Thermoresponsive Nanostructured Surfaces Generated by the

Oct 7, 2014 - Langmuir−Schaefer Method Are Suitable for Cell Sheet Fabrication ... and detachment were fabricated by the Langmuir−Schaefer method...
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Thermoresponsive Nanostructured Surfaces Generated by the Langmuir−Schaefer Method Are Suitable for Cell Sheet Fabrication Morito Sakuma,†,‡ Yoshikazu Kumashiro,*,‡ Masamichi Nakayama,‡ Nobuyuki Tanaka,‡ Kazuo Umemura,† Masayuki Yamato,‡ and Teruo Okano*,‡ †

Department of Physics, Graduate School of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku, Tokyo 162-8601, Japan Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University (TWIns), 8-1 Kawadacho, Shinjuku, Tokyo, 162-8666, Japan

‡

S Supporting Information *

ABSTRACT: Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir−Schaefer method. Block copolymers composed of polystyrene and PIPAAm (St-IPAAms) having various chain lengths and compositions were synthesized by reversible addition−fragmentation chain transfer radical polymerization. The St-IPAAm Langmuir film at an air−water interface was horizontally transferred onto a hydrophobically modified glass substrate while regulating its density. Atomic force microscopy images clearly visualized nanoscaled sea−island structures on the surface. By adjusting both the composition of St-IPAAms and the density of immobilized PIPAAms, a series of thermoresponsive surfaces was prepared to control the strength, rate, and quality of cell adhesion and detachment through changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was more rapidly recovered on the optimized surfaces than on conventional PIPAAm surfaces. These unique PIPAAm surfaces are suggested to be useful for controlling the strength of cell adhesion and detachment.

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functions of the tissue or organ.15−17 For cell sheet recovery, PIPAAm-modified surfaces are synthesized by several methods, including electron beam irradiation and surface-initiated reversible addition−fragmentation chain transfer radical (RAFT) polymerization.11−13 These studies have reported that the thickness, composition, and density of grafted PIPAAms are important factors for appropriately fabricating and harvesting cell sheets. In addition, physical polymer coating methods, such as spin coating and casting, have also been developed for controlling cell adhesion and detachment.14,18,19 The use of physical polymer coating methods for these purposes also provides information on the relationship between cell adhesion/detachment and polymer composition because the polymer composition is defined in the bulk and solution states. A Langmuir film is one of the most widely studied physical polymer coatings.20−24 A Langmuir film is formed by dropping amphiphilic molecules onto an air−water interface. Then, the Langmuir film can be transferred onto a solid surface with physical or chemical adsorption, and the molecular conformation on a solid surface can be controlled using vertical and horizontal transfer methods.25−31 The density of the Langmuir film can be precisely regulated by compressing the air−water interface. Recently, this method has also proven to be effective

INTRODUCTION Stimuli-responsive polymers, which rapidly change their configuration in response to external stimuli, have been applied in a wide range of scientific fields, such as drug delivery, bioseparation, and tissue engineering.1−3 Configurational changes to stimuli-responsive polymer-modified surfaces induce drastic alterations of the physical and chemical surface characteristics. A surface modified with poly(N-isopropylacrylamide)s (PIPAAms), which shrinks and stretches in response to temperature in aqueous media, is one of the most widely applied stimuliresponsive surface materials.4−10 Because PIPAAm-modified surfaces show hydrophobic/hydrophilic changes across the lower critical solution temperature (LCST) range of PIPAAm molecules (approximately 32 °C), the mode of cell adhesion on the surfaces can be actively controlled by changing the temperature. Cells can adhere on a PIPAAm-modified surface at 37 °C because PIPAAm molecules shrink and the surface becomes hydrophobic. After reducing the temperature below the LCST of PIPAAm molecules, the surface is in a hydrophilic state, and the adhering cells can spontaneously detach themselves without enzyme or chemical treatment. When the cells are cultured to confluence at 37 °C, a two-dimensional cell sheet structure having cell−cell junctions and adhesion proteins is maintained after reducing the temperature.11−14 Because the detached cell sheet has extracellular matrix (ECM) on its basal side, it can be used to transplant a biological tissue or organ without suturing and to reconstruct the lost structures and © 2014 American Chemical Society

Received: August 13, 2014 Revised: October 5, 2014 Published: October 7, 2014 4160

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Biomacromolecules

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Figure 1. Preparation of a Langmuir film-transferred surface. (A) Polystyrene-block-PIPAAm (St-IPAAm) chloroform solution was dropped onto the trough. Surface pressure was measured at the end of the compression. (B) Barriers compressed St-IPAAm molecules on the interface to a target area of 50 cm2. (C) After compression, a hydrophobically modified cover glass substrate was horizontally placed on the interface. sulfanylpentanoic acid (ECT) was prepared as a chain transfer agent with as described by Moad et al.35 and Convertine et al.36 Styrene (153.6 mmol), ECT (0.2 mmol), and ACVA (0.04 mmol) were dissolved in 40 mL of 1,4-dioxane, and the solution was degassed by a freeze−pump−thaw cycle three times. Then, the polymerization was performed at 70 °C for 15 h. After the polymerization, the polymer product was obtained by precipitating with an excess of ether and then dried in vacuo. Then, the St-IPAAm molecule was synthesized from the synthesized PSt macro RAFT agent. For example, IPAAm (2.63 mmol), PSt macro RAFT agent (0.021 mmol), and ACVA (0.004 mmol) were dissolved in 4.32 mL of 1,4-dioxane. Then, the polymerization was performed at 70 °C for 15 h after degassing. The synthesized St-IPAAm molecule was obtained in the same manner as that for the PSt macro RAFT agent. The molecular weight and polymer composition of St-IPAAm molecules were characterized by 1H NMR (INOVA 400) (Varian, Palo Alto, CA) and gel permeation chromatography (GPC) (HLC8320GPC) (Tosoh, Tokyo) with two sequentially connected columns (TSKgel Super Aw2500, TSKgel Super AW3000 and TSKgel Super AW4000) (Tosoh) at 40 °C using DMF containing 50 mmol/L LiCl as an eluent (flow rate, 0.6 mL/min), and the columns were calibrated with polystyrene of a defined molecular weight. Preparation of Langmuir Film-Transferred Surfaces with StIPAAms. Langmuir films of St-IPAAm molecules were prepared by

for fabricating nanoscaled sea−island structures by utilizing amphiphilic block copolymers.32−34 The nanostructures are assumed to consist of hydrophobic segments as a core and hydrophilic segments as a shell. In addition, the number of nanostructures on a surface is regulated by controlling the area per molecule (Am) of the block copolymer at the interface. In this study, block copolymers composed of polystyrene (P(St)) and PIPAAm (St-IPAAm) were synthesized, and a Langmuir film of St-IPAAms with various polymer compositions was fabricated. The Langmuir film with a specific Am was horizontally transferred on a hydrophobically modified glass substrate. The physical properties of the St-IPAAm Langmuir film-transferred surface (St-IPAAm surface) were characterized by various surface analyses. In addition, cell adhesion and detachment on the prepared surface, promoted by temperature change, was evaluated, and the optimal conditions for cell sheet harvesting are also discussed.

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EXPERIMENTAL SECTION

Materials. N-Isopropylacrylamide (IPAAm) was kindly provided by Kohjin (Tokyo, Japan). Azobis(4-cyanovaleric acid) (ACVA) was purchased from Wako Pure Chemicals (Osaka, Japan) and recrystallized from methanol. Styrene, 1,3,5-trioxane was purchased from Sigma-Aldrich Japan (Tokyo). 1,4-Dioxane (Wako), N,N-dimethylformamide (DMF) (Wako), and the other solvents were used without further treatment. Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s modified phosphate-buffered saline (PBS), and trypan blue solution were purchased from Sigma (St. Louis, MO), streptomycin and penicillin were from GIBCO BRL (Gaithersburg, MD), fetal bovine serum (FBS) was from Moregate Biotech (Australia), and bovine carotid artery endothelial cells (BAECs) were from Health Science Research Sources Bank (Osaka, Japan). Cover glasses (size, 24 × 50 mm; thickness, 0.12−0.17 mm) were from Matsunami Glass Industry (Osaka, Japan). Synthesis and Characterization of Polystyrene-block-poly(Nisopropylacrylamide). Block copolymers composed of polystyrene (P(St)) and poly(N-isopropylacrylamide) (St-IPAAms) with specific molecular weights were synthesized by two-step reversible addition− fragmentation chain transfer radical (RAFT) polymerization. For synthesizing the PSt macro RAFT agent, 4-cyano-4-(ethylsulfanylthiocarbonyl)

Table 1. Synthetic Results of Polystyrene-block-poly(Nisopropylacrylamide)s codea

CTAb (mmol/L)

IPAAmc (mol/L)

molecular weightd

Mw/Mnd

St-IPAAm110 St-IPAAm170 St-IPAAm240 St-IPAAm480

5.0 5.0 4.0 4.8

2.86 4.32 5.86 7.27

26 000 32 800 40 700 67 900

1.14 1.31 1.33 1.50

a

The nomenclature for the block copolymers composed of polystyrene (P(St)) and poly(N-isopropylacrylamide) (PIPAAm) is shown as St-IPAAmx, where x indicates the PIPAAm composition. b CTA, chain transfer agent. cIPAAm, N-isopropylacrylamide. dWeightaverage molecular weight (Mw), number-average molecular weight (Mn), and their distributions were determined by gel permeation chromatography calibrated with polystyrene. 4161

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adsorb the film. Then, the substrate was horizontally removed and used for the following experiments after drying for 1 day. Characterization of Langmuir Film-Transferred Surfaces of St-IPAAms. The topographic images of the Langmuir film-transferred surface (St-IPAAm surface) were obtained by an atomic force microscope (AFM) (NanoScope V) (Veeco, Santa Barbara, CA) in tapping mode using a phosphorus-doped silicon cantilever (RFESP; Veeco) with a spring constant of 3 N/m and resonant frequency of 70−90 kHz. The amount of PIPAAms transferred was determined by an attenuated total reflection Fourier-transform infrared spectroscope (ATR/FT-IR) (NICOLET 6700 FT-IR) (Thermo Scientific, Waltham, MA). The absorption peaks derived from glass (Si−O) and PIPAAm (CO) were observed at around 1000 and 1650 cm−1, respectively. The amount of PIPAAms on the glass was determined by the peak intensity ratio of I1650/I1000 using a calibration line prepared from a series of known amounts of PIPAAm cast on the hydrophobic glass. The relative atomic compositions of St-IPAAm surfaces were measured by an X-ray photoelectron spectroscope (XPS) (K-Alpha) (Thermo Fisher Scientific, Yokohama, Japan) (the takeoff angle was 90°). After survey spectra were acquired, the surface atomic compositions were calculated using integrated peak areas. Cell Adhesion and Detachment Assay. For BAECs cultivation, DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin was prepared. BAECs were cultured on tissue culture polystyrene (TCPS) at 37 °C under a humidified atmosphere with 5% CO2 and 95% air. BAECs cultured to confluence were recovered from TCPS with 0.25% trypsin-EDTA and then seeded on St-IPAAm surfaces at a concentration of 1.0 × 104 cells/cm2 at 37 °C. After incubating at 37 °C under 5% CO2, adherent BAECs were observed 24 h after seeding by a phase contrast microscope (ECLIPSE TE2000-U) (Nikon, Tokyo), and the number of adherent cells was counted. Then, to investigate the effects of temperature on cell detachment from the St-IPAAm surfaces, BAECs were incubated at 20 °C for 2 h. Cell Sheet Fabrication. A sheet of BAECs was fabricated on the St-IPAAm surfaces with various Am and St-IPAAm molecules in a manner similar to that for the single cell experiment. BAECs were seeded on St-IPAAm surfaces at a concentration of 1.0 × 105 cells/cm2 at 37 °C. After being cultured to confluence at 37 °C for 3 days, the confluent cells were incubated at 20 °C. The recovered cell sheet was visually observed, and images were captured by a digital camera (CX-6) (Ricoh, Tokyo). The viability of the cell sheet was evaluated by trypan blue staining.

Figure 2. Surface pressure−area per molecule (π−Am) isotherms of a polystyrene-block-PIPAAm (St-IPAAm) Langmuir film at an air−water interface. The y axis shows the surface pressure of the film, and the x axis shows the area per molecule. The solid, dotted, long dasheddotted, and dashed lines indicate π−Am isotherms of the films of St-IPAAm110, St-IPAAm170, St-IPAAm240, and St-IPAAm480 molecules, respectively. A description of the polymer nomenclature on the surface is given in Table 1. using a Langmuir-trough apparatus (Figure 1) by a method similar to that in a previous study.37 St-IPAAm molecules dissolved in chloroform solution (1.0 mg/mL) were carefully dropped onto a trough (size, 580 × 145 mm) filled with purified water at 22 °C. After waiting 5 min to allow the complete evaporation of chloroform, both barriers were moved horizontally to compress the St-IPAAm molecules at the interface. The compression rate of the barriers was maintained at 0.5 mm/s until the target area of 50 cm2 was reached. The surface pressure (π)-area per molecule (Am) isotherms were measured during compression with a platinum Wilhelmy plate (perimeter, 39.24 mm). A theoretical value of Am was calculated using the following equation

Am =

AM w cNAV

(1)

where A is the area of the interface, Mw is the molecular weight of St-IPAAms, c is the concentration of the chloroform solution, NA is Avogadro’s number, and V is the volume of the dropping polymer solution. After the compression, the interface was kept for 5 min in a specific area of 50 cm2, and then a hexyltrimethoxysilane-modified hydrophobic cover-glass substrate (size: 25 × 24 mm) was placed on the Langmuir film in a parallel fashion for 5 min in order to robustly

Figure 3. Atomic force microscope topographic images (1 × 1 μm) of Langmuir film-transferred surfaces. The upper row shows a surface with an area per molecule (Am) of 3 nm2/molecule, the middle row shows a surface with an Am of 10 nm2/molecule, and the lower row shows a surface with an Am of 40 nm2/molecule. Columns containing images A−C, D−F, G−I, and J−L show the surfaces of St-IPAAm110, St-IPAAm170, StIPAAm240, and St-IPAAm480, respectively. Image M shows the surface of hydrophobic hexyltriethoxysilane-modified surface as a control. 4162

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Fabrication of Langmuir Films of the St-IPAAms at an Air−Water Interface. Langmuir films with various areas per molecule (Am) were fabricated at an air−water interface by dropping St-IPAAm molecules dissolved in a chloroform solution. Figure 2 shows the surface pressure−area per molecule (π−Am) isotherms of St-IPAAm Langmuir films at an air−water interface. The theoretical value of Am was calculated by the equation described in the Experimental Section. First, the gas phase was observed at 0 mN/m after dropping the chloroform solution. After starting the compression of the barriers, an initial phase transition of the film from the gas phase to the liquid phase was observed (0−30 mN/m). Through the liquid−solid phase of approximately 30 mN/m above 5 nm2/molecule, a second phase transition of the film was observed (the solid phase), and the film moved to the collapsed phase with further compression (above 45 mN/m). Under compression, the St-IPAAm480 Langmuir film reached the liquid−solid phase at a density of 160 nm2/molecule, whereas the other films reached this phase below 60 nm2/ molecule. The larger St-IPAAm molecules reached the liquid phase at larger Am because the St-IPAAm chains became intermolecularly entangled at the interface. At a density of 5 nm2/ molecule, all Langmuir films moved to the solid phase, and the St-IPAAm480 Langmuir film rapidly entered into the collapsed phase. These results indicated that the phase of the Langmuir film was precisely controlled by the synthetic molecules and the compression. Characterization of Langmuir Film-Transferred Surfaces. A prepared St-IPAAm Langmuir film was transferred onto a hydrophobically modified glass substrate at 3, 10, and 40 nm2/molecule, and the transferred surfaces (St-IPAAm surfaces) were characterized by AFM. Figure 3 shows AFM topographic images (1 × 1 μm) of St-IPAAm surfaces with various Am and St-IPAAm molecules. Nanostructures (hydrophobic core/hydrophilic shell) were observed on St-IPAAm surfaces, whereas such structures were hardly observed on a bare hydrophobic substrate. The bright yellow area based on nanoscaled sea−island structures was increased with decreasing Am. The total areas on St-IPAAm110 surfaces at 40 and 3 nm2/molecule

Table 2. Quantitative Evaluation of St-IPAAm Surfaces relative atomic composition (%)c

codea St-IPAAm110_3 St-IPAAm110_10 St-IPAAm110_40 St-IPAAm170_3 St-IPAAm170_10 St-IPAAm170_40 St-IPAAm240_3 St-IPAAm240_10 St-IPAAm240_40 St-IPAAm480_3 St-IPAAm480_10 St-IPAAm480_40

amount of PIPAAm molecules on the Langmuir−Schaefer surface (μg/cm2)b 1.28 0.66 0.30 1.54 0.87 0.41 1.81 1.06 0.46 2.53 1.91 0.63

± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.09 0.02 0.10 0.03 0.05 0.19 0.10 0.04 0.18 0.28 0.04

C

N

O

Si

70.7 63.2 38.3 78.9 58.2 21.2 77.6 69.8 29.4 78.9 73.7 35.3

4.6 3.8 2.0 7.2 4.5 1.4 8.2 6.0 2.1 8.2 8.4 2.9

16.4 22.0 40.6 10.7 25.2 53.0 11.5 16.9 45.7 11.4 14.2 41.7

8.2 11.0 19.1 3.0 12.0 24.4 2.6 7.1 22.6 1.4 3.6 20.1

a

The nomenclature for the St-IPAAm surfaces is shown as St-IPAAmx_y, where x and y indicate the PIPAAm composition and area per molecule, respectively. bEstimated by attenuated total reflection Fourier-transform infrared spectroscopy. The data are the average of three measurements. cMeasured by X-ray photoelectron spectroscopy. The takeoff angle was set at 90°.

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RESULTS AND DISCUSSION Characterization of Block Copolymers Composed of Polystyrene and Poly(N-isopropylacrylamide). Block copolymers composed of polystyrene and poly(N-isopropylacrylamide) (St-IPAAms) with specific molecular weights were synthesized by RAFT polymerization. The obtained block polymers characterized by 1H NMR and GPC are summarized in Table 1. The molecular weights of St-IPAAms were 26 000, 32 800, 40 700, and 67 900, with narrow molecular weight distributions. The conversion ratios of the reaction between the PSt macro RAFT agent and PIPAAm were determined to be approximately 17% and more than 85%, respectively.

Figure 4. Microscopy photographs of the BAECs adhering to Langmuir film-transferred surfaces 24 h after seeding. The upper row shows cells adhering to the surface with an area per molecule (Am) of 3 nm2/molecule, the middle row shows cells adhering with an Am of 10 nm2/molecule, and the lower row shows cells adhering with an Am of 40 nm2/molecule. Columns containing images A−C, D−F, G−I, and J−L show the BAECs adhering to the surface on St-IPAAm110, St-IPAAm170, St-IPAAm240, and St-IPAAm480, respectively. Image M shows adhering cells on the surface of hydrophobically modified glass as the control. Table 1 provides an explanation of the nomenclature of the polymers on the surface. 4163

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were approximately 0.12 and 0.57 μm2, respectively. Similar phenomena were observed on the other surfaces, with the exception of the St-IPAAm480 surface at 3 nm2/molecule because the phase of the St-IPAAm480 Langmuir film at 3 nm2/molecule was the collapsed phase and the nanostructures may have been fused. The diameter of island structures was found to be approximately 20 nm. AFM topographic images confirmed that the morphologies of the nanoscaled sea−island structures were dependent on Am and the polymer compositions of the St-IPAAm molecules. The relative atomic compositions of St-IPAAm surfaces were measured by XPS (Table 2). The composition ratio of silicon atoms derived from the glass substrate (takeoff angle was 90°) was found to be approximately 20% at 40 nm2/molecule, and the ratio decreased to nearly 0% at 3 nm2/molecule. As the ratio of silicon decreased with decreasing Am (increasing density), the substrate was densely packed with the Langmuir film at small Am, and it was assumed that the exposure of the glass substrate was reduced. The ratio of silicon on the St-IPAAm110 surface at 3 nm2/molecule was higher than that on the St-IPAAm480 surface. Since the length of PIPAAm in St-IPAAm110 was shorter than that in St-IPAAm480, the glass substrate might have been partially exposed on the St-IPAAm110 surface. The angle-dependent relative atomic compositions were also measured by angularly resolved XPS (see Figure S2 in the Supporting Information). The ratio of silicon and oxygen was gradually decreased to lower takeoff angles, indicating that thin Langmuir films were transferred onto the hydrophobically modified glass substrates. The results of quantitative evaluation of St-IPAAm surfaces by ATR/FT-IR measurements are summarized in Table 2. The amounts of PIPAAm transferred onto the St-IPAAm110 surfaces at 3, 10, and 40 nm2/molecule were determined to be 1.28 ± 0.02, 0.66 ± 0.09, and 0.30 ± 0.02 μg/cm2, respectively. On the basis of the amounts of PIPAAm determined by ATR/ FT-IR, the resulting values of Am on St-IPAAm110 surfaces were calculated and found to be 3.3, 6.5, and 14.3 nm2/ molecule. At the liquid−solid and gas phases, the resultant values of Am were lower than the theoretical values. Since the volume of nanostructures at the interface did not reach the saturation state at the liquid−solid and gas phases, it would be expected that St-IPAAm molecules were able to move freely at the interface followed by a small amount of transference. On the other hand, the resultant values transferred from the solid phase were found to be close to the theoretical values. The robust modification of St-IPAAm molecules on the hydrophobically modified glass substrate was also evaluated by ATR/ FT-IR. To confirm the stability of St-IPAAm molecules on the surface, St-IPAAm surfaces were washed with purified water. The amounts of PIPAAm on the washed St-IPAAm surfaces were found to be nearly the same as those on the surfaces without washing, indicating that St-IPAAm molecules were robustly adsorbed on the glass substrate in aqueous media. Cell Adhesion and Detachment on Langmuir FilmTransferred Surfaces. We next examined the adhesion and detachment of BAECs on St-IPAAm surfaces with various Am and St-IPAAm molecules having various PIPAAm chain lengths. Microphotographs of adhering BAECs and the time courses of cell adhesion on St-IPAAm surfaces after 24 h of incubation at 37 °C are shown in Figures 4 and 5A,C,E. The number of BAECs adhering to the St-IPAAm surfaces at 40 nm2/molecule after 24 h of incubation at 37 °C was 0.8−0.9 × 104 cells/cm2, which was similar than that on the control

Figure 5. BAEC adhesion and detachment assay on a Langmuir filmtransferred surface. The upper, middle, and lower graphs show the number of BAECs adhering to St-IPAAm110 (closed circles), St-IPAAm170 (closed triangles), St-IPAAm240 (closed squares), and St-IPAAm480 (closed diamonds) surfaces with areas per molecule (Am) of 3 nm2/molecule (upper), 10 nm2/molecule (middle), and 40 nm2/molecule (lower). The number of BAECs on hydrophobically modified surfaces is also shown in all graphs as a control (open circles). The graphs on the left and right show the cell adhesion process at 37 °C and the cell detachment process when reducing the temperature from 37 to 20 °C for 120 min, respectively.

hydrophobic surface. The number of adhering BAECs decreased with decreasing Am, and the number on the St-IPAAm surfaces at 3 nm2/molecule was below 0.2 × 104 cells/cm2, except in the case of the St-IPAAm110 surface. Notably, the number of 4164

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Figure 6. BAEC sheet recovery of a Langmuir film-transferred surface with various densities and PIPAAm chain lengths. “Good” represents intact cell sheet recovery, and the number shown in parentheses indicates the recovery time after reducing the temperature. “Hardly recovered” means that the cells could be cultured to confluence and the cultured cells showed little or no detachment from the surface even after a reduction in temperature. “Weak adhesion” means that cells were not cultured to confluence at 37 °C for 3 days.

BAECs adhering to the St-IPAAm110 surface at 3 nm2/ molecule was decreased between 12 and 24 h. The initial surface attachment of BAECs that occurred within 12 h was not fully maintained, probably because the adhesion force between BAECs and the St-IPAAm110 surface was weakened. Figure 5B,D,F also shows the cell detachment occurring after a reduction in temperature from 37 to 20 °C for 2 h. All BAECs detached at 3 nm2/molecule within 30 min of incubation. At 10 nm2/molecule, rapid detachment of BAECs was also observed on the St-IPAAm240 and St-IPAAm480 surfaces after 30 min of incubation, whereas adhesion of BAECs (more than 0.2 × 104 cells/cm2) was still observed on the St-IPAAm110 and St-IPAAm170 surfaces. On the other hand, BAECs were gradually detached from the St-IPAAm110, St-IPAAm170, and St-IPAAm240 surfaces at 40 nm2/molecule, and complete cell detachment was observed only at the St-IPAAm480 surface after 2 h. These results showed that (1) higher Am (lower density) is effective for increasing the adhesion of BAECs at 37 °C and (2) the PIPAAm chain length of St-IPAAm molecules is the dominant factor underlying the acceleration of the cell detachment rate. After culturing BAECs to confluence at 37 °C with various Am and St-IPAAm molecules, we attempted to recover a twodimensional cell structure (cell sheet) by reducing the temperature, and the microphotographs of the resulting cell sheets are summarized in Figure 6. BAEC sheet recoveries were observed on the St-IPAAm240 and St-IPAAm480 surfaces at 40 nm2/molecule at 46 ± 11 and 26 ± 5 min, respectively. Although BAECs were cultured to confluence on St-IPAAm110 and St-IPAAm170 surfaces at 40 nm2/molecule, there was little or no detachment of the adherent BAECs in response to temperature reduction. At 10 nm2/molecule, recovery of the BAEC sheet was observed only on the St-IPAAm170 surface at 33 ± 3 min. At 3 nm2/molecule, BAECs could not be cultured to full confluence on the St-IPAAm surfaces even by 3 days of incubation at 37 °C because the adhesion of single BAECs was strongly inhibited on the surfaces (Figure 5). We speculate that the mechanisms underlying the physical properties of the St-IPAAm surfaces may be as follows. From the results of the AFM observations and XPS measurements, the surface structure could be as described in the illustration in

Figure 1. When the St-IPAAm molecules were dropped on an air−water interface, the molecules formed micelle-like structures (hydrophobic core/hydrophilic shell) during the evaporation of chloroform because PSt segments spontaneously formed as an inner core. One type of micelle-like structure in particular was sufficiently distant from the neighboring structure and thus the structures of this type exhibited less interaction with each other at high Am (>40 nm2/molecule). However, these structures began to come into contact during compression (10−40 nm2/molecule). Finally, the micelle-like structures fused into larger structures (