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Terminally Functionalized Thermoresponsive Polymer Brushes for Simultaneously Promoting Cell Adhesion and Cell Sheet Harvest Hironobu Takahashi,† Naoki Matsuzaka,†,‡ Masamichi Nakayama,† Akihiko Kikuchi,‡ Masayuki Yamato,† 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 ‡ Department of Materials Science and Technology, Graduate School of Industrial Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan ABSTRACT: For preparing cell sheets effectively for cell sheet-based regenerative medicine, cell-adhesion strength to thermoresponsive cell culture surfaces need to be controlled precisely. To design new thermoresponsive surfaces via a terminal modification method, thermoresponsive polymer brush surfaces were fabricated through the surface-initiated reversible addition−fragmentation chain transfer (RAFT) radical polymerization of N-isopropylacrylamide (IPAAm) on glass substrates. The RAFT-mediated grafting method gave dithiobenzoate (DTB) groups to grafted PIPAAm termini, which can be converted to various functional groups. In this study, the terminal carboxylation of PIPAAm chains provided high cell adhesive property to thermoresponsive surfaces. Although cell adhesion is generally promoted by a decrease in the grafted PIPAAm amount, the decrease also decelerated thermally-induced cell detachment, whereas the influence of terminal modification was negligible on the cell detachment. Consequently, the terminally modified PIPAAm brush surfaces allowed smooth muscle cells (SMCs) to simultaneously adhere strongly and detach themselves rapidly. In this study, SMCs were unable to reach a confluent monolayer on as-prepared PIPAAm brush surfaces (grafted amount: 0.41 μg/cm2) without terminal carboxylation due to their insufficient cell-adhesion strength. On the other hand, though a decrease in the PIPAAm amount allowed SMCs to form a confluent cell monolayer on the PIPAAm brush surface, the SMCs were unable to be harvested as a monolithic cell sheet by low-temperature culture at 20 °C. Because of their unique property, only terminal-carboxylated PIPAAm brush surfaces achieved rapid harvesting of complete cell sheets by low-temperature culturing.

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INTRODUCTION In a wide variety of tissue engineering researches, “cell sheet engineering” has been established as one of the most effective tissue reconstruction technologies.1,2 A tissue-like cellular monolayer, called “cell sheet”, has been developed as a new tool for regenerative medicine and already applied to human clinical studies (e.g., cornea reconstruction, the treatment of esophageal ulcerations after endoscopic submucosal dissection).3−5 Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) grafted cell-culture substrates allow us to fabricate cell sheets through thermoresponsive alternations across their lower critical solution temperature (LCST) at 32 °C in an aqueous medium.6−8 Because cell sheets can be harvested intact with associated extracellular matrix (ECM) only by reducing cell culture temperature below the LCST, they are transplantable effectively to the damaged sites of tissues/organs as a regenerative medical treatment. With the progress of cell sheet technology for the various kinds of tissues/organs,9−11 thermoresponsive cell culture substrates are now required to be designed on “on-demand” for each cell type, because the various types of cells show individual cell adhesion/detachment behaviors. From this viewpoint, the several kinds of thermoresponsive surfaces previously have been prepared through various PIPAAm grafting methods including electron beam (EB)-induced graft polymerization,12,13 surface-initiated living radical polymerization,14,15 and other techniques.16 © 2011 American Chemical Society

In general, the thermoresponsive property of PIPAAmgrafted surfaces has been adjusted by changing grafted PIPAAm amount regardless of the grafting method, because PIPAAm grafting reduces their surface hydrophobicity and results in decreasing cell-adhesion strength to the surfaces.8,14,15 Therefore, PIPAAm amount is often attempted to be decreased for enhancing cell adhesion onto PIPAAm-grafted surface and obtaining a confluent cell monolayer. However, this method includes a serious “dilemma” that decrease of PIPAAm also induced poor efficiency in thermally-induced cell detachment during the cell sheet preparation process. This study focused on finding a breaking-through solution for resolving the “dilemma” via a new fabrication method for preparing thermoresponsive surfaces and demonstrated that the terminal carboxylation of PIPAAm brushes gave both the acceleration of cell adhesion and rapid harvest of cell sheets. Reversible addition−fragmentation chain transfer (RAFT) radical polymerization process as a recently developed living radical polymerization is well-known to provide polymers with precisely controlled molecular weights17,18 and has also been applied for fabricating thermoresponsive PIPAAm brushes with various well-controlled chain lengths.15 Uniquely, it also provides another advantage for polymer surface engineering. Received: November 2, 2011 Revised: November 25, 2011 Published: November 28, 2011 253

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atmosphere. After being washed with Milli-Q water, Mal- or MalC2H4COOH-terminated PIPAAm brush surfaces (Mal-PIPAAm and COOH-PIPAAm) were obtained. Mal-C3H7 was conjugated to PIPAAm termini via the same manner as described above using THF as a solvent, and Mal-C3H7-terminated PIPAAm brush surfaces (C3H7-PIPAAm) were obtained. Characterization of PIPAAm Brush Surfaces. Grafted amounts of PIPAAm on glass surfaces were determined by an attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy system (Spectrum One; Perkin-Elmer, Salem, MA) equipped with a germanium ATR crystal (Harrick Scientific Corporation, Pleasantville, NY), as reported previously.15 Briefly, the peak intensity at near 1650 cm−1 originated from the amide carbonyl group of IPAAm was normalized by the peak intensity from Si−O−Si bond of glass substrates at around 1000 cm−1, and the grafted PIPAAm amounts were determined from the intensity ratio using a calibration curve. The calibration curve was made from PIPAAm with known quantity cast on initiator-immobilized surfaces. Data are averaged from three separate samples and shown with their standard deviation. Wettability of PIPAAm brush surfaces was determined by a static contact angle measurement instrument (DSA100; KRÜ SS, Hamburg, Germany).35 Water droplets were placed onto the surfaces at 37 °C to examine the wettability using a sessile drop method. Data are averaged from three separate experiments for each surface and shown with their standard deviation. Protein Adsorption Assay. Protein adsorption on a series of PIPAAm brush surfaces was examined by coloration with anti-Fn antibody. PIPAAm brush surfaces were incubated with Fn (0.5 μg/ mL) and BSA (1 mg/mL) in PBS at 37 °C for 24 h.36 After being washed thoroughly with PBS, all samples were incubated with HRPconjugated anti-Fn antibody (a dilution of 1:2000 in PBS) for 2 h at 37 °C to detect selectively Fn adsorbed on the surfaces. Following washing with PBS, SureBlue TMB Microwell Peroxidase Substrate (400 μL) was added to the surfaces for visualizing the amounts of adsorbed Fn. After 10 min of color development, the reaction was finalized by adding equal volume of TMB Stop Solution, and then absorbance at 450 nm was measured to evaluate the relative amounts of Fn on the surfaces. Data were averaged from three separate experiments for each surface and shown with their standard deviation. Cell Adhesion and Detachment Assay. SMCs were cultured in smooth muscle cell growth medium on tissue culture polystyrene (TCPS) dishes at 37 °C, 5% CO2. PIPAAm-grafted glass substrates were cut and placed on TCPS dishes (35 mm in diameter). Cultured cells (at passage 3−7) were trypsinized and seeded onto PIPAAm brush surfaces at a density of 1 × 104 cells/cm2. After being incubated at 37 °C, 5% CO2, adherent cells were observed at 3, 6, 12, and 24 h post seeding by a phase contrast microscope (ECLIPSE TE2000-U; Nikon, Tokyo). Following a 24 h incubation, adherent cells were incubated at 20 °C, 5% CO2, and the number of spreading cells was counted at 10 min, 30 min, 1 h, and 2 h after reducing the temperature to 20 °C. For a series of PIPAAm brush surfaces, three separated experiments were carried out, and the averaged data were shown in the adhesion and detachment profiles. Cell Sheet Preparation. For cell sheet harvesting, SMCs were seeded at 5 × 104 cells/cm2 onto the four types of PIPAAm brush surfaces (as-prepared PIPAAm, Mal-PIPAAm, COOH-PIPAAm, and C3H7-PIPAAm) and as-prepared PIPAAm surfaces with decreased grafted amount (Decreased-PIPAAm), followed by incubation at 37 °C for 24 h. The adherent cells were observed microscopically, and then the cells were incubated at 20 °C to induce thermoresponsive cell detachment.15 Cell sheet harvesting was observed microscopically and visually at various time periods. Recovery periods of SMC sheets from the surfaces were determined from at least three times reproducible results.

RAFT process using dithiobenzoate (DTB)-based chain tranfer agent (CTA) gives chain-transfer active DTB groups to resultant polymer termini.19,20 These terminal DTB groups allow various functional groups to be conjugated to the termini.21−23 For few decades, on the other hand, a number of studies have investigated that terminal groups of self-assembled monolayer (SAM) influence protein adsorption and cell adhesion significantly to various biomaterial surfaces.24−32 From this viewpoint, thermoresponsive polymer surfaces were also expected to be functionalized only by the terminal modification of grafted polymer brushes prepared via RAFTmediated grafting methods. To date, grafted PIPAAm amount is required to be adjusted for control of thermoresponsive properties in conventional polymer grafting methods. As a new fabrication technique without changing PIPAAm chains themselves, the terminal modification of PIPAAm brushes is promising technique for regulating cellular behavior on thermoresponsive surfaces in the cell sheet technology.

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EXPERIMENTAL SECTION

Materials. IPAAm was kindly gifted by Kohjin (Tokyo, Japan) and purified by recrystallization from n-hexane. 3-Aminopropyltriethoxysilane (APTES), a silane coupling agent, and 1-(ethoxycarbonyl)-2ethoxy-1,2-dihydroquinoline (EEDQ), a condensing agent, were purchased from Shin-Etsu Chemical (Tokyo) and Tokyo Chemical Industries (Tokyo), respectively. 4,4′-Azobis(4-cyanovaleric acid) (V501), N-propylmaleimide (Mal-C3H7), 2-aminoethanol, sodium hydrosulfite, 1,4-dioxane, N,N-dimethylformamide (DMF), tetrahydrofuran (THF), and acetone were supplied from Wako Pure Chemical Industries (Osaka, Japan). Glass coverslips (size: 24 × 50 mm, 0.2 mm in thickness) were obtained from Matsunami Glass (Osaka). Human aortic smooth muscle cells (SMCs) and smooth muscle cell growth medium (SmGM-2) were purchased from Lonza (Walkersville, MD), and the cells were used at passage 4−8 for cell culture studies. Dulbecco’s phosphate buffered saline (PBS), bovine serum albumin (BSA), maleimide (Mal), and 3-maleimidopropionic acid (Mal-C2H4COOH) were supplied from Sigma-Aldrich (St Louis, MO). Fibronectin (Fn) and horseradish peroxidase (HRP) conjugated anti-Fn antibody were purchased from BD Biosciences (Bedford, MA) and Abcam (Cambridge, MA), respectively. A solution of substrate for colorimetric HRP-based detection, SureBlue Reserve TMB Microwell Peroxidase Substrate (1-Component), and TMB Stop Solution were obtained from KPL (Gaitherburg, MD). A chain transfer agent, 4cyanopentanoic acid dithiobenzoate, was synthesized by a previously reported procedure with a slight modification.15 Water used in this study was purified by a water purification system, Milli-Q A10 (Millipore, Billerica, MA) unless otherwise mentioned. Preparation of Thermoresponsive PIPAAm Brush Surfaces. PIPAAm was grafted on initiator-immobilized surfaces through a surface-initiated RAFT polymerization process as reported previously.15 Briefly, a carboxylated azo-initiator V-501 was immobilized on amine-modified glass substrates using a condensing agent, EEDQ. The initiator-immobilized substrates were immersed in 1,4-dioxane containing IPAAm (1.0 mol/L) and CTA (0.5, 1.0, or 2.0 mmol/L), and then the reaction solutions were heated at 70 °C for 20 h in a nitrogen atmosphere. The resultant PIPAAm-grafted glass substrates were washed with acetone thoroughly and then dried in a vacuum oven. Terminal Group Modification of Thermoresponsive Polymer Brushes. RAFT-mediated grafting method provides CTA-related DTB groups to the ω-position of grafted PIPAAm chains. The DTB groups of PIPAAm brushes were converted to various functional groups through a terminal aminolysis and subsequent coupling reactions with a variety of maleimide derivatives as reported previously.33,34 DTB-terminated PIPAAm brush surfaces were immersed in PBS solution (pH 7.4) containing Mal or MalC2H4COOH (30 mmol/L), 2-aminoethanol (10 mmol/L), and sodium hydrosulfite (1 mmol/L) for 20 h at 20 °C in a nitrogen

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RESULTS Effect of Terminal Functionalization on Cell Adhesion. In our RAFT-mediated grafting method, the amount of grafted PIPAAm can be adjusted by changing CTA 254

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Scheme 1. Synthetic Pathway for Terminal Group Conversion of Poly(N-isopropylacrylamide) (PIPAAm) Brushesa

a

Terminal dithiobenzoate (DTB) groups (red circle) on as-prepared PIPAAm brush surfaces were converted to various maleimide derivatives (green diamond; maleimide for Mal-PIPAAm, 3-maleimidopropionic acid for COOH-PIPAAm, and N-propylmaleimide for C3H7-PIPAAm).

concentration.15 As reported previously, the polymer amounts were determined to be 0.41 ± 0.02 and 0.22 ± 0.01 μg/cm2 with 0.5 and 1.0 mmol/L CTA, respectively. When prepared with 2.0 mmol/L CTA, the grafted amount was further reduced by increasing CTA and was undetectable correctly due to the detection limit. Considering results in our previous study, the grafted amount was estimated to be less than 0.17 μg/cm2 with 2.0 mmol/L CTA. In this study, as-prepared DTB-terminated PIPAAm brush surface (grafted PIPAAm: 0.41 μg/cm2) was modified terminally, as shown in Scheme 1. Terminal DTB groups of PIPAAm brushes were converted to three different maleimide derivatives (Mal, Mal-C2H5COOH, and Mal-C3H7) through an aminolysis as reported previously,33,34 and three types of terminally modified PIPAAm brush surfaces were obtained (Mal-terminated PIPAAm was “Mal-PIPAAm”; MalC2H5COOH-terminated PIPAAm, “COOH-PIPAAm”; and Mal-C3H7-terminated PIPAAm, “C3H7-PIPAAm”). Mal-PIPAAm and C3H7-PIPAAm were prepared as the controls to determine that only carboxyl group influences cell adhesion on PIPAAm brush surfaces. Although the conversion rates were unable to be examined precisely in this study, the excess maleimide derivatives probably resulted in conversion of all DTB groups to the maleimide groups on PIPAAm termini.33,34 Figure 1 shows SMC adhesion profiles on the four types of PIPAAm brush surfaces for 24 h post cell seeding. The number of adherent cells on COOH-PIPAAm surfaces was much larger than those on as-prepared PIPAAm, Mal-, and C3H7-PIPAAm surfaces. Because the adherent SMCs spread significantly only on COOH-PIPAAm surfaces (Figure 2), these results indicated that SMCs were unable to be cultured on other PIPAAm brush surfaces (grafted amount: 0.41 μg/cm2). This was due to the highly grafted PIPAAm and a resultant increased surface hydrophilicity.15 As reported previously, other cell types including bovine carotid artery endothelial cell (BAEC) and normal human dermal fibroblast (NHDF) can adhere and proliferate normally on the as-prepared PIPAAm brush surfaces.15,34,37 However, in the case of some other cell types such as SMC, grafted PIPAAm is often required to be decreased to obtain sufficient cell adhesion.8,14,15 In this study, on the other hand, terminal carboxylation enhanced cell adhesion significantly on PIPAAm brush surfaces without

Figure 1. Cell adhesion profiles on PIPAAm brush surfaces with various terminal groups (◆: initiator-immobilized surface as the control, ○: COOH-PIPAAm, △: Mal-PIPAAm, ●: C3H7-PIPAAm, □: as-prepared PIPAAm). Smooth muscle cells were seeded at a density of 1 × 104 cells/cm2 and then incubated at 37 °C for 24 h. The relative numbers of adherent cells on the respective surfaces were calculated as the numbers of cells on initiator-immobilized surface at 24 h was assumed to be 100%.

changing PIPAAm amount, resulting in high cell adhesion, spreading, and proliferation on the PIPAAm brush surfaces in spite of its highly grafted amount. Terminal Group Dependence for Protein Adsorption. Protein adsorption is often considered for designing cellrelated biomaterial surfaces, because cell adhesion is generally modulated by adsorbed proteins on surfaces. In particular, celladhesive proteins (e.g., Fn) play critical roles in cell adhesion.25,36 Figure 3 shows that the amount of adsorbed Fn onto a series of PIPAAm brush surfaces prepared in this study under the competitive adsorption condition. The amount of Fn recognized by anti-Fn antibody was obviously affected by the terminal groups of PIPAAm brushes (Figure 3A), agreeing with the result of cell adhesion study (Figures 1 and 2) and indicating that terminal carboxylation induced to raise the number of cell-binding sites of Fn adsorbed on the surface. As a result, even in the case of the highly grafted PIPAAm (0.41 μg/ cm2), the terminal carboxylation allowed SMCs to adhere significantly on PIPAAm brush surfaces. In contrast to the 255

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adsorption, no difference in surface wettability was found among the four types of PIPAAm brush surfaces (contact angles at 37 °C (cos θ): 0.28 ± 0.05 for as-prepared PIPAAm surface, 0.39 ± 0.05 for Mal-PIPAAm, 0.36 ± 0.03 for COOHPIPAAm, and 0.40 ± 0.04 for C3H7-PIPAAm). This indicates that the terminal modification induced no change in surface hydrophobicity/hydrophilicity. Due to increase of the surface hydrophilicity, PIPAAm grafting is generally known to suppress protein adsorption and cell adhesion on the surfaces. From this viewpoint, the grafted amount of PIPAAm was decreased for enhancing protein adsorption on PIPAAm-grafted surfaces (denoted as Decreased-PIPAAm). In fact, compared with initiator-immobilized surface, PIPAAm grafting induced suppression of Fn adsorption on the as-prepared PIPAAm surface (0.41 μg/cm2). However, by decreasing grafted PIPAAm, the suppression became negligible (Figure 3B). As discussed in previous studies, protein adsorption was enhanced by decreasing grafted PIPAAm amount in this study.8,14,15 Importantly, on the other hand, terminal carboxylation also increased Fn adsorption recognized by anti-Fn antibody at the same level found on Decreased-PIPAAm surface. The terminal group effects on cell adhesion and protein adsorption were observed only on COOH-PIPAAm surface in this study (Figure 1−3). Since terminal maleimide and propyl groups gave no difference between as-prepared PIPAAm surface and terminally modified PIPAAm surfaces, terminal carboxylation is necessary to obtain the unique effects. Thermally Regulated Cell Detachment from Polymer Brush Surfaces. Figure 4A indicates that (1) SMC adhesion

Figure 2. Microscopic photographs of adherent smooth muscle cells on four types of PIPAAm surfaces with various terminal groups after a 24-h incubation at 37 °C. Cells were seeded at 1 × 104 cells/cm2 onto as-prepared PIPAAm brush surface “PIPAAm”, and terminally modified PIPAAm brush surfaces “Mal-PIPAAm”, “C3H7-PIPAAm”, and “COOH-PIPAAm”. Scale bar: 100 μm.

Figure 4. Cell adhesion and detachment profiles on carboxylterminated PIPAAm brush surfaces (COOH-PIPAAm; ○), asprepared PIPAAm brush surfaces with decreased grafted amount (Decreased-PIPAAm; ■), and initiator-immobilized surface (◆). Smooth muscle cells were incubated for 24 h at 37 °C (A) and then incubated for 2 h at 20 °C (B). Adherent cells were observed microscopically, and their numbers were averaged from at least three separate procedure. The bars indicate the standard deviations of means.

Figure 3. Adsorbed amounts of fibronectin (Fn) on PIPAAm brush surfaces under competitive adsorption condition with bovine serum albumin (BSA). As-prepared PIPAAm brush surface was compared with terminally modified PIPAAm brush surfaces (Mal-terminated PIPAAm, “Mal-PIPAAm”; Mal-C2H5COOH-terminated PIPAAm, “COOH-PIPAAm”; and Mal-C3H7-terminated PIPAAm, “C3H7PIPAAm”) (A) or with unmodified PIPAAm brush surfaces with decreased grafted amount (Decreased-PIPAAm) and initiatorimmobilized surface (Initiator-surface) (B). Fn and BSA were incubated with each surface in PBS for 24 h at 37 °C, and then HRP-conjugated anti-Fn antibody was reacted specifically with adsorbed Fn on the surfaces. The intensity on as-prepared PIPAAm brush surface was normalized to be 100%. *p < 0.05.

on as-prepared PIPAAm surface was promoted by decreasing grafted PIPAAm and (2) the resultant cell adhesion profiles were nearly equal to that on COOH-PIPAAm surfaces. Because PIPAAm grafting decreases protein adsorption and cell adhesion onto surfaces,8,14,15 the results in Figure 4A agreed with previous studies and the protein adsorption study in Figure 3. The SMC adhesion profiles showed no difference on between COOH-PIPAAm surface and Decreased-PIPAAm surface. However, thermoresponsive cell detachment was decelerated only by decreasing PIPAAm amount (Figure 4B). This deceleration was probably caused by the shorter chain of PIPAAm and the higher protein adsorption on the Decreased-

result of Figure 3A, though surface water-wettability is also known to be an important parameter to modulate protein 256

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Figure 5. Microscopic photographs of adherent cells at 2 h after reducing temperature to 20 °C on initiator-immobilized surface (Initiator-surface), and PIPAAm brush surfaces with decreased grafted amount (Decreased-PIPAAm) and with terminal carboxylation (COOH-PIPAAm). Scale bar: 100 μm.

Figure 6. Microscopic photographs of smooth muscle cells on as-prepared PIPAAm brush surfaces and terminally modified PIPAAm brush surfaces. The terminal groups of PIPAAm brushes (A) were converted to (B) Mal, (C) Mal-C3H7, or (D) Mal-COOH. Cells were seeded at 5 × 104 cells/cm2 onto PIPAAm brush surfaces (A−D) with various terminal groups and (E) with decreased grafted amount. The photographs were taken after incubation at 37 °C for 24 h. Scale bar: 100 μm.

culture.15,34 However, SMCs were often observed to peel off from the surface, even at 37 °C prior to becoming confluent. This was probably due to a delicate balance between cell-to-cell interactions and cell-to-surface interactions. In fact, the adherent SMCs shown in Figure 6A−C peeled off from the surfaces before reaching a confluent monolayer even under normal cell culture condition at 37 °C. On the other hand, by decreasing grafted PIPAAm amount or terminal carboxylation of PIPAAm brushes, SMC monolayers were able to be obtained after 24-h incubation at 37 °C (Figure 6D,E). Therefore, only these two types of PIPAAm brush surfaces were used for preparing cell sheets in this study. As shown in Figure 7A, cell sheet detachment was observed on COOH-PIPAAm surface within 30 min post low-temperature treatment, whereas cell monolayer remained on Decreased-PIPAAm surface for at least 3 h-incubation at 20 °C (Figure 7B). As a result, SMC sheets were harvested completely only from COOH-PIPAAm surfaces by reducing culture temperature (Figure 7C). As described above and reported previously, insufficient PIPAAm grafting often gives no cell sheet harvest by the low-temperature treatment. Therefore, terminal functionalization without the adjustment of grafted PIPAAm amount has a potential to provide a wide variety of cell sheets such as SMC sheet.

PIPAAm surface. Previous studies also report that the decrease of grafted PIPAAm induces a slow cell detachment from various types of PIPAAm-grafted surfaces.14,15 After low-temperature treatment for 2 h, cell morphology obviously showed the difference in thermoresponsive property between on the two types of PIPAAm brush surfaces (Figure 5). In contrast to the previous method, in this study, cell adhesion was affected only by terminal functionalization without any changes in grafted PIPAAm amount. As a result, COOH-PIPAAm surface achieved rapid cell detachment without the deceleration of cell detachment, compared with Decreased-PIPAAm surface. Consequently, strong cell adhesion and rapid cell detachment were simultaneously realized by the modification of polymer termini with carboxyl groups, compared with other PIPAAm brush surfaces. Cell Sheet Harvest from Terminal-Carboxylated Polymer Brush Surface. To harvest cell sheets by temperature changes, cultured cells are required to reach confluency on thermoresponsive surfaces. After being seeded at a density of 5 × 104 cell/cm2, SMC monolayer was generally observed on bare glass surfaces. However, because PIPAAm grafting suppressed cell adhesion, SMCs adhered at less than 50% confluency on as-prepared PIPAAm, Mal-PIPAAm and C3H7PIPAAm surfaces due to their highly grafted PIPAAm (0.41 μg/cm2; Figure 6A−C). This indicated that a long culturing term was required to obtain confluent cell monolayers on these surfaces. In previous studies, for example, BAEC and NHDF reached confluency on these surfaces by long-term cell

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DISCUSSION A number of research groups have reported the effect of terminal groups on protein adsorption and cell adhesion onto SAM surfaces and concluded that carboxyl-terminated SAM 257

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adhesion.25,36 Therefore, to discuss cell adhesion on biomaterials, the adsorption of Fn is often considered as one parameter influencing directly cell adhesion on the surfaces. Furthermore, previous studies reported that terminal carboxylation on SAM surfaces influences the conformation and replacement behavior of adsorbed Fn on material surfaces, inducing high cell adhesion on the SAM surfaces. Specifically, Garcia’s group and some others have demonstrated that the terminal groups of SAM surfaces influence the orientation of cell binding domains in adsorbed Fn (e.g., RGD cell binding motifs), and particularly terminal hydroxyl and carboxyl groups on SAM surfaces certainly play an important role in cell adhesion and spreading.25,26,28 Additionally, in cell culturing process, various kinds of proteins adsorb competitively onto cell-culture substrates. Therefore, the separating ability of each serum protein in medium from surfaces is also an important for cell adhesion and spreading. From this viewpoint, the previous studies have reported replacement of preadsorbed albumin with celladhesive proteins on cell culture surfaces.24,29 In particular, terminal-carboxylated SAM exhibits higher albumin elutability, compared with other terminally modified SAM. Consequently, cell-adhesive proteins such as Fn adsorbed efficiently in exchange for serum proteins such as albumin. 24 The complicated relationships between terminal groups and cell adhesion were reported by many research groups27,36 and were also found in this study. In spite of no difference in the amount of simply adsorbed Fn (data not shown), the amount of Fn recognized by anti-Fn antibody under the competitive

Figure 7. Microscopic photographs of smooth muscle cell on terminal carboxylated PIPAAm brush surface (COOH-PIPAAm) (A) and asprepared PIPAAm brush surface with deceased grafted amount (Decreased-PIPAAm) (B) after low-temperature treatment at 20 °C within 30 min (A) and 3 h (B). (C) Photograph of a cell sheet harvested from COOH-PIPAAm surface by 30 min low-temperature treatment. Cells were seeded at a density of 5 × 104 cells/cm2, and then incubated for 24 h at 37 °C before reducing temperature. Scale bar: (A, B) 100 μm, (C) 5 mm.

allowed cells to adhere strongly on the surfaces.24−32 In the process of protein adsorption onto cell culture surfaces, celladhesive proteins (e.g., Fn) play critical roles to mediate cell

Figure 8. Schematic illustration of thermally regulated cell sheet preparation using PIPAAm brush surfaces with decreasing grafted amount and terminal functionalization. Because highly grafted PIPAAm suppresses cell adhesion, the polymer grafting induces a difficulty in preparing cell sheet (e.g., as-prepared PIPAAm surface). Whereas cell adhesion can be enhanced by decreasing grafted PIPAAm amount, the short chains of PIPAAm brushes often decelerate cell sheet harvest (e.g., Decreased-PIPAAm). In this study, terminal carboxylation of PIPAAm brushes induced both acceleration of cell adhesion and rapid cell sheet harvest (e.g., COOH-PIPAAm). 258

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the dilemma and achieving simultaneously high cell-adhesive property and rapid cell sheet detachment.

adsorption condition was larger significantly on COOHPIPAAm surface than those on other PIPAAm brush surfaces (Figure 3A). Consequently, the terminal carboxylation enhanced cell adhesion without reducing grafted PIPAAm amount. In addition, since no cell adhesion was affected on the controls, Mal-IPAAm and C3H7-PIPAAm, the presence of carboxyl groups was found to be important for simultaneously promoting cell adhesion and cell detachment. Furthermore, as described above, some other functional groups (e.g., hydroxyl group, amine group) also have a potential to promote cell adhesion.27 Therefore, terminal functionalization with some different groups is probably effective for harvesting various kinds of cell sheets. With progress in cell sheet technology, various cell types are potentially used for cell sheet-based regenerative medicine, thus PIPAAm-grafted surfaces need to be prepared according to each cell type. To date, since the decrease of grafted amount allowed cells to adhere significantly on PIPAAm-grafted surfaces, this method has been generally used for giving high cell-adhesive property to thermoresponsive polymer surfaces. However, the decreasing also induced a poor efficiency in cell sheet detachment.14,15 This study demonstrated that the terminal functionalization overcomes conclusively this “dilemma” between cell adhesiveness and cell sheet detachment in cell sheet preparation. The unique property provided by terminal carboxylation is summarized in schematic illustration (Figure 8). Some cell types require long-term incubation at 37 °C to reach confluency or are unable to be confluent on PIPAAmgrafted surfaces such as the case of SMCs with as-prepared PIPAAm brush surfaces (grafted amount: 0.41 μg/cm2) in this study. To overcome the difficulty in cell sheet preparation, the amount of grafted PIPAAm is often required to be decreased. Whereas the PIPAAm reduction allows various kinds of cells to adhere strongly and proliferate rapidly on surfaces such as the Decreased-PIPAAm surface in this study, adherent cells need long-term low-temperature treatment at 20 °C for detaching themselves from the surface or are unable to be harvested as a monolithic cell sheet, due to their high cell-to-surface interactions. The terminal carboxylation accelerated SMC adhesion remarkably without adjustment of grafted PIPAAm amount. On the other hand, SMCs were unable to adhere and proliferate significantly on the as-prepared PIPAAm brush surface. Although a small number of cells were observed to adhere and spread on the as-prepared surface, the majority of them often peeled off from the surface automatically. As well as the Decreased-PIPAAm surface, COOH-PIPAAm surface allowed SMCs to adhere and spread on the thermoresponsive surface. However, importantly, the chain length of PIPAAm was never manipulated for enhancing the cell adhesive property of the PIPAAm-grafted surface. SMCs, therefore, were able to reach confluency even on the highly grafted PIPAAm surface (grafted amount: 0.41 μg/cm2), and SMC sheets were able to be harvested rapidly from the surfaces without the poor responsibility originated from the short PIPAAm chain. Furthermore, this method also enhanced adhesion of some other cells (e.g., normal human renal proximal tubule epithelial cell) which show low adhesiveness on PIPAAm-grafted surfaces (data not shown). Only terminal carboxylated PIPAAm brush surfaces allowed them to reach a confluent monolayer, whereas they were unable to proliferate normally on other types of PIPAAm brush surfaces in this study. This surface functionalization technique is simple and effective for breaking through

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CONCLUSIONS This study reported a novel approach for improving thermoresponsive cell sheet harvest. Terminal functionalization of PIPAAm brushes has been successfully performed through terminal aminolysis and subsequent thiol-maleimide chemistry. Particularly, PIPAAm brushes modified with carboxyl groups simply showed a high cell-adhesive property. Since this terminal modification promoted cell adhesion without the decrease of grafted PIPAAm amount, PIPAAm brush surfaces showed both the acceleration of cell adhesion and the rapid detachment of cell sheets. This unique property allowed newly applicable cell sheets to be prepared from various cell types which were hardly detached and show low cell-adhesion strength. Moreover, this method was able to functionalize terminally PIPAAm brushes with various functional groups and bioactive moieties. This functionalization technique is widely useful for thermoresponsive surface and can promise to advance cell sheet technology.

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AUTHOR INFORMATION Corresponding Author *Tel.: +81-3-5367-9945 (6201). Fax: +81-3-3359-6046. E-mail: [email protected].

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ACKNOWLEDGMENTS This work was partially supported by Formation of Innovation Center for Fusin of Advanced Technologies in the Special Coordination Funds for Promoting Science and Technology “Cell Sheet Tissue Engineering Center (CSTEC)” and Grantin-Aid for Scientific Research (B; 20300169) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

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