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Culture of mammalian cells on polypyrrole-coated ITO as a biocompatible electrode
Synthetic
ELSEVIER
Culture of mammalian T.Aokia),
M.Taninoa),
Metals
71 (1995) 2229-2230
cells on polypyrrole-coated
K.Sanuia),
N.Ogataa),
K.Kumakurab),
IT0 as a biocompatible T.Okanoc),
YSakuraic)
electrode
and M.Watanabed)
a)Dept. of Chem., bkife Sci. Inst., Sophia University, 7-1 Kioi-cho, Chiyoda, Tokyo 102, Japan C)Inst. of Biomed. Eng., Tokyo Women’s Medical College 8 Kawada-cho, Shinjuku, Tokyo 162, Japan d)Dept. of Chem., Yokohama National University 156 Tokiwadai, Hodogaya, Kanagawa 240, Japan Abstract Primary culture of the chromaffin cells on polypyrrole-coated indium-tin oxide was performed and the influence of polypyrroles having some kinds of dopants on cell function was investigated. While the cells cultured on indium-tin oxide showed decrease of catecholamine release, those on polypyrrole-coated iridium-tin oxide maintained tbe cell function of release of catecholamines. Furthermore, no influence of various dopants on the secretory function from the cultured cells was observed. To prepare polypyrrole on electrode as a cell-culture substrate was considered to be available for supporting the cell function. 1. INTRODUCTION We are interested in having communication with mammalian cells, tissue and organs. Especially, it is interesting to construct a sensor for receiving communicational signals from nerve cells, since the nervous system governs reaction to stimuli, processes information and generates various patterns of signals to control complex behaviors. An electrochemical detection is thought to be useful for receiving the signals such as catecholamines, because it is suitable for real-time and/or high sensitive detections of the biological substances and catecholamines are electrochemically oxidized. We pay attentions here to achieve the real-time monitoring system for the communicational signals from the cells cultured on an electrode. Namely, by culturing the cells on an electrode, signals such as neurotransmittances from the cells on it may be directly and electrochemically detected, This research aims evaluating whether an electrode as a sensor is suitable for the cell-culture substratum or not. On the other hands, polypyrrole is readily obtained on an electrode by electrochemical polymerization and its electrochemical behavior depends on the mobility of a doping anion. Therefore, it is also of interest to study that the relationship between polypyrrole and mammalian cells. Although the interactions between polypyrrole and erythrocytesl) or endothelial cell& have been reported, the influence of an electrode surface on the cell functions is not understood. In the preliminary study3), both indium-tin oxide (ITO) and polypyrrole were picked up 0379-6779/95/%09.50 0 1995 Elsevier Science S.A. All rights reserved SSDI 0379-6779(94)03235-X
and the primary culture of adrenal chromaffm cells on the polypyrrole-coated IT0 and immunostaining of the cells with anti-tyrosinhydroxylase antibodies were performed. It was found that the cells adhered on the polymer film and survived, without significant morphological changes. In this paper, the influence of electrodes on cell function was evaluated by catecholamine(CA) assay, since the chromaffin cells synthesize and release CA. 2. MATERIALS
AND METHODS
Polypyrrole for cell-culture substratum was prepared by electro-oxidation polymerization in the aqueous solution containing O.lM of freshly distilled pyrrole and 2wt% of poly(acrylic acid) (PAAc) or poly(styrene sulphonate) sodium salt (PSS). Electrodepositions were carried out at a constant current of 1.19mA/cm2 using IT0 electrodes. Adrenal chromaffin cells were isolated from bovine adrenal glands by collagenase digestion4). The chromal5n cells were plated on each substratum in modified eagle medium (DMEM) Dulbecco’s supplemented with 10% fetal calf serum (FCS). Culture dishes were incubated in a humidified incubator with 5% CO2 / 95% air at 37°C for 7days. The culture medium was changed every 3days and the cells were observed by phase-contrast microphotographs. After stimulating the cultured cells by adding acetylcholine (ACh) solution into each well, CAs were extracted with 0.4M perchlori& acid and assayed by high-performance liquid chromatography connecting an electrochemical detection.
T. Aoki et al. / Synthetic Metals 71 (1995) 2229-2230
2230
Fin.1 Microphotographs of the chromaffin 7&ys. Magnificat& 150 x.
cells cultured on both (a) PPy/PSS and (1~)PPyfPAAc coated IT0 substrata for
15 :3. RE:SLLTS
ANI) DISCUSSION
G-
; -
PPy/PSS
:
Since the chromaffin cells have been widely studied in terms of their catecholamine secretory function, the cells were used in this study. In the previous study5), it was found that polypyrrole protected the chromaffin
/ sodium chloride (PPy/Cl-) cells from the toxicity of ITO.
However, PPy/Cl- had a rough surface and the secretory rate from the cells on it exhibited a large error in the data. Therefore, polypyrrole films having polyanions were tested for cell culture. Fig.1 shows the photomicrograph of the chromaffin cells culturerl on PPyJPSS and PPyiPAAcfor 7ciays. The chromaffin cells exhibited good adhesions on them without appreciable differences in between morphologies of the cells on these materials and those on collagen as a control. It was noted that both PPy/PSS- and PPy/PAAc- gave no cell-shape changes. It is known that the chromaffin cells show a secretory response to ACh. The secretory function of CA from the cultured chromaffin cells was investigated. Fig.2 shows the influence of substrata on the dose-response curve for ACh-evoked release of CA. Release rate of the cells cultured on PPy/pSS- was significantly higher value than that on ITO. These results reveal that the cells on IT0 show a poor responsiveness to ACh 5 ) and that those on PPyiPSS- maintain the secretory function. In conclusion, it is noted that when the cells adhered the foreign materials such as electrodes, the secretory function depends on the surfaces of electrodes and that PPy plays a role to maintain the secretory function of the cells cultured on It.
6
4 5 -Log(AC h/M)
3
Fig.2 The influence of substrate on dose-response curves for ACh-evoked release CA from cultured cells for ‘Idays.
I. H. Shinohara, J. Kojima, M. Yaolta and M. Aizawa, Bioelectrochem. Bioenerg., 22, (1989) 23. 2. J. Y. Wang, R. Langer and D. G. Ingher, Proc. Natl. Acad. Sci. USA, 91, (1994) 3201. 3. T. Aoki, K. Kitagawa, M. Watanabe, K. Sanui, N. Ogata, M. Imaizumi, K. Kumakura, T. Okano and Y. Sakurai, Polym. Prepr., Jpn (Eng Ed.), 41, (1992) E201. 4. M. Ohara-Imaizumi, Y. Miyakawa and K. Kumakura, Neurosci. L&t., 93, (1988) 294. 5. T. Aoki, et al., in preparation.
ELSEVIER
Culture of mammalian T.Aokia),
M.Taninoa),
Metals
71 (1995) 2229-2230
cells on polypyrrole-coated
K.Sanuia),
N.Ogataa),
K.Kumakurab),
IT0 as a biocompatible T.Okanoc),
YSakuraic)
electrode
and M.Watanabed)
a)Dept. of Chem., bkife Sci. Inst., Sophia University, 7-1 Kioi-cho, Chiyoda, Tokyo 102, Japan C)Inst. of Biomed. Eng., Tokyo Women’s Medical College 8 Kawada-cho, Shinjuku, Tokyo 162, Japan d)Dept. of Chem., Yokohama National University 156 Tokiwadai, Hodogaya, Kanagawa 240, Japan Abstract Primary culture of the chromaffin cells on polypyrrole-coated indium-tin oxide was performed and the influence of polypyrroles having some kinds of dopants on cell function was investigated. While the cells cultured on indium-tin oxide showed decrease of catecholamine release, those on polypyrrole-coated iridium-tin oxide maintained tbe cell function of release of catecholamines. Furthermore, no influence of various dopants on the secretory function from the cultured cells was observed. To prepare polypyrrole on electrode as a cell-culture substrate was considered to be available for supporting the cell function. 1. INTRODUCTION We are interested in having communication with mammalian cells, tissue and organs. Especially, it is interesting to construct a sensor for receiving communicational signals from nerve cells, since the nervous system governs reaction to stimuli, processes information and generates various patterns of signals to control complex behaviors. An electrochemical detection is thought to be useful for receiving the signals such as catecholamines, because it is suitable for real-time and/or high sensitive detections of the biological substances and catecholamines are electrochemically oxidized. We pay attentions here to achieve the real-time monitoring system for the communicational signals from the cells cultured on an electrode. Namely, by culturing the cells on an electrode, signals such as neurotransmittances from the cells on it may be directly and electrochemically detected, This research aims evaluating whether an electrode as a sensor is suitable for the cell-culture substratum or not. On the other hands, polypyrrole is readily obtained on an electrode by electrochemical polymerization and its electrochemical behavior depends on the mobility of a doping anion. Therefore, it is also of interest to study that the relationship between polypyrrole and mammalian cells. Although the interactions between polypyrrole and erythrocytesl) or endothelial cell& have been reported, the influence of an electrode surface on the cell functions is not understood. In the preliminary study3), both indium-tin oxide (ITO) and polypyrrole were picked up 0379-6779/95/%09.50 0 1995 Elsevier Science S.A. All rights reserved SSDI 0379-6779(94)03235-X
and the primary culture of adrenal chromaffm cells on the polypyrrole-coated IT0 and immunostaining of the cells with anti-tyrosinhydroxylase antibodies were performed. It was found that the cells adhered on the polymer film and survived, without significant morphological changes. In this paper, the influence of electrodes on cell function was evaluated by catecholamine(CA) assay, since the chromaffin cells synthesize and release CA. 2. MATERIALS
AND METHODS
Polypyrrole for cell-culture substratum was prepared by electro-oxidation polymerization in the aqueous solution containing O.lM of freshly distilled pyrrole and 2wt% of poly(acrylic acid) (PAAc) or poly(styrene sulphonate) sodium salt (PSS). Electrodepositions were carried out at a constant current of 1.19mA/cm2 using IT0 electrodes. Adrenal chromaffin cells were isolated from bovine adrenal glands by collagenase digestion4). The chromal5n cells were plated on each substratum in modified eagle medium (DMEM) Dulbecco’s supplemented with 10% fetal calf serum (FCS). Culture dishes were incubated in a humidified incubator with 5% CO2 / 95% air at 37°C for 7days. The culture medium was changed every 3days and the cells were observed by phase-contrast microphotographs. After stimulating the cultured cells by adding acetylcholine (ACh) solution into each well, CAs were extracted with 0.4M perchlori& acid and assayed by high-performance liquid chromatography connecting an electrochemical detection.
T. Aoki et al. / Synthetic Metals 71 (1995) 2229-2230
2230
Fin.1 Microphotographs of the chromaffin 7&ys. Magnificat& 150 x.
cells cultured on both (a) PPy/PSS and (1~)PPyfPAAc coated IT0 substrata for
15 :3. RE:SLLTS
ANI) DISCUSSION
G-
; -
PPy/PSS
:
Since the chromaffin cells have been widely studied in terms of their catecholamine secretory function, the cells were used in this study. In the previous study5), it was found that polypyrrole protected the chromaffin
/ sodium chloride (PPy/Cl-) cells from the toxicity of ITO.
However, PPy/Cl- had a rough surface and the secretory rate from the cells on it exhibited a large error in the data. Therefore, polypyrrole films having polyanions were tested for cell culture. Fig.1 shows the photomicrograph of the chromaffin cells culturerl on PPyJPSS and PPyiPAAcfor 7ciays. The chromaffin cells exhibited good adhesions on them without appreciable differences in between morphologies of the cells on these materials and those on collagen as a control. It was noted that both PPy/PSS- and PPy/PAAc- gave no cell-shape changes. It is known that the chromaffin cells show a secretory response to ACh. The secretory function of CA from the cultured chromaffin cells was investigated. Fig.2 shows the influence of substrata on the dose-response curve for ACh-evoked release of CA. Release rate of the cells cultured on PPy/pSS- was significantly higher value than that on ITO. These results reveal that the cells on IT0 show a poor responsiveness to ACh 5 ) and that those on PPyiPSS- maintain the secretory function. In conclusion, it is noted that when the cells adhered the foreign materials such as electrodes, the secretory function depends on the surfaces of electrodes and that PPy plays a role to maintain the secretory function of the cells cultured on It.
6
4 5 -Log(AC h/M)
3
Fig.2 The influence of substrate on dose-response curves for ACh-evoked release CA from cultured cells for ‘Idays.
I. H. Shinohara, J. Kojima, M. Yaolta and M. Aizawa, Bioelectrochem. Bioenerg., 22, (1989) 23. 2. J. Y. Wang, R. Langer and D. G. Ingher, Proc. Natl. Acad. Sci. USA, 91, (1994) 3201. 3. T. Aoki, K. Kitagawa, M. Watanabe, K. Sanui, N. Ogata, M. Imaizumi, K. Kumakura, T. Okano and Y. Sakurai, Polym. Prepr., Jpn (Eng Ed.), 41, (1992) E201. 4. M. Ohara-Imaizumi, Y. Miyakawa and K. Kumakura, Neurosci. L&t., 93, (1988) 294. 5. T. Aoki, et al., in preparation.