Biomacromolecules 2005, 6, 970-974
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In Situ Control of Cell Adhesion Using Photoresponsive Culture Surface Jun-ichi Edahiro,† Kimio Sumaru,*,† Yuichi Tada,†,§ Katsuhide Ohi,† Toshiyuki Takagi,† Mitsuyoshi Kameda,† Toshio Shinbo,‡ Toshiyuki Kanamori,† and Yasuo Yoshimi§ Research Center of Advanced Bionics, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan, Institute for Materials and Chemical Process, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan, and Department of Industrial Chemistry, Shibaura Institute of Technology, 3-9-14 Shibaura Minato-ku, Tokyo 108-8548, Japan Received October 20, 2004; Revised Manuscript Received December 14, 2004
A photoresponsive culture surface (PRCS) allowing photocontrol of cell adhesion was prepared with a novel polymer material composed of poly(N-isopropylacrylamide) having spiropyran chromophores as side chains. Cell adhesion of the surface was drastically enhanced by the irradiation with ultraviolet (UV) light (wavelength: 365 nm); after subsequent cooling and washing on ice, many cells remained in the irradiated region, whereas most cells were removed from the nonirradiated region. The cell adhesion of the PRCS, which had been enhanced by previous UV irradiation, was reset by the visible light irradiation (wavelength 400-440 nm) and the annealing at 37 °C for 2 h. Also it was confirmed that the regional control of cell adhesion was induced several times by repeating the same series of operations. Further, living cell patterning with the 200 µm line width was produced readily by projecting UV light along a micropattern on the PRCS on which the living cells had been seeded uniformly in advance. By using a fluorescent probe that stains living cells only, it was confirmed that the cells maintained sufficient viability even after UV light irradiation followed by cooling and washing. Introduction In recent years, various medical technologies such as cell therapy and regeneration medicine1-3 have been studied actively aiming at clinical applications. In such situations, development of new methodology is needed to analyze intercellular interaction and to select living cells according to their functions or species from a cell population in which several kinds of cells are mixed. The functional cell culture surface on which cell adhesion of adherent cells can be controlled has the potential to realize this methodology and has become one of the central topics of research in biochemical engineering. By means of the cell culture surface modified with a gel of a thermoresponsive polymer, poly(N-isopropylacrylamide) (pNIPAAm), Okano et al. developed a technique to recover the cultured and proliferated cells, simply by lowering the temperature. pNIPAAm is dissolved in water at temperatures below the lower critical solution temperature (LCST), whereas it dehydrates and precipitates at temperatures above LCST. On a surface modified with this polymer, cells adhered well at a temperature above the LCST, whereas they did not adhere at a temperature below the LCST.4 According to this principle, a cellular sheet could be recovered intact * To whom correspondence should be addressed. E-mail: k.sumaru@ aist.go.jp. † Research Center of Advanced Bionics, AIST. ‡ Institute for Materials and Chemical Process, AIST. § Shibaura Institute of Technology.
together with membranal proteins and extra cellular matrix (ECM) without the use of conventional detaching agents such as trypsin and EDTA and have been applied to a variety of purposes.5-6 However, this method cannot directly be applied to the manipulation of individual cells because it is almost impossible for a temperature stimulus to affect only in the restricted micrometer-scale region. On the other hand, as a method to change cell adhesion property in the local region, Nakayama et al. developed a cell culture surface on which cell adhesion was enhanced just by the irradiation of ultraviolet light before cell seeding.7 However, ultraviolet light with the wavelength of 300 nm or shorter, which lethally damages cells, is required to isomerize the leuco-chromophore contained in their system as a photoresponsive component. Therefore, this technique is not applicable to the manipulation of living cells. To manipulate living cells adhering on the photoresponsive culture surface (PRCS), the cell adhesion property of PRCS must be significantly changed by irradiating with a light at a certain wavelength and intensity, which is harmless to living cells, on the cell adhering region where living cells have already existed. In addition, reversible control of cell adhesion is desired because a complicated patterning of several species of cells would be formed easily by repeating the regional cell attachment and detachment. In this study, using a thermoresponsive polymer modified with a photoresponsive chromophore,8-12 we prepared a novel PRCS allowing regional and reversible control of cell adhesion by
10.1021/bm0493382 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/21/2005
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Figure 1. Chemical structure of pNSp-NIPAAm and photoisomerization scheme of NSp chromophore.
photoirradiation. Based on this PRCS, we intended to establish a novel technique to manipulate a large number of living cells at a time. Experimental Section Fixed-Point Microscopic Observation and Micropatterned Photoirradiation. In the following experiments, a successive observation of cell distribution in a certain microscope field was necessary during a series of operations such as light-irradiation, washing, and incubation. Therefore, a fixed-point observation system composed of a motorized XY-stage ProScan (PRIOR) and a confocal laser scanning microscope FluoView300 (Olympus) was used in this study. In addition, for a micro-patterned photoirradiation and subsequent observation of the irradiated region, a proprietary micropatterned photoirradiation apparatus was installed to the fixed-point observation system. Preparation of the Photoresponsive Culture Surface (PRCS). A pNIPAAm-based copolymer (pNSp-NIPAAm, Mw ) 17 300) containing (5.4 mol %) acrylamide monomer with nitrospiropyran (NSp) residue was used as a component of a photoresponsive material. Since NSp is isomerized into a colored zwitterionic structure (opened-ring form) by ultraviolet light irradiation and is isomerized back into a colorless nonionic structure (closed-ring form) by visible light,13-16 it provides the cell culture surface with reversible photoresponsive property (Figure 1). To retain pNSp-NIPAAm, which is basically watersoluble, at the culture surface stably, the PRCS was prepared in the following procedure. Polymer blend (pNSp-NIPAAm: poly(methyl methacrylate) (Mw ) 55 000) ) 20:80 w/w%) was dissolved in 1,2-dichloroethane at a concentration of 0.2 w/w%. Then 400 µL of the solution was poured onto a circular glass substrate with the diameter of 25 mm, which had been hydrophobicized with dichlorodimethylsilane and was dried in air for 3 days. After annealing at 120 °C for 2
Figure 2. Schematic illustration of experimental procedure for cell adhesion control on the photoresponsive culture surface.
h, the substrate was clamped at the base of a substrate holder to form a cell incubator for experimental convenience. Cell Strain and Culture Condition. CHO-K1 cells (Riken Bioresource Center, Tsukuba, Japan) were seeded onto the PRCS at about 2.0 × 105 cells/cm2 and cultured in the minimum essential medium Eagle R-modification (Sigma) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 units/mL penicillin, 100 µg of streptomycin and glutamine at 37 °C for 24h in a humidified atmosphere of 5% CO2/ 95% air. Cell Adhesion Control on the PRCS by Photoirradiation and Temperature Change. A scheme of the experimental procedure was shown in Figure 2. At first, PRCS on which cells adhered evenly (Figure 2A) was irradiated regionally by ultraviolet light (365 nm, 30 mW/cm2, abbreviated as UV hereinafter), which isomerizes NSp into the opened-ring form, at room temperature for 5 min. After photoirradiation, the whole area of PRCS was cooled on ice for 20 min and was washed evenly with ice-cold D-PBS buffer solution (Sigma) to remove detached and partially detached cells (Figure 2B). We call this cooling and washing process hereinafter “the low-temperature washing”. Micrographs of the PRCS were taken for 4 different microscope fields in each UV-irradiated and nonirradiated region. Adhering cell density was determined from the number of the cells remaining on the PRCS. Average values and standard errors of adhering cell density were calculated in each condition. Second, to remove the cells remaining in the UV-irradiated region even after the low-temperature washing, the PRCS was irradiated for 5 min with visible light (400-440 nm, 24 mW/cm2, abbreviated as VIS hereinafter) that isomerizes NSp in opened-ring form into closed-ring form, and then incubated at 37 °C for 2 h to induce dehydration of pNSp-
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NIPAAm (from Figure 2B-C). As control experiments, either VIS irradiation or incubation at 37 °C for 2 h were exclusively applied. After these operations followed by the low-temperature washing (from Figure 2C-D), each PRCS was observed by microscope. Third, after removing most of the cells from the PRCS (Figure 2D), CHO-K1 cells, which had been prestained by Cell Tracker Green CMFDA (Invitrogen, USA) to be distinguished from previously adhering cells, were reseeded onto the same PRCS uniformly and cultured at 37 °C for 24 h (Figure 2E). The region of the PRCS, which had not been irradiated with UV in the former UV-irradiation procedure, was irradiated with UV for 5 min. After the low-temperature washing (Figure 2F), the PRCS was observed by microscope. Formation of Micrometer-Scale Cell Patterning. As a demonstration of regional control of cell adhesion, formation of micrometer-scale cell patterning was examined as described below. PRCS on which cells had been seeded uniformly in the whole area was irradiated with UV in a pattern “?” (question mark) that was 2 by 3 mm in size with a minimum line width of approximately 200 µm by means of a micropatterned photoirradiation apparatus. After lowtemperature washing, cells that remained on the PRCS were stained with CMFDA and were observed by microscope. Results and Discussion Selective Cell Detachment by Photoirradiation and Temperature Change. Figure 3A shows the PRCS 24 h after cell seeding, indicating that CHO-K1 cells adhered uniformly on the whole area of the PRCS. After regional UV irradiation on the PRCS and the low temperature washing, the cells remained only in the UV-irradiated region (Figure 3B, inside of the yellow rectangle). As a result, it was confirmed that the regional control of cell adhesion based on photoirradiation was successfully achieved. Figure 4 shows the cell adhesion density in UV-irradiated and nonirradiated regions before and after the regional UV irradiation followed by low-temperature washing. As a result, in the UV-irradiated region, 65% of adhering cells remained on the PRCS, which was about 20 times as much as that in the nonirradiated region. Figure 3A clearly showed that cells adhered well at standard culture conditions (37 °C). However, the cell adhesion property was significantly reduced after low temperature washing only in the nonirradiated region (Figure 3B, outside of the yellow rectangle). These results suggested that the cell adhesion property of the PRCS in nonirradiated region was reduced due to the hydration of pNSp-NIPAAm in the PRCS at low temperature as in the case of pNIPAAm.4 On the other hand, in the UV-irradiated region (Figure 3B, inside of the yellow rectangle), cell adhesion was significantly enhanced, and as a result, cell detachment was strongly inhibited during the low-temperature washing. Although the mechanism of cell adhesion enhancement brought by UV irradiation has not been revealed yet, it was suggested that there was some attractive interaction between NSp in a twitterionic form and the surface of an animal cell composed of phospholipids, which are also twitterionic.
Figure 3. Microscopic images of photoresponsive culture surface (A) before and (B) after regional UV irradiation followed by the lowtemperature washing, and (C) after second regional UV irradiation followed by the low-temperature washing. Yellow rectangles indicate UV-irradiated regions.
Reversibility of the Cell Adhesion Property. After the VIS irradiation followed by incubation at 37 °C for 2 h, the cells immobilized in the preceding UV irradiation were removed in the subsequent low-temperature washing. Additionally, also in case that VIS irradiation was applied after incubation at 37 °C for 2 h, i.e., the reverse order to the above, the cells were detached effectively. However, when either the VIS irradiation or the incubation at 37 °C for 2 h was exclusively applied, effective cell detachment was not induced. These results indicated that the dehydration of pNSp-NIPAAm prior to low temperature washing as well as the isomerization of NSp into the closed-ring form is necessary to release the cells effectively from the PRCS,
In Situ Control of Cell Adhesion
Figure 4. Adhering cell density on the UV-irradiated and nonirradiated region before (gray bar) and after (white bar) cell detachment. Error bars show standard deviation (n ) 4).
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senting the character “?”, whereas most cells were detached from the nonirradiated region representing the background. This result clearly showed that cell adhesion was controlled with the precision of a submillimeter scale and also that the change in cell adhesion was not an artifact caused by the variation of the washing operation or by inhomogeneity of the PRCS itself. The resulting cell pattern shown in Figure 5 was not sharply outlined. However, it was mainly due to the aberration of the optical system used in the experiment, and this problem can be improved readily by using an optical system with a higher precision. Figure 5 also shows high viability of adhering cells after the UV irradiation and the low temperature washing because the cells were stained by CMFDA, which is a fluorescent probe staining only living cells.17 It was observed that adhering cells in the UV-irradiated region proliferated during incubation for 24 h at 37 °C after UV irradiation. Additionally, in the preliminary examination using CHO-K1 cells cultured on a tissue culture polystyrene dish, it was confirmed that UV irradiation in the same condition as used in this study did not lead growth suppression or heteromorphology. These results indicated that the cells irradiated with UV in this research maintained sufficient viability. Conclusion
Figure 5. Micrometer-scale living cell patterning produced on a photoresponsive culture surface.
where cell adhesion had been enhanced once by the UV irradiation. Although the mechanism is still unclear, we suppose that the adherent cells were removed in the hydrating process of dehydrated pNSp-NIPAAm chain having no twitterionic NSp. After removing cells as described above, reseeded cells adhered on the whole area of PRCS at 37 °C. Moreover, after UV irradiation on the other region (Figure 3C, inside of the yellow rectangle) than previously UV-irradiated region (Figure 3C, outside of the yellow rectangle) followed by the low temperature washing, both the cell adhesion in the freshly UV-irradiated region and the cell detachment in the non-UV-irradiated region were effectively induced (Figure 3C). These results indicated that the preceding UV irradiation and the low temperature washing before the resetting procedure described above did not affect the subsequent photocontrol of cell adhesion. In other words, the cell adhesion property of the PRCS was completely initialized by VIS irradiation and the incubation at 37 °C for 2 h. Additionally, it was also indicated that the change in cell adhesion property of the PRCS was not brought about by any irreversible process such as dissolution of constituent polymers or photolysis of the chromophore. Formation of Micrometer-Scale Living Cell Patterning. Figure 5 shows the result of living cell patterning produced by the local light irradiation after uniform cell seeding. Cell adhesion was enhanced in the UV-irradiated region repre-
By using a proprietary photoresponsive polymer, we developed a novel functional cell culture surface on which cell adhesion property can significantly be changed by regional photoirradiation. As a result of systematic experiments using living cells, regional control of cell adhesion was successfully achieved by means of this PRCS. Since this photocontrol can be applied even on the region where living cells have already existed, this method enables the objective cells to be recovered or unwanted cells to be removed selectively from adhering and proliferated cell population after identifying them in the microscopic observation. Additionally, due to the reversibility of cell adhesion control, several species of cells can be arranged freely in the predefined order by repeating both cell detachment and cell adhesion processes. As a cell manipulation method with high flexibility, the laser-trapping method has already been in practical use.18 In contrast to this method, which can treat only one single cell at a time, the new method developed in this study can manipulate a large number of adhering cells in a parallel and simultaneous manner by projecting the light in a 2-dimensional micropattern. We believe that this is a promising technique to manipulate living cells that makes a clear departure from existing methods and will provide a novel and powerful tool in the research field of cell technology. Acknowledgment. This work was supported by the Industrial Technology Research Grant Program in 2002 from the New Energy Development Organization (NEDO) of Japan. References and Notes (1) Giordano, R.; Lazzari, L.; Rebulla, P. Vox Sang. 2004, 87, 65. (2) Atala, A. RejuVenation Res. 2004, 7, 15.
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(3) Taylor, D. A. Int. J. Cardiol. 2004, 95, S8-S12; Suppl. 1. (4) Okano, T.; Yamada, N.; Sakai, H.; Sakurai, Y. J. Biomed. Mater. Res. 1993, 27, 1243. (5) Yamato, M.; Konno, C.; Utsumi, M.; Kikuchi A.; Okano, T. Biomaterials 2002, 23, 561. (6) Nandkumar, M. A.; Yamato, M.; Kushida, A.; Konno, C.; Hirose, M.; Kikuchi, A.; Okano, T. Biomaterials 2002, 23, 1121. (7) Nakayama, Y.; Furumoto, A.; Kidoaki, S.; Matsuda, T. Photochem. Photobiol. 2003, 77, 480. (8) Irie, M.; Kungwatchakun, D. Macromolecules 1986, 19, 2476. (9) Kro¨ger, R.; Menzel, H.; Hallensleben, M. L. Macromol. Chem. Phys. 1994, 195, 2291. (10) Sumaru, K.; Kameda, M.; Kanamori, T.; Shinbo, T. Macromolecules 2004, 37, 4949. (11) Kameda, M.; Sumaru, K.; Kanamori, K.; Shinbo, T. Langmuir 2004, 20, 9315.
Edahiro et al. (12) Sumaru, K.; Kameda, M.; Kanamori, T.; Shinbo, T. Macromolecules 2004, 37, 7854. (13) Dagan, L. D.; Tibbon, S. M.; Katz, E.; Willner, I. Angew. Chem., Int. Ed. Engl. 1995, 34, 1604. (14) Shipway, A. N.; Willner, I. Acc. Chem. Res. 2001, 34, 421-432. (15) Fissi, A.; Pieroni, O.; Angelini, N.; Lenci, F. Macromolecules 1999, 32, 7116. (16) Rosario, R.; Gust, D., Hayes, M.; Jahnke, F. Langmuir 2002, 18, 8062 (17) Poole, C. A.; Brookes, N. H.; Gilbert, B. W.; Beaumont, A.; Crowther, L.; Scott, L.; Merrilees, M. J. Connect. Tissue Res. 1996, 33, 233. (18) Satomaeda, M.; Uchida, M.; Granfer, F.; Tashiro, H. DeVel. Biol. 1994, 162, 77.
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