higher ratio of NHDFs was able to be recovered in medium during the 10 °C

Figure 10 shows a separation of a mixture of HUVECs and NHDFs by a multistep temperature change. At 37 °C, both cells were adhered on IPB-5, and the external temperature was reduced to 10 °C for recovering NHDFs. After 30 min incubation at 10 °C, the temperature was elevated to 20 °C for 3430

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Figure 9. Morphologies of green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) and NHDFs adhered on and detached from IPB-5 in endothelial cell culture medium. (A and C) Cell morphologies at 37 °C for adhesion, and at 20 and 10 °C for recovery, respectively. (B and D) Cell adhesion and detachment profiles of 20 and 10 °C, respectively. The green circles and the orange squares represent GFPHUVECs and NHDFs, respectively.

systems. Additionally, cell separation using thermo-responsive polymer brush required a relatively longer time than cell sorting systems. However, in terms of cell purity, no highly purified cells are required for fabricating tissues, because animal and human tissues primitively contain a mixture of various types of cells. Additionally, separated cells using the thermo-responsive copolymer brush has a high activity without cell surface modification, which is important for the transplantation of fabricated tissues. Moreover, the separation can be performed by only changing the external temperature of the prepared copolymer brush surfaces. These advantages of the proposed cell separation are valuable for current tissue engineering and regenerative medicine. The results obtained from these investigation indicated that precisely designed P(IPAAm-co-BMA) brush, prepared through the surface-initiated ATRP, was able to separate a mixture of cells by multistep temperature change. Therefore, hydrophobized thermoresponsive copolymer brush surfaces would be applicable to cell separation by using cell temperature sensitivity for detachment. The prepared surfaces would be useful as materials for a cell separation tool such as cell-separation column chromatography, microfluidics, or combined fields flow fractionation (FFF) and adsorption chromatography.63,64

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CONCLUSIONS Dense P(IPAAm-co-BMA) brushes having various BMA compositions were grafted onto glass surfaces, and these prepared surfaces were investigated as cell separation materials. Characterization of prepared surfaces indicated that dense hydrophobized thermoresponsive copolymer brushes were successfully grafted on glass surfaces through surface-initiated ATRP. In cell-adhesion and -detachment profiles, the cell adhesion ratio on copolymer brush surfaces increased with increasing BMA composition in grafted copolymer brushes, probably due to the enhanced adsorption of cellular proteins. Upon cell detachment from hydrophobized thermoresponsive copolymer brushes at 10 or 20 °C, HUVECs and NHDFs exhibited different cells detachment profiles at specific temperatures. Effective detachments of HUVECs and NHDFs were

Figure 10. (A) GFP-HUVECs and NHDFs cell adhesion on and detachment from IPB-5 in endothelial cells culture medium. The green circles and the orange squares represent GFP-HUVECs and NHDFs, respectively. Cells adhesion was performed at 37 °C for 24 h; then the cells were incubated for initial 30 min at 10 °C and after a subsequent period at 20 °C for recovering adhered cells. (B) GFP-HUVECs and NHDFs morphology adhesion on and detachment from IPB-5.

incubation period, and then a subsequent incubation period at 20 °C provided a higher ratio of HUVECs in medium. Compared with conventional cell sorting systems such as FACS and MACS, each cell fraction separated by hydrophobized thermoresponsive copolymer brush contained a small amount of other cells, and the purity was lower than those of cell sorting 3431

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observed at 20 and 10 °C, respectively, because of the balance between the hydration degree of grafted copolymer and cellular metabolism for detachment. Using these cells’ intrinsic properties, HUVECs and NHDFs were separated by a multistep temperature change. Mixtures of HUVECs and NHDFs were adhered on a hydrophobized thermoresponsive copolymer brush at 37 °C, and then external temperature was reduced to 10 °C for recovery of NHDFs. NHDFs detached themselves promptly from the copolymer brush, while HUVECs still adhered. Subsequently, the temperature was elevated to 20 °C, and adhered HUVECs were recovered. The high ratio of NHDF and HUVEC were obtained in incubation at 10 and 20 °C, respectively. Therefore, precisely designed thermoresponsive copolymer brush was able to separate cells by using difference in cells’ temperature sensitivity for detachment.

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ASSOCIATED CONTENT

* Supporting Information S

Standard curve for estimating amount of grafted copolymer, XPS peak deconvolution of XPS C1s peaks, fibronectin adsorption on prepared copolymer brush, cellular metabolic activity on detachment, and cell activity determined by alamar blue assay. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-3-5367-9945 ext. 6201. Fax: +81-3-3359-6046. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Part of the present research was financially supported by the “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),” initiated by the Council for Science and Technology Policy from the Japan Society for the Promotion of Science (JSPS), Grants-in-Aid for Young Scientists (B) no. 24760580 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. We thank Ms. Ayaka Kimura for valuable suggestions and discussion and Dr. Norio Ueno for English editing.

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