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Electrical characterization of human mesenchymal stem cell growth on microelectrode
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Microelectronic Engineering 85 (2008) 1272–1274 www.elsevier.com/locate/mee
Electrical characterization of human mesenchymal stem cell growth on microelectrode Sungbo Cho *, Hagen Thielecke Biohybrid Systems, Fraunhofer Institute for Biomedical Engineering, Ensheimerstr. 48, 66386 St. Ingbert, Germany Received 7 October 2007; accepted 7 January 2008 Available online 12 January 2008
Abstract To support the development of stem cell therapies with in vitro assays, non-destructive methods are required for the quality control of stem cell culture. In this article, it was investigated whether an electrode based chip with electrical impedance spectroscopy can be used to characterize the growth of human mesenchymal stem cells on electrodes without any chemical marker. From finite element method simulations, the electrical characteristics of cell layer under weak alternating electric fields were investigated with respect to the different cell/ cell or cell/substrate gap, and modelled as an equivalent circuit. The impedance spectra were measured during the long-term cultivation of human mesenchymal stem cells on platinum electrodes. By fitting analyses the equivalent circuit to measured spectra, an extra cellular resistance reflecting the cell growth was extrapolated. Ó 2008 Elsevier B.V. All rights reserved. Keywords: Finite element method; Impedance spectroscopy; Stem cell chip
1. Introduction Stem cells can renew themselves and differentiate into a diverse range of specific cell types [1]. Stem cell therapy is a promising medical technique to repair the damaged tissues and to regenerate the function of living organs. These self-renewal and unlimited potency of stem cells have been investigated by in vitro clonogenic assays [2]. Recently, the label-free in vitro assays are increasingly required due to the guarantee of cell culture conditions [3]. As a label-free method to characterize the cells, Giaever and Keese [4] have pioneered the electrical monitoring of cell behaviours on planar microelectrodes by using electrical impedance spectroscopy (EIS). The electrical characterization of cells has been used to quantify the morphological change of cells under chemical and physical stimulations [5], to test the cytotoxicity [6], or to *
Corresponding author. Tel.: +49 6894 980 274. E-mail address: [email protected] (S. Cho). URL: http://www.ibmt.fraunhofer.de (S. Cho).
0167-9317/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2008.01.004
diagnose cells infected with viruses [7]. On application of stem cell therapies, non-destructive methods for the quality control of stem cell culture are required. In this article, it was investigated whether EIS can be used to monitor the growth of human mesenchymal stem cells (hMSCs) on electrodes. For the long-term cultivation of hMSCs, the feasibility of impedance monitoring with electrode based chip was tested. To investigate the electrical characteristics of cell layer on electrodes, the finite element method (FEM) simulation was used. The measured impedance spectra were interpreted from fitting analyses with equivalent circuit derived from the FEM simulation. 2. FEM simulation and equivalent circuit Cells have an ultra thin membrane of bi-layer lipids and adhere to specific molecules on surfaces with clefts of tens to hundreds of nano-meter [8]. To design a model of cell layer for FEM simulation, it was assumed that the cells have a cylindrical shape with equivalent cell/sub-
S. Cho, H. Thielecke / Microelectronic Engineering 85 (2008) 1272–1274
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Fig. 1. Schematic model of cell monolayer on electrode for FEM simulation (not scaled), r and er: conductivity and dielectric constant, respectively (a), and equivalent circuit (b), CPEel: constant phase element for electrode impedance, Re: extra cellular resistance, CPEm: constant phase element for impedance of cell membrane, Rs: spreading resistance.
strate (h) and cell/cell gap (g) as Fig. 1a. Further, it was assumed that the cell and medium are homogenous, source-free and linear volume dielectric and that the potential distribution satisfies the generalized Laplace’s equation. The conductivity and dielectric constant of cytoplasm and cell membrane were cited from [9,10]. The current and impedance were simulated for equidistant cells on an electrode with radius of 500 lm by FEM (used software: FlexPDE, PDE Solutions Inc., Sunol, USA). The parameters h and g were varied. In the low frequency range, the current flow was affected by the gaps (h and g) due to the low conductivity of cell membrane. With increase of frequency, the current was able to penetrate the intra cellular space, and the magnitude of reactance caused by the potential distributed over the dielectric membrane was increased. Thus, the equivalent circuit of cell layer was modelled by a parallel connected extra cellular resistance (Re) and a constant phase element for cell membrane (CPEm) as Fig. 1b. A constant phase element CPEel and a resistance Rs were for the electrode impedance and for the spreading resistance, respectively. In the Nyquist plots of Fig. 2, the symbolic simulated impedances of cell layer were well fitted with circuit of Re and parallel CPEm. The resistance at the low frequencies was increased with the decrease of h or g. However, the effect of different h or g on the impedance decreased with increase of frequency. 3. Experiments and analysis For experiments, the electrode based chip (Biohybrid Systems of Fraunhofer IBMT, St. Ingbert, Germany)
was used. The chip consisted of a culture dish integrated with a 4 4 array of circular platinum electrodes with radius of 500 lm and chip carrier [7]. The planar electrode substrate of chip was fabricated by MEMS technology [11]. While hMSCs were cultivated on the electrode based substrate at 5% CO2 and 37 °C in an incubator Heraeus BB 6220 (Heraeus-Christ, Hanau, Germany), the impedance was measured from 100 Hz to 1 MHz by using an impedance analyzer Solartron 1260 (Solartron Analytical, Farnborough, UK). The level of potential was 10 mV. The medium in the chip was refreshed two times per one week. During the cultivation, the applied hMSCs adhered and spread onto the platinum electrodes. The impedance spectra measured without (No hMSCs) or with cells cultivated for 460 h (hMSCs (460 h)) were shown in the Bode plots of Fig. 3a. From the spectra, it was shown that the electrode impedance mainly contributes to the total impedance at the low frequencies below hundreds of Hz. At enough high frequency, the impedance magnitude of ‘hMSCs (460 h)’ was higher than that of ‘No hMSCs’ and decreased at the higher frequencies than hundreds of kHz. By fitting the equivalent circuit of Fig. 1b to the impedance spectra measured from different three pairs of electrodes, the extrapolated Re during the cultivation was shown in Fig. 3b. Re increased with fluctuation until 420 h of cultivation and afterwards only fluctuated without significant increases. At this time, the hMSCs proliferated enough and the density of cells on the electrode area did not increase more.
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Fig. 2. Nyquist plots of simulated impedance (FEM) with the model of Fig. 1a on an electrode with radius of 500 lm, and fitted graph (fit) with an equivalent circuit of a parallel connected resistance Re and a constant phase element CPEm with respect to cell/substrate gap (h) (a) or cell/cell gap (g) (b).
4. Discussion The platinum electrode based chip with EIS showed the feasibility and stability for the electrical characterization of hMSCs growth on electrodes during the long-term cultivation. Therefore, it is expected that the chip can be used also for non-invasive monitoring of hMSCs differentiation. The cellular adhesion did affect the flow of low frequency current due to the low conductivity of cell membrane. However, the high frequency current was able to penetrate the cell membrane and to reveal the passive cell membrane capacitance short-circuiting the membrane resistance. Thus, Re reflecting the cellular adhesion was increased correspondingly to the growth of hMSCs on the electrodes during the cultivation. As the cells proliferated on the electrode until confluence, Re was on a constant level but still fluctuated. To increase the efficiency for the treatment of cells and test substances, the electrode based cell chip will be combined with micro fluidic systems.
Fig. 3. Measured spectra (measure) without hMSCs (No hMSCs) or with cells cultured for 460 h (hMSCs (460 h)) on a pair of electrodes with radius of 500 lm and fitted graph (fit) with the equivalent circuit of Fig. 1b (a), and extra cellular resistance (Re) extrapolated by fitting analyses with three different experiments (b).
References [1] A.J. Becker, E.A. McCulloch, J.E. Till, Nature 197 (1963) 452–454. [2] A.J. Friedenstein, U.F. Deriglasova, N.N. Kulagina, A.F. Panasuk, S.F. Rudakowa, E.A. Luria, I.A. Ruadkow, Experimental Hematology 2 (1974) 83–92. [3] T.S. Hug, Assay and Drug Development Technologies 1 (2003) 479– 488. [4] I. Giaever, C. Keese, Nature 366 (1993) 591–592. [5] S. Arndt, J. Seebach, K. Psathaki, H.J. Galla, J. Wegener, Biosensors and Bioelectronics 19 (2004) 583–594. [6] C. Xiao, J.H.T. Luong, Toxicology and Applied Pharmacology 206 (2005) 102–112. [7] S. Cho, S. Becker, H. von Briesen, H. Thielecke, Sensors and Actuators B: Chemical 123 (2007) 978–982. [8] K.F. Giebel, C. Bechinger, S. Herminghaus, M. Riedel, P. Leiderer, U. Weiland, M. Bastmeyer, Biophysical Journal 76 (1999) 509–516. [9] K. Asami, Y. Takahashi, S. Takashima, Biophysical Journal 58 (1990) 143–148. [10] S. Cho, H. Thielecke, Biosensors and Bioelectronics 22 (2007) 1764– 1768. [11] I.W. Rangelow, P. Hudek, Microelectronic Engineering 27 (1995) 471–474.
Microelectronic Engineering 85 (2008) 1272–1274 www.elsevier.com/locate/mee
Electrical characterization of human mesenchymal stem cell growth on microelectrode Sungbo Cho *, Hagen Thielecke Biohybrid Systems, Fraunhofer Institute for Biomedical Engineering, Ensheimerstr. 48, 66386 St. Ingbert, Germany Received 7 October 2007; accepted 7 January 2008 Available online 12 January 2008
Abstract To support the development of stem cell therapies with in vitro assays, non-destructive methods are required for the quality control of stem cell culture. In this article, it was investigated whether an electrode based chip with electrical impedance spectroscopy can be used to characterize the growth of human mesenchymal stem cells on electrodes without any chemical marker. From finite element method simulations, the electrical characteristics of cell layer under weak alternating electric fields were investigated with respect to the different cell/ cell or cell/substrate gap, and modelled as an equivalent circuit. The impedance spectra were measured during the long-term cultivation of human mesenchymal stem cells on platinum electrodes. By fitting analyses the equivalent circuit to measured spectra, an extra cellular resistance reflecting the cell growth was extrapolated. Ó 2008 Elsevier B.V. All rights reserved. Keywords: Finite element method; Impedance spectroscopy; Stem cell chip
1. Introduction Stem cells can renew themselves and differentiate into a diverse range of specific cell types [1]. Stem cell therapy is a promising medical technique to repair the damaged tissues and to regenerate the function of living organs. These self-renewal and unlimited potency of stem cells have been investigated by in vitro clonogenic assays [2]. Recently, the label-free in vitro assays are increasingly required due to the guarantee of cell culture conditions [3]. As a label-free method to characterize the cells, Giaever and Keese [4] have pioneered the electrical monitoring of cell behaviours on planar microelectrodes by using electrical impedance spectroscopy (EIS). The electrical characterization of cells has been used to quantify the morphological change of cells under chemical and physical stimulations [5], to test the cytotoxicity [6], or to *
Corresponding author. Tel.: +49 6894 980 274. E-mail address: [email protected] (S. Cho). URL: http://www.ibmt.fraunhofer.de (S. Cho).
0167-9317/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2008.01.004
diagnose cells infected with viruses [7]. On application of stem cell therapies, non-destructive methods for the quality control of stem cell culture are required. In this article, it was investigated whether EIS can be used to monitor the growth of human mesenchymal stem cells (hMSCs) on electrodes. For the long-term cultivation of hMSCs, the feasibility of impedance monitoring with electrode based chip was tested. To investigate the electrical characteristics of cell layer on electrodes, the finite element method (FEM) simulation was used. The measured impedance spectra were interpreted from fitting analyses with equivalent circuit derived from the FEM simulation. 2. FEM simulation and equivalent circuit Cells have an ultra thin membrane of bi-layer lipids and adhere to specific molecules on surfaces with clefts of tens to hundreds of nano-meter [8]. To design a model of cell layer for FEM simulation, it was assumed that the cells have a cylindrical shape with equivalent cell/sub-
S. Cho, H. Thielecke / Microelectronic Engineering 85 (2008) 1272–1274
1273
Fig. 1. Schematic model of cell monolayer on electrode for FEM simulation (not scaled), r and er: conductivity and dielectric constant, respectively (a), and equivalent circuit (b), CPEel: constant phase element for electrode impedance, Re: extra cellular resistance, CPEm: constant phase element for impedance of cell membrane, Rs: spreading resistance.
strate (h) and cell/cell gap (g) as Fig. 1a. Further, it was assumed that the cell and medium are homogenous, source-free and linear volume dielectric and that the potential distribution satisfies the generalized Laplace’s equation. The conductivity and dielectric constant of cytoplasm and cell membrane were cited from [9,10]. The current and impedance were simulated for equidistant cells on an electrode with radius of 500 lm by FEM (used software: FlexPDE, PDE Solutions Inc., Sunol, USA). The parameters h and g were varied. In the low frequency range, the current flow was affected by the gaps (h and g) due to the low conductivity of cell membrane. With increase of frequency, the current was able to penetrate the intra cellular space, and the magnitude of reactance caused by the potential distributed over the dielectric membrane was increased. Thus, the equivalent circuit of cell layer was modelled by a parallel connected extra cellular resistance (Re) and a constant phase element for cell membrane (CPEm) as Fig. 1b. A constant phase element CPEel and a resistance Rs were for the electrode impedance and for the spreading resistance, respectively. In the Nyquist plots of Fig. 2, the symbolic simulated impedances of cell layer were well fitted with circuit of Re and parallel CPEm. The resistance at the low frequencies was increased with the decrease of h or g. However, the effect of different h or g on the impedance decreased with increase of frequency. 3. Experiments and analysis For experiments, the electrode based chip (Biohybrid Systems of Fraunhofer IBMT, St. Ingbert, Germany)
was used. The chip consisted of a culture dish integrated with a 4 4 array of circular platinum electrodes with radius of 500 lm and chip carrier [7]. The planar electrode substrate of chip was fabricated by MEMS technology [11]. While hMSCs were cultivated on the electrode based substrate at 5% CO2 and 37 °C in an incubator Heraeus BB 6220 (Heraeus-Christ, Hanau, Germany), the impedance was measured from 100 Hz to 1 MHz by using an impedance analyzer Solartron 1260 (Solartron Analytical, Farnborough, UK). The level of potential was 10 mV. The medium in the chip was refreshed two times per one week. During the cultivation, the applied hMSCs adhered and spread onto the platinum electrodes. The impedance spectra measured without (No hMSCs) or with cells cultivated for 460 h (hMSCs (460 h)) were shown in the Bode plots of Fig. 3a. From the spectra, it was shown that the electrode impedance mainly contributes to the total impedance at the low frequencies below hundreds of Hz. At enough high frequency, the impedance magnitude of ‘hMSCs (460 h)’ was higher than that of ‘No hMSCs’ and decreased at the higher frequencies than hundreds of kHz. By fitting the equivalent circuit of Fig. 1b to the impedance spectra measured from different three pairs of electrodes, the extrapolated Re during the cultivation was shown in Fig. 3b. Re increased with fluctuation until 420 h of cultivation and afterwards only fluctuated without significant increases. At this time, the hMSCs proliferated enough and the density of cells on the electrode area did not increase more.
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Fig. 2. Nyquist plots of simulated impedance (FEM) with the model of Fig. 1a on an electrode with radius of 500 lm, and fitted graph (fit) with an equivalent circuit of a parallel connected resistance Re and a constant phase element CPEm with respect to cell/substrate gap (h) (a) or cell/cell gap (g) (b).
4. Discussion The platinum electrode based chip with EIS showed the feasibility and stability for the electrical characterization of hMSCs growth on electrodes during the long-term cultivation. Therefore, it is expected that the chip can be used also for non-invasive monitoring of hMSCs differentiation. The cellular adhesion did affect the flow of low frequency current due to the low conductivity of cell membrane. However, the high frequency current was able to penetrate the cell membrane and to reveal the passive cell membrane capacitance short-circuiting the membrane resistance. Thus, Re reflecting the cellular adhesion was increased correspondingly to the growth of hMSCs on the electrodes during the cultivation. As the cells proliferated on the electrode until confluence, Re was on a constant level but still fluctuated. To increase the efficiency for the treatment of cells and test substances, the electrode based cell chip will be combined with micro fluidic systems.
Fig. 3. Measured spectra (measure) without hMSCs (No hMSCs) or with cells cultured for 460 h (hMSCs (460 h)) on a pair of electrodes with radius of 500 lm and fitted graph (fit) with the equivalent circuit of Fig. 1b (a), and extra cellular resistance (Re) extrapolated by fitting analyses with three different experiments (b).
References [1] A.J. Becker, E.A. McCulloch, J.E. Till, Nature 197 (1963) 452–454. [2] A.J. Friedenstein, U.F. Deriglasova, N.N. Kulagina, A.F. Panasuk, S.F. Rudakowa, E.A. Luria, I.A. Ruadkow, Experimental Hematology 2 (1974) 83–92. [3] T.S. Hug, Assay and Drug Development Technologies 1 (2003) 479– 488. [4] I. Giaever, C. Keese, Nature 366 (1993) 591–592. [5] S. Arndt, J. Seebach, K. Psathaki, H.J. Galla, J. Wegener, Biosensors and Bioelectronics 19 (2004) 583–594. [6] C. Xiao, J.H.T. Luong, Toxicology and Applied Pharmacology 206 (2005) 102–112. [7] S. Cho, S. Becker, H. von Briesen, H. Thielecke, Sensors and Actuators B: Chemical 123 (2007) 978–982. [8] K.F. Giebel, C. Bechinger, S. Herminghaus, M. Riedel, P. Leiderer, U. Weiland, M. Bastmeyer, Biophysical Journal 76 (1999) 509–516. [9] K. Asami, Y. Takahashi, S. Takashima, Biophysical Journal 58 (1990) 143–148. [10] S. Cho, H. Thielecke, Biosensors and Bioelectronics 22 (2007) 1764– 1768. [11] I.W. Rangelow, P. Hudek, Microelectronic Engineering 27 (1995) 471–474.