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Micropatterning of Cells Using Modulated Magnetic Fields Tsunehisa Kimura,*,†,‡ Yukiko Sato,† Fumiko Kimura,‡,§ Masakazu Iwasaka,§ and Shoogo Ueno§ Department of Applied Chemistry, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, Tokyo 192-0397, Japan, Tsukuba Magnet Laboratory, National Institute for Materials Science, 3-13 Sakura, Tsukuba, Ibaraki 305-0003, Japan, and Department of Biomedical Engineering, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Received October 8, 2004. In Final Form: December 14, 2004 A new technique of cell micropatterning was presented. Mouse osteoblast cells (MC3T3-E1) were seeded on a substrate whose surface was exposed to a periodically modulated magnetic field (a line pattern with a 200- or 600-µm pitch) produced by a field modulator inserted into a homogeneous magnetic field of 1 T generated by an electromagnet. The cells were trapped consistent with the line profile of the modulated field. The trapping efficiency was enhanced by adding Mn(II)EDTA (paramagnetic) to the cultivation medium. The cells were subsequently incubated in the magnetic field. The same technique was applied to whole blood to pattern red blood cells.
Introduction The micropatterning of cells is one of the most important issues in cell biology and its applications.1-5 A variety of methods of micropatterning have been reported, including photolithography,6-8 soft lithography called microcontact printing,9-12 and so on.13-16 In these methods, biological, chemical, and physical modifications of the substrate surface are made in order to encourage site-specific cell adhesion. Usually, the surface treatment requires complex multiple steps. Magnetic trapping17,18 provides another route to cell micropatterning. In the previous paper,18 we demonstrated * Corresponding author. Phone: +81 426 77 2845. Fax: +81 426 77 2821. E-mail:
[email protected]. † Tokyo Metropolitan University. ‡ National Institute for Materials Science. § The University of Tokyo. (1) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425-1428. (2) Bhatia, S. N.; Balis, U. J.; Yarmush, M. L.; Toner, M. FASEB J. 1999, 13, 1883-1900. (3) Ito, Y. Biomaterials 1999, 20, 2333-2342. (4) Kaji, H.; Takii, Y.; Nishizawa, M.; Matsue, T. Biomaterials 2003, 24, 4239-4244. (5) Lee, K.-B.; Kim, D. J.; Lee, Z.-W.; Woo, S. I.; Choi, I. S. Langmuir 2004, 20, 2531-2535. (6) Kleinfeld, D.; Kahler, K. H.; Hockberger, P. E. J. Neurosci. 1988, 8, 4098-4120. (7) Rohr, S.; Fluckiger-Labrada, R.; Kucera, J. P. Eur. J. Physiol. 2003, 446, 125-132. (8) Revzin, A.; Rajagopalan, P.; Tilles, A. W.; Berthiaume, F.; Yarmush, M. L.; Toner, M. Langmuir 2004, 20, 2999-3005. (9) Kumar, A.; Whitesides, G. M. Appl. Phys. Lett 1993, 63, 2002. (10) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363-2376. (11) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5992-5996. (12) Oliva, A. A., Jr.; James, C. D.; Kingman, C. E.; Graighead, H. G.; Banker, G. A. Neurochem. Res. 2003, 28, 1639-1648. (13) Kaji, H.; Kanada, M: Oyamatsu, D.; Matsue, T.; Nishizawa, M. Langmuir 2004, 20, 16-19. (14) Zheng, H.; Berg, M. C.; Rubner, M. F.; Hammond, P. T. Langmuir 2004, 20, 7215-7222. (15) Yamato, M.; Konno, C.; Utsumi, M.; Kikuchi, A.; Okano, T. Biomaterials 2002, 23, 561-567. (16) Dillmore, W. S.; Yousaf, M. N.; Mrksich, M. Langmuir 2004, 20, 7223-7231. (17) Winkleman, A.; Gudiksen, K. L.; Ryan, D.; Whitesides, G. M.; Greenfield, D.; Prentiss, M. Appl. Phys. Lett. 2004, 85, 2411-2413. (18) Kimura, T.; Yamato, M.; Nara, A. Langmuir 2004, 20, 572-574.
that polystyrene microspheres (20 µm) suspended in a liquid are trapped into line patterns through the application of a modulated magnetic field generated by a field modulator inserted into a uniform magnetic field of ∼1 T. The advantage of this method is that no chemical or physical modification of the substrate surface is required. Any material can be used as the substrate. In addition, it is not necessary to modify the particles to be trapped, for example, by attaching ferromagnetic materials to the particles. The method presented here is applicable to any diamagnetic particles including biological particles such as the cells considered in the present study. The disadvantage of this method is that a field modulator is necessary to obtain the formation of the desired pattern. The substrate to be put on the modulator should be thin (thinner than the typical scale of the pattern size used) so that the modulated field can be effective over the substrate surface. Furthermore, in some cases, paramagnetic compounds should be added to the cultivation medium to enhance the trap. In this study, we report on the micropatterning of mouse osteoblast cells achieved by seeding the cells under a microscopically modulated magnetic field, followed by incubation. In addition, red blood cells are trapped from whole blood, being micropatterned in lines. Experimental Section Mouse osteoblast cells (MC3T3-E1 obtained from the RIKEN Cell Bank (Japan)) were cultured to confluence in 25-cm2 culture flasks containing a medium composed of Dulbecco’s modified Eagle’s medium (DMEM) (Sigma D-6046) supplemented with 10% fetal bovine serum (FBS) (Sigma F9423) and 1% antibiotic/ antimycotic (Gibco 15240-062) at 37 °C under humidified 5% CO2-95% air. The cells were trypsinized and dispersed in a 3-mL medium to obtain a cell suspension. The cells (1-mL suspension) were then seeded on a specially prepared plastic dish containing a mixture of 10 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES)-buffered medium and 0.1 M Mn(II)EDTA aqueous solution. The composition of the HEPES-buffered medium and Mn(II)EDTA solution was varied from 10:0 to 10:5 by volume. The dish was immediately exposed to the magnetic field, with the temperature kept at 37 °C by water circulation. The plastic dish used in this study was specially prepared as follows. A hole (15-mm diameter) was cut in the bottom of a
10.1021/la047517z CCC: $30.25 © 2005 American Chemical Society Published on Web 12/30/2004
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Figure 1. Schematic diagram showing the setup of a specially prepared plastic dish with a thin substrate film in the bottom, placed in close contact with the field modulator (left). The position of the modulator with respect to the direction of the field is shown (right). plastic dish (40-mm diameter). A sheet of Kapton film (8 µm thick), a sheet of Saran Wrap (10 µm thick), or a cover glass (160 µm thick) was glued to the outer surface of the bottom to cover the hole. This specially prepared dish was necessary because the modulated field persists only over a distance of about half the pitch of the modulator (100-300 µm in the present case), which is far thinner than the dish bottom (1200 µm); the modulation does not reach the bottom surface of the dish. The field modulators used were similar to that used in the previous work.18 They are composed of alternating aluminum and iron sheets 100 or 300 µm thick, forming a layer structure of periodicity of 200 or 600 µm. The modulator was fixed in the center of two pole pieces (5 cm apart) of an electromagnet, generating a horizontal field of 1 T (see Figure 1 in ref 18). A dish containing the cell suspension was placed on the modulator so that the substrate (Kapton film, Saran Wrap, or a cover glass) was in close contact with the surface of the modulator and exposed to the magnetic field of 1 T (Figure 1) for various periods of time, then removed from the magnetic field, and observed under a microscope. Human blood was collected from one of the authors just before the experiment, and ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-2Na) (1.5 mg/mL of blood) was added as an anticoagulant. A 60-µL mixture of an equal volume of the blood and an aqueous solution of 0.12 M manganese(II) chloride was poured onto a sheet of Kapton film (8 µm), which was mounted on the field modulator and placed in the magnetic field (1 T). The in situ observation was then made with a charge-coupled device (CCD) camera.
Figure 2. Micropatterning of mouse osteoblast cells on a glass substrate using a 600-µm field modulator: (a) 10 min after seeding; (b) after incubation for 6 h under the magnetic field. The distance between two lines is 600 µm. The composition of the HEPES-buffered medium and Mn(II)EDTA solution was 10:1. Enlargements of parts a and b are shown in parts a′ and b′, respectively.
Figure 3. Micropatterning of mouse osteoblast cells using a 200-µm field modulator, taken 10 min after seeding on a Saran Wrap substrate. The distance between two lines is 200 µm. The composition of the HEPES-buffered medium and Mn(II)EDTA solution was 10:1. The cells were Giemsa stained before microscopic observation.
Results and Discussion Before the patterning experiment, the cell culture was performed at various concentrations of Mn(II)EDTA without the magnetic field to estimate the tolerance of the cells to Mn(II)EDTA. The compositions of the HEPESbuffered medium and the Mn(II)EDTA solution were varied from 10:0 to 10:5. Cells grown in the 10:5 medium observed 24 h after seeding did not show any appreciable difference from those grown in the 10:0 (no Mn(II)EDTA) medium. We did not check the maximum dosage, but the 10:5 composition was sufficient to make the cells diamagnetic enough relative to the medium. The experiments were performed at a composition of 10:1. Figure 2 shows the cells patterned using the 600-µm pitch modulator. Figure 2a shows the patterning observed 10 min after seeding. Figure 2b shows the patterning observed after incubation for 6 h. The patterning was also possible with the 200-µm modulator (Figure 3). It is obvious from Figure 2b and b′ that the initially patterned cells were grown in the subsequent incubation under the influence of the field. The viability of the cells was not assessed, but the change of cell shape occurring during the incubation for 6 h clearly demonstrates that the cells are alive.
Figure 4. Schematic diagram of the modulated field profile at a distance of 100 µm above the modulator surface of the 600-µm modulator.
Figure 4 is a schematic diagram of the modulated field generated under the current experimental setup, as shown in Figure 1. Modulation does not persist for a long distance from the modulator surface. The persistence distance is about half the pitch of the modulator. Therefore, only cells floating within the persistence distance can be trapped. If a cell gets close to the substrate surface, it is strongly pulled by the magnetic force and attracted to the place where the field strength is weak. In the current setup of the modulator, where the field is horizontal, the field
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Figure 5. Micropatterning of red blood cells using a 600-µm modulator. A mixture of whole blood and 0.12 M manganese(II) chloride aqueous solution (1:1 by volume) was used. The substrate was Kapton. The distance between two lines is 600 µm. The streaks in the aluminum layers are due to the boundaries of the 100-µm aluminum foil; a 300-µm aluminum layer is composed of three sheets of the 100-µm aluminum foil.
strength is weak above the iron layers. Therefore, the cells are selectively seeded above the iron layers. The trapping speed is proportional to the magnetic force, µ0-1V∆χ∇B2/2. Here, µ0 is the magnetic permeability of the vacuum, V is the volume of a cell, ∆χ ) χc - χm, with χc and χm being the magnetic susceptibilities of the cell and the medium, respectively, B is the field strength, and ∇B2 is the field gradient created by the field modulator. The diamagnetic susceptibility of the cells (χc) and that of the medium (χm) might be very close to each other, and hence, |∆χ| is small. Trapping might become possible only by making the difference |∆χ| larger. For this purpose, we added Mn(II)EDTA (paramagnetic) to the medium. The trapping distribution19 is governed by the Boltzmann factor, exp(µ0-1V∆χ(B2 - B02)/2kBT), where kB is the Boltzmann constant, T is the temperature, and B and B0 denote the field strengths at the top and bottom of the field profile, respectively. Using the typical values assumed for the present experiment (V ) (10 µm)3, ∆χ ) -10-6, ∇B2/2 ) B∆B/∆x ) 0.75 × 0.5/(300 × 10-6) ) 1250 T2/m, B ) 1 T, B0 ) 0.5 T, and T ) 300 K), we estimate the force acting on a cell to be -1 pN and the Boltzmann factor to be zero. Figure 5 shows the patterning of red blood cells with the modulated field (600-µm pitch). The red blood cell is diamagnetic in the oxidated state, while it is paramagnetic (19) Kimura, T. Polym. J. 2003, 35, 823-843.
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in the deoxidated state. From Figure 5, we observe that the cells are trapped above the iron layers. This means ∆χ ) χc - χm < 0. The patterning was completed almost instantaneously after the magnetic field was turned on. When the magnetic field was turned off, the pattern disappeared, which is in contrast to the patterning of osteoblast cells that attach tightly onto the substrate when seeded. The use of high gradient magnetic separation (HGMS) to separate red blood cells has been reported.20,21 This technique is also used for the magnetophoresis of red blood cells.22,23 It has been reported that the red blood cell undergoes magnetic alignment with its disk plane parallel to the field, and the alignment is saturated around 4 T.24 The trapped cells could align, but the alignment is not very high because the field strength above the iron layer is estimated to be ∼0.1 T. Other components of whole blood might be trapped, but they were not covered in the present study. Conclusions A new technique of cell micropatterning that uses a microscopically modulated magnetic field was presented. Using this technique, we demonstrated the patterning of mouse osteoblast cells and red blood cells. Although this technique has some disadvantages such as limitations on the thickness of the substrate, the use of paramagnetic compounds, and the preparation of field modulators, it provides a facile means of cell micropatterning. In the present study, manganese(II) compounds were used to make the surrounding medium paramagnetic relative to the cells, with resultant enhancement of the trapping efficiency. However, the addition of paramagnetic compounds may not be required if higher fields are used. Further study of this matter is under way. Acknowledgment. This work was partially supported by a Grant-in-Aid for Scientific Research on Priority Area “Innovative utilization of strong magnetic fields” (Area 767, no. 15085207) from MEXT of Japan. LA047517Z (20) Takayasu, M.; Duske, N.; Ash, S. R.; Friedlaender, F. J. IEEE Trans. Magn. 1982, MAG-18, 1520-1522. (21) Melville, D.; Paul, F.; Roath. S. IEEE Trans. Magn. 1975, JAG11, 1701-1704. (22) Watarai, H.; Namba, M. J. Chromatogr., A 2002, 961, 3-8. (23) Zborowski, M.; Ostera, G. R.; Moore, L. R.; Milliron, S.; Chalmers, J. J.; Schechter, A. N. Biophys. J. 2003, 84, 2638-2645. (24) Higashi, T.; Yamagishi, A.; Takeuchi, T.; Kawaguchi, N.; Sagawa, S.; Onishi, S.; Date, M. 1993, Blood 82, 1328-1334.
