Article pubs.acs.org/Biomac
Thermoresponsive Cationic Copolymer Brushes for Mesenchymal Stem Cell Separation 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: Thermoresponsive, cationic, copolymer brushes poly(N-isopropylacrylamide(IPAAm)-co-N,N-dimethylaminopropylacrylamide-co-N-tert-butylacrylamide(tBAAm)) and poly(IPAAm-co-3-acrylamidopropyl trimethylammonium chloride-cotBAAm) were prepared on glass substrates through surfaceinitiated atom transfer radical polymerization. Prepared copolymer brushes were investigated as thermally modulated cell separation materials. Densely packed cationic copolymer brushes were formed on the glass substrates, and the positive charge density was modulated by controlling the composition of cationic moieties and species. During observation of cell adhesion and detachment properties on copolymer brushes, human bone marrow mesenchymal stem cells (hbmMSC) exhibited thermally modulated cell adhesion and detachment, while other bone-marrowderived cells did not adhere. Using these properties, hbmMSC could be purified from mixtures of human bone-marrow-derived cells simply by changing the external temperature. Therefore, the prepared cationic copolymer brush is useful for separation of hbmMSC.
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ature.25 Various types of bioseparation systems using PIPAAm and its copolymers have been investigated, such as thermoresponsive chromatography for separating biological compounds, peptides, and pharmaceutical proteins. Thermoresponsive chromatography systems use a PIPAAm-modified chromatographic stationary phase, such as silica beads,26,27 monolithic silica,28 or polystyrene beads29 as packing materials, whose surface hydrophobicity can be modulated by external temperature change. Thus, interaction between analytes and the stationary phase can be modulated by changing the temperature, leading to separation without requiring organic solvents in the mobile phase, thereby protecting the biological activity of analytes. A variety of techniques to modify chromatography stationary phases with PIPAAm have been investigated, such as coupling reactions,26 radical polymerization,30 and surface-initiated atom transfer radical polymerization (ATRP).27 Surface-initiated ATRP is particularly effective because it can achieve the grafting of densely packed polymer brushes on the surface.31−37 In PIPAAm-modified stationary phases, these densely packed PIPAAm brushes exhibit strong interactions with analytes because of the large amounts of PIPAAm available on the surface.
INTRODUCTION Cell-based regenerative medicine through transplantation of cells into the human body has become a promising therapy for patients because it offers new therapeutic effects for patients who cannot be treated through conventional therapies that involve chemically synthesized drugs. To improve these therapies, various cell delivery technologies have been developed, such as direct injection,1 transplantation of constructed tissues using biodegradable scaffolds,2,3 and cell sheets prepared in thermoresponsive cell culture dishes.4−6 At the same time, cell separation technologies are also important for effective therapy because therapies using cell injection require purified cell suspensions. In addition, when fabricating tissues and organs using tissue engineering technologies, the cell content in each part of the structure and the approaches used for cell purification and mixing influence the function of the prepared tissues.7−9 Various cell separation methods have been developed to date.10−15 From the medical applications viewpoint, simple and safe methods are required for cell separation rather than complicated methods. To investigate a new cell separation method and meet that requirement, we focused on thermoresponsive surfaces prepared by grafting poly(N-isopropylacrylamide) (PIPAAm) and its copolymer. PIPAAm has been widely used in biomedical applications16−24 because it has the unique property that its hydrophobic/ hydrophilic characteristics depend on the external temper© XXXX American Chemical Society
Received: October 31, 2014 Revised: December 16, 2014
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DOI: 10.1021/bm501591s Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 1. Scheme showing the preparation of thermoresponsive cationic copolymer brush grafted glass surfaces through surface-initiated ATRP as thermoresponsive interfaces for cell separation.
medicine46,47 and several reports have indicated that positive charges on substrates promote adhesion of MSC.48,49 The potential of the prepared surfaces for use as cell-separating materials that separate cells simply through changing the external temperature was investigated.
In the same way, several approaches to using PIPAAm and its copolymer brushes for cell separation have been developed. B cell and T cell separation using the difference in their cell adhesion to a PIPAAm brush was performed by adjusting the length of the PIPAAm brush on glass beads packed into a cell separation column.38 Cell separation for cardiovascular tissue engineering has also been performed using the difference in cell detachment rate from a PIPAAm brush with reducing temperature.39 Furthermore, various specific temperatures for cell detachment from a P(IPAAm-co-butyl methacrylate) copolymer brush have been identified for commonly used cardiovascular cells, and this behavior is used for cell separation.40 These cell separation systems allow for cell separation without modification of cells while maintaining cells activity simply by changing temperature. Incorporation of ionic groups within polymer brushes is effective in improving cell separation efficiency because cells have specific, mainly negatively charged, electrostatic properties.41,42 If cationic charges could be introduced into a PIPAAm brush, differences in cell adhesion and detachment behaviors would be enhanced, leading to more effective separation. In this study, we prepared thermoresponsive cationic copolymer brushes by introducing two types of cationic group, the tertiary43 and quaternary amines,44,45 and varied their compositions to modulate the positive charge density in a thermoresponsive copolymer. In particular, to investigate an effective cationic brush structure for cell separation that allows for thermally modulated cell adhesion and detachment, we prepared relatively short cationic copolymer brushes with a relatively low cationic monomer composition and large hydrophobic monomer content compared with those of previous reports.43,44 Cell adhesion and detachment behaviors of human bone marrow mesenchymal stem cells (hbmMSC) and other types of human bone-marrow-derived cells were observed on the prepared copolymer brush surface because MSC are widely used in various types of regenerative
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EXPERIMENTAL SECTION
Materials. N-Isopropylacrylamide, N,N-dimethylaminopropylacrylamide (DMAPAAm), and 3-acrylamidopropyl trimethylammonium chloride (APTAC) were kindly provided by Kohjin (Tokyo, Japan). IPAAm was purified by recrystallization from n-hexane. DMAPAAm was purified by distillation. The polymerization inhibitor in APTAC was removed by passing it through an inhibitor removal column (Sigma-Aldrich, St. Louis, MO). N-tert-Butylacrylamide (tBAAm) was purchased from Tokyo Chemical Industry (Tokyo, Japan) and recrystallized from acetone. Formaldehyde, sodium hydroxide, formic acid, tris(2-aminoethyl)amine (TREN), methanol, acetone, toluene, 2propanol, CuCl, and α-chloro-p-xylene ethylenediamine-N,N,N′,N′tetraacetic acid (EDTA) were purchased from Wako Pure Chemicals (Tokyo, Japan). Tris(2-N,N-dimethylaminoethyl)amine (Me6TREN) was synthesized according to previous reports.50 Glass coverslips (24 × 50 mm, 0.2 mm in thickness) were obtained from Matsunami Glass (Osaka, Japan). ((Chloromethyl)phenylethyl)trimethoxysilane (mixed m,p isomers) was purchased from Gelest (Morrisville, PA). Tissue culture polystyrene dishes (TCPS) (Falcon 3002) were purchased from BD Bioscience (Billerica, MA). Cells and cell culture media were purchased from Takara Bio (Ohtsu, Japan). Green fluorescent protein (GFP)-expressing neonatal normal human dermal fibroblasts (GFPNHDF) were obtained from Angio-Proteomie (Boston, MA). Human bone marrow mesenchymal stem cells (hbmMSC) were obtained from RIKEN Bio Resource Center (Tsukuba, Japan) and stained green using a dye for cell tracking (Cells Tracker Green, Carlsbad, CA). Preparation of ATRP Initiator Modified Glass. ATRP initiator was immobilized on glass coverslips, as shown in Figure 1. Glass coverslips were cleaned by oxygen-plasma irradiation for 180 s (intensity: 400 W, oxygen pressure: 0.1 mmHg) in a plasma dry cleaner (PX-1000) (March Plasma Systems, Concord, CA,). The cleaned glass coverslips were placed in a separator flask, which was humidified at 60% for 2 h. ATRP initiator solution was prepared by B
DOI: 10.1021/bm501591s Biomacromolecules XXXX, XXX, XXX−XXX
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Table 1. Elemental Analyses of Thermoresponsive Cationic Copolymer Brush-Grafted Glass Surfaces by X-ray Photoelectron Spectroscopy at a Take-Off angle of 15° atom (%) code
a
initiator-modified glass IPDtB-3 IPDtB-5 IPAtB-3 IPAtB-5 IPtB-0 calcd of IPAAmb calcd of DMAPAAmb calcd of APTACb calcd of tBAAmb
monomer feed ratio (mol %) IPAAm/DMAPAAm or APTAC/tBAAm 77/3/20 75/5/20 77/3/20 75/5/20 80/0/20
C
N
O
Si
Cl
N/C ratio
25.2 68.9 67.8 71.5 70.9 65.0 75.0 72.7 75.0 77.8
0.2 10.4 9.74 9.85 9.74 9.18 12.5 18.2 16.7 11.1
49.1 14.4 15.3 13.5 13.8 17.1 12.5 9.09 8.33 11.1
25.1 5.96 7.02 4.80 5.33 8.38
0.44 0.38 0.23 0.32 0.24 0.36
0.01 0.150 0.144 0.138 0.137 0.141 0.167 0.250 0.222 0.143
a
All sample surfaces were abbreviated as IPDtB-X or IPAtB-X, where X is the feed composition of DMAPAAm or APTAC (Table 1). bTheoretical atomic composition of each monomer.
dissolving 2-(m/p-chloromethylphenyl) ethyltrimethoxysilane in toluene (46.3 mM in 700 mL) and poured in the flask. The reaction proceeded under continuous stirring for 18 h at room temperature. The initiator-modified glass coverslips were rinsed with toluene and acetone and dried in a vacuum oven at 110 °C. Thermoresponsive Copolymer Modification by ATRP. Thermoresponsive cationic copolymer brushes possessing tertiary or quaternary amine groups were grafted to the initiator-modified glass surface through ATRP, as shown in Figure 1. A typical ATRP procedure was as follows. IPAAm (29.4 g, 260 mmol), DMAPAAm (1.58 g, 10.1 mmol), and tBAAm (8.58 g, 67.5 mmol) were dissolved in 450 mL of 2-propanol. Total monomer concentration was set to 750 mM. DMAPAAm or APTAC feed composition was varied to 3 or 5 mol %. tBAAm feed composition was set at 20 mol %. The solution was deoxygenated by argon gas bubbling for 2 h. Me6TREN (765 mg, 3.3 mmol) and CuCl (295 mg, 3 mmol) were added to the monomer solution under an argon atmosphere. Initiator-modified glass coverslips were placed on a 500 mL separator flask, and the flask and monomer solution were then placed into a glovebox. Argon gas purging and vacuum were applied until the oxygen concentration in the glovebox was below 0.5%. The monomer solution was poured in the flask, and α-chloro-p-xylene (39.48 μL, 0.3 mmol) was added to the reaction solution. The ATRP reaction proceeded for 16 h under continuous stirring at 25 °C. After the reaction, the copolymer-modified glass coverslips were rinsed with acetone, methanol, EDTA solution, and water and dried in a vacuum oven for 2 h at 50 °C. The reaction solution of ATRP containing free copolymer initiated from α-chloro-pxylene was dialyzed against EDTA solution and water using a cellulose dialysis membrane (MWCO 1 kDa) with daily changes of water. The purified solution containing copolymer was lyophilized, and copolymer was obtained. Characterization of Copolymer and Copolymer-Modified Surface. Elemental analysis of copolymer-modified glass surfaces was performed by X-ray photoelectron spectroscopy (XPS) (K-Alpha, ThermoFisher, Waltham, MA). Excitation X-rays were produced from a monochromatic Al Kα1,2 source at a takeoff angle of 15°. Wide scans were recorded to analyze all existing elements on the surface, and a high-resolution narrow scan analysis was performed for the peak deconvolution of carbon C 1s signals. The amount of thermoresponsive copolymer grafted was measured by attenuated total reflection Fourier transform infrared spectroscopy (ATR/FT-IR) (Nicolet 6700, Thermo Fisher Scientific). Glass exhibited absorption from Si−O at 1000 cm−1. Absorption of the amide carbonyl derived from the copolymer appeared at 1650 cm−1. The peak intensity ratio (I1650/I1000) was used to determine the amount of copolymer on the glass substrate (Figure S1 in the Supporting Information). Calibration curves for each copolymer were prepared from a series of known amounts of copolymer cast on glass coverslips (Figure S2 in the Supporting Information).
Zeta potential of copolymer-modified glass surfaces were measured by a laser zeta electrometer (ELS-8000, Otsuka Electronics, Osaka, Japan). Prepared copolymer brush-modified glasses were placed onto the measurement cell in the electrometer; then, the copolymer brush surfaces were washed with Milli-Q water and distilled PBS (salt concentration 10 mM). The zeta potential of the copolymer brush was measured at 37 °C using polystyrene latex as a monitor particle. Number-average molecular weights (Mn) and polydispersity index (PDI) values of the copolymers were determined by a gel permeation chromatography system (GPC-8020: columns TSKgel SuperAW2500, SuperAW3000, and SuperAW4000, Tosho, Tokyo, Japan). N,NDimethylformamide containing 50 mM lithium chloride was used as the mobile phase at a flow rate of 1.0 mL/min. Column temperature was set at 45 °C, and the elution profile was monitored by refractometer. A calibration curve was obtained using polyethylene glycol standards. Graft density of the copolymer was obtained using the following equation
graft density =
mC · NA Mn
(1)
where mC is the amount of grafted copolymer on the silica bead surface (g/m2), NA is Avogadro’s number, and Mn is the numberaverage molecular weight of the grafted copolymer. Each monomer content in the copolymers was determined by 1H NMR (UNITYINOVA 400 MHz spectrometer, Varian, Palo Alto, CA) using deuterium oxide as the solvent. Phase-transition profiles of copolymers in phosphate-buffered saline (PBS) were observed using a UV−vis spectrometer (V-660, JASCO, Tokyo, Japan). The temperature-dependent optical transmittance change of copolymer PBS solution at 600 nm was observed at a heating rate of 0.10 °C/min. The lower critical solution temperature was defined at the temperature corresponding to 90% transmittance. Cell Culture and Cell Adhesion Behavior. Cell adhesion and detachment behaviors on prepared cationic copolymer brush surfaces were observed using four types of bone-marrow-derived cells: hbmMSC, human bone marrow stromal cells (hbmSC), human bone marrow mononuclear cells (hbmMNC), and human bone marrow CD34+ progenitor cells (hbmCD34+) and NHDF for comparison. NHDF was used for comparison to represent other types of adhesive cells because fibroblast cells are widely used in tissue engineering. NHDF and hbmMSC were cultured on conventional TCPS dishes with fibroblast cell medium (FGM) and Dulbecco’s modified Eagle medium (DMEM), respectively. Other bone-marrowderived cells, hbmSC, hbmMNC, and hbmCD34+, were used as received. Cell culture media and conditions are summarized in Table S1 in the Supporting Information. These cells were suspended in each medium, then seeded on the prepared copolymer brush-modified glass coverslips at 3.3 × 104 cells/mL and incubated at 37 °C in a C
DOI: 10.1021/bm501591s Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules Table 2. Characterization of Thermoresponsive Cationic Copolymer Brush-Modified Glass Substrates IPAAm/DMAPAAm or APTAC/tBAAm (molar ratio)b codea
in feed
in copolymer
IPDtB-3 IPDtB-5 IPAtB-3 IPAtB-5 IPtB-0
77/3/20 75/5/20 77/3/20 75/5/20 80/0/20
74.2/0.68/25.1 73.4/2.33/24.3 72.3/3.17/24.7 71.5/5.20/23.3 77.5/0/22.5
amount of copolymer (μg/cm2)c
Mnd
Mw/Mnd
grafted density (chains/nm2)
phase transition temperature (°C)e
± ± ± ± ±
11300 8800 8200 6500 10200
1.17 1.37 1.45 1.44 1.33
1.05 1.04 0.705 1.21 0.16
20.9 25.6 25.7 45.8 19.1
1.98 1.51 0.96 1.31 2.17
0.115 0.112 0.204 0.116 0.191
zeta potential (mV)f 11.7 19.8 16.2 18.9 −24.3
± ± ± ± ±
4.3 5.7 2.6 5.2 3.0
a
All sample surfaces were abbreviated as IPDtB-X, where X is the feed composition of DMAPAAm or APTAC (Table 1). bDetermined by 1H NMR measurement. cDetermined by ATR/FT-IR measurement. dDetermined by GPC using DMF containing 50 mmol/L LiCl. eDefined as the temperature giving 90% transmittance. fDetermined by laser zeta electrometer.
retention times and smaller estimated molecular weights.45 Thus, when calculating the graft density using eq 1, a lower molecular weight leads to relatively high graft densities. In fact, the IPtB-0 surface, with neutral copolymer brush surfaces, exhibited a relatively low graft density compared with that of cationic copolymer brushes. The phase-transition profile of the copolymer was observed using copolymer in PBS solution (Figure 2). All copolymers
humidified atmosphere of 5% CO2 for 24 h to observe cell adhesion. They were then transferred to another incubator controlled at 20 °C and again incubated for 4 h to observe cell detachment. Cell morphology was observed and photographed at predetermined times with a phase-contrast microscope (ECLIPSE TE2000-U) (Nikon, Tokyo, Japan) and a digital camera (OXM1200C) (Nikon, Tokyo). GFP-NHDF and fluorescently stained hbmMSC were observed using a fluorescence-microscope (ECLIPSE TE2000-U) and a digital camera (AxioCam HRc, Carl ZEISS, Oberkochen, Germany). Adhering cells were counted on the microphotographs in multiple areas. Percentage cell adhesion is presented as the mean and standard deviation (SD) of three measurements.
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RESULTS AND DISCUSSION Characterization of Cationic Copolymer Brush. To investigate the elemental composition of the prepared copolymer brush modified surfaces, we performed XPS measurement, and the data are summarized in Table 1. All samples were named using initial monomers and the feed composition of cationic monomers. Thus, “IP”, “D”, “A”, and “tB” denote IPAAm, DMAPAAm, APTAC, and tBAAm, respectively. After the copolymerization, the nitrogen composition attributed to the copolymer increased, indicating that copolymer was successfully grafted onto the glass surface through surface-initiated ATRP. Also, the silicon composition attributed to the basal glass decreased after copolymerization. A certain amount of silicon composition was observed on the copolymer brush-modified surfaces, even though copolymer covered the glass substrates, because the modified copolymer brush layers were thin, leading to detection of the basal glass substrate. In addition, chloride composition slightly decreased after ATRP. A previous report on acrylamide polymer brushes indicated that the terminal chloride of the polymer brush is difficult to detect by XPS because the chloride atom is buried into polymer brush.37 Also, loss of chloride would occur in the termination reaction during ATRP. In this study, these factors also decreased chloride composition of the copolymer brush surfaces compared with that of the initiator-modified surface. The characteristics of the copolymer brushes are summarized in Table 2. 1H NMR measurement of copolymers revealed that the composition of tBAAm was slightly higher than that of IPAAm and DMAPAAm, probably because of differences in reactivity of each monomer during the ATRP procedure. A larger graft density was observed than in our previous reports regarding PIPAAm or its copolymer brushes,39,40 probably because of the smaller molecular weight of the copolymer, as estimated by gel permeation chromatography (GPC). Our previous reports indicated that thermoresponsive cationic copolymers interact with the GPC column, resulting in longer
Figure 2. Phase-transition profiles of the thermoresponsive cationic copolymer in PBS. The closed squares represent IPtB-0; the open circles, IPDtB-3; the open triangles, IPDtB-5; the closed circles, IPAtB3; and the closed triangles, IPAtB-5. The abbreviations for the copolymers are defined in Table 1
exhibited sharp phase-transition profiles because they consisted entirely of acrylamide monomers. The IPAtB-5 copolymer exhibited a high transition temperature, which exceeds the cell cultivation temperature of 37 °C. Thus, the IPAtB-5 copolymer brush-modified surface was not suitable for thermally modulated cell adhesion and detachment. The observed zeta potential of the copolymer brushes correlated to the cationic monomer composition and basicity of the monomers (DMAPAAm < APTAC).45 Cell Adhesion and Detachment on Copolymer Brushes. Cell adhesion and detachment behaviors on the prepared copolymer brush were observed using hbmMSC and NHDF because hbmMSC are widely used as a cell source in regenerative medicine. Also, the commercially available cell culture dishes, with cationic charges, exhibited strong hbmMSC adhesion. NHDF was used for comparison to represent other types of adhesive cells because fibroblast cells are widely used in tissue engineering. Figures 3−6 show the adhesion/detachment D
DOI: 10.1021/bm501591s Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 3. Adhesion and detachment profiles on the IPtB-0 copolymer brush for hbmMSCs (○) and NHDFs (▲) (A) and cell morphology at 37 and 20 °C, observed after 24 and 4 h incubations, respectively (B). Copolymer brush codes are defined in Table 1
Figure 4. Adhesion and detachment profiles on the IPDtB-3 copolymer brush for hbmMSCs (○) and NHDFs (▲) (A) and cell morphology at 37 and 20 °C, observed after 24 and 4 h incubations, respectively (B). Copolymer brush codes are defined in Table 1
Figure 5. Adhesion and detachment profiles on the IPDtB-5 copolymer brush for hbmMSCs (○) and NHDFs (▲) (A) and cell morphology at 37 and 20 °C, observed after 24 and 4 h incubations, respectively (B). Copolymer brush codes are defined in Table 1
profiles and cells morphologies on prepared copolymer brushes for hbmMSC and NHDF. On noncationic copolymer brush IPtB-0 (Figure 3), hbmMSC and NHDF tended to adhere on the copolymer brush because of the relatively strong hydrophobicity of the copolymer. However, at reduced temperature, these cells did not effectively detach from the copolymer brush,
a behavior that we attribute to the excessive hydrophobicity, and this result indicates that the cell recovery rate from the copolymer brush surface is not sufficient for cell separation applications. Also, relatively similar adhesion rates between NHDF and hbmMSC were observed, indicating that separation of these cells using difference in adhesion and detachment rate E
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Figure 6. Adhesion and detachment profiles on the IPAtB-3 copolymer brush for hbmMSCs (○) and NHDFs (▲) (A) and cell morphology at 37 and 20 °C, observed after 24 and 4 h incubations, respectively (B). Copolymer brush codes are defined in Table 1
Figure 7. hbmMSCs and GFP-NHDFs adhesion and detachment profiles on the IPDtB-3 copolymer brush in the same culture medium (A) and cell morphology at 37 and 20 °C, observed after 12, 24, and 4 h incubations, respectively (B). Copolymer brush codes are defined in Table 1
approximately half of the adhered cells were detached even at 37 °C. Also, cell morphologies on IPAtB-3 showed that cells were not effectively spread on the surface for adhesion because the strong cationic copolymer brush IPAtB-3 was relatively hydrophilic compared with the weak cationic copolymer brushes IPDtB-3 or IPDtB-5.45 To investigate the mechanism behinds the differences in cell adhesion properties for hbmMSC and NHDFs on cationic copolymer brushes, we characterized the cell properties such as zeta potential and the amount of the sialic acid present (Figure S3 in the Supporting Information). However, there are no significant differences between these cell types. We therefore believe that other factors influence the differences in adhesion behavior, such as differences in extracellular matrix or cell membrane proteins. Using the difference in cell adhesive properties on IPDtB-3 brush surfaces, a mixture of hbmMSC and NHDF cells was separated (Figure 7). To distinguish each cell type, we used GFP-NHDF. A mixture of these cells was seeded and incubated using the cell culture medium for hbmMSC. After incubation for 24 h at 37 °C, almost all hbmMSC were adhered to the copolymer brush, while only 40% of NHDF adhered. After decreasing the temperature, almost all cells detached and were recovered from the surfaces. The results indicate that hbmMSC
would be difficult. On IPDtB-3 (Figure 4), hbmMSC adhered effectively at 37 °C and effectively detached at 20 °C. On the contrary, NHDF did not adhere extensively at 37 °C. This is probably attributed to the enhanced adhesion of hbmMSC on the cationic copolymer brush. Thus, the difference in detachment properties between hbmMSC and NHDF should be useful for cell separation. In addition, almost all adhered cells on copolymer brushes were detached from the cationic copolymer brush. At low temperature, cationic copolymer brushes hydrate and become hydrophilic, leading to reduction of cell adhesion. Although a certain level of electrostatic interaction between the cell surface and copolymer brush remains, the increased hydrophilic properties of the brush dominate cell detachment. IPDtB-5 also exhibited enhanced hbmMSC adhesion (Figure 5). However, NHDF adhesion was also enhanced on the cationic copolymer brush, and the difference in the adhesion ratio between hbmMSC and NHDF was smaller than that for IPDtB-3. Also, similar detachment profiles were observed between these cells. Thus, the IPDtB-5 copolymer brush surface is not suitable for separation of these cells. On IPAtB-3, hbmMSC were relatively well adhered on the copolymer brush surfaces during the initial incubation period until ∼12 h. However, with incubation for 24 h, F
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Figure 8. Adhesion and detachment profiles of human bone marrow-derived cells on (A) IPtB-0, (B) IPDtB-3, and (C) IPDtB-5 copolymer brushes. Copolymer brush codes are defined in Table 1
Figure 9. Adhesion and detachment profiles of all human bone-marrow-derived cells on IPDtB-5 copolymer brushes (A) and cell morphologies at 37 and 20 °C, observed after 24 and 4 h incubations, respectively (B). Copolymer brush codes are defined in Table 1
can be purified from other types of adhering cells by incubation at 37 °C for 24 h and then reducing the temperature to 20 °C. In addition, to ensure that the recovered hbmMSC were not differentiated, hbmMSC recovered from IPDtB-3 were stained with the undifferentiated marker STRO-1. STRO-1 was indeed observed, indicating that the recovered hbmMSC retained differentiation potency (Figure S4 in the Supporting Information). We also investigated the cell adhesion behaviors of other human bone-marrow-derived cells because the difference in cell adhesive properties on the cationic copolymer brush was expected to be useful for purification of hbmMSC from other bone-marrow-derived cells. Figure 8 shows that cell adhesion and detachment behaviors of human bone-marrow-derived cells, while the cell morphologies on the cationic copolymer brushes are shown in Figure S5 in the Supporting Information. Human bone-marrow-derived cells, hbmSC, hbmCD34+, and hbmMNC, scarcely adhered on the cationic copolymer brush, while hbmMSC was adhered at 37 °C and detached at 20 °C. These results indicate that hbmMSC can be purified from other human bone-marrow-derived cells. Therefore, a mixture of human bone marrow-derived cells was seeded onto IPDtB-5. For distinguishing hbmMSC from other types of hbm-derived cells, hbmMSCs were stained green using a dye for cell tracking
(Cells Tracker Green). Figure 9 shows the adhesion and detachment profile for hbmMSC and other hbm-derived cells. hbmMSC adhered on IPDtB-5 at 37 °C, while other types of cells scarcely adhered. After reducing the temperature to 20 °C, hbmMSC detached from the copolymer brush. This result demonstrates that hbmMSC can be purified from human bone marrow cells using the cationic copolymer brush simply by changing temperature. Thermoresponsive copolymer brushes possessing cationic moieties are effective for thermally modulated hbmMSC adhesion and detachment, with a certain level of specificity, attributed to their cationic properties and moderate hydrophilicity. Also, recovered cells from copolymer brush would maintain their function and activities.40,51 Therefore, these copolymer brushes are expected to be useful for purification of hbmMSC from bone marrow or separation of undifferentiated hbmMSC and differentiated cells.
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CONCLUSIONS
Thermoresponsive cationic copolymer brushes, P(IPAAm-coDMAPAAm-co-tBAAm) and P(IPAAm-co-APTAC-co-tBAAm), were prepared through surface-initiated ATRP on glass surfaces, and the prepared brushes were investigated as cell separation materials. Characterization of the brushes indicated G
DOI: 10.1021/bm501591s Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
(8) Matsuura, K.; Masuda, S.; Haraguchi, Y.; Yasuda, N.; Shimizu, T.; Hagiwara, N.; Zandstra, P. W.; Okano, T. Biomaterials 2011, 32, 7355−7362. (9) Sekine, H.; Shimizu, T.; Hobo, K.; Sekiya, S.; Yang, J.; Yamato, M.; Kurosawa, H.; Kobayashi, E.; Okano, T. Circulation 2008, 118, S145−S152. (10) Giddings, J. C.; Barman Bhajendra, N.; Liu, M.-K. Separation of Cells by Field-Flow Fractionation. In Cell Separation Science and Technology; American Chemical Society: Washington, DC, 1991; Vol. 464, pp 128−144. (11) Chianéa, T.; Assidjo, N. E.; Cardot, P. J. P. Talanta 2000, 51, 835−847. (12) Kataoka, K. High-Capacity Cell Separation by Affinity Selection on Synthetic Solid-Phase Matrices. In Cell Separation Science and Technology; American Chemical Society: Washington, DC, 1991; Vol. 464, pp 159−174. (13) Kataoka, K.; Sakurai, Y.; Hanai, T.; Maruyama, A.; Tsuruta, T. Biomaterials 1988, 9, 218−224. (14) Moldavan, A. Science 1934, 80, 188−189. (15) Leary James, F.; Ellis Steven, P.; McLaughlin Scott, R.; Corio Mark, A.; Hespelt, S.; Gram Janet, G.; Burde, S. High-Resolution Separation of Rare Cell Types. In Cell Separation Science and Technology; American Chemical Society: Washington, DC, 1991; Vol. 464, pp 26−40. (16) Hoffman, A. S. Adv. Drug Delivery Rev. 2002, 54, 3−12. (17) Kikuchi, A.; Okano, T. Prog. Polym. Sci. 2002, 27, 1165−1193. (18) Gil, E. S.; Hudson, S. M. Prog. Polym. Sci. 2004, 29, 1173−1222. (19) Kikuchi, A.; Okano, T. J. Controlled Release 2005, 101, 69−84. (20) Ulbricht, M. Polymer 2006, 47, 2217−2262. (21) Hoffman, A. S.; Stayton, P. S. Prog. Polym. Sci. 2007, 32, 922− 932. (22) Mano, J. F. Adv. Eng. Mater. 2008, 10, 515−527. (23) Mori, T.; Maeda, M. Langmuir 2003, 20, 313−319. (24) Kim, Y.-J.; Ebara, M.; Aoyagi, T. Angew. Chem., Int. Ed. 2012, 51, 10537−10541. (25) Heskins, M.; Guillet, J. E. J. Macromol. Sci. A 1968, 2, 1441− 1455. (26) Kanazawa, H.; Yamamoto, K.; Matsushima, Y.; Takai, N.; Kikuchi, A.; Sakurai, Y.; Okano, T. Anal. Chem. 1996, 68, 100−105. (27) Nagase, K.; Kobayashi, J.; Kikuchi, A.; Akiyama, Y.; Kanazawa, H.; Okano, T. Langmuir 2007, 23, 9409−9415. (28) Nagase, K.; Kobayashi, J.; Kikuchi, A.; Akiyama, Y.; Kanazawa, H.; Okano, T. Langmuir 2011, 27, 10830−10839. (29) Mizutani, A.; Nagase, K.; Kikuchi, A.; Kanazawa, H.; Akiyama, Y.; Kobayashi, J.; Annaka, M.; Okano, T. J. Chromatogr. A 2010, 1217, 522−529. (30) Yakushiji, T.; Sakai, K.; Kikuchi, A.; Aoyagi, T.; Sakurai, Y.; Okano, T. Anal. Chem. 1999, 71, 1125−1130. (31) Matyjaszewski, K.; Xia, J. Chem. Rev. 2001, 101, 2921−2990. (32) Edmondson, S.; Osborne, V. L.; Huck, W. T. S. Chem. Soc. Rev. 2004, 33, 14−22. (33) Balamurugan, S.; Mendez, S.; Balamurugan, S. S.; O’Brien, M. J., II; López, G. P. Langmuir 2003, 19, 2545−2549. (34) Tu, H.; Heitzman, C. E.; Braun, P. V. Langmuir 2004, 20, 8313−8320. (35) Iwata, R.; Suk-In, P.; Hoven, V. P.; Takahara, A.; Akiyoshi, K.; Iwasaki, Y. Biomacromolecules 2004, 5, 2308−2314. (36) Yoshikawa, C.; Goto, A.; Tsujii, Y.; Fukuda, T.; Kimura, T.; Yamamoto, K.; Kishida, A. Macromolecules 2006, 39, 2284−2290. (37) Xiao, D.; Wirth, M. J. Macromolecules 2002, 35, 2919−2925. (38) Nagase, K.; Mukae, N.; Kikuchi, A.; Okano, T. Macromol. Biosci. 2012, 12, 333−340. (39) Nagase, K.; Kimura, A.; Shimizu, T.; Matsuura, K.; Yamato, M.; Takeda, N.; Okano, T. J. Mater. Chem. 2012, 22, 19514−19522. (40) Nagase, K.; Hatakeyama, Y.; Shimizu, T.; Matsuura, K.; Yamato, M.; Takeda, N.; Okano, T. Biomacromolecules 2013, 14, 3423−3433. (41) Makino, K.; Fukai, F.; Kawaguchi, T.; Ohshima, H. Colloids Surf., B 1995, 5, 221−226.
that a densely packed copolymer brush was grafted onto the glass surfaces, and their positive charge density was modulated by changing the cationic monomer composition and species. NHDF and hbmMSC were seeded and cell adhesion and detachment behaviors were observed to investigate cell adhesive properties. hbmMSC exhibited strong adhesive properties on cationic copolymer brush compared with NHDF, and adhered hbmMSC were detached from cationic copolymer brushes at reduced temperature. For the separation of hbmMSC from other bone marrow-derived cells, a mixture of human bone marrow cells comprising hbmMSC, hbmSC, hbmCD34+, and hbmMNC was seeded onto the cationic copolymer brush. Only hbmMSC adhered to the brush at 37 °C, while other cells scarcely adhered and hbmMSC was detached from the brush after reducing the temperature to 20 °C. These results indicate that the prepared cationic copolymer brush will be useful for purification of hbmMSC from bone marrow and separation of hbmMSC and other somatic cells.
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ASSOCIATED CONTENT
S Supporting Information *
FTIR spectra, the standard curves used to estimate the amount of grafted copolymer, cell culture conditions, zeta potential of cells, sialic acid amount of cells, image of STRO-1 stained MSC, and cell morphologies of human bone-marrow-derived cells on copolymer brushes. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*K.N.: Tel: +81-3-5367-9945, ext. 6201. Fax: +81-3-3359-6046. E-mail:
[email protected]. *T.O.: E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Part of the present research was financially supported by “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), Japan. Grants-inAid for Scientific Research (C) No. 26420714 from the Japan Society for the Promotion of Science (JSPS), Japan. We thank Ms. Ayaka Kimura for valuable suggestions and discussion.
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REFERENCES
(1) Menasché, P.; Hagège, A. A.; Scorsin, M.; Pouzet, B.; Desnos, M.; Duboc, D.; Schwartz, K.; Vilquin, J.-T.; Marolleau, J.-P. Lancet 2001, 357, 279−280. (2) Langer, R.; Vacanti, J. Science 1993, 260, 920−926. (3) Shinoka, T.; Breuer, C. Yale J. Biol. Med. 2008, 81, 161−166. (4) Nishida, K.; Yamato, M.; Hayashida, Y.; Watanabe, K.; Yamamoto, K.; Adachi, E.; Nagai, S.; Kikuchi, A.; Maeda, N.; Watanabe, H.; Okano, T.; Tano, Y. N. Engl. J. Med. 2004, 351, 1187−1196. (5) Sawa, Y.; Miyagawa, S.; Sakaguchi, T.; Fujita, T.; Matsuyama, A.; Saito, A.; Shimizu, T.; Okano, T. Surg. Today 2012, 42, 181−184. (6) Ohki, T.; Yamato, M.; Ota, M.; Takagi, R.; Murakami, D.; Kondo, M.; Sasaki, R.; Namiki, H.; Okano, T.; Yamamoto, M. Gastroenterology 2012, 143, 582−588.e2. (7) Sekiya, S.; Shimizu, T.; Yamato, M.; Kikuchi, A.; Okano, T. Biochem. Biophys. Res. Commun. 2006, 341, 573−582. H
DOI: 10.1021/bm501591s Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules (42) Makino, K.; Fukai, F.; Hirata, S.; Ohshima, H. Colloids Surf., B 1996, 7, 235−238. (43) Nagase, K.; Kobayashi, J.; Kikuchi, A.; Akiyama, Y.; Kanazawa, H.; Okano, T. Biomaterials 2011, 32, 619−627. (44) Tamura, A.; Nishi, M.; Kobayashi, J.; Nagase, K.; Yajima, H.; Yamato, M.; Okano, T. Biomacromolecules 2012, 13, 1765−1773. (45) Nagase, K.; Geven, M.; Kimura, S.; Kobayashi, J.; Kikuchi, A.; Akiyama, Y.; Grijpma, D. W.; Kanazawa, H.; Okano, T. Biomacromolecules 2014, 15, 1031−1043. (46) Wei, X.; Yang, X.; Han, Z.-p.; Qu, F.-f.; Shao, L.; Shi, Y.-f. Acta Pharmacol. Sin. 2013, 34, 747−754. (47) Salem, H. K.; Thiemermann, C. Stem Cells 2010, 28, 585−596. (48) Lu, H.; Guo, L.; Kawazoe, N.; Tateishi, T.; Chen, G. J. Biomater. Sci., Polym. Ed. 2009, 20, 577−589. (49) Semenov, O. V.; Malek, A.; Bittermann, A. G.; Vörös, J.; Zisch, A. H. Tissue Eng., Part A 2009, 15, 2977−2990. (50) Ciampolini, M.; Nardi, N. Inorg. Chem. 1966, 5, 41−44. (51) Kushida, A.; Yamato, M.; Konno, C.; Kikuchi, A.; Sakurai, Y.; Okano, T. J. Biomed. Mater. Res. 1999, 45, 355−362.
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DOI: 10.1021/bm501591s Biomacromolecules XXXX, XXX, XXX−XXX
