Fabrication of polyHEMA grids by micromolding in capillaries for cell patterning and single-cell arrays Fang Ye,1 Binghe Ma,1 Jie Gao,1 Li Xie,2 Chen Wei,1 Jin Jiang1 1
Key Laboratory of Micro/Nano Systems for Aerospace, Ministry of Education and School of Mechanical Engineering, Northwestern Polytechnical University, Xi’an 710072, People’s Republic of China 2 Key Laboratory of Space Bioscience and Biotechnology, Institute of Special Environmental Biophysics, School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, People’s Republic of China Received 10 May 2014; revised 16 August 2014; accepted 1 October 2014 Published online 12 November 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33300 Abstract: Control of cell adhesion and growth by microfabrication technology and surface chemistry is important in an increasing number of applications in biotechnology and medicine. In this study, we developed a method to fabricate (2hydroxyethyl methacrylate) (polyHEMA) grids on glass by micromolding in capillaries (MIMIC). As a non-fouling biomaterial, polyHEMA was used to inhibit the nonspecific bonding of cells, whereas the glass surface provided a cell adhesive background. The polyHEMA chemical barrier was directly obtained using MIMIC without surface modification, and the microchannel networks used for capillarity were easily achieved by reversibly bonding the polydimethylsiloxane (PDMS)mold and the glass. After fabrication of the polyHEMA micropattern, individual cytophilic microwells surrounded by cytophobic sidewalls
were presented on the glass surface. The polyHEMA micropattern proved effective in controlling the shape and spreading of cells, and square-shaped mouse osteoblast MC3T3-E1 cells were obtained in microwell arrays after incubation for 3 days. Moreover, the widths of the microwells in this micropattern were optimized for use as single-cell arrays. The proposed method could be a convenient tool in the field of drug screenC 2014 Wiley Periing, stem cell research, and tissue engineering. V odicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 1375– 1380, 2015.
Key Words: MIMIC, polyHEMA, chemical micropattern, cell patterning, single-cell array
How to cite this article: Ye F, Ma B, Gao J, Xie L, Wei C, Jiang J. 2015. Fabrication of polyHEMA grids by micromolding in capillaries for cell patterning and single-cell arrays. J Biomed Mater Res Part B 2015:103B:1375–1380.
INTRODUCTION
Cell patterning has been a useful tool for the investigation of cellular responses to various stimuli and the development of cell-based biosensors for the functional characterization and detection of drugs, pathogens, toxicants, odorants, and other chemicals.1–3 Accordingly, studies of cell behavior for understanding biological and immunological responses through the precise control of cell shape and size are highly important. In particular, regular arrays of single cells have drawn increasing attention. Many approaches have been developed for the creation of high-quality cell patterns, including photolithography, microcontacting printing (mCP), and stencil-assisted patterning. Most of these techniques are effective and are applicable to the high-throughput formation of individual cell growth regions. In general, adhesive materials, such as extracellular matrix proteins (e.g., fibronectin) are commonly used to increase cell attachment by either adsorption or covalent grafting.4,5 However, the adhesive proteins used to bind the cells to the surface might be desorbed, denatured or dissociated.6,7 Some reports have developed a possible solution to this problem by fabricating a non-fouling
micropattern on the substrate as a substitute. Revzin et al. presented a photolithography approach to fabricate poly (ethylene glycol)-diacrylate (PEG-DA) microwells for use in high-density cell arrays.8 Mandal et al. patterned antiadhesive polymer brushes based on poly(N-isopropylacrylamide) on a glass surface via deep UV photolithography for single cell studies.9 Microfluidic patterning (in particular, MIMIC) offers a much simpler approach than the above methods because it provides good control over the density of the solution of modified materials.1 Although this method has often been used to produce line-like cell patterns, our work has been conducted on isolated adhesive cells. This article describes the first use of MIMIC to achieve a polyHEMA micropattern for cell patterning and single-cell culture. A rapid approach to patterning polyHEMA for the adhesion and growth of isolated cells using MIMIC is described. A PDMS mold with recessed microstructures was necessary, and the mold was bonded to a clean coverslip to construct microchannel networks. These microchannels were quickly filled with a prepared ethanol solution containing polyHEMA using capillary force without surface treatment. Because of the inherent properties of polyHEMA, the cells preferred to adhere to
Correspondence to: B. Ma; e-mail: [email protected]
C 2014 WILEY PERIODICALS, INC. V
1375
FIGURE 1. A schematic diagram of the method used for microfluidic patterning (lFLP). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
isolated glass regions after random cell seeding. Furthermore, the use of MC3T3-E1 cells cultured on the modified substrate for single-cell arrays was investigated. MATERIALS AND METHODS
Preparation of materials Choice of anti-adhesive materials. Poly(hydroxyethyl methacrylate) is generally considered biocompatible and finds widespread use in tissue engineering for ophthalmic devices,10,11 cartilage replacements,12 and three-dimensional scaffolds.13,14 This material has a property that is even more attractive than biocompatibility for us; this material has proven effective at preventing the cell adhesion of several cell types, including NIH3T3 cells, 3Y1 cells,15 W-18 cells, and A31 cells.16 Additionally, fluorescein isothiocyanate (FITC)labeled bovine serum albumin (BSA) was used to evaluate protein adsorption on polyHEMA, and the amount of adsorbed FITC–BSA was determined from the green color intensity in the fluorescence photographs took by an inverted fluorescence microscope (TE2000). We can draw a conclusion that the adsorption of FITC–BSA protein on polyHEMA is poor from the color intensities on polyHEMA (analyzed by Image Pro Plus 6.0).Therefore, we chose polyHEMA for use as an anti-adhesive material for MC3T3-E1 cells.
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YE ET AL.
Cleaning of glass substrates. Coverslips (22 3 22 mm2) were first cleaned using a surfactant, rinsed with water and dried at 80 C in an oven. The coverslips were then soaked in potassium dichromate overnight (or at least for 6 h), washed using abundant deionized water and dried completely under nitrogen. Preparation of the polyHEMA solution. The ethanolic solution of poly (2-hydroxyethyl methacrylate) (polyHEMA, Sigma– Aldrich) was prepared before fabrication of the polymer micropattern. polyHEMA was dissolved in ethanol (70 mg mL21) and maintained at room temperature for 2 days until fully dissolved. Fabrication of the polyHEMA micropattern Silicon microstructures prepared using traditional photolithography was necessary for fabrication of the polyHEMA micropattern [Figure 1(a)]. A poly(dimethylsiloxane) (PDMS, Sylgard 184, Dow Corning, USA) mold containing complementarily recessed microstructures was obtained using prepared silicon microstructures; a 10:1 mix ratio of PDMS base and curing agent was used, and the material was cured for 1 h at 80 C [Figure 1(b)]. The height of the microstructures in the PDMS was 5 lm. Then, the PDMS mold was
FABRICATION OF polyHEMA GRIDS
ORIGINAL RESEARCH REPORT
FIGURE 2. Micrographs of the polyHEMA micropattern and the PDMS microstructure. (a) A SEM image of the PDMS microstructure showing the absence of polyHEMA residue (3200). (b) A SEM image of a poly(HEMA) grid on glass containing a high-density array of 80 3 80 lm2 microwells with 20-lm cell-resistant polyHEMA side walls (3200). (c) A SEM micrograph of the polyHEMA micropattern at higher magnification (3900).
carefully cut into 10 3 10 mm2 blocks to ensure that the microchannel networks that were formed later would have open ends. When the block was placed on coverslip for tens of seconds, the PDMS mold and the glass were reversibly bonded without surface modification. In this way, microchannel networks were formed between the PDMS mold and glass for solution feeding [Figure 1(c)]. Next, the ethanolic solution of polyHEMA was placed at the open ends of the molds and allowed to spontaneously fill the void spaces by capillary action for several minutes [Figure 1(d)]. Finally, a polyHEMA micropattern was obtained on the coverslip after complete evaporation of the ethanol (at least 12 h of evaporation at room temperature) [Figure 1(e)]. Detailed information regarding cell seeding [Figure 1(f)] and cell patterning [Figure 1(g)] will be presented in the following text. Cell culture Mouse osteoblastic MC3T3-E1 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and penicillin–streptomycin. Trypsin (0.25%) was used to detach the cells from the culture flasks, and the cells were centrifuged at 1,000 rpm for 7 min.Before cell seeding, the polyHEMA micropattern on the coverslips was sterilized using ultraviolet light for 2 h and then immersed in sterilized phosphate buffered saline (PBS) for 24 h. Next, osteoblastic cells were loaded onto the micropatterned surfaces in 12-well culture plates (Nunc) at a cell seeding density of 3 3 104 cell cm22. The cells were allowed to grow at 37 C in a humidified 5% CO2 incubator for 24 and 72 h. Immunofluorescence After incubation on micropatterned coverslips, the MC3T3E1 cells were fixed with 4% paraformaldehyde and permeabilized using 0.1% Triton X-100. Cell actin and nuclei were
stained with phalloidin-FITC (5 lg mL21, Sigma–Aldrich) and Hoechst 33258 (2 lg mL21, Sigma–Aldrich), respectively. Fluorescence photographs of stained MC3T3-E1 cells were recorded using a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). Cell area and numbers were calculated from fluorescence images of the cell actin and nuclei. Scanning electron microscopy Topological analysis was performed using a scanning electron microscope (VEGA 3 LMU, TESCAN, Czech Republic). To improve image quality, the polyHEMA micropattern on the glass was sputtered with gold (at least 10 nm) before imaging. Statistical analysis For every experiment, at least three non-overlapping pictures were taken of each region, covering about half of the entire region, and the number of cells in each microwell was counted (cell counts within each region were added). Data for each well size was averaged across multiple experiments. RESULTS AND DISCUSSIONS
Fabrication of the polyHEMA micropattern Chemically micropatterned surfaces have opened new possibilities to address fundamental questions related to cell– substrate interactions and the regulation of cell function by controlling cell shape and positioning.17 We manufactured an anti-adhesive micropattern on glass surfaces for cell patterning and single cell culture using MIMIC. In our study, a highly ordered polyHEMA micropattern was fabricated on glass using MIMIC. Several factors played important roles in the fabrication process. First, stable contact performance between the PDMS mold and flat glass
TABLE I. Geometric Parameters of the Microwells and the Walls for the Three Patterns Data Point 1 2 3
Original Design of the Silicon Microstructures (mm)
Measurements of the PDMS Mold (mm)
Measurements of the polyHEMA Micropattern (mm)
a 5 20, b 5 20 a 5 50, b 5 20 a 5 80, b 5 20
a 5 18.16, b 5 20.78 a 5 46.90, b 5 20.95 a 5 75.76, b 5 20.43
a 5 23.03, b 5 16.38 a 5 51.43, b 5 16.96 a 5 84.25, b 5 16.32
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | OCT 2015 VOL 103B, ISSUE 7
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FIGURE 3. Viability of MC3T3-E1 cells confined within the polyHEMA micropattern after 3 days of incubation. (a) Immunofluorescence image of the cell pattern on 50 3 50 lm2 microwells surrounded by 20-lm polyHEMA walls (F-actin filaments in green and nuclei in blue). (b) Spindleshaped cells in a glass culture flask. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
was required. Because the surface of PDMS has a low interfacial free energy (gPDMS/air5 21.6 dyne cm21) that provides stable adhesion with the support substrate, the PDMS surface and the glass were reversibly bonded. While the solution filled the microchannels by capillary action, the bonding strength afforded an excellent seal between the PDMS and the glass without allowing solution leakage.18,19 Second, the solution filled the microchannel network rapidly through capillary action. In general, the dominant contribution to the interfacial free energy of wetting the capillary came from the wetting of the glass surface. Organic solvents such as ethanol, which have lower surface tension, increase the filling speed and the filling distance. Accordingly, modification of the surface of the PDMS mold by plasma oxidation was unnecessary. After the polyHEMA grid patterned on the coverslip had dried, the polymer micropattern adhered more strongly to the glass than to the PDMS, and it was easy to peel the PDMS mold from the glass. To verify this conclusion, the surface of the polyHEMA micropattern and the PDMS mold were carefully examined using a scanning electron microscope. No polyHEMA residue was seen on the surface of the PDMS mold in the SEM images [Figure 2(a)], and the polyHEMA micropattern on the glass substrate was defect-free [Figure 2(b,c)].
Except for the micrographs of the polyHEMA grid, the dimensions of the silicon microstructures, PDMS microstructures, and polyHEMA microstructures were recorded in details. More attention was focused on quantifying the pattern fidelity. For cell patterning, the width of the cytophilic microwells decides the actual area available for cell growth and spreading; this size is clearly influenced by the polyHEMA walls in our study. Table I lists the geometric parameters measured using SEM. a, b represent the dimensions of the microwells and the walls on the glass surface. In Table I, the dimension loss of the wall during the twice-repeated pattern transfer can be observed. First, the PDMS mold shrinks upon curing during the microstructure transfer from silicon to PDMS.20 The first part of the dimension loss of a and b is unavoidable. Second, the mass of evaporated ethanol was responsible for the dimension loss of b during the formation of the polyHEMA micropattern. The second part of the dimension loss of b was almost constant at 4.0– 4.4 mm when the channel width is 20 mm. Therefore, we suggest that a micropattern design with large microwells and narrow side walls will contribute to further improving the pattern fidelity. This fabrication method can be used to control cell/colony shape and positioning at the microscale (1–500 mm) level.17
FIGURE 4. Comparison of cell area and cell lengths for MC3T3-E1 cells spread in microwells and in a glass culture flask after 3 days of incubation.
Cell patterning Cell patterning on desired areas is an important technique for the development of biosensors, bioelectronic devices, and tissue engineering. An orderly polyHEMA micropattern is first applied to control the shape and spreading of cells. The effectiveness of the polyHEMA grid in controlling cell positioning and spreading was investigated using MC3T3-E1 osteoblastic cells. The cells were cultured on the prepared micropattern and in a glass culture flask under equal seeding density, incubation time, and experimental environment. When the cells were loaded on the polyHEMA grid by random seeding, they preferentially attached to the glass regions within several minutes. After incubation on a chemical micropattern for 72 h, we found that isolated MC3T3-E1 cells were growing and spreading in the microwells [Figure 3(a)];
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YE ET AL.
FABRICATION OF polyHEMA GRIDS
ORIGINAL RESEARCH REPORT
FIGURE 5. MC3T3-E1 cell arrays confined within different widths of microwells (dotted line) in polyHEMA micropatterns after 24 h of incubation (F-actin filaments in green and nuclei in blue). (a) Immunofluorescence image of cell arrays in 80 3 80 lm2 microwells. (b) Immunofluorescence image of cells in 20 3 20 lm2 microwells. (c) Immunofluorescence image of single cell arrays in 50 3 50 lm2 microwells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the shapes of osteoblastic cells on the polyHEMA grid were clearly different to those of the cells in the flask [Figure 3(b)]. To evaluate these morphological differences, the cell areas and cell lengths were analyzed further (Figure 4). The area of cells cultured on the micropattern or in the flask for 3 days were both
Key Laboratory of Micro/Nano Systems for Aerospace, Ministry of Education and School of Mechanical Engineering, Northwestern Polytechnical University, Xi’an 710072, People’s Republic of China 2 Key Laboratory of Space Bioscience and Biotechnology, Institute of Special Environmental Biophysics, School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, People’s Republic of China Received 10 May 2014; revised 16 August 2014; accepted 1 October 2014 Published online 12 November 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33300 Abstract: Control of cell adhesion and growth by microfabrication technology and surface chemistry is important in an increasing number of applications in biotechnology and medicine. In this study, we developed a method to fabricate (2hydroxyethyl methacrylate) (polyHEMA) grids on glass by micromolding in capillaries (MIMIC). As a non-fouling biomaterial, polyHEMA was used to inhibit the nonspecific bonding of cells, whereas the glass surface provided a cell adhesive background. The polyHEMA chemical barrier was directly obtained using MIMIC without surface modification, and the microchannel networks used for capillarity were easily achieved by reversibly bonding the polydimethylsiloxane (PDMS)mold and the glass. After fabrication of the polyHEMA micropattern, individual cytophilic microwells surrounded by cytophobic sidewalls
were presented on the glass surface. The polyHEMA micropattern proved effective in controlling the shape and spreading of cells, and square-shaped mouse osteoblast MC3T3-E1 cells were obtained in microwell arrays after incubation for 3 days. Moreover, the widths of the microwells in this micropattern were optimized for use as single-cell arrays. The proposed method could be a convenient tool in the field of drug screenC 2014 Wiley Periing, stem cell research, and tissue engineering. V odicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 1375– 1380, 2015.
Key Words: MIMIC, polyHEMA, chemical micropattern, cell patterning, single-cell array
How to cite this article: Ye F, Ma B, Gao J, Xie L, Wei C, Jiang J. 2015. Fabrication of polyHEMA grids by micromolding in capillaries for cell patterning and single-cell arrays. J Biomed Mater Res Part B 2015:103B:1375–1380.
INTRODUCTION
Cell patterning has been a useful tool for the investigation of cellular responses to various stimuli and the development of cell-based biosensors for the functional characterization and detection of drugs, pathogens, toxicants, odorants, and other chemicals.1–3 Accordingly, studies of cell behavior for understanding biological and immunological responses through the precise control of cell shape and size are highly important. In particular, regular arrays of single cells have drawn increasing attention. Many approaches have been developed for the creation of high-quality cell patterns, including photolithography, microcontacting printing (mCP), and stencil-assisted patterning. Most of these techniques are effective and are applicable to the high-throughput formation of individual cell growth regions. In general, adhesive materials, such as extracellular matrix proteins (e.g., fibronectin) are commonly used to increase cell attachment by either adsorption or covalent grafting.4,5 However, the adhesive proteins used to bind the cells to the surface might be desorbed, denatured or dissociated.6,7 Some reports have developed a possible solution to this problem by fabricating a non-fouling
micropattern on the substrate as a substitute. Revzin et al. presented a photolithography approach to fabricate poly (ethylene glycol)-diacrylate (PEG-DA) microwells for use in high-density cell arrays.8 Mandal et al. patterned antiadhesive polymer brushes based on poly(N-isopropylacrylamide) on a glass surface via deep UV photolithography for single cell studies.9 Microfluidic patterning (in particular, MIMIC) offers a much simpler approach than the above methods because it provides good control over the density of the solution of modified materials.1 Although this method has often been used to produce line-like cell patterns, our work has been conducted on isolated adhesive cells. This article describes the first use of MIMIC to achieve a polyHEMA micropattern for cell patterning and single-cell culture. A rapid approach to patterning polyHEMA for the adhesion and growth of isolated cells using MIMIC is described. A PDMS mold with recessed microstructures was necessary, and the mold was bonded to a clean coverslip to construct microchannel networks. These microchannels were quickly filled with a prepared ethanol solution containing polyHEMA using capillary force without surface treatment. Because of the inherent properties of polyHEMA, the cells preferred to adhere to
Correspondence to: B. Ma; e-mail: [email protected]
C 2014 WILEY PERIODICALS, INC. V
1375
FIGURE 1. A schematic diagram of the method used for microfluidic patterning (lFLP). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
isolated glass regions after random cell seeding. Furthermore, the use of MC3T3-E1 cells cultured on the modified substrate for single-cell arrays was investigated. MATERIALS AND METHODS
Preparation of materials Choice of anti-adhesive materials. Poly(hydroxyethyl methacrylate) is generally considered biocompatible and finds widespread use in tissue engineering for ophthalmic devices,10,11 cartilage replacements,12 and three-dimensional scaffolds.13,14 This material has a property that is even more attractive than biocompatibility for us; this material has proven effective at preventing the cell adhesion of several cell types, including NIH3T3 cells, 3Y1 cells,15 W-18 cells, and A31 cells.16 Additionally, fluorescein isothiocyanate (FITC)labeled bovine serum albumin (BSA) was used to evaluate protein adsorption on polyHEMA, and the amount of adsorbed FITC–BSA was determined from the green color intensity in the fluorescence photographs took by an inverted fluorescence microscope (TE2000). We can draw a conclusion that the adsorption of FITC–BSA protein on polyHEMA is poor from the color intensities on polyHEMA (analyzed by Image Pro Plus 6.0).Therefore, we chose polyHEMA for use as an anti-adhesive material for MC3T3-E1 cells.
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YE ET AL.
Cleaning of glass substrates. Coverslips (22 3 22 mm2) were first cleaned using a surfactant, rinsed with water and dried at 80 C in an oven. The coverslips were then soaked in potassium dichromate overnight (or at least for 6 h), washed using abundant deionized water and dried completely under nitrogen. Preparation of the polyHEMA solution. The ethanolic solution of poly (2-hydroxyethyl methacrylate) (polyHEMA, Sigma– Aldrich) was prepared before fabrication of the polymer micropattern. polyHEMA was dissolved in ethanol (70 mg mL21) and maintained at room temperature for 2 days until fully dissolved. Fabrication of the polyHEMA micropattern Silicon microstructures prepared using traditional photolithography was necessary for fabrication of the polyHEMA micropattern [Figure 1(a)]. A poly(dimethylsiloxane) (PDMS, Sylgard 184, Dow Corning, USA) mold containing complementarily recessed microstructures was obtained using prepared silicon microstructures; a 10:1 mix ratio of PDMS base and curing agent was used, and the material was cured for 1 h at 80 C [Figure 1(b)]. The height of the microstructures in the PDMS was 5 lm. Then, the PDMS mold was
FABRICATION OF polyHEMA GRIDS
ORIGINAL RESEARCH REPORT
FIGURE 2. Micrographs of the polyHEMA micropattern and the PDMS microstructure. (a) A SEM image of the PDMS microstructure showing the absence of polyHEMA residue (3200). (b) A SEM image of a poly(HEMA) grid on glass containing a high-density array of 80 3 80 lm2 microwells with 20-lm cell-resistant polyHEMA side walls (3200). (c) A SEM micrograph of the polyHEMA micropattern at higher magnification (3900).
carefully cut into 10 3 10 mm2 blocks to ensure that the microchannel networks that were formed later would have open ends. When the block was placed on coverslip for tens of seconds, the PDMS mold and the glass were reversibly bonded without surface modification. In this way, microchannel networks were formed between the PDMS mold and glass for solution feeding [Figure 1(c)]. Next, the ethanolic solution of polyHEMA was placed at the open ends of the molds and allowed to spontaneously fill the void spaces by capillary action for several minutes [Figure 1(d)]. Finally, a polyHEMA micropattern was obtained on the coverslip after complete evaporation of the ethanol (at least 12 h of evaporation at room temperature) [Figure 1(e)]. Detailed information regarding cell seeding [Figure 1(f)] and cell patterning [Figure 1(g)] will be presented in the following text. Cell culture Mouse osteoblastic MC3T3-E1 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and penicillin–streptomycin. Trypsin (0.25%) was used to detach the cells from the culture flasks, and the cells were centrifuged at 1,000 rpm for 7 min.Before cell seeding, the polyHEMA micropattern on the coverslips was sterilized using ultraviolet light for 2 h and then immersed in sterilized phosphate buffered saline (PBS) for 24 h. Next, osteoblastic cells were loaded onto the micropatterned surfaces in 12-well culture plates (Nunc) at a cell seeding density of 3 3 104 cell cm22. The cells were allowed to grow at 37 C in a humidified 5% CO2 incubator for 24 and 72 h. Immunofluorescence After incubation on micropatterned coverslips, the MC3T3E1 cells were fixed with 4% paraformaldehyde and permeabilized using 0.1% Triton X-100. Cell actin and nuclei were
stained with phalloidin-FITC (5 lg mL21, Sigma–Aldrich) and Hoechst 33258 (2 lg mL21, Sigma–Aldrich), respectively. Fluorescence photographs of stained MC3T3-E1 cells were recorded using a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). Cell area and numbers were calculated from fluorescence images of the cell actin and nuclei. Scanning electron microscopy Topological analysis was performed using a scanning electron microscope (VEGA 3 LMU, TESCAN, Czech Republic). To improve image quality, the polyHEMA micropattern on the glass was sputtered with gold (at least 10 nm) before imaging. Statistical analysis For every experiment, at least three non-overlapping pictures were taken of each region, covering about half of the entire region, and the number of cells in each microwell was counted (cell counts within each region were added). Data for each well size was averaged across multiple experiments. RESULTS AND DISCUSSIONS
Fabrication of the polyHEMA micropattern Chemically micropatterned surfaces have opened new possibilities to address fundamental questions related to cell– substrate interactions and the regulation of cell function by controlling cell shape and positioning.17 We manufactured an anti-adhesive micropattern on glass surfaces for cell patterning and single cell culture using MIMIC. In our study, a highly ordered polyHEMA micropattern was fabricated on glass using MIMIC. Several factors played important roles in the fabrication process. First, stable contact performance between the PDMS mold and flat glass
TABLE I. Geometric Parameters of the Microwells and the Walls for the Three Patterns Data Point 1 2 3
Original Design of the Silicon Microstructures (mm)
Measurements of the PDMS Mold (mm)
Measurements of the polyHEMA Micropattern (mm)
a 5 20, b 5 20 a 5 50, b 5 20 a 5 80, b 5 20
a 5 18.16, b 5 20.78 a 5 46.90, b 5 20.95 a 5 75.76, b 5 20.43
a 5 23.03, b 5 16.38 a 5 51.43, b 5 16.96 a 5 84.25, b 5 16.32
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | OCT 2015 VOL 103B, ISSUE 7
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FIGURE 3. Viability of MC3T3-E1 cells confined within the polyHEMA micropattern after 3 days of incubation. (a) Immunofluorescence image of the cell pattern on 50 3 50 lm2 microwells surrounded by 20-lm polyHEMA walls (F-actin filaments in green and nuclei in blue). (b) Spindleshaped cells in a glass culture flask. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
was required. Because the surface of PDMS has a low interfacial free energy (gPDMS/air5 21.6 dyne cm21) that provides stable adhesion with the support substrate, the PDMS surface and the glass were reversibly bonded. While the solution filled the microchannels by capillary action, the bonding strength afforded an excellent seal between the PDMS and the glass without allowing solution leakage.18,19 Second, the solution filled the microchannel network rapidly through capillary action. In general, the dominant contribution to the interfacial free energy of wetting the capillary came from the wetting of the glass surface. Organic solvents such as ethanol, which have lower surface tension, increase the filling speed and the filling distance. Accordingly, modification of the surface of the PDMS mold by plasma oxidation was unnecessary. After the polyHEMA grid patterned on the coverslip had dried, the polymer micropattern adhered more strongly to the glass than to the PDMS, and it was easy to peel the PDMS mold from the glass. To verify this conclusion, the surface of the polyHEMA micropattern and the PDMS mold were carefully examined using a scanning electron microscope. No polyHEMA residue was seen on the surface of the PDMS mold in the SEM images [Figure 2(a)], and the polyHEMA micropattern on the glass substrate was defect-free [Figure 2(b,c)].
Except for the micrographs of the polyHEMA grid, the dimensions of the silicon microstructures, PDMS microstructures, and polyHEMA microstructures were recorded in details. More attention was focused on quantifying the pattern fidelity. For cell patterning, the width of the cytophilic microwells decides the actual area available for cell growth and spreading; this size is clearly influenced by the polyHEMA walls in our study. Table I lists the geometric parameters measured using SEM. a, b represent the dimensions of the microwells and the walls on the glass surface. In Table I, the dimension loss of the wall during the twice-repeated pattern transfer can be observed. First, the PDMS mold shrinks upon curing during the microstructure transfer from silicon to PDMS.20 The first part of the dimension loss of a and b is unavoidable. Second, the mass of evaporated ethanol was responsible for the dimension loss of b during the formation of the polyHEMA micropattern. The second part of the dimension loss of b was almost constant at 4.0– 4.4 mm when the channel width is 20 mm. Therefore, we suggest that a micropattern design with large microwells and narrow side walls will contribute to further improving the pattern fidelity. This fabrication method can be used to control cell/colony shape and positioning at the microscale (1–500 mm) level.17
FIGURE 4. Comparison of cell area and cell lengths for MC3T3-E1 cells spread in microwells and in a glass culture flask after 3 days of incubation.
Cell patterning Cell patterning on desired areas is an important technique for the development of biosensors, bioelectronic devices, and tissue engineering. An orderly polyHEMA micropattern is first applied to control the shape and spreading of cells. The effectiveness of the polyHEMA grid in controlling cell positioning and spreading was investigated using MC3T3-E1 osteoblastic cells. The cells were cultured on the prepared micropattern and in a glass culture flask under equal seeding density, incubation time, and experimental environment. When the cells were loaded on the polyHEMA grid by random seeding, they preferentially attached to the glass regions within several minutes. After incubation on a chemical micropattern for 72 h, we found that isolated MC3T3-E1 cells were growing and spreading in the microwells [Figure 3(a)];
1378
YE ET AL.
FABRICATION OF polyHEMA GRIDS
ORIGINAL RESEARCH REPORT
FIGURE 5. MC3T3-E1 cell arrays confined within different widths of microwells (dotted line) in polyHEMA micropatterns after 24 h of incubation (F-actin filaments in green and nuclei in blue). (a) Immunofluorescence image of cell arrays in 80 3 80 lm2 microwells. (b) Immunofluorescence image of cells in 20 3 20 lm2 microwells. (c) Immunofluorescence image of single cell arrays in 50 3 50 lm2 microwells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the shapes of osteoblastic cells on the polyHEMA grid were clearly different to those of the cells in the flask [Figure 3(b)]. To evaluate these morphological differences, the cell areas and cell lengths were analyzed further (Figure 4). The area of cells cultured on the micropattern or in the flask for 3 days were both
