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Micropatterning of HeLa Cells on Glass Substrates and Evaluation of Respiratory Activity Using Microelectrodes Matsuhiko Nishizawa,* Kimiyasu Takoh, and Tomokazu Matsue* Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan Received October 22, 2001. In Final Form: January 18, 2002 The micropatterns of mammalian cells (HeLa cells) were prepared on glass substrates, and the respiration of the patterned cells was studied by microelectrode techniques, mainly by scanning electrochemical microscopy (SECM). The cellular patterns on a micrometer scale were prepared by microcontact printing of an extracellular matrix protein, fibronectin, onto a hydrophobic glass plate. The oxygen concentration in the vicinity of the patterned cells was mapped by scanning a Pt microelectrode, and the obtained SECM images proved that the cells in patterns were living with the uptake of oxygen. HeLa cells in the band patterns were well spread, while the cells in the small island patterns were restricted in their shape. The respiratory activities of these cells were evaluated by measuring the difference in the oxygen concentration between the bulk solution and the cell surface, and it was shown that a spreading cell consumed a significantly larger amount of oxygen than a round cell.
Introduction In vitro assays using cultured cells have been commonly carried out for evaluating the cytotoxic effects of chemicals as an alternative to animal experiments. On the other hand, sensors having cell-based sensing elements have recently attracted a great deal of attention as a relatively new approach in the design of biosensors.1-5 There are various methods for monitoring the changes in the physiology of cultured cells,1 e.g., pH monitoring,2 impedance measurement,3 and extracellular potential recording.4,5 We and other groups have recently been studying the possible use of scanning electrochemical microscopy (SECM)6-9 as a noninvasive tool to monitor the cellular status, in which the tip of a microelectrode is scanned near the cells to map the local distribution of electroactive species such as oxygen.10-17 The SECM image of the oxygen * Corresponding authors. (1) Bousse, L. Sens. Actuators, B 1996, 34, 270-275. (2) McConnell, H. M.; Owicki, J. C.; Parce, J. W.; Miller, D. L.; Baxter, G. T.; Wada, H. G.; Pitchford, S. Science 1992, 257, 1906-1912. (3) Giaever, I.; Keese, C. R. Nature 1993, 366, 591-592. (4) Jung, D. R.; Cuttino, D. S.; Pancrazio, J. J.; Manos, P.; Cluster, T.; Sathanoori, R. S.; Aloi, L. E.; Coulombe, M. G.; Czarnaski, M. A.; Borkholder, D. A.; Kovacs, T. A.; Bey, P.; Stenger, D. A.; Hickman, J. J. J. Vac. Sci. Technol., A 1998, 16, 1183-1188. (5) Pancrazio, J. J.; Bey Jr, P. P.; Cuttino, D. S.; Kusel, J. K.; Borkholder, D. A.; Shaffer, K. M.; Kovacs, G. T. A.; Stenger, D. A. Sens. Actuators, B 1998, 53, 179-185. (6) Bard, A. J.; Fan, F.-R. F.; Mirkin, M. V. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel-Dekker: New York, 1994; Vol. 18. (7) Bard, A. J.; Fan, F. R. F.; Kwak, J.; Lev, O. Anal. Chem. 1989, 61, 132-138. (8) Engstrom, R. C.; Pharr, C. M. Anal. Chem. 1989, 61, 1099A1104A. (9) Wijayawardhana, C. A.; Wittstock, G.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 2000, 72, 333-338. (10) Lee, C. M.; Kwak, J. Y.; Bard, A. J. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1740-1743. (11) Tsionsky, M.; Cardon, Z. G.; Bard, A. J.; Jackson, R. B. Plant Physiol. 1997, 113, 895-901. (12) Yasukawa, T.; Kondo, Y.; Uchida, I.; Matsue, T. Chem. Lett. 1998, 767-768. (13) Liu, B.; Rotenberg, S. A.; Mirkin, M. V. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9855-9860. (14) Liu, B.; Cheng, W.; Rotenberg, S. A.; Mirkin, M. V. J. Electroanal. Chem. 2001, 500, 590-597. (15) Yasukawa, T.; Kaya, T.; Matsue, T. Chem. Lett. 1999, 975-976. (16) Yasukawa, T.; Kaya, T.; Matsue, T. Anal. Chem. 1999, 71, 46374641.
concentration around a cell directly reflects the respiration activity and can serve as a measure of cellular status.12 To make further progress in the development of the SECMbased cellular sensing systems, it will be undoubtedly required to place cells in predetermined locations (on the device chip) with defined shapes and sizes. The patterning of living cells has been extensively attempted by a number of research groups, since the spatial control of mammalian cell adhesion and growth is a critical issue in many areas of biotechnology. Microcontact printing (µCP) would be the most established method to form patterns of cells.18-20 This method uses an elastomeric stamp made of poly(dimethylsiloxane) (PDMS) for making patterns of typically the self-assembled monolayer (SAM) of thiols. The patterned SAM of the thiolated poly(ethylene glycol) showed an excellent blocking effect to the adsorption of extracellular matrix (ECM) proteins and thus to the attachment of cells,21 resulting in patterns of a variety of anchorage-dependent mammalian cells.22-27 The µCP based on the SAMs of thiols requires the precoating of the substrate surface by a thin film of Au, Ag, or Cu.28 Mrksich et al. have utilized the Au film as an electrode and achieved the electrochemical control of cell adhesion.26 Unfortunately, such a conductive substrate is (17) Shiku, H.; Shiraishi, T.; Ohya, H.; Matsue, T.; Abe, H.; Hoshi, H.; Kobayashi, M. Anal. Chem. 2001, 73, 3751-3758. (18) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550-575. (19) Kane, R. S.; Takayama, S.; Ostumi, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363-2376. (20) Mrksich, M. Chem. Soc. Rev. 2000, 29, 267-273. (21) Lo´pez, G. P.; Albers, M. W.; Schreiber, S. L.; Carroll, R.; Peralta, E.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 5877-5878. (22) Singhvi, R.; Kumar, A.; Lo´pez, G. P.; Stephanopoulos, G. N.; Wang, D. I. C.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696-698. (23) Mrksich, M.; Chen, C. S.; Xia, Y.; Dike, L. E.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10775-10778. (24) Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.; Whitesides, G. M. Exp. Cell Res. 1997, 235, 305313. (25) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425-1428. (26) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Proc. Natl. Acad. Sci. USA 2001, 98, 5992-5996. (27) Amirpour, M. L.; Ghosh, P.; Lackowski, W. M.; Crooks, R. M.; Pishko, M. V. Anal. Chem. 2001, 73, 1560-1566. (28) Finklea, H. O. In Electroanalytical Chemistry; Bard, A. J., Rubinstein, I., Eds.; Marcel Dekker: New York, 1996; Vol. 19, pp 109-335.
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often troublesome for SECM imaging due to redox cycling between the microelectrode probe and the substrate.6 Recently the control of axonal growth of neuronal cells has been achieved by the microcontact printing of biomolecules such as laminin and polylysine directly onto glass substrates without the use of SAM of thiols.29-32 Other soft lithographic techniques using microfluidic channels33,34 and microwells35 have also been developed to pattern cells on glass and plastic surfaces. These techniques enable the patterning of cells on the insulating substrates which is required for the SECM studies. This paper describes the electrochemical studies of the respiratory activity of mammalian cells (HeLa cells) patterned on a glass substrate, as the first attempt to combine the cell patterning and SECM with the objective of developing an electrochemical bioassay system. The research can be divided to two sections: (1) the preparation of cell patterns on the glass plate by the µCP method; (2) the evaluation of the respiratory activity of the patterned cells by SECM imaging of the oxygen concentration around the cells. The SECM studies proved that the micropatterned HeLa cells were alive with uptake of oxygen. The control of the degree of cell spreading by patterning and its effects on the cellular respiratory activity will also be discussed. Experimental Section Fibronectin (FN) (from human plasma, Wako Pure Chemical Industries), bovine serum albumin (BSA) (Wako Pure Chemical Industries), n-octadecyltrichlorosilane (Tokyo Kasei), CF3(CF2)3(CH2)2SiCl3 (Shinetsu Chem. Ind., Ltd.), PDMS (KE-106) (Shinetsu Chem. Ind., Ltd.), and all other chemicals were used as received. HeLa cells, a human cervix epithelial cell line, were cultured in Petri dishes (Falcon) with RPMI1640 medium (Gibco) containing 10% fatal bovine serum (Gibco), 50 µg mL-1 penicilin, and 50 µg mL-1 streptomicyne, in a humidified incubator (37 °C in an atmosphere of 5% CO2). The cells were trypsinized in a 0.25% trypsin solution and seeded (3 × 105 cells mL-1) onto the culture plates such as the protein-patterned substrates. As schematically described in Figure 1, the pattern of FN was prepared by the µCP method. The template for the PDMS microstamp was a photoresist pattern (9 µm thickness) on a glass slide. The liquid prepolymer of PDMS was poured over the template pattern and cured at 100 °C for 1 h, with another glass slide on (Figure 1a) to make the back face of the stamp flat. The surfaces of both the template substrate and the cover plate were prefluorinated by CF3(CF2)3(CH2)2SiCl3 for the ease of removing the cured stamp. The resulting thin PDMS stamp (200-300 µm thickness) was cut into 2 × 2 cm2 pieces, stuck on a clean glass plate of the same size, and heated at 80 °C for 1 h to reinforce the adhesion (Figure 1b). The stamp was treated with O2 plasma (100 W, 30 min) to make its surface hydrophilic. Immediately after the plasma treatment, a 0.4 mL FN aqueous solution (2.8 mg mL-1) was applied onto the stamp surface and dried for 15 min. The resulting FN-inked stamp was put on a hydrophobically pretreated glass plate and pressed using a 25 g weight. The weighted staff (Figure 1c) was incubated for 1 h at 37 °C. After being thoroughly rinsed with PBS, the substrate was further (29) Kam, L.; Shain, W.; Turner, J. N.; Bizios, R. Biomaterials 2001, 22, 1049-1054. (30) James, C. D.; Davis, R.; Meyer, M.; Turner, A.; Turner, S.; Withers, G.; Kam, L.; Banker, G.; Craighead, H.; Isaacson, M.; Turner, J.; Shain, W. IEEE Trans. Biomed. Eng. 2000, 47, 17-21. (31) Branch D. W.; Wheeler, B. C.; Brewer G. J.; Leckband, D. E. IEEE Trans. Biomed. Eng. 2000, 47, 290-300. (32) Scholl, M.; Spro¨ssler, C.; Denyer, M.; Krause, M.; Nakajima, K.; Maelicke, A.; Knoll, W.; Offenha¨usser, A. J. Neurosci. Methods 2000, 104, 65-75. (33) Folch, A.; Ayon, A.; Hurtado, O.; Schmidt, M. A.; Toner, M. J. Biomech. Eng. 1999, 121, 28-34. (34) Folch, A.; Toner, M. Biotechnol. Prog. 1998, 14, 388-392. (35) Ostuni, E.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 2828-2834.
Figure 1. Schematic diagram describing the procedure used to prepare the FN patterns on a glass plate. A thin PDMS stamp (200-300 µm thickness) was produced using a photoresist pattern as the template (a, b). Immediately after the O2 plasma treatment for stamp surface, a droplet of FN aqueous solution was applied and dried. The pattern of FN was then transferred to the glass surface pretreated with alkylsilane (c). After being rinsed thoroughly with PBS, the substrate was further incubated in a 0.3 mg mL-1 BSA solution to cover the remainder with BSA (d). See text in the Experimental section for details. incubated in BSA/PBS solution (0.3 mg mL-1) to cover the remainder with BSA molecules. The SECM imaging was carried out in a buffered isotonic solution composed of 10 mM HEPES, 150 mM NaCl, 4.2 mM KCl, 2.7 mM MgCl2, 1 mM NaH2PO4, and 11.2 mM glucose. The Pt microelectrode for SECM was fabricated as previously reported.12,36 A fine Pt wire was first electrochemically etched, inserted into a glass capillary, and shielded by thermal fusing of the glass. Finally, the tip of the capillary was carefully polished to give a disk-type microelectrode. The radius of the Pt disk at the tip was typically 5.0 µm, as determined from the ferrocyanide voltammograms, while the tip radius including the insulating glass part was ca. 30 µm. By using a motor-driven xyz stage controlled by PC, the tip of the microelectrode was first in contact with the surface of the substrate, retracted to make a 10 µm separation, and then scanned in the x-y plane over the patterned cells at the scan rate of 9.8 µm s-1. The potential of the microelectrode was held at -0.5 V vs Ag/AgCl during scans to monitor the reduction current for oxygen. Prior to the scanning of tip, we have routinely stabilized the electrode surface in the medium solution until the steady O2 reduction current was observed to minimize the gradual change of the electrode activity during the imaging. The time required to obtain an image of 150 × 200 µm2 at the spatial resolution of 5 µm was about 10 min. The quartz crystal microbalance (QCM) was used with a commercial system (Seiko EG & G, QCA917) set in an incubator (5% CO2, 37 °C). A QCM sensor (a shear mode 9 MHz AT-cut quartz crystal Au electrode, 0.2 cm2) was first coated with the SAM of undecanethiol and further with adsorption layers of proteins such as FN and BSA. The protein-modified QCM sensors were stabilized in a culture medium (ca. 0.5 mL), and then 20 µL of the cell suspension (70 × 104 cells mL-1) was gently added for monitoring the cell adhesion. (36) Matsue, T.; Koike, S.; Uchida, I. Biochem. Biophys. Res. Commun. 1993, 197, 1283-1287.
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Figure 3. Optical micrographs of (a) the surface of PDMS stamp used for microcontact printing of proteins and (b) the pattern of HeLa cells prepared via the series of procedures explained in Figure 1. The FN patterning and the blocking treatment with BSA were conducted prior to the HeLa cultivation.
Figure 2. (a) Time courses of resonant frequency upon seeding HeLa cells, taken for the FN-adsorbed (solid curve) and the BSA-adsorbed (dotted curve) QCM chips. A 20 µL cell suspension (70 × 104 cells mL-1) was gently added to the QCM chamber set in a CO2 incubator. (b) Optical microscope image showing the selective attachment of HeLa cells onto the fibronectincoated region (the left side) of a glass plate.
Results and Discussion Prior to the micropatterning of cells, we carried out preliminary experiments with the objective to characterize the proteins, fibronectin (FN) and bovine serum albumin (BSA), as the building blocks for the patterns of the HeLa cells. FN is a well-known extracellular matrix protein, which generally promotes the adhesion of anchoragedependent mammalian cells.37 On the other hand, BSA has been widely used as a blocking protein, especially in immunoassay,38,39 to inhibit the nonspecific adsorption of other proteins. Figure 2a depicts the QCM responses observed upon seeding HeLa cells onto the FN-adsorbed (solid curve) and the BSA-adsorbed (dotted curve) QCM chips. Though a quantitative analysis has not been established, the decrease in resonant frequency can be thought to correlate with the cell adhesion,40 and thus the results evidently show the expected functions of FN and BSA on the adsorption of the HeLa cell: FN promotes the cell adhesion; BSA resists the adhesion of HeLa by blocking further adsorption of the ECM proteins from the serum in the culture medium. These effects of FN and BSA on the adhesion of the HeLa cells were then examined by preliminary macroscale patterning. Figure 2b shows the (37) Hynes, R. O. Fibronectins; Springer-Verlag: New York, 1990. (38) Heineman, W. R.; Brain, H. H. Anal. Chem. 1985, 57, 1321A1331A. (39) Kasai, S.; Yokota, A.; Zhou, H.; Nishizawa, M.; Niwa, K.; Onouchi, T.; Matsue, T. Anal. Chem. 2000, 72, 5761-5765. (40) Janshoff, A.; Galla, H.-J.; Steinem, C. Angew. Chem., Int. Ed. 2000, 39, 4004-4032.
HeLa cells cultured at the boundary between the FNadsorbed (left) and the BSA-adsorbed (right) regions, showing the ideally selective adhesion of HeLa cells to the FN-adsorbed surface. The culture plate used in this experiment was prepared by putting a small droplet of FN aqueous solution (4 µL, 2.8 mg mL-1) on a hydrophobically pretreated glass substrate for 1 h, followed by immersing the substrate into a 0.3 mg mL-1 BSA/PBS solution. It was clearly shown that BSA molecules adsorbed on the remainder of the FN-adsorbed region as a background inert to cell adhesion. The results obtained from these experiments indicate that the stepwise procedure, in which the microcontact printing of FN is followed by BSA treatment to the entire surface, would be applicable to prepare a pattern of HeLa cells on a micrometer scale. Figure 3 shows the optical microscope images of (a) the surface of the PDMS stamp having an array of 5 square islands and (b) the pattern of HeLa cells on a culture plate prepared via the series of procedures explained in Figure 1. The protein-patterned glass plate was incubated in a cell suspension (3 × 105 cells mL-1) for 1 h, followed by a further incubation for 6 h in the cell-free culture medium. The incubation in the beginning was for the relatively quick cell attachment to the FN islands, and the subsequent incubation was for the cell spreading within each island. The pattern on the PDMS stamp was successfully represented as a cell pattern, and it is impressive that 1, 2, and 3 cells are spreading within each of the smallest three islands. The cell pattern was stable for at least 2 days under the cultivation conditions. To obtain such a cell pattern on glass, a few modifications were made to the usual µCP procedure, as described in the Experimental Section. The O2 plasma treatment, which was necessary for inking the stamp with aqueous solution, weakened the sticky nature of the PDMS. Therefore, a suitable pressure was needed to transfer a sufficient amount of proteins to the surface (Figure 1c). To apply a uniform pressure during the protein transfer, we cured PDMS with the sandwich assembly, and the resulting flat stamp was supported by a glass plate (Figure 1a,b). We have examined
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Figure 4. (a) Optical micrographs of island and band patterns of HeLa cells and (b) SECM images for the enclosed parts of the patterns, taken in a HEPES-based saline solution. A Pt microelectrode, the potential of which was set at -0.5 V, was scanned at 10 µm above the substrate at the scan rate of 9.8 µm s-1.
that the present method with FN/BSA couple is satisfactorily available for the patterning of myocardial cells from chick embryo, while the bovine vascular endothelial cell was not be patterned probably due to its active migration. Figure 4 shows (a) optical microscope images of the island and band patterns of the HeLa cells and (b) SECM images for the enclosed parts of these patterns. The SECM measurements were conducted in a HEPES-based saline solution by scanning a Pt microelectrode at 10 µm above the substrate. The images with low oxygen reduction current (dark areas) in Figure 4b coincided with the location of the patterned HeLa cells in the optical microscope images (Figure 4a). The addition of respiratory inhibitors such as KCN significantly paled these images, as we have already demonstrated in our previous work for the adhering cells randomly to a culture dish.12 These facts indicate that the dark areas in Figure 4b represent mainly the O2 consumption by cellular respiration, proving that the patterned HeLa cells are living with the uptake of oxygen. The effect of shielding the O2 diffusion by the cell surface seems to be minor during these SECM imaging, undoubtedly due to the high permeability of O2 to the cell membranes. Since the respiratory activity is a direct measure of the cellular status, the substrate with the patterned cells may serve as a cell chip for bioassay. As can be seen in Figure 4, HeLa cells in the band patterns were spreading almost freely, while the cells in the small island patterns appear to be restricted in their shape. To evaluate the cellular status depending on the cell shape with respect to the respiratory activity, we prepared a substrate containing both island (50 × 50 µm2) and band (50 µm width) patterns of HeLa cells, as shown in Figure 5a. Both the island and band patterns can be recognized in the SECM image (Figure 5b), in which the island patterns showed clearer contrast than the band patterns. It should be noted, however, that the present SECM image may also contain the effect of cell shape itself as well as the respiratory activity. Since the height of the microelectrode probe was kept 10 µm above the substrate during the imaging, the distance between the electrode tip and the cell surface depends on the degree of humping of each cell. The cells in the island patterns were round (cell height, ca. 8 µm) and thus close to the probe tip, resulting in the image with a higher contrast compared with the case of spreading cells (cell height, ca. 6 µm) in the band patterns. In this work, therefore, we attempt to evaluate the respiratory activity of cells by the
Figure 5. (a) Optical micrograph and (b) SECM image of island and band patterns of HeLa cells formed on a glass substrate. Experimental conditions are the same as that in Figure 4. (c) Profile of the oxygen concentration in the vicinity of a round cell in the island pattern (O) and a spreading cell in the band pattern (b). A microelectrode probe was scanned every 2 µm in the z-axis direction by measuring O2 reduction current. The obtained current values were converted to oxygen concentration using the value in bulk solution of 0.22 mM.
profile of the oxygen concentration along the z axis in the vicinity of the cells. Figure 5c shows typical profiles obtained for a cell in the island pattern (O) and for a cell in the band pattern (b). A thinner microelectrode probe (ca. 1 µm radius Pt disk at the tip) was approached to these cells with measuring the O2 reduction current until the electrode tip touched the cell surface and then extracted. The deviations between the data measured during the forward and backward scans are less than 10% of the ∆C (the O2 concentration difference between the bulk and cell surface). The results indicate that the spreading cell was consuming a significantly larger amount of oxygen than the round cell. If we assume that the cells are hemispherical, the oxygen consumption rate (F) can be expressed by F ) 2πrhD∆C, where rh is the hight of cell and D is the diffusion coefficient of oxygen (2.18 × 10-5 cm2 s-1).17 The resulting F values are 3.0 and 4.7 fmol s-1 for the round and spreading cells, respectively, while this is a rough estimation assuming hemispherical shape for the cells. It has been recently demonstrated that the cell shape is one of the important factors regulating cellular functions including cell cycle progres-
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sion22 and apoptosis.25 The cells in the small islands seem to be in the confluent state, while the cells in the band patterns are subconfluent and probably still growing. Such a difference in the cellular status would be the main origin of the observed shape-dependent respiratory activities. This information would be useful in the future fabrication of cell chips to optimize the size and the shape of the cellular patterns. We are planning further systematic studies on respiratory activity using an array of patterns including the isolated single cells. Conclusion The SECM imaging of the patterned living cells has been reported for the first time. The microcontact printing of the cell adhesive protein successfully enabled the formation of HeLa cell patterns on insulating substrates without the use of SAMs of thiols. We have also examined
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that the present simple method with the FN/BSA couple is available for the patterning of myocardial cells from chick embryo, even though the experiments are still preliminary. Although this paper focused on the SECMrelating experiments, the patterning cells on a microelectrode array substrate was also possible and should be attractive for the whole-cell bioelectronics. Acknowledgment. This study was supported by Industrial Technology Research Grant Program in 2000 from NEDO and partly by Grant-in-Aids for Scientific Research on Priority Area (No. 11227201) from the Ministry of Education, Science, and Culture of Japan. The HeLa cells were donated by the Institute of Development, Aging and Cancer, Tohoku University. LA011576K
