HeLa Cell Adhesion on Various Collagen-Grafted Surfaces Yi Zhu,† Zhonghan Wu,‡ Zuming Tang,† and Zuhong Lu*,† National Laboratory of Molecular and Biomolecular Electronics, Southeast University, Nanjing 210096, China, and Department of Physics, Southeast University, Nanjing 210096, China Received May 31, 2002
Abstract: Cell adhesion is important to develop cell microarrays and biocompatible materials. Collagen has been reported to be able to improve cell adhesion. In this paper, two collagen coating methods (collagen grafted directly on the substrate and chitosan-modified substrate) were carried out, on which the adhesive behaviors of HeLa cells were studied. An atomic force microscope and a surface potential meter were used to characterize morphologies and electric polarization of these surfaces. It was found that surface electric polarization and the its durability and surface topography were key factors to cell adhesion. Collagen (1 mg/mL) grafted on 1% chitosan-modified surface showed the best adhesion of HeLa cell. This work might be helpful to the practical application of cell microarray chips. Keywords: HeLa cell • surface modification • cell adhesion • collagen
1. Introduction Cell microarrays, containing a number of different living cells on a single substrate, have been attracted much attention in the field of high-throughput drug screening and genomic and proteomic research.1-3 But there are still many difficulties in keeping the concentration, viability, and natural physiological state of cells on the substrate. In vitro cell adhesion is one of the essential factors for cell growth and recovery. Improving cell adhesion by surface modification has been studied in the field of developing biocompatible materials. The methods of surface modification can be classified into biochemical and physical routes. As to the former, specific peptides or proteins were immobilized on the material’s surface to build up the interaction between cell receptors and immobilized biomolecules.4-7 As to the latter, it was to adjust physical and chemical properties of material’s surface, such as electric charges and hydrophobicity, to increase cell adhesion.8-10 Collagen coating was found to be able to improve cell adhesion. Different methods to prepare collagen coating were developed that affected cell adhesion to different extents.11 Some showed that the physical interaction between cells and collagen coating has a significant effect on cell adhesion.12 Up to now, the mechanism of cell adhesion on collagen coating * To whom correspondence should be addressed. Tel: +86 025 3792245. Fax: +86 025 3619983. E-mail:
[email protected]. † National Laboratory of Molecular and Biomolecular Electronics. ‡ Department of Physics. 10.1021/pr020007a CCC: $22.00
2002 American Chemical Society
has not been thoroughly clear. Previously, we reported that collagen is a bio-electret that exhibits persistent dielectric polarization.13 In this paper, both collagen coatings on glass and substrates chemically modified with chitosan were prepared, and their morphologies and polarization properties were studied. The HeLa cell is a classical and representative tumor cell line; thus, we focused on HeLa cell adhesion properties. We had also tested other tumor cells (such as A549, MKN-45, MCF-7, MCG803) and obtained similar results. Our results showed that surface electric polarization, its durability, and substrate surface topography were key factors to cell adhesion, and we extended these results to our cell microarray chips. Recently, we have been developing high-throughput tumor cell microarray chips for antitumor drug initial screening. The chips included different tumor cell lines on a plastic chip with highdensity glass microwells connecting with each other by microfluidic channels on a cap plate. Collagen was grafted on the wells’ surface of microwells, and tumor cells adhered to the wells. Drugs flowed through those microfluidic channels to microwells, and the detection used cell-based assays. Our cell microarray chip will therefore be a practical tool for studying the rate of drugs against tumor cells and will be simple and inexpensive with better repetition.14
2. Material and Methods 2.1. Substrate Treatment. Glass slides of 1 cm2 were cleaned with “piranha solution” in an ultrasonic bath for 1 h, rinsed in Millipore H2O, and dried in a stream of nitrogen. Collagen coatings on glass slides were fabricated by two methods. In one method, washed glass slides were silanized in 5% APTES (Sigma) for 1 h, incubated in 5% glutaraldehyde (Sigma) for 2 h, reduced in 20 mM NaBH4 for 20 min, and finally grafted in different concentrations of 0.01, 0.1, 1, and 10 mg/mL collagen (Worthington) solution for 2 h at 37 °C. In the other method, washed glass slides were adhered to the spinner by doublesided adhesive tape, added with 100 µL of 1% chitosan (Sigma) solution, and spin-coated at 2000 rpm/min for 30 s, and the above procedure was followed. All treated slides were cleaned five times with pH 7.4 PBS and Millipore H2O, dried in a stream of nitrogen, and ultraviolet sterilized over 4 h before further applications. 2.2. Cell Culture and Image. HeLa cell cultured at 37 °C with 5% CO2 in RPMI1640 (GibcoBRL) with 10% fetal bovine serum (Hyclone), 100 units/mL of penicillin G sodium, and 100 µg/ mL streptomycin. A 1 mL solution with approximately 10 000 cells was seeded onto the above slides in a 24-well plate and incubated at 37 °C with a 5% CO2 environment for 12 h. Cells were treated for 2 s with 10 mM KCl and fixed for 30 min in Journal of Proteome Research 2002, 1, 559-562
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HeLa Cell Adhesion on Collagen-Grafted Surfaces
technical notes
Figure 1. AFM topographic images of collagen-treated substrates. (1) and (2) indicate the slides with and without chitosan treatment, respectively. The AFM images of a, b, c, d and e are the silanized or chitosan-modified slides without collagen treatment and with treatment of collagen concentrations of 0.01, 0.1, 1, and 10 mg/mL, respectively. The scan size is 1 µm × 1 µm. Height is coded by color in the sacle from 0 to 30 nm. 560
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Figure 2. Optical microscopic pictures of HeLa cells adhered on various substrate surfaces in 12 h. The substrates are the silanated glass slide (a), 1% chitosan coating slides without collagen grafting (b), and with grafting in collagen solutions of 0.01 (c), 0.1 (d), 1 (e), and 10 mg/mL (f). Magnification: × 200. Scale bars: 100 µm. Table 1. Surface Polarization of Various Substrates (Unit: V, Mean ( SD) collagen
nonchitosan chitosan
0 mg/mL
0.01 mg/mL
0.1 mg/mL
1 mg/mL
10 mg/mL
0.81 ( 0.017 2.08 ( 0.0031
-0.055 ( 0.067 -0.055 ( 0.079
-0.065 ( 0.082 -0.085 ( 0.075
-0.09 ( 0.065 -0.12 ( 0.0025
-0.07 ( 0.092 -0.11 ( 0.0087
1% paraformaldehyde before staining with 0.25 µg/mL DAPI for 1 h in darkness, counted, and imaged with a Nikon microphot-FXA 3036 at 200× magnification. Afterward, we decanted the medium, washed it three times in sterilized D-Hanks solution, cultured the cells in serum-free RPMI1640. Cells were counted and serum-free RPMI1640 was refreshed every 12 h.
3. Results and Discussion 3.1. Surface Topography. Surfaces of all treated slides were imaged by Nanoscope III a AFM (Digital Instruments, Santa Barbara, CA) and operated in the tapping mode under ambient conditions. Figure 1(1) gives topographic views of both the glass slide (a) and slides grafted in different concentrations [0.01 (b), 0.1 (c), 1 (d), and 10 mg/mL (e)] of collagen solution. The surface of glass slide was crude and rather rough, and many microcracks were also conspicuously observed. This type of surface was not favorable to cell adhesion.15 Collagen modification produced more mounted surfaces on which cells adhered faster than on smoother surfaces.15 Figure 1(2) gives topographic views of both the glass slide coated with chitosan (a) and that further grafted in different concentrations [0.01 (b), 0.1 (c), 1 (d), and 10 mg/mL (e)] of collagen solution. Chitosan was proved to be beneficial to cell culture.16 However, chitosan coating was rather coarse, and numerous irregularly shaped particles were observed on the surface of coating, on which cells adhered with low efficiency.17 After being grafted with collagen, the surfaces of substrates became more regular and produced relatively thicker and more mounted collagen coatings, with enlarged HeLa cells adhesive area.
3.2. Surface Polarization Measurement. Electric polarizations of the above samples were monitored with a surface potential meter (SP-216, Japan), and the data were listed in the Table 1. It showed that positive charged surfaces were identified for both silanized and chitosan modified glass slides before collagen grafting. The surface potential of 1% chitosan modified slide had more positive value than that of silanized glass slide. Others were turned to negative polarization after collagen grafting. When the concentration of the collagen solution was increased, the negative surface potential became larger. It reached to a maximum at the concentration of 1 mg/ mL. After collagen grafting, surface polarization of previously chitosan-modified slides was higher than that of the corresponding silanized slides, which was probably due to higher negative polarization of the chitosan-collagen interaction.11 3.3. HeLa Cell Adhesion. HeLa cell adhesion was viewed with a Nikon microphot-FXA 303 as shown in Figure 2. The results are summarized in Figure 3. HeLa cells adhesion number on both collagen grafting glass surfaces and collagen grafting 1% chitosan coating surfaces was larger than that on glass slide surface (p < 0.025)and on 1% chitosan coating surface (p < 0.05). The number of adhesive HeLa cells on collagen grafting 1% chitosan coating surfaces was significantly larger than that on collagen grafting glass surfaces with the corresponding concentration (p < 0.05), and cells adhesion number on 1% chitosan coating surface was larger than that on silanized glass surface (p < 0.05). Cell adhesion numbers on 1 mg/mL collagen grafting 1% chitosan coating surface increased more significantly than those on others (p < 0.025). The negative surface electric Journal of Proteome Research • Vol. 1, No. 6, 2002 561
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HeLa Cell Adhesion on Collagen-Grafted Surfaces
4. Conclusions
Figure 3. HeLa cell adhesion on various substrates in 12 h.
Collagen coating on substrates will improve HeLa cell adhesion and extend adhesion time. We have optimized the collagen modification procedure and found that Hela cells showed the best adhesive characteristics on the sample of 1% chitosan-modified glass slide grafting in 1 mg/mL collagen solution. Microstructure and negative electric polarization of samples might enhance HeLa cell adhesion and extend their adhesion time. The large positive electric polarization of the 1% chitosan modified glass slide produced a thick collagen coating during collagen grafting process. Rougher surfaces of chitosan-modified glasses produced a relatively larger surface area, and increased depositing quantity of collagen. The negative surface polarization of collagen coating and its durability are also the key effects to cell adhesion. The present technical researches would help to increase cell concentration and its viability of cell microarray chips.
References
Figure 4. Long-term adhesion of HeLa cells on the 1% chitosanmodified glass with grafting in collagen solutions of various concentrations.
Figure 5. Long-term adhesion of HeLa cells on the silanated glass with grafting in collagen solutions of various concentrations.
polarization and relative rough surface might be the reasons for the high efficiency in cell adhesion. 3.4. Experiment of Serum-Free Culture. Serum proteins adsorb on substrate surfaces and affect subsequent cell functions such as adhesion,5 so adhesive HeLa cells were maintained in serum-free RPMI1640. The medium was refreshed every 12 h and examined under microscope. The adhesive cells were counted, and the data was plotted in Figures 4 and 5. From Figures 4 and 5, the adhesive cell number rapidly decreased with time. The long-term adhesion (referring to the time when the number of adhesive cells decreased by half) of HeLa cells was about 48 h for 1 mg/mL collagen-grafted 1% chitosan substrate and only 36 h for the others. The high negative surface polarization, its durability, and surface topography could be the reason. Therefore, we cultured HeLa cells in serum-free RPMI1640 to screen antitumor drugs for 24 h before application on cell chips. 562
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