Development of a Regenerable Cell Culture System That Senses and

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Langmuir 2005, 21, 4043-4049

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Development of a Regenerable Cell Culture System That Senses and Releases Dead Cells Shuhei Okajima,† Yasuyuki Sakai,‡,§ and Takeo Yamaguchi*,† Department of Chemical System Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan Received December 7, 2004. In Final Form: February 10, 2005 We developed a rapidly regenerable cell culture system in which the cell culture substrate detects cell death and selectively releases the dead cells. This culture material was achieved by combining a detector that responds to the signal from the dead cells and an actuator to release the dead cells. Benzo-18-crown6-acrylamide (BCAm) with a pendant crown ether receptor was used as the sensor to recognize cellular signals and N-isopropylacrylamide (NIPAM) was used as the actuator. This copolymer of NIPAM and BCAm can respond to potassium ions and change its nature from hydrophobic to hydrophilic at the culture temperature of 37 °C. Living cells concentrate potassium ion internally; when cells die, potassium ions are released. The polymer surface recognizes the potassium ions released from the dead cells, the NIPAM hydrates, and the dead cells are selectively detached. This in vitro culture system is a novel one in which artificial culture materials work cooperatively with cellular metabolism by responding to this signal from the cells, thereby realizing in vitro tissue regeneration partly mimicking the mechanisms of in vivo homeostasis.

Introduction bioreactors1

organs2-4

and bioartificial In recent years, mimicking various cellular functions have been developed, although there remain unsolved problems such as their inefficacy over long periods. The main reason for this is the substantial difference between living tissue and artificial materials; that is, whether the latter are capable of spontaneous regeneration and repair. When cells attached to artificial materials die, they release inflammatory substances that damage adjacent living cells, leading to expansion of the inflammatory response5 throughout the tissue. We here describe a novel cell culture system that promotes detachment of dead cells from the cell layers cultured on the material in response to signals from the dead cell. When cells die, potassium ions are released. Normally, the intracellular concentration of potassium ions is 140 mM, while extracellularly it is 5 mM.6 When cells die, * To whom correspondence may be addressed. Telephone number: +81-3-5841-7345. Fax number: +81-3-5841-7227. E-mail address: [email protected]. † Department of Chemical System Engineering, The University of Tokyo. ‡ Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo. § Institute of Industrial Science, The University of Tokyo. (1) Sajc, L.; Grubisic, D.; Vunjak-Novakovic, G. Bioreactors for plant engineering: an outlook for further research. Biochem. Eng. J. 2000, 4 (2), 89-99. (2) Langer, R.; Vacanti, J. P. Tissue engineering. Science 1993, 260, 920-926. (3) Tan, J.; Saltzman, W. M., Biomaterials with hierarchically defined micro- and nanoscale structure. Biomaterials 2004, 25, 3593-3601. (4) Lee, K. Y.; Mooney, D. J. Hydrogels for tissue engineering. Chem. Rev. 2001, 101, 1869-1879. (5) Haslett, C.; Savill, J. S.; Whyte, M. K. B.; Stern, M.; Dransfield, I.; Meagher, L. C. Granulocyte apoptosis and the control of inflammation. Philos. Trans. R. Soc. London, Ser. B 1994, 345, 327-333. (6) Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. D. Molecular Biology of the Cell, 3rd ed.; Garland Publishing: New York, 1994; p 508.

because of the breakdown of the ATP-ADP energy transformation cycle, they cannot maintain this high internal potassium concentration through active transport and potassium ions are therefore released. In this study, a novel regenerable cell culture system was developed, as shown in Figure 1. Cells are cultured on the material’s surface,7,8 upon which a specific ion recognition polymer is grafted. Recently, hydrogels or membranes that respond to stimuli9,10 such as pH,11-13 glucose,14,15 antigens,16 and molecular signals17 have been (7) Yamaguchi, T.; Ito, T.; Sato, T.; Shinbo, T.; Nakao, S. Development of a fast response molecular recognition ion gating membrane. J. Am. Chem. Soc. 1999, 121, 4078-4079. (8) Ito, T.; Hioki, T.; Yamaguchi, T.; Shinbo, T.; Nakao, S.; Kimura, S. Development of a molecular recognition ion gating membrane and estimation of its pore size control. J. Am. Chem. Soc. 2002, 124, 78407846. (9) Sumaru, K.; Kameda, M.; Kanamori, T.; Shinbo, T. Characteristic phase transition of aqueous solution of poly(N-isopropylacrylamide) functionalized with spirobenzopyran. Macromolecules 2004, 37, 49494955. (10) Zhao, B.; Brittain, W. J. Polymer brushes: surface-immobilized macromolecules. Prog. Polym. Sci. 2000, 25, 677-710. (11) Mika, A. M.; Childs, R. F.; Dickson, J. M.; McCarry, B. E.; Gagnon, D. R. A new class of polyelectrolyte-filled microfiltration membranes with environmentally controlled porosity. J. Membr. Sci. 1995, 108 (1-2), 37-56. (12) Mika, A. M.; Childs, R. F. Acid/base properties of poly(4vinylpyridine) anchored within microporous membranes. J. Membr. Sci. 1999, 152 (1), 129-140. (13) Liu, G. J.; Lu, Z. H.; Duncan, S. Porous membranes of polysulfonegraft-poly(tert-butyl acrylate) and polysulfone-graft-poly(acrylic acid): Morphology, pH-gated water flow, size selectivity, and ion selectivity. Macromolecules 2004, 37, 4218-4226. (14) Ishihara, K.; Kobayashi, M.; Shionohara, I. Control of insulin permeation through a polymer membrane with responsive function for glucose. Makromol. Chem., Rapid Commun. 1983, 4, 327-331. (15) Kataoka, K.; Miyazaki, H.; Bunya, M.; Okano, T.; Sakurai, Y. Totally synthetic polymer gels responding to external glucose concentration: Their preparation and application to on-off regulation of insulin release. J. Am. Chem. Soc. 1998, 120, 12694-12695. (16) Miyata, T.; Asami, N.; Uragami, T. A reversibly antigenresponsive hydrogel. Nature 1999, 399, 766-769. (17) Hollman, A. M.; Bhattacharyya, D. Controlled permeability and ion exclusion in microporous membranes functionalized with poly(Lglutamic acid). Langmuir 2002, 18, 5946-5952.

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Figure 1. Design concept of the proposed new cell culture material. Cells are cultured on the surface of the polymer membrane, which changes its surface hydrophilic properties in the presence of a specific ion (potassium ion in this case). When cells die, potassium ions are released from the dead cells, the polymer recognizes the ion signal, and the NIPAM is hydrated. As a result, dead cells are selectively removed from the surface of the membrane. After the dead cell detaches, the polymer returns to the dehydrated state and the tissue is regenerated by the proliferation of surrounding living cells.

proposed. In this study, this cell culture material can respond to potassium ions as a signal released from dead cells. A designed and synthesized polymer having such characteristics is a copolymer of N-isopropylacrylamide (NIPAM) and benzo-18-crown-6-acrylamide (BCAm)18,19 with a pendant crown ether receptor. The crown pendant polymer is the sensor that recognizes the cellular signals, and NIPAM is the actuator. NIPAM has a lower critical solution temperature (LCST) of about 32 °C in water, and its hydrophilic and hydrophobic properties are altered by changes in temperature. NIPAM is hydrated below its LCST and dehydrated above its LCST, thus showing a dramatic phase change. When the crown ether captures a specific ion, the LCST shifts to a higher temperature7,20 because the guest-host complex breaks hydrogen bonds.21 Consequently, when the crown ether receptor captures potassium ions, the grafted copolymer reversibly swells and shrinks at living body temperature, which is located between the two LCSTs.7 Moreover, linear-grafted polymers show much higher mobility than cross-linked gels because of fast diffusion in the gel.7 As a result, ion-gating membranes can behave very quickly. NIPAM-grafted surfaces have often been used to promote recovery of cell layers cultured on artificial substrates without conventional enzymatic treatments, as the nature of the polymer can be controlled by changes in temperature.22-24 Cells can adhere to the hydrophobic surface, while they are unable to do so on the highly hydrated hydrophilic surface.25 Consequently, cells become detached when the temperature decreases to below the LCST. (18) Ungaro, R.; Haj, B. E.; Smid, J. Substituent effects on stability of cation complexes of 4′-substituted monobenzo crown ethers. J. Am. Chem. Soc. 1976, 98, 5198-5202. (19) Yagi, K.; Ruiz, J. A.; Sanchez, M. C. Cation binding-properties of polymethacrylamide derivatives of crown ethers. Makromol. Chem., Rapid Commun. 1980, 1, 263-268. (20) Irie, M.; Misumi, Y.; Tanaka, T. Stimuli-responsive polymers chemical-induced reversible phase-separation of an aqueous-solution of poly(N-isopropylacrylamide) with pendent crown-ether groups. Polymer 1993, 34, 4531-4535. (21) Ito, T.; Sato, Y.; Yamaguchi, T.; Nakao, S. Response mechanism of a molecular recognition ion gating membrane. Macromolecules 2004, 37, 3407-3414. (22) Yamada, N.; Okano, T.; Sakai, H.; Karikusa, F.; Sawasaki, Y.; Sakurai, Y. Thermoresponsive polymeric surfaces - control of attachment and detachment of cultured-cells. Makromol. Chem. Rapid Commun. 1990, 11, 571-576. (23) Takezawa, T.; Mori, Y.; Yoshizato, K. Cell-culture on a thermoresponsive polymer surface. Biotechnology 1990, 8, 854-856. (24) Okano, T.; Yamada, N.; Sakai, H.; Sakurai, Y. A novel recoverysystem for cultured-cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide). J. Biomed. Mater. Res. 1993, 27, 1243-1251. (25) Tamada, Y.; Ikada, Y. Fibroblast growth on polymer surfaces and biosynthesis of collagen. J. Biomed. Mater. Res. 1994, 28, 783-789.

Therefore, when cells die the polymer surface recognizes the release of potassium ions from the dead cells, the NIPAM hydrates, and the dead cells are selectively detached at constant temperature. This research is the first to use an approach in which the material responds to signals from the cells themselves. If this material can respond to cellular signals, resultant biomaterials will achieve functions highly similar to those of the living body. Furthermore, following the detachment of dead cells, tissue is regenerated through the proliferation of surrounding living cells. After the released potassium ions have diffused into the culture medium, the NIPAM chain returns to its original hydrophobic state and surrounding living cells can then multiply. In this research, first, a target ion species was added to the culture medium, and then we examined whether living cells became detached from the polymer membrane in response to the specific ion signal. After that, cell death was induced by irradiation with strong parallel ultraviolet light, and we confirmed the detachment of dead cells. Subsequently, we confirmed that potassium ions were the key signal for detachment of dead cells by adding linear poly-BCAm into the culture medium to capture the released potassium ions. Furthermore, detachment of dead cells was also achieved by adding lithium ions, as toxic agents, to damage cell membranes. Experimental Section Preparation of Cell Culture Membrane Substrate and Linear Poly-BCAm. Porous polyethylene membrane (porous PE membrane; maximum pore size ) 0.28 µm, thickness ) 100 µm) and nonporous polyethylene film (PE film; thickness ) 20 µm) were used as substrates. Plasma-graft polymerization was employed to grow linear grafted polymers onto the membrane or film.26,27 N-Isopropylacrylamide (NIPAM) was kindly provided by Kohjin Co. (Tokyo, Japan). BCAm was synthesized according to reported procedures.18,19 NIPAM-grafted membranes or films were prepared by irradiating the substrate with an argon plasma at 30 W for 60 s. The plasma-treated substrate was placed in contact with a 5% aqueous solution of monomer at 30 °C. The NIPAM-co-BCAm grafted membranes or films were prepared by irradiating the substrate as above, exposing the membrane or film to air for 60 s, and then placing them in contact with a 5% aqueous solution of monomer (NIPAM:BCAm ) 85:15) at 80 °C. Then, each membrane was washed with 50% ethanol solution. Linear poly-BCAm was synthesized using radical polymerization in dehydrated N,N-dimethylformamide (DMF) using 1.0 (26) Yamaguchi, T.; Nakao, S.; Kimura, S. Plasma-graft filling polymerization - preparation of a new type of pervaporation membrane for organic liquid-mixtures. Macromolecules 1991, 24, 5522-5527. (27) Yamaguchi, T.; Nakao, S.; Kimura, S. Evidence and mechanisms of filling polymerization by plasma-induced graft polymerization. J. Polym. Sci., Part A1 1996, 34, 1203-1208.

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Figure 2. Phase-contrast photographs of cells cultured on PE, PE-g-NIPAM, and PE-g-NIPAM-co-BCAm films. Cells were cultured for 11 days. mol % AIBN. The BCAm monomer was 100%. The synthesized polymer was purified by reprecipitation from diethyl ether. Cell Culture. Grafted and base substrates were cut into disks (diameter 20 mm) and put into 12-well culture plates. The A549 lung carcinoma cell line28 was used as a model epithelial cell because it remains as a monolayer upon reaching confluence. A549 cells were obtained from the Riken Bioresource Center (Tsukuba, Japan) and were routinely cultured in Dulbecco’s Modified Eagle medium (DMEM; Sigma, Chemical, St. Louis, MO) supplemented with 10% fetal bovine serum (Filtron, Altona, Australia), 25 mM hydroxyethylpiperazine-N′ 2-ethanesulfonic acid (HEPES; Dojindo, Kumamoto, Japan), 100 units of penicillin/ mL (Wako, Osaka, Japan), 100 µg of streptomycin/mL (Wako), and 0.25 µg of amphotericin B/mL (Sigma). Cells were subcultured using 0.25% trypsin (Gibco BRL, Grand Island, NY) in phosphatebuffered saline (PBS; Sigma). The cells were cultured on the substrates at 37 °C in 5% CO2 and 95% air, and the culture medium was changed every 1-2 days. Detachment of Living Cells by Potassium Ion Signals. Culture medium containing potassium ions for cell detachment was prepared. Its osmolality was measured and maintained in a range of 310-330 mOsm/kg, a range in which the osmolality should not cause serious toxicity. This was achieved by simple dilution of the potassium ion containing culture medium with Mill-Q water. As the control, culture medium containing sodium ion was prepared. Cells were cultured on porous PE membrane, porous PE-g-NIPAM membrane, and porous PE-g-NIPAM-coBCAm membrane for 8 days. Then, 1 mL of culture medium for cell detachment was added to cells on each membrane. After another 1 day, floating cells were collected from the culture medium by centrifugation. Cells that remained on the membranes were collected by trypsinization from the surface. Viable cell number was measured using an AP assay,29 and the cell detachment ratio was calculated from the detached and attached cell numbers. Cells were also detached by changing the temperature by 10 °C, and the cell detachment ratio was measured in the same manner. Detachment of Dead Cells following UV Irradiation. First, we confirmed the live cell ratio after UV irradiation. Cells were cultured on 12-well culture plates for 8 days and then irradiated for different times with strong parallel ultraviolet light produced by a high-voltage mercury lamp (250 W, 42 mW/cm2, Ushio Optical Modulx SX-UID250HUV). Cell cultivation was then continued, and live cell ratios were measured over time using the AP assay. Second, we checked for the detachment of dead cells. Cells were cultured to confluence on a series of synthesized membranes. Cell death was induced by irradiating with UV. After 1 day, detached cells and cells remaining on the membrane were collected by trypsinization. Attached and detached cell numbers were measured using a hemocytometer and trypan blue staining, and detached dead cell ratios calculated. Finally, we observed the detached cell morphologies on the synthesized films. Cells were cultured on PE film, PE-g-NIPAM (28) Giard, D. J.; Aaronson, S. A.; Todaro, G. J.; Arnstein, P.; Kersey, J. H.; Dosik, H.; Parks, W. P. In-vitro cultivation of human tumors establishment of cell lines derived from a series of solid tumors. J. Natl. Cancer Inst. 1973, 51, 1417-1423. (29) Yang, T. T.; Sinai, P.; Kain, S. R. An acid phosphatase assay for quantifying the growth of adherent and nonadherent cells. Anal. Biochem. 1996, 241 (1), 103-108.

film, or PE-g-NIPAM-co-BCAm film. After that, cell death was induced. Detached cells were observed by phase contrast microscopy (IX70; Olympus, Tokyo, Japan). Addition of Free Poly-BCAm. Cells were cultured on PEg-NIPAM-co-BCAm membranes. Linear poly-BCAm can dissolve in water, herein referred to as free poly-BCAm. Once cell death was induced by UV irradiation, free poly-BCAm was added into the culture medium, and the potassium ions released from the dead cells were selectively captured by the free poly-BCAm. After 1 day, detached dead cell ratios were measured using a hemocytometer and trypan blue staining. Cell Death Induced by the Addition of Lithium Ions. Cell death was induced by adding lithium ions, to demonstrate that dead cells will be detached following any method of inducing cell death. Lithium ions were added to cells to upset the osmotic balance. Cells were cultured on 12-well culture plates for 8 days, and culture medium containing lithium ions was added for 1 day. Cell cultivation was then continued in normal culture medium. Live cell ratios were measured over time using the AP assay. As for UV irradiation, detached dead cell ratios were measured after 1 day. In addition, we observed the morphology of the cells detached from the synthesized films.

Results and Discussion Cell Culture on the Synthesized Films. Cells were cultured on PE, PE-g-NIPAM, and PE-g-NIPAM-co-BCAm films for 11 days. The morphology of the cultured cells is shown in Figure 2. There is no difference between the cells on these films. It is well-known that NIPAM monomer23 and crown ether monomer30 have toxicity to cells; however, grafted NIPAM-co-BCAm polymer is not harmful to cells. It is considered that NIPAM-co-BCAm polymer is not taken up by cells. Indeed, NIPAM-coBCAm-grafted surfaces are more suitable for cell proliferation than PE films, particularly because NIPAM-coBCAm has a contact angle of around 70°,25 suitable for cell attachment at 37 °C. Living Cell Detachment by Potassium Ion Signals. Cells were cultured on porous PE membrane, porous PEg-NIPAM membrane, and porous PE-g-NIPAM-co-BCAm membrane for 8 days. In the first test, the temperature was decreased to 10 °C. The results are shown in Figure 3. Cells were detached from the NIPPM-based polymer grafted membranes. This is because the NIPAM chains were hydrated as temperature decreased to below the LCST.24 In the second test, a target ion species was added to the culture medium, and we examined whether cells become detached from the polymer membrane in response to the specific ion signals. Culture medium containing 100 mM potassium chloride was added to a series of synthesized membranes upon which cells had been cultured. As shown (30) Gad, S. C.; Conroy, W. J.; McKelvey, J. A.; Turney, R. A. Behavioral and neuropharmacological toxicology of the macrocyclic ether 18-crown-6. Drug Chem. Toxicol. 1978, 1, 339-353.

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Figure 3. Detached cell ratios in response to various signals. Cells were cultured on porous base PE membrane, porous PE-gNIPAM membrane, or porous PE-g-NIPAM-co-BCAm membrane, and ions were added at 37 °C or the temperature was changed to 10 °C. Cell numbers were measured by AP assay. Bars represent standard deviations. n ) 6.

in Figure 3, over 70% of the living cells were detached from the porous PE-g-NIPAM-co-BCAm membrane, whereas few cells were detached from the PE-g-NIPAM or base PE membranes. The crown ether captures the added potassium ion, and the LCST is shifted to a higher temperature range.21 Consequently, the polymer surface is changed to a hydrophilic state that does not support cell attachment.25 The porous PE-g-NIPAM membrane did not change its properties at a constant temperature of 37 °C, and the addition of 100 mM sodium ion caused the detachment of only a few cells from all membranes. This is because the crown ether used in this experiment cannot capture sodium ions and, therefore, the porous PE-g-NIPAM-co-BCAm membrane is not hydrated, leading to maintained cell attachment. From these results, it was clear that cells could be detached from the ion recognition membrane by adding potassium ion at a constant temperature. Cell Death by UV Irradiation. Cell death was induced using a short period of treatment with UV irradiation.31 Cells cannot maintain high internal potassium concentration after induction of necrosis, and therefore, potassium ions are released. Figure 4 shows the live cell ratios over time following UV irradiation. When cells were irradiated with UV for 1 min, the live cell ratio was increased after first decreasing. In the case of irradiation times of 2, 3, and 5 min, the live cell ratios gradually decreased with time. This is thought to be because of damage to cell membranes, proteins, and nuclei, with induction of necrosis, and because the tissue could not then repair. In this study, UV irradiation for 3 min was also considered to influence the materials. Dead Cell Detachment following UV Irradiation. Cell death was induced using UV light exposure for 3 min, and cell detachment experiments were performed. Figure 5 shows the dead cell detachment results. The detached dead cell ratio is the ratio of the number of detached dead cells to the total number of dead cells. Most dead cells were detached from the porous PE-g-NIPAMco-BCAm membrane, whereas few cells were detached from the porous PE-g-NIPAM or base PE membranes. The crown ether captures the released potassium ion from dead cells, and the LCST is shifted to a higher temperature range.7,8,21 The intracellular concentration of potassium ions is 140 mM, while extracellularly it is 5 mM.6 When potassium ions released from dead cells (31) Kato, R.; Hasegawa, K.; Hidaka, Y.; Kuramitsu, S.; Hoshino, T. Characterization of a thermostable DNA photolyase from an extremely thermophilic bacterium, Thermus thermophilus HB27. J. Bacteriol. 1997, 179, 6499-6503.

Figure 4. Live cell ratios after UV irradiation. Cells were irradiated with UV light for (b) 1 min, (0) 2 min, (2) 3 min, or (]) 5 min. Live cell ratios were measured using the AP assay. Bars represent standard deviations. n ) 6.

diffuse uniformly, the concentration of potassium ions is very low, so the ion recognition polymer cannot respond. The lowest response concentration of potassium ion is about 40 mM.21 From calculations, the concentration needed for a response near the dead cells appears to occur for only a short time. However, this ion recognition membrane can respond very quickly, because the linear grafted polymer has been applied.7 Therefore, the response occurs before the potassium ion diffuses away and the local concentration is reduced. Consequently, the polymer surface is changed to a hydrophilic state that does not support cell attachment, and dead cells detach rapidly. Some living cells were also detached from the porous PEg-NIPAM-co-BCAm membrane. This is because living cells surrounding dead cells are also detached by the hydration of the NIPAM chains. Cells were cultured on PE-g-NIPAM and PE-g-NIPAMco-BCAm films. In this experiment, a nonporous film was used as a transparent material allowing microscopic observation. Figure 6 shows phase contrast microscopy images of cell morphologies after UV irradiation. At 20 h after UV irradiation, the cells started to detach, but only from the PE-g-NIPAM-co-BCAm films. In contrast, very few cells were detached from the PE-g-NIPAM films. After 30 h, almost all cells on the PE-g-NIPAM-co-BCAm films were detached, although some cells remained attached on the surface of the PE-g-NIPAM films. Living cells

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Figure 5. (A) Cell abundance ratio. (B) Detached dead cell ratio after UV irradiation. Cells were cultured on porous PE membrane, porous PE-g-NIPAM membrane, or porous PE-g-NIPAM-co-BCAm membrane. Part A shows the ratio of attached or detached cells, and living or dead cells, to total cells. Part B shows detached dead cells to total dead cells. Bars represent standard deviations. n ) 6.

Figure 6. Phase contrast micrographs of cells after UV irradiation. Cells were cultured on PE-g-NIPAM and PE-g-NIPAM-coBCAm films.

establish a spreading epithelial morphology.32 In the case of the PE-g-NIPAM films, attached cells were observed to assume such spreading forms. Therefore, it was visually clear that cells could be rapidly detached from NIPAMco-BCAm film. Confirmation of the Key Signal for Dead Cell Detachment. The potassium ions released from dead cells can be captured by adding free poly-BCAm to the culture medium. In this culture system, the substance selectively captured by the poly-BCAm is potassium ions only. Figure 7 shows detached dead cell ratios following UV light irradiation and the addition of different concentrations of poly-BCAm. The detached dead cell ratios decreased with increasing concentrations of poly-BCAm. When poly(32) Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y., Mechanism of Cell Detachment from Temperature-Modulated, Hydrophilic-Hydrophobic Polymer Surfaces. Biomaterials 1995, 16 (4), 297303.

BCAm was added to over 60 mM, the result was almost the same as for the PE-g-NIPAM membranes, which do not have sensors (Figure 5). Therefore, when cells died but the potassium ions were sequestered, few dead cells were detached from the PE-g-NIPAM-co-BCAm membrane. In other words, when the crown ether sensor of NIPAM-co-BCAm recognizes potassium ions, NIPAM chains are hydrated and dead cells are detached from the NIPAM-co-BCAm membranes. We thus confirmed that potassium ions are the key for dead cell detachment. Cell Death Induced by Adding Lithium Ions. We confirmed that dead cells were detached from the porous PE-g-NIPAM-co-BCAm membrane when the potassium ion signal was released from dead cells. In this experiment, we applied a second method of inducing cell death to demonstrate that dead cells can be detached by any method of inducing cell death. Lithium ion was chosen as the toxic

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Figure 7. Dead cell detachment following UV irradiation, as a function of the added free poly-BCAm concentration. Cells were cultured on porous PE-g-NIPAM-co-BCAm membranes. Detached dead cell ratio is the ratio of the detached dead cell number to the total dead cell number. Bars represent standard deviations. n ) 6.

Figure 8. Living cell ratio after adding lithium ion. The culture medium containing (b) 100 mM, (0) 150 mM, or (2) 200 mM lithium ion was added. The cell number was measured with time by the AP assay. Bar represents standard deviations. n ) 6.

agent because it damages cell membranes by changing the osmotic balance over long periods of treatment.6 NIPAM chains are not affected by lithium ions, unlike

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organic agents, and it is known that 18-crown-6 does not capture lithium ions7,8,21 because of their size. Cell death was induced by adding lithium ions to the culture medium. Cells could not maintain the osmotic pressure, cell membranes were broken, and potassium ion was released from the dead cells. Figure 8 shows the live cell ratio over time after adding lithium. In the case of lithium ion at 100 mM, the cells were regenerated within 4 days. However, when over 150 mM lithium ion was added, cells gradually died. Cells were dying locally in the cultured cell sheet, and cell death was finally induced in almost all cells. Following this experiment, we did the cell detachment experiments with 150 mM lithium ion, as few dead cells were detached naturally. Dead Cell Detachment Induced by Adding Lithium Ion. Cell death was induced by adding lithium chloride. The results are shown in Figure 9. Almost all dead cells were detached from the porous PE-g-NIPAMco-BCAm membrane, whereas few cells were detached from the porous PE-g-NIPAM or base PE membranes. This is because the crown ether captures the released potassium ion signal from the dead cells, the LCST is shifted to a higher temperature range,7,8,21 and the NIPAM chains are hydrated. Cells were cultured on PE-g-NIPAM and PE-g-NIPAMco-BCAm films. Figure 10 shows the cell morphologies seen by phase contrast microscopy after the addition of lithium ions. One day after adding lithium ions, cells were detached from the PE-g-NIPAM-co-BCAm films. In contrast, very few cells were detached from the PE-g-NIPAM films, and most of the cells were attached to the surface of the PE-g-NIPAM films. After 2 days, cells were gradually detached from the PE-g-NIPAM films; however, the difference is clear. These micrographs represent the results shown in Figure 9 that almost all dead cells were detached. From these results, dead cells can be detached selectively from the ion recognition membrane following induction of cell death by the two methods that release potassium ions. Therefore, when the NIPAM-co-BCAm membrane responds to the potassium ion signal, dead cells can be detached rapidly, regardless of the method by which cell death was induced. When cultured cells are damaged and cells die locally, rapid repair is expected by the active and selective removal of dying cells in each damaged part of the PE-g-NIPAM-co-BCAm surface. Therefore, this material will be able to inhibit the release of inflammatory substances33-38 from damaged cells. This material can be applied in many fields, as it can respond very quickly and cell detachment occurs under wide-ranging conditions.

Figure 9. (A) Cell abundance ratio. (B) Detached dead cell ratio after adding lithium ions. Cells were cultured on porous PE membrane, porous PE-g-NIPAM membrane, or porous PE-g-NIPAM-co-BCAm membrane. Part A shows the ratios of attached or detached cells, and living or dead cells, to total cells. Part B shows the ratio of detached dead cells to total dead cells. Bars represent standard deviations. n ) 6.

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Figure 10. Phase contrast micrographs of cells after the addition of lithium ions. Cells were cultured on PE-g-NIPAM and PE-g-NIPAM-co-BCAm films.

Conclusion Cells can be cultured on grafted NIPAM-co-BCAm copolymer material, which does not have toxic effects. Living cells can be detached from the PE-g-NIPAM-coBCAm membrane by the addition of potassium ions, which is a signal from dead cells. Cell death was induced by UV irradiation. Cells gradually die locally, and cell death is induced in almost all damaged cells. Dead cells were selectively detached from the PE-g-NIPAM-co-BCAm membranes immediately after cell death. Detachment was induced in response to (33) Ohzato, H.; Yoshizaki, K.; Nishimoto, N.; Ogata, A.; Tagoh, H.; Monden, M.; Gotoh, M.; Kishimoto, T.; Mori, T. Interleukin-6 as a new indicator of inflammatory status - detection of serum levels of interleukin-6 and c-reactive protein after surgery. Surgery 1992, 111 (2), 201-209. (34) Burvall, K.; Palmberg, L.; Larsson, K. Effects by 8-bromocyclicAMP on basal and organic dust-induced release of interleukin-6 and interleukin-8 in A549 human airway epithelial cells. Respir. Med. 2003, 97 (1), 46-50. (35) Hetland, R. B.; Cassee, F. R.; Refsnes, M.; Schwarze, P. E.; Lag, M.; Boere, A. J. F.; Dybing, E. Release of inflammatory cytokines, cell toxicity and apoptosis in epithelial lung cells after exposure to ambient air particles of different size fractions. Toxicol. in Vitro 2004, 18 (2), 203-212. (36) Tilg, H.; Dinarello, C. A.; Mier, L. W. IL-6 and APPs: antiinflammatory and immunosuppressive mediators. Immunol. Today 1997, 18, 428-432.

potassium ion release from the dead cells. We also confirmed, by adding free poly-BCAm, that the crown ether selectively captures potassium ions. Cell death was also induced by adding lithium ions. It was thus clear that dead cells could be detached from the ion recognition membrane following two methods of cell death induction that release a potassium ion signal. This ion recognition membrane can respond very quickly, and the response occurs before the potassium ions diffuse and the local concentration falls under that needed for a response. Therefore, this material can be applied in many fields, as cell detachment occurs in wide-ranging conditions. This study demonstrates a pioneering approach in which a synthetic substrate has been designed to recognize cellular signals and to work actively within living systems in the novel field of tissue engineering. LA046994E (37) Xing, Z.; Gauldie, J.; Cox, G.; Baumann, H.; Jordana, M.; Lei, X. F.; Achong, M. K. IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J. Clin. Invest. 1998, 101, 311-320. (38) Benbaruch, A.; Michiel, D. F.; Oppenheim, J. J. Signals and receptors involved in recruitment of inflammatory cells. J. Biol. Chem. 1995, 270, 11703-11706.