Cell Patterning Using a Template of Microstructured Organosilane

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Cell Patterning Using a Template of Microstructured Organosilane Layer Fabricated by Vacuum Ultraviolet Light Lithography Munehiro Yamaguchi,† Koji Ikeda,† Masaaki Suzuki,*,† Ai Kiyohara,‡ Suguru N. Kudoh,‡ Kyoko Shimizu,§ Toshio Taira,§ Daisuke Ito,|| Tsutomu Uchida,|| and Kazutoshi Gohara|| †

Advanced Industrial Science and Technology (AIST), 2-17-2-1, Tsukisamu-Higashi, Toyohira-ku, Sapporo, 062-8517 Japan School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, 669-1337 Japan § Primary Cell Co., Ltd., Kita 21 Nishi 12, Kita-ku, Sapporo, 001-0021 Japan Division of Applied Physics, Faculty of Engineering, Hokkaido University, Kita 13 Nishi 8, Kita-ku, Sapporo, 060-8628 Japan

)

‡

ABSTRACT: Micropatterning techniques have become increasingly important in cellular biology. Cell patterning is achieved by various methods. Photolithography is one of the most popular methods, and several light sources (e.g., excimer lasers and mercury lamps) are used for that purpose. Vacuum ultraviolet (VUV) light that can be produced by an excimer lamp is advantageous for fabricating material patterns, since it can decompose organic materials directly and efficiently without photoresist or photosensitive materials. Despite the advantages, applications of VUV light to pattern biological materials are few. We have investigated cell patterning by using a template of a microstructured organosilane layer fabricated by VUV lithography. We first made a template of a microstructured organosilane layer by VUV lithography. Cell adhesive materials (poly(Dlysine) and polyethyleneimine) were chemically immobilized on the organosilane template, producing a cell adhesive material pattern. Primary rat cardiac and neuronal cells were successfully patterned by culturing them on the pattern substrate. Long-term culturing was attained for up to two weeks for cardiac cells and two months for cortex cells. We have discussed the reproducibility of cell patterning and made suggestions to improve it.

1. INTRODUCTION Micropatterning has become increasingly important in cellular biology, and techniques to arrange cells on a desired area are very useful for biotechnology. For instance, they are applicable to drug screening,1,2 stem cell differentiation,3 and tissue engineering.4,5 They also provide very powerful tools for fundamental study (e.g., cell-to-cell interaction). Various kinds of cells can be studied using patterning techniques. Among them, neuronal and cardiac cells have attracted particular interest, since they communicate through special chemical or electrical connections. These connections are essential to their functions, and patterning techniques can provide powerful tools for studying them. Also, control of axonal growth is an important subject for neuroscience and tissue engineering (e.g., treatment of spinal cord injury). Techniques that are easily accessible for biologists, highly reliable, and compatible with conventional biological experimental systems are desired for the application of patterning to cell biology. In addition, long-term culturing is necessary to study cell communication and network formation especially for neurons. A number of pioneering studies were conducted in the 1960s, 1970s, and 1980s. Carter employed a patterned Pd metal thin layer as the cell adhesive material for mouse fibroblasts in order to investigate cell motility.6 Letourneau investigated the growth cone elongation of sensory ganglia on grid-like patterns of Pd films formed on various surfaces.7 Hammarback et al. examined r 2011 American Chemical Society

the neurite outgrowth of dorsal root ganglia on patterned laminin formed by irradiation of the laminin layer with UV light.8 Klebe demonstrated patterns of various cell lines formed on an adhesive protein layer fabricated by using an ink jet printer.9 Hirono et al. observed the directional growth of the axons of dorsal root ganglia on grating substrates.10 Kleinfeld et al. published a particular paper on the patterning of primary neurons.11 They fabricated the patterns of several kinds of amino- and alkyl-silanes on glass or silicon substrates by UV photolithography using resist, and obtained a pattern with a minimum width of 5 μm. They also analyzed the effect of the amino silane structure and serum on cell adhesion. Thereafter, patterning studies have greatly increased. Moreover, recently proposed novel techniques 12
20 and materials 21
28 for patterning have enhanced this research field. Most methods and their applications have been introduced in recent review papers.29
36 Each method has inherent advantages and disadvantages. There is no generally applicable method to pattern all cell types. Photolithography, one of the most popular patterning techniques, is used to micropattern biological molecules37,38 and cells.39 Microcontact printing5,40
44 is often used to pattern cells, in addition Received: March 15, 2011 Revised: August 26, 2011 Published: September 07, 2011 12521

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47 The methods are useful and rather simple, once stamps or molds are obtained. However, photolithographic techniques with resists are generally necessary to fabricate stamps and molds. Photolithography is a versatile method for cell patterning. Until now, many studies have been conducted to pattern neuronal11,23,48
66 and cardiac cells.1,67,68 Several light sources were used for pattern formation; for example, conventional UV lamps with a wavelength exceeding 300 nm were used in combination with a photoresist11,48
53,67,68 or photosensitizer.69 The photoresist is generally not biocompatible, and some researchers have pointed out the necessity of handling it in a clean room.70,71 Excimer lasers (193 nm)1,23,54
66 and mercury lamps71,72 have also been used as light sources for pattern formation without photoresists. The excimer laser can ablate organic materials, but it needs higher energy for materials with low absorption. Sometimes, it is used in combination with a beam homogenizer to obtain excellent patterns.63,73 A mercury lamp can etch organic material, since it emits both 185 and 254 nm light, but the efficiency is low. An excimer lamp that can emit vacuum ultraviolet (VUV) light with a wavelength of generally less than 180 nm has some advantages for patterning organic materials. The advantages of using VUV (excimer lamp) are as follows: (1) It can directly decompose most organic materials with its high photon energy and with the existing small amount of oxygen, eliminating the need for photo resist and photosensitive materials.77 The high efficiency of the excimer lamp for micropatterning is reportedly due to the shortening of the wavelength.74 UV lights such as mercury lamp can also decompose organic materials, but the decomposition rate is extremely slow. (2) It can be used for both etching organic materials and changing surface chemical species.75 For instance, alkyl groups can be converted to carboxyl groups with VUV irradiation and this phenomenon is very useful for surface patterning. (3) The excimer lamp is not as expensive as the excimer laser, and it is almost maintenance-free. It can treat a rather large area (10 cm) and emits monochromated light with high efficiency. Monochromatic property makes us easy to conduct fundamental studies (e.g., reaction mechanism). The drawbacks are as follows: (1) We need a vacuum system, at least a simple vacuum system such as a rotary pump. However, Hong et al.76 demonstrated a sophisticated system performing irradiation at atmospheric pressure. (2) A conventional lens system is generally not suitable because transparent materials for VUV are quite limited. (3) In general, a gaseous species plays an important role in the reaction, and we need to pay special attention to it for example, the contact between the metal mask and sample. Thus, UV photolithography using VUV light is promising for patterning and microstructured material formation. It has been used to pattern organosilanes77 and to fabricate metal patterns78
83 and has been extended to fabricating a micropattern of mesoporous silica.84 However, applications of excimer lamps to pattern biological materials are very few.85,86 In the present study, we attempted to arrange cells using a microstructured organosilane layer fabricated by VUV as a template. Organosilane compounds are frequently employed for the cell adhesive layer itself or as base materials for immobilizing other cell adhesive materials. Aminosilanes have been used as the cell adhesive layer for neuronal cells,11,56 but only diethylenetriaminosilane (trimethoxysilylpropyldiethylenetriamin, DETA) seems to produce sufficiently mature neuronal cells,65 and it is preferable

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to employ other cell adhesive materials or peptide for neuronal cell patterning.51,64 Cell lines that can be passaged indefinitely and express a reasonably stable phenotype are used in many biological studies (e.g., drug screening2 and cell-material interaction87). They have also frequently been used in cell patterning experiments.3,9,12
14,20
24,26,28,39,42,45 In the field of neuroscience, many neuronal cell lines have been established and are more easily cultured than primary cells; however, almost no cell lines exhibit the well-defined neuronal properties routinely found in primary neuronal cultures.88 Hence, in the field of neuroscience, especially for research on neuronal cell interaction and network, primary cells (i.e., those taken directly from the animal) should be used. We aim to pattern primary cells for future applications. In this research, we fabricated a template of a microstructured organosilane layer by VUV lithography and, then, patternimmobilized two cell-adhesive materials by chemically modifying the template. Finally, we examined the cell patterning of primary neuronal and cardiac cells on those patterns.

2. EXPERIMENTAL DETAILS 2.1. Materials. Aminopropyltriethoxysilane (APTES) was purchased from Chisso Corporation and 2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane (PEG-silane, SIM6492.7, CH3-O-(CH2-CH2-O) n-(CH2)3-Si-(OCH3)3, n = 6
9, MW 460
590) was purchased from GELEST. Glutaric anhydride was purchased from Wako Pure Chemical Industries. N-Hydroxysuccinimide (NHS), absolute dimethylfolmamide (DMF), N,N0 -dicyclohexylcarbodiimide (DCC), poly(D-lysine) hydrobromide (PDL, P7280, MW 30 000
70 000), Polyethyleneimine (PEI, P3143, MW 750 000), Anti-MAP2 mouse IgG, and Antineurofilament 200kD rabbit IgG were purchased from Sigma-Aldrich. Fluorescein isothiocyanate (FITC) was purchased from Pierce Chemical Company. Alexa Fluor 488-labeled antimouse IgG and Alexa Fluor 546-labeled antirabbit IgG were purchased from Molecular Probes. Hoechst 33342 was purchased from Dojindo Lab. They were used without any further purification. Glass coverslips (Matsunami Glass Ind., Ltd., 22 mm j) were used as substrates. Stainless steel mask (striped pattern, width of metal part: 142 ( 3 μm, width of hole part: 90 ( 2 μm, thickness: 50 μm) was fabricated by Koken Chemical Co., Ltd. by using the photoetching method. 2.2. Apparatus. An excimer lamp (UER20
172A, Ushio) was used as a source of VUV light (172 nm). Light power density was measured using an ultraviolet radiometer (UIT-150, Ushio) equipped with a photosensor (VUV-S172, Ushio). The surface compositions of organosilane layers were analyzed by an X-ray photoelectron spectroscope (XPS) equipped with a monochromatic Al Kα ray (EscaLab220XLi, VG). A flood gun was used for analyzing electrically insulating samples (glass substrates), and binding energies were corrected by adjusting the saturated hydrocarbon (C
C) peak to 285.0 eV. The contact angle was measured by the sessile drop method (CA-A, Kyowa Interface Science Co., Ltd.). Fluorescent images were observed using a highly sensitive color CCD camera (VB6010, KEYENCE) combined with an ordinary microscope. 2.3. Preparation of Carboxyl-Terminated Silane Compound. Silane coupling agents bearing carboxyl and hydroxyl groups have not been commercialized, since these groups are incompatible with a chloro or alkoxysilyl group. Carboxyl-terminated silane compound (3-(triethoxylsilylpropyl-carbamoyl)-butyric acid, APTES-COOH) was prepared by adding 5.1 g glutaric anhydride to 20 mL APTES solution of absolute DMF (50 v/v%) with vigorous stirring. After the solution was cooled to near-room temperature, 3.5 mL diisopropylamine was added 12522

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Scheme 1. Schematic View of Fabricating Cell Adhesive Material Patterns

dropwise and stirred overnight in a nitrogen atmosphere.89 The APTESCOOH was stored in a refrigerator until use. 2.4. Formation of Patterned Organosilane Layer. Our method is briefly illustrated in Scheme 1. Substrates were cleaned with Piranha solution consisting of a 7:3 ratio of concentrated H2SO4 and 30% H2O2 aqueous solution (caution: this mixture reacts violently with organic materials and must be handled with extreme care) for 3 h at 100 °C. They were further cleaned by irradiating with 172 nm light in air for 10 min at a distance of 1 mm between substrate and VUV lamp window. They were then immersed in 1% APTES-COOH solution of absolute DMF for 15 to 24 h at room temperature. Thirty-four substrates were treated at once in a 15-cm-diameter laboratory dish. After silanization, the substrates were rinsed in DMF, then blow-dried and heat-treated at 120 °C for 30 min (Scheme 1a). The APTES-COOH layer was pattern-etched by irradiating 172 nm light through a metal mask at 75 Pa for 3 to 45 min (Scheme 1b,c). The power density ranged from 8.5 to 9.7 mW/cm2 during the experiment. The experiment setup of the etching is described elsewhere.86 The substrates were set in a vacuum chamber, and a metal mask was placed on them. We did not use quartz glass in most cases. Instead, a metal frame was placed on the mask in order to ensure tight contact between the metal mask and the substrate. In some cases, a second organosilane layer (PEG-silane) was deposited on the etched area using a solution method (Scheme 1f). We deposited a PEG-silane layer under two conditions (soft and hard). Some of the patterned substrates were immersed in 1% absolute DMF solution of PEG-silane for 1 h at room temperature (soft treatment). Others were immersed in 3% absolute toluene solution of PEG-silane with 1% triethylamine added for 24 h at 60 °C (hard treatment).

2.5. Immobilization of Cell Adhesive Materials on a Patterned Organosilane Layer. We employed two cell adhesive materials, PDL and PEI. The patterned organosilane layers were chemically modified in order to immobilize PDL and PEI. They were immersed in a solution of 0.5 g NHS and 1.0 g DCC in 10 mL anhydrous DMF for one night at room temperature. They were then rinsed three times with DMF and blow-dried. With this treatment, carboxyl groups of the APTESCOOH layer were converted to succinimide ester groups that could bind chemically to amino groups of PDL and PEI (Scheme 1d,g). Patterns of cell adhesive materials were derived by immersing the chemically modified substrates in 0.01 wt % solution of PDL in carbonate buffer (pH 8) or in 0.01 wt % solution of PEI in phosphate buffer (pH 6.4) for 1 h (Scheme 1e,h). They were then rinsed with buffer solution and MilliQ water three or more times. 2.6. Cell Culture. We examined two kinds of primary neuronal cells and one primary cardiac cell. Rat cortex cells (MB-X0304) were purchased from Sumitomo Bakelite Co., Ltd. Before culturing experiments, they were defrosted by keeping them at room temperature for 30 min. Suspensions of cortex cells were prepared by using a commercial dispersion kit (MB-X9901). They were dispersed on the patterned glass substrate and cultured using a commercial culturing medium (MBX9501, Sumitomo Bakelite Co. Ltd.) at a cell density of 250 cells/mm2. This medium contains rat normal glial conditioned medium and supplements but is serum-free. The basis of this medium is DME/F-12. After cell culturing, some of the patterned samples were fixed by adding glutaraldehyde to the culture medium (1.5%) and letting them stand overnight. They were then washed and stained immunocytochemically. The method used was basically the same as described previously.90 In brief, axons were stained red by detecting neurofilament 12523

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Figure 1. XPS spectra of the C1s region of the APTES (black) and APTES-COOH (red) layer formed on a glass surface by immersing the glass substrate in 1% APTES or APTES-COOH in absolute DMF solution for 24 h. The binding energy was corrected by adjusting the peak of the hydrocarbon chain (C
C) to 285.0 eV. 200 kD (NF200) using anti-NF200 rabbit IgG as the primary antibody and 0.4% Alexa Fluor 546-labeled antirabbit IgG as the secondary antibody. Dendrite and cell bodies were stained green by detecting microtubule associated protein 2 (MAP2) using anti-MAP2 mouse IgG as the primary antibody and 0.4% Alexa Fluor 488-labeled antirabbit IgG as the secondary antibody. Nuclei were stained blue by incubating with 2 μg/mL Hoechst 33342 for 10 min. Rat hippocampal neurons were dissected from Wister rats on embryonic day 18 (E18). They were cultured in Neurobasal medium containing 2% B27 supplement, 100 units/mL penicillin streptomycin, and 5 μg/mL insulin at a cell density of 50 cells/mm2. Rat cardiac cells harvested three days after birth were supplied by Primary Cell Co., Ltd. They were cultured in DMEM/F12 medium containing 10% bovine fetal serum, 100 units/mL penicillin, and 100 mM streptomycin. They were dispersed on the substrates at a cell density of 156 cells/mm2. All the cells were cultured at 37 °C in 5% CO2 and 95% air at saturated humidity, and half of the culture medium was renewed twice a week.

3. RESULTS AND DISCUSSION 3.1. Fabrication of Patterned Organosilane Layer. Organosilane compounds are very practical materials that can introduce functional groups on material surfaces by forming a selfassembled layer on the surface. Of these compounds, aminosilanes are frequently used in such biological applications as constructing biomedical devices.91
93 Although amino-silane compounds are crucial for such applications, several researchers have pointed out the difficulty of forming self-assembled monolayers with distinct and uniform structures, mainly due to the strong interaction between amino groups and surface silanol groups.94
96 The formation processes of amino-silane layers are very complex and sensitive to reaction conditions. We examined APTESCOOH in which the terminal group was changed from an amino group to a carboxyl group. Figure 1 plots the C1s XPS spectra of APTES and APTES-COOH layers formed on a glass surface. A peak at 288.1 eV was observed in the spectrum of APTESCOOH, and it was confirmed that carboxyl groups were introduced on the surface.

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Figure 2. Changes of the surface composition of the APTES-COOH layer with VUV irradiation as determined by XPS. The initial composition was 36.2 atom % C, 6.6 atom % N, 18.9 atom % Si, and 38.7 atom % O. black box, C; red ball, N; blue triangle, Si; green inverted triangle, O.

We checked the surface composition of the APTES-COOH layers on the glass substrate by XPS. It was very difficult to obtain the same surface compositions of the APTES-COOH layers on the glass substrate for unknown reasons. For instance, the APTES-COOH layer whose surface composition was 36.2 atom % (atomic %) carbon (C), 6.6 atom % nitrogen (N), 18.9 atom % silicon (Si), and 38.7 atom % oxygen (O) was derived by immersing the glass substrate in 1% APTES-COOH solution for 24 h at room temperature. The other APTES-COOH layer, whose surface composition was 50.6 atom % C, 8.7 atom % N, 12.4 atom % Si, and 28.3 atom % O, was obtained by immersing the substrate in 1% APTES-COOH solution for 15 h at room temperature. If the amounts of APTES-COOH on the glass substrates differ, the optimum etching times for pattern formation should differ. Therefore, we determined the optimum etching times for APTES-COOH layers with different surface compositions by XPS measurement and observation of fluorescent images of the resulting cell adhesive material patterns. We irradiated the APTES-COOH layer whose initial composition was 36.2 atom % C, 6.6 atom % N, 18.9 atom % Si, and 38.7 atom % O with VUV light at different times without a mask, and studied the change of surface composition and contact angle. Figure 2 depicts the change of surface composition of the APTES-COOH layer by VUV irradiation as determined by XPS. The C and N decreased with increasing irradiation time and became nearly zero above 180 s. Figure 3 depicts the change of contact angle against Milli-Q water by VUV irradiation time. The contact angle of the APTES-COOH surface was 21.5°. This result is in agreement with the reported values of 24°97 and 20°37 for a carboxylic surface. The contact angle drastically decreased from 21.5° to 2.9° with 30 s irradiation, and 2.9° was almost the lower limit of measurement. It remained almost unchanged with further irradiation of VUV. We also pattern-etched the APTES-COOH layer at various irradiation times and applied PEG (soft) treatment. The patterned substrates were then chemically modified and PEI or PDL were immobilized on them as described in the Experimental section. The obtained patterns were stained with FITC. Figure 4 presents examples of the fluorescent images of PEI-PEG (soft) prepared at different VUV irradiation times. At an irradiation time of 135 s, clear pattern images were observed for both the 12524

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Figure 3. Change of the contact angle with VUV irradiation.

Figure 4. Examples of the fluorescent images of cell adhesive material patterns prepared at various VUV irradiation times. PEI-PEG (soft) Photosensitivity: ISO 1600. Exposure time: 10 s. Scale bar: 200 μm. (a) 135 s, edge. Selectivity value: 1.4. Width: 156 ( 8 μm. (b) 135 s, center. Selectivity value: 1.4. Width: 149 ( 8 μm. (c) 180 s, edge. Selectivity value: 1.3. Width: 146 ( 8 μm. (d) 180 s center. Selectivity value: 1.2. Width: 145 ( 13 μm. (e) 225 s, edge. Selectivity value: 1.2. Width: 131 ( 10 μm. (f) 225 s, center. Selectivity value: 1.2. Width: 122 ( 13 μm.

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Figure 5. Changes of the surface composition of the APTES-COOH layer with VUV irradiation as determined by XPS. The initial composition was 50.6 atom % C, 8.7 atom % N, 12.4 atom % Si, and 28.3 atom % O. black box, C; red ball, N; blue triangle, Si; green inverted triangle, O.

pattern edge and the center. The selectivity ratio (a ratio of the fluorescence intensity in the unexposed and exposed regions of the image) was 1.4 for both the pattern edge and the center. The widths of PEI lane estimated by signal intensity line profile were 156 ( 8 μm for the edge and 149 ( 8 μm for the center. At 180 s, clear images were obtained, with somewhat less image contrast at the center. The selectivity ratios were 1.3 for the edge and 1.2 for the center. The widths were 146 ( 8 μm for the edge and 145 ( 13 μm for the center. At 225 s, pattern images were still observed; however, the contrast was less (selectivity ratio: 1.2) for both the edge and the center. The widths were 131 ( 10 μm for the edge and 122 ( 13 μm for the center, and a slight narrowing of lanes was observed especially at the center suggesting somewhat overetching. For PDL-PEG (soft), similar fluorescent images were observed. Even though clear images were obtained and the contact angle reached nearly zero at 135 s, we determined that the optimum etching time was 180 s in this case, because most of the C and N appeared to be removed at 180 s. In another APTES-COOH preparation experiment, an APTESCOOH layer with surface composition of 50.6 atom % C, 8.7 atom % N, 12.4 atom % Si, and 28.3 atom % O was obtained. Figure 5 indicates the change of the surface composition of the APTESCOOH layer by irradiation with VUV. The C and N content decreased with increasing irradiation time; however, 29.2 atom % C and 5.0 atom % N remained on the surface at 180 s irradiation. The C and N content decreased rapidly until 180 s, then gradually decreased and finally became nearly zero at 1800 s. The etching rate seemed to decrease with increasing irradiation time. For this layer, we determined that the optimum etching time should be 1800 s by XPS data alone, since the C and N content became nearly zero. Papra et al.98 calculated the thickness of the PEG-silane layer from XPS measurement based on Beer’s law, which states that the photoelectron current decreases exponentially with thickness. It is also assumed that the film/air interfaces are perfectly flat and that the film has a uniform density. If we apply this method to the APTES-COOH film, the thickness (t) can be calculated from the integral equation Z %C ¼ %CðAPTES-COOHÞ 0

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t

e
x=λ dx λ

ð1Þ

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Figure 6. Photographs of rat cardiac cells cultured on the PEI pattern substrates. Cell density: 156 cells/mm2. 6 DIV. Scale bar: 200 μm. (a) PEI-glass. (b) PEI-PEG (soft). (c) PEI-PEG (hard).

where %C is the C1s signal fraction in the XPS spectrum, % C(APTES-COOH) is the carbon fraction in the APTES-COOH molecule, and λ is the escape length of the C1s photoelectron in the APTES-COOH layer. Solving integral eq 1, t can be calculated by  t ¼
λ ln 1


 %C %CðAPTES-COOHÞ

ð2Þ

The escape length λ for APTES-COOH was unknown. However, if we assumed the same value of the PEG layer (3.4 nm),99,100 the thickness of this layer was 2.9 nm and that of the former APTES-COOH was 1.4 nm. The molecular length of APTESCOOH calculated by GAMESS101 was 1.26 nm (from Si to O (
OH) atom). These thicknesses corresponded to those of mono- and double layers. It is noteworthy that the thickness ratio of these films, which is 2, does not depend on the value of λ. The required etching time to eliminate most C and N from the surface was thus ten times longer when the thickness of APTES-COOH increased twice. C and N are removed by VUV light as volatile oxide species (e.g., CO and NO), but Si cannot be volatile and remained as silicon oxide on the surface.102 When the organosilane layer reached a certain thickness, it might form an inorganiclike structure as VUV irradiation proceeded. This structure is not as easily etched as the initial organic structure, resulting in a decreased etching rate. The etching rate of organosilane layer with

VUV irradiation drastically decreases, probably when the thickness of the layer exceeded monolayer. In some cases, PEG-silane was further deposited on the patterned APTES-COOH layer. PEG-silanes were less reactive than amino-silane.103 The PEG-silane surfaces that formed on the glass substrates were analyzed by XPS, and the C contents of the surfaces were 10 atom % for soft treatment and 17 atom % for hard treatment. The thicknesses estimated by applying eq 1 to these PEG layer were 0.39 nm for soft treatment and 0.43 nm for hard treatment employing the value of 3.4 nm as the escape length λ of C1s photoelectrons in PEG99,100 and assuming that the average length of PEG chain in the PEG-silane was (CH2
CH2
O)7.5. The molecular length of PEG-silane (from Si to C (CH3) atom) calculated by GAMESS101 was 2.83 (n = 6) and 3.91 nm (n = 9) and the obtained PEG layer was found to be very thin even with the hard treatment. Finally, the PDL and PEI patterns were derived as described in the experimental details both with and without PEG-silane. 3.2. Culturing of Rat Cardiac Cells. Cardiac cells were cultured on various patterned substrates except a substrate of PDL-PEG (soft). Figures 6 and 7 present the photos of cardiac cells cultured on 6 DIV (day(s) in vitro) on PEI and PDL pattern substrate. No pattern was observed, and swelled island clusters were uniformly distributed on the PEI-glass pattern substrate (Figure 6a). A pattern was observed on the PEI-PEG (soft) substrate and a clearer pattern was observed on PEI-PEG (hard) (Figure 6b,c). For PDL, patterns 12526

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Figure 7. Photographs of rat cardiac cells cultured on the PDL pattern substrates. Scale bar: 200 μm. 6 DIV. (a) PDL-glass. (b) PDL-PEG (hard).

Figure 8. Pattern formation of rat cardiac cells on the PEI and PEG-silane (hard) substrate. Scale bar: 100 μm. Cell density: 156 cells/mm2. (a) 1 DIV. (b) 2 DIV. (C) 6 DIV. (d) 8 DIV.

were observed on both PDL-glass and PDL-PEG (hard), and the effect of PEG coating was not specific (Figure 7). The effect of PEG coating was clearly observed in PEI but not in PDL. PEG effectively prevents binding of proteins and cells and it also prevents binding of PEI to the glass surface during pattern preparation. In our pattern formation (Scheme 1e), cell adhesive materials may nonspecifically bind to the glass area. Since PEI has strong nonspecific binding ability to glass, a very small amount of

PEI might remain on the glass area after rinsing, and cardiac cells might be attached to the glass area; no pattern formation was observed on the PEI-glass pattern. The PDL employed has lower molecular weight and less binding ability to glass than PEI; therefore, a cell pattern was observed on the PDL-glass substrate. During culturing, PEG-silane prevented protein and cell adhesion. Results indicated that hard treatment tended to prevent cell adhesion more than soft treatment. 12527

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Figure 9. Rat cortex cells cultured on the PDL and glass pattern. Scale bar: 200 μm. Cell density: 250 cells/mm2. (a) 4 DIV. (b) 18 DIV. (c) 25 DIV. (d) 63 DIV.

Figure 8 presents the growth of cardiac cells on the pattern substrate of PEI and PEG-silane (hard). Cells were dispersed uniformly on the substrate (0 DIV, not shown). The next day, cells were arranged and a clear pattern appeared (Figure 8a). Many cells had adhered and started to elongate in the PEI lanes and few cells had adhered in the PEG-silane lane. On 2 DIV (Figure 8b), many cells kept growing in the PEI lane and some cells exhibited spontaneous pulsing, proving that they got their original maturity. However, some cells had adhered to the PEGsilane lane. As culturing proceeded (6 DIV), swelled island cell clusters formed on the PEI lane and the PEG lane was covered by thin widely spread cells (Figure 8c). These cells were more clearly observed to grow on 8 DIV (Figure 8d). The cell clusters in the PEI lane demonstrated harmonic pulsing, whereas the widely spread thin cells in the PEG-silane lane did not. These cells were confined to a hard and flat substrate with widespread shape, and may have difficulty changing their shapes when pulsing. Alternatively, these cells maybe fibroblastic cells that were usually contained in primary cell suspension and propagate well in a serum-containing medium. To identify the cell species, further studies such as immunocytochemical staining will be necessary. On 14 DIV, many cells were observed to die and the pattern was greatly deformed; however, living cells still exhibited pulsing for a long period (∼10 s). Cardiac cells were also cultured on the PEI-coated substrate that was prepared by a similar method using organosilane. These cells demonstrated similar adhesion behavior and swelled island cell cluster formation as culturing

proceeded, and cell clusters distributed uniformly on the surface. Such a cluster formation was not observed when the cells were cultured on a fibronectin-coated glass substrate. This behavior is considered to be characteristic of PEI and PDL substrates when cardiac cells are cultured on them. In the early stage of culturing, PEG-silane seemed to effectively prevent cell adhesion; however, cells started to bind to the PEG area as culturing proceeded, probably due to the adsorption of serum proteins existing in the medium. As described in the previous section, the PEG-silane layer was very thin even in the hard condition, and refinement of the PEG coating will be necessary to improve long-term pattern durability for cardiac cells. 3.3. Culturing of Rat Cortex Cells. Rat cortex cells were cultured on various pattern substrates. For cardiac cells, a cell pattern was observed for many substrates; however, the success rate was rather low for neuronal cells. Excellent patterns were observed on PDL-glass, PEI-PEG (hard), and PEI-PEG (soft) substrates. Figure 9 depicts rat cortex cells cultured on a pattern substrate of PDL and glass. The cells were arranged according to the PDL lane. By 4 DIV, they had stretched their axons along the pattern. As the culturing proceeded, neuronal cells extended their axons and connected with each other, resulting in a clearer pattern (Figure 9b). On 25 DIV, the neuronal network continued to develop, maintaining an excellent pattern. They could be cultured more than 64 days maintaining the basic pattern line, while the pattern became narrower and some cells could be observed out of 12528

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Figure 10. Rat cortex cells in and out of pattern. 12 DIV. Scale bar: 100 μm. (a) Out of pattern. (b) In pattern.

Figure 12. Rat hippocampal cells cultured on the PEI-PEG-silane (soft) substrate. 50 cells/mm2, 13 DIV. Scale bar: 100 μm. Figure 11. Fluorescent image of the neuronal cell pattern stained axons in red, dendrite and cell body in green, and nucleus in blue. Scale bar: 200 μm.

the PDL lane. Unlike the cardiac cells, there were fewer cells on the glass lane until 25 DIV, probably because the employed culture medium basically did not contain serum that adsorbs to the glass surface and promotes cell adhesion, in addition to the fact that primary neuronal cells are generally difficult to bind to glass substrates. Figure 10 presents the neuronal cells and their axons on and outside the pattern area. The cells extended their axons randomly outside the pattern area. Inside the pattern, many axons ran along the pattern direction, suggesting that even a width of 140 μm effectively controls the outgrowth direction of neurons. In particular, axons tended to run straightforward along the pattern edge (Figure 10b). This tendency is also clearly demonstrated in Figure.11. Axons (red in the figure) clearly ran along the pattern edge. These observations seem consistent with the finding of Wheeler et al.104 that axons tend to run along the border of the cell adhesive and the nonadhesive region. 3.4. Culturing of Rat Hippocampal Neurons. Rat hippocampal cells were cultured on the substrates of PEI-PEG (hard), PEIPEG (soft), and PDL-glass substrate, and cell patterns were observed. Figure 12 depicts the representative result on the PEI-PEG (soft)

substrate on 13 DIV. The neurons were arranged on the pattern lane, and most axons ran along the pattern direction. In addition, thick axons were observed mostly at the border of the celladhesive and nonadhesive region. A few cells were observed in the nonadhesive region, and some axons bridged over the lane. This result indicates that a very thin PEG-silane layer prevented neuronal cell adhesion effectively but not completely. 3.5. Improvement of Cell Pattern Reproducibility. To improve the success rate of cell patterning, we stained pattern substrates with dilute FITC (1.25 μM, 5 min) and observed the fluorescent images and then cultured rat cortex cells on the stained substrates. We sometimes observed strange fluorescent images for pattern substrates that were prepared under the same condition but with different experiment runs (silane coating and VUV pattern etching). When the fluorescent image correctly reflected the metal mask pattern with good contrast, we obtained an excellent cell pattern (Figure 13). When the fluorescent image did not correctly reflect the metal mask pattern, the cells were arranged according to the fluorescent pattern, or they did not adhere well on the substrates. Prestaining with dilute FITC did not interrupt the adhesion and proliferation of rat cortex cells. Using such prestained substrates, we expect to improve the success rate of cell patterning. Sometimes the fluorescent images of the pattern substrates did not correctly reflect the metal mask pattern. It was assumed that a 12529

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was achieved for at least two weeks, and it was possible to maintain the pattern up to two months with the rat cortex cells. We found that formation of a definite organosilane monolayer is a prerequisite to take advantage of VUV lithography. We demonstrated that the prestaining method effectively increased the success rate, and we suggested several methods to improve the reproducibility of the cell patterning.

’ AUTHOR INFORMATION Corresponding Author

*Tel: +81-11-857-8953, Fax: +81-11-857-8900. E-mail: [email protected].

’ REFERENCES Figure 13. Rat cortex cell pattern on the prestained PDL-glass substrate on 7 DIV and the fluorescent image (inset) taken before culturing. Photosensitivity: ISO 1600. Exposure time: 30 s. Scale bar: 100 μm.

small deviation of thickness might cause a mismatch of thickness and etching time, since the etching rate was very sensitive to the organosilane layer thickness. VUV light is an effective tool for etching organosilane layers, but this advantage can only be applied for monolayer. It is a prerequisite to fabricate definite organosilane monolayers to improve the reproducibility of pattern formation. Gas-phase reaction105
107 was reported to be an excellent method for such monolayer formation and is one possible solution to the problem, while there are no reports on the formation of a carboxylterminated organosilane monolayer. Using a monoalkoxyl silane compound (e.g., 3-aminopropyldimethylethoxysilane) can also solve this problem, since it is difficult for such a compound to form a polymerized structure.106
109 Gaseous species play an important role in VUV etching.77 Firm contact between the metal mask and the sample is very important for obtaining an excellent pattern. For example, Figure 4e,f demonstrates that the pattern tended to be narrower in the center of the mask than at the edge. This was probably due to the fact that the metal frame weight did not press down as well at the center of the mask as at the edge part and there might have been very little space above the sample at the center. It is necessary to ensure firm contact between the mask and the sample. Use of a stencil mask is one possible solution to this problem.110 The resolution of this technique probably depends on the resolution of organosilane templates. Possible minimum pattern size of the metal mask is about 30
50 μm. Sugimura et al.111 and Hong et al.76 reported the fabrication of an organosilane pattern with resolution of 1
2 μm using a quartz photomask and 172 nm light. The resolution of this technique can be 1
2 μm.

4. CONCLUSIONS We fabricated APTES-COOH layers by a liquid method and patterned these layers by VUV photolithography. The optimum etching time was determined mainly by XPS measurement and depended on the layer thickness. Cell adhesive materials (PDL and PEI) were chemically immobilized on these patterned organosilane layers. Primary rat cardiac cells and cortex and hippocampal cells were successfully arranged simply by culturing them on these patterns. The results suggest that our pattern substrates are applicable to a wide variety of cells and media. Long-term culturing

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