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Intracellular calcium-expression-display (ICED) device operated by compressive stimulation of cells
Microelectronic Engineering 98 (2012) 703–706
Contents lists available at SciVerse ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
Intracellular calcium-expression-display (ICED) device operated by compressive stimulation of cells Tae Kyung Kim, Ok Chan Jeong ⇑ Department of Biomedical Engineering, Inje University, Gimhae 621-749, Republic of Korea
a r t i c l e
i n f o
Article history: Available online 15 June 2012 Keywords: Cell chip Compressive stress Intracellular calcium-expression-display (ICED)
a b s t r a c t The effects of steady compressive stimulation on intracellular calcium expression in MG-63 human osteoblast-like bone cells were examined using a fabricated micro cell chip with a microchannel array. A computer-controlled pneumatic system was used to create compressive stress as a source of mechanical stimulation without fluid flow in the microchannel. Intracellular calcium levels were observed using laser-scanning fluorescence microscopy and showed multiple peaks of intracellular calcium expression over time. To visualize the mechanosensory properties of live cells suggested by the observed periodic intracellular calcium expression, we designed and fabricated a seven-segment-type of intracellular calciumexpression-display (ICED) device. The device consisted of a glass cell-culture substrate, two polydimethylsiloxane layers for cell introduction and pneumatic stimulation, and seven individually controllable pneumatic actuators. Compressive stimulation was applied to the cells via deformation of the pneumatic actuator diaphragm. The application of external pressure to the ICED device resulted in a green fluorescence representation of intracellular calcium expression in the form of an ‘‘8’’. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Physical and mechanical stimuli, such as compression, shear stress, strain, stretching, and hydraulic force, are important in cell proliferation and differentiation [1–3]. Microelectromechanics research has led to the design and fabrication of a micro cell stimulator for applying compressive stress to human mesenchymal stem cells [4]. In addition, a macro system for shear and compression stimulation has been developed [3]. Compared with a typical macro system, microfluidics technologies have several advantages: they minimize fluid volumes and the numbers of cell culture surfaces and samples required, reduce procedure times, and allow for the use of microincubators in cell chips [4]. Thus, micro cell chips are more effective than, and preferable to, macro systems for studying the effects of mechanical stimulation on cells [5]. Studies examining the role of cyclic mechanical stimulation in cell differentiation have provided evidence of its importance; however, the reported macro- and micro-system experiments vary greatly in their magnitudes of applied stimulation. Furthermore, the cell refractory period should be taken into consideration when selecting the duration of the mechanical stimulus [6]. A quantitative study of the optimal or appropriate magnitude and period of mechanical stimulation is necessary. ⇑ Corresponding author. E-mail address: [email protected] (O.C. Jeong). 0167-9317/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mee.2012.05.035
In the present study, a micro cell chip with a polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, Inc.) microchannel array and a glass cell-culture substrate was fabricated using a replica molding [7] and bonding process. To determine the appropriate duration for mechanical stimuli, we observed intracellular calcium expression in MG-63 human osteoblast-like bone cells under compressive stimulation in the micro cell chip. We found that periodic compressive force applied using this chip could induce periodic increases in intracellular calcium levels in the enclosed cells. We then fabricated an intracellular calcium-expression-display (ICED) device operated by compressive stimulation to demonstrate mechanotransduction in the cells. The cells were successfully induced to blink using individually controllable compressive stimulators.
2. Experimental methods 2.1. Micro cell chip The fabricated micro cell device consisted of a PDMS fluidicchannel array for applying compressive stress and a 120 lm-thick cover glass that served as a cell-culture substrate and permitted optical observation (Fig. 1). The microchannel was 1 mm wide, 0.4 mm high, and 15 mm long. The cell chip was fabricated from PDMS using soft lithography and pierced to produce two fluidic ports. The chip was then bonded
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T.K. Kim, O.C. Jeong / Microelectronic Engineering 98 (2012) 703–706
2.2. Experimental system The experimental set-up was comprised of a lab-designed pneumatic supplying system and a microscope. A computer-controlled electromagnetic valve was used to control the application frequency of external compressed air. The supplied air pressure was monitored over time using a precision pressure sensor. Intracellular calcium levels in the cells on the cell chip were observed using laser-scanning fluorescence microscopy (LSM 510 META; Carl Zeiss, Oberkochen, Germany). The excitation wavelength of Argon-ion laser for the green-fluorescent calcium indicator was 488 nm and the observed wavelength range were 500–570 nm. 3. Results and discussion 3.1. Compressive stress-induced intracellular calcium expression Fig. 1. Fabricated micro cell chip consisting of a polydimethylsiloxane (PDMS) channel array and a glass cell-culture substrate. Compressive stress is applied via by compressed air through the pneumatic ports, which are indicated by a circled ‘‘P’’.
to the glass substrate using atmospheric pressure plasma technology [8]. There are some advantages to the use of atmospheric pressure plasma like dry process, simple treatment process in the air, and several seconds of the surface modification [9]. After fabrication, the cell chip was sterilized by ultraviolet irradiation, and the glass surface was coated with fibronectin. The chip was seeded with MG-63 cells (1.0 105 cells/mL), incubated for 24 h, and stained with 10 M Fluo-4/AM, a fluorescent calcium indicator. To apply pure compressive stress without fluid flow to cells seeded on the glass substrate, compressive pressure was applied equally and simultaneously to the two cell/media ports in the micro channels.
Captured fluorescence images showing the levels of intracellular calcium before and after the application of a 25 kPa compressive load to the micro cell chip are shown in Fig. 2. The fluorescence intensity of the cells increased dramatically when the compressive stress was applied and then decreased over time. Moreover, the steady mechanical stimulation resulted in multiple peaks of intracellular calcium expression over time, similar to the case of the fluid-flow induced shear stress [10]. This phenomenon may has been related to the duration of intracellular calcium expression and release from intracellular stores into the cytoplasm as a result of extracellular mechanical stress [11,12]. 3.2. ICED device The periodicity of the cellular response to steady compressive stimulation led us to fabricate and test a seven-segment ICED
Fig. 2. Relative intracellular calcium levels in a micro cell culture indicated by green fluorescence. Individual cells (a-1) in the observation area (900 lm 900 lm) and the corresponding surface plots (a-2) are shown at rest (b-1) and after the application of compressive stress (b-2). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
T.K. Kim, O.C. Jeong / Microelectronic Engineering 98 (2012) 703–706
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Fig. 3. The seven-segment intracellular calcium-expression-display (ICED) device. (a) A mounted device. (b) Enlarged view of a seven-segment cell chamber.
Fig. 4. Images of (a) the deformed diaphragm of the pneumatically driven compressive stimulator and (b) the corresponding three-dimensional fluorescence profile indicating intracellular calcium expression.
device for visualizing the changes in intracellular calcium expression in response to compressive stimulation (Fig. 3a). The ICED device contained a PDMS chamber for cell introduction, a PDMS chamber for pneumatic stimulation having seven individually controllable pneumatic channels, and a glass cell-culture substrate. To construct the replica mold for PDMS structures, a photosensitive epoxy (SU-8) mold was fabricated through typical photolithography process. The process temperature and time for soft bake and post expose bake were 65 °C for 6 min and 95 °C for 10 min, respectively. A PDMS was mixed, stirred, and degassed in a vacuum chamber. The mixing ratios of base polymer to curing agent was 10:1. The prepared liquid PDMS mixture was poured into the SU-8 molds, spun, and cured at 75 °C for 10 min [13]. The depth, width, and length of the SU-8 mold for cell chambers were 40, 300, and 860 lm, respectively. The channels for the pneumatic and fluidic connections near the cell chambers were 40 lm wide. The PDMS diaphragm for applying mechanical compressive stress was 60 lm thick. After the pneumatic stimulation layer was peeled off the SU-8 mold, surfaces of PDMS layers for both cell chamber and pneumatic stimulation were treated with atmospheric pressure plasma for several seconds. The pneumatic stimulation layer was aligned and lowered onto the plasma-treated PDMS layer for the cell chamber. The stacked PDMS layers were placed on a hot-plate and then heated at 120 °C for 30 min. After
the bonding process was complete, the structure was peeled off the SU-8 mold and 20 holes were punched out to form the pneumatic and fluidic ports. The bonded PDMS layers were then bonded to a cover glass substrate for cell seeding and growth using the sequential bonding process like atmospheric plasma treatment and directing heating process [14]. An enlarged view of the seven-segment cellular device and its cross-sectional view (AA0 ) are shown in Fig. 3b. Images of the seven-segment device with diaphragms deformed in the shapes of a an ‘‘8’’ by 125 kPa of applied pressure are shown in Fig. 4a. The corresponding green fluorescence images indicating intracellular calcium expression are shown in Fig. 4b. To visualize the mechanosensory properties of the cells, the fluorescence intensity was converted into an interactive three-dimensional surface plot using ImageJ. This plot showed that most of the cells responded well to steady compressive stimulation, although their response times varied. 3.3. Simulation of compressive stress in the ICED Because the ICED device transferred compressive stress to cells through the deformation of the PDMS diaphragm, the actual amount of compressive stress on the cells in the device was difficult to determine. However, to estimate the fluid pressure on the
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Fig. 5. Fluid structure interaction analysis of the effect of the pneumatically driven compressive stimulators on ICED device. The nonlinear fluidic pressure distribution induced by the structural deformation of the pneumatic actuator was simulated using FEMLAB. (a) Model of the ICED device. (b) Deformation of PDMS diaphragm induced by a 125 kPa load. (c) Pressure distribution of fluid in the cell chamber. (d) Results of the final analysis of structural deformation of the diaphragm and the pressure distribution in the fluid film.
cells, the multiphysical effects of the compressive stimulation were simulated using the FEMLAB software package (Comsol, Inc., Burlington, MA, USA). Fig. 5 shows the application of compressed air to the device (Fig. 5a), the deformation of the diaphragm (Fig. 5b), and the subsequent application of compressive stress or pressure to the cells seeded in the bottom of the cell chamber (Fig. 5c). The maximum displacement of the stimulator diaphragm was 36.6 lm, and the generated pressure of the fluid in the chamber was 365.5 Pa. It was equivalent to the maximum compressive stress for stimulating cells. From simulation results, the actual compressive stress experienced by the cells was much lower than the pressure applied to the stimulator since most of the applied pressure was used to structurally deform the thick PDMS diaphragm.
4. Conclusions This paper presents a visualization of mechanosensory properties in live MG-63 cells using microfluidics technology and an ICED device fabricated using soft lithography. First, a micro cell chip containing a microchannel array was fabricated to apply compressive stimulation to the cells, and intracellular calcium levels were measured. The cells were found to be sensitive to the external mechanical compressive stress. Under steady compressive stimulation, the calcium levels varied cyclically. Next, an ICED device with a seven-segment display similar to those used in electrical
engineering was fabricated and successfully used to demonstrate the visualization of mechanosensory properties in cells. Acknowledgments This research was supported by the Basic Science Research Program of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2009-0076333). References [1] C.R. Jacobs, C.E. Yellowley, B.R. Davis, Z. Zhou, J.M. Cimbalan , H.J. Donahue, J. Biomech. 31 (1998) 969–976. [2] A.M. Sorkin, K.C. Dee, M.L. Knothe Tate, Am. J. Physiol. Cell Physiol. 287 (2004) C1527–C1536. [3] E.H. Frank, M.S. Jin, A.M. Loening, M.E. Levenston, A.J. Grodzinsky, J. Biomech. 33 (2000) 1523–1527. [4] W.Y. Sim, S.W. Park, S.H. Park, B.H. Min, S.R. Park, S.S. Yang, Lab Chip 7 (2007) 1775–1782. [5] X.E. Guo, E. Takai, X.J. Jiang, Q. Xu, G.M. Whitesides, J.T. Yardley, C.T. Hung, E.M. Chow, T. Hantschel, K.D. Costa, Mol. Cell Biomech. 3 (3) (2006) 95–107. [6] S.W. Donahue, H.J. Donahue, C.R. Jacobs, J. Biomech. 36 (2003) 35–43. [7] Y. Xia, G.M. Whitesides, Annu. Rev. Mater. Sci. 28 (1998) 153–184. [8] S.M. Hong, S.H. Kim, J.H. Kim, H.I. Hwang, J. Phys: Conf. Ser. 34 (2006) 656–666. [9] H.T. Kim, O.C. Jeong, Microelectron. Eng. 88 (2011) 2281–2285. [10] J.H. Jeon, T.K. Kim, S.H. Park, J.W. Shin, O.C. Jeong, Proc. Micro Total Anal. Syst., Cheju, Korea, 2009. pp. 1156–1158. [11] M.J. Berridge, P. Lipp, M.D. Bootman, Nat. Rev. Mol. Cell Biol. (2000) 11–21. [12] M. Zayzafoon, J. Cell. Biochem. (2006) 56–70. [13] O.C. Jeong, S. Konishi, J. Micromech. Microeng. 18 (2008) 085017. [14] H.T. Kim, J.K. Kim, O.C. Jeong, Jpn. J. Appl. Phys. 50 (2011) 06GL04.
Contents lists available at SciVerse ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
Intracellular calcium-expression-display (ICED) device operated by compressive stimulation of cells Tae Kyung Kim, Ok Chan Jeong ⇑ Department of Biomedical Engineering, Inje University, Gimhae 621-749, Republic of Korea
a r t i c l e
i n f o
Article history: Available online 15 June 2012 Keywords: Cell chip Compressive stress Intracellular calcium-expression-display (ICED)
a b s t r a c t The effects of steady compressive stimulation on intracellular calcium expression in MG-63 human osteoblast-like bone cells were examined using a fabricated micro cell chip with a microchannel array. A computer-controlled pneumatic system was used to create compressive stress as a source of mechanical stimulation without fluid flow in the microchannel. Intracellular calcium levels were observed using laser-scanning fluorescence microscopy and showed multiple peaks of intracellular calcium expression over time. To visualize the mechanosensory properties of live cells suggested by the observed periodic intracellular calcium expression, we designed and fabricated a seven-segment-type of intracellular calciumexpression-display (ICED) device. The device consisted of a glass cell-culture substrate, two polydimethylsiloxane layers for cell introduction and pneumatic stimulation, and seven individually controllable pneumatic actuators. Compressive stimulation was applied to the cells via deformation of the pneumatic actuator diaphragm. The application of external pressure to the ICED device resulted in a green fluorescence representation of intracellular calcium expression in the form of an ‘‘8’’. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Physical and mechanical stimuli, such as compression, shear stress, strain, stretching, and hydraulic force, are important in cell proliferation and differentiation [1–3]. Microelectromechanics research has led to the design and fabrication of a micro cell stimulator for applying compressive stress to human mesenchymal stem cells [4]. In addition, a macro system for shear and compression stimulation has been developed [3]. Compared with a typical macro system, microfluidics technologies have several advantages: they minimize fluid volumes and the numbers of cell culture surfaces and samples required, reduce procedure times, and allow for the use of microincubators in cell chips [4]. Thus, micro cell chips are more effective than, and preferable to, macro systems for studying the effects of mechanical stimulation on cells [5]. Studies examining the role of cyclic mechanical stimulation in cell differentiation have provided evidence of its importance; however, the reported macro- and micro-system experiments vary greatly in their magnitudes of applied stimulation. Furthermore, the cell refractory period should be taken into consideration when selecting the duration of the mechanical stimulus [6]. A quantitative study of the optimal or appropriate magnitude and period of mechanical stimulation is necessary. ⇑ Corresponding author. E-mail address: [email protected] (O.C. Jeong). 0167-9317/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mee.2012.05.035
In the present study, a micro cell chip with a polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, Inc.) microchannel array and a glass cell-culture substrate was fabricated using a replica molding [7] and bonding process. To determine the appropriate duration for mechanical stimuli, we observed intracellular calcium expression in MG-63 human osteoblast-like bone cells under compressive stimulation in the micro cell chip. We found that periodic compressive force applied using this chip could induce periodic increases in intracellular calcium levels in the enclosed cells. We then fabricated an intracellular calcium-expression-display (ICED) device operated by compressive stimulation to demonstrate mechanotransduction in the cells. The cells were successfully induced to blink using individually controllable compressive stimulators.
2. Experimental methods 2.1. Micro cell chip The fabricated micro cell device consisted of a PDMS fluidicchannel array for applying compressive stress and a 120 lm-thick cover glass that served as a cell-culture substrate and permitted optical observation (Fig. 1). The microchannel was 1 mm wide, 0.4 mm high, and 15 mm long. The cell chip was fabricated from PDMS using soft lithography and pierced to produce two fluidic ports. The chip was then bonded
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T.K. Kim, O.C. Jeong / Microelectronic Engineering 98 (2012) 703–706
2.2. Experimental system The experimental set-up was comprised of a lab-designed pneumatic supplying system and a microscope. A computer-controlled electromagnetic valve was used to control the application frequency of external compressed air. The supplied air pressure was monitored over time using a precision pressure sensor. Intracellular calcium levels in the cells on the cell chip were observed using laser-scanning fluorescence microscopy (LSM 510 META; Carl Zeiss, Oberkochen, Germany). The excitation wavelength of Argon-ion laser for the green-fluorescent calcium indicator was 488 nm and the observed wavelength range were 500–570 nm. 3. Results and discussion 3.1. Compressive stress-induced intracellular calcium expression Fig. 1. Fabricated micro cell chip consisting of a polydimethylsiloxane (PDMS) channel array and a glass cell-culture substrate. Compressive stress is applied via by compressed air through the pneumatic ports, which are indicated by a circled ‘‘P’’.
to the glass substrate using atmospheric pressure plasma technology [8]. There are some advantages to the use of atmospheric pressure plasma like dry process, simple treatment process in the air, and several seconds of the surface modification [9]. After fabrication, the cell chip was sterilized by ultraviolet irradiation, and the glass surface was coated with fibronectin. The chip was seeded with MG-63 cells (1.0 105 cells/mL), incubated for 24 h, and stained with 10 M Fluo-4/AM, a fluorescent calcium indicator. To apply pure compressive stress without fluid flow to cells seeded on the glass substrate, compressive pressure was applied equally and simultaneously to the two cell/media ports in the micro channels.
Captured fluorescence images showing the levels of intracellular calcium before and after the application of a 25 kPa compressive load to the micro cell chip are shown in Fig. 2. The fluorescence intensity of the cells increased dramatically when the compressive stress was applied and then decreased over time. Moreover, the steady mechanical stimulation resulted in multiple peaks of intracellular calcium expression over time, similar to the case of the fluid-flow induced shear stress [10]. This phenomenon may has been related to the duration of intracellular calcium expression and release from intracellular stores into the cytoplasm as a result of extracellular mechanical stress [11,12]. 3.2. ICED device The periodicity of the cellular response to steady compressive stimulation led us to fabricate and test a seven-segment ICED
Fig. 2. Relative intracellular calcium levels in a micro cell culture indicated by green fluorescence. Individual cells (a-1) in the observation area (900 lm 900 lm) and the corresponding surface plots (a-2) are shown at rest (b-1) and after the application of compressive stress (b-2). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
T.K. Kim, O.C. Jeong / Microelectronic Engineering 98 (2012) 703–706
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Fig. 3. The seven-segment intracellular calcium-expression-display (ICED) device. (a) A mounted device. (b) Enlarged view of a seven-segment cell chamber.
Fig. 4. Images of (a) the deformed diaphragm of the pneumatically driven compressive stimulator and (b) the corresponding three-dimensional fluorescence profile indicating intracellular calcium expression.
device for visualizing the changes in intracellular calcium expression in response to compressive stimulation (Fig. 3a). The ICED device contained a PDMS chamber for cell introduction, a PDMS chamber for pneumatic stimulation having seven individually controllable pneumatic channels, and a glass cell-culture substrate. To construct the replica mold for PDMS structures, a photosensitive epoxy (SU-8) mold was fabricated through typical photolithography process. The process temperature and time for soft bake and post expose bake were 65 °C for 6 min and 95 °C for 10 min, respectively. A PDMS was mixed, stirred, and degassed in a vacuum chamber. The mixing ratios of base polymer to curing agent was 10:1. The prepared liquid PDMS mixture was poured into the SU-8 molds, spun, and cured at 75 °C for 10 min [13]. The depth, width, and length of the SU-8 mold for cell chambers were 40, 300, and 860 lm, respectively. The channels for the pneumatic and fluidic connections near the cell chambers were 40 lm wide. The PDMS diaphragm for applying mechanical compressive stress was 60 lm thick. After the pneumatic stimulation layer was peeled off the SU-8 mold, surfaces of PDMS layers for both cell chamber and pneumatic stimulation were treated with atmospheric pressure plasma for several seconds. The pneumatic stimulation layer was aligned and lowered onto the plasma-treated PDMS layer for the cell chamber. The stacked PDMS layers were placed on a hot-plate and then heated at 120 °C for 30 min. After
the bonding process was complete, the structure was peeled off the SU-8 mold and 20 holes were punched out to form the pneumatic and fluidic ports. The bonded PDMS layers were then bonded to a cover glass substrate for cell seeding and growth using the sequential bonding process like atmospheric plasma treatment and directing heating process [14]. An enlarged view of the seven-segment cellular device and its cross-sectional view (AA0 ) are shown in Fig. 3b. Images of the seven-segment device with diaphragms deformed in the shapes of a an ‘‘8’’ by 125 kPa of applied pressure are shown in Fig. 4a. The corresponding green fluorescence images indicating intracellular calcium expression are shown in Fig. 4b. To visualize the mechanosensory properties of the cells, the fluorescence intensity was converted into an interactive three-dimensional surface plot using ImageJ. This plot showed that most of the cells responded well to steady compressive stimulation, although their response times varied. 3.3. Simulation of compressive stress in the ICED Because the ICED device transferred compressive stress to cells through the deformation of the PDMS diaphragm, the actual amount of compressive stress on the cells in the device was difficult to determine. However, to estimate the fluid pressure on the
706
T.K. Kim, O.C. Jeong / Microelectronic Engineering 98 (2012) 703–706
Fig. 5. Fluid structure interaction analysis of the effect of the pneumatically driven compressive stimulators on ICED device. The nonlinear fluidic pressure distribution induced by the structural deformation of the pneumatic actuator was simulated using FEMLAB. (a) Model of the ICED device. (b) Deformation of PDMS diaphragm induced by a 125 kPa load. (c) Pressure distribution of fluid in the cell chamber. (d) Results of the final analysis of structural deformation of the diaphragm and the pressure distribution in the fluid film.
cells, the multiphysical effects of the compressive stimulation were simulated using the FEMLAB software package (Comsol, Inc., Burlington, MA, USA). Fig. 5 shows the application of compressed air to the device (Fig. 5a), the deformation of the diaphragm (Fig. 5b), and the subsequent application of compressive stress or pressure to the cells seeded in the bottom of the cell chamber (Fig. 5c). The maximum displacement of the stimulator diaphragm was 36.6 lm, and the generated pressure of the fluid in the chamber was 365.5 Pa. It was equivalent to the maximum compressive stress for stimulating cells. From simulation results, the actual compressive stress experienced by the cells was much lower than the pressure applied to the stimulator since most of the applied pressure was used to structurally deform the thick PDMS diaphragm.
4. Conclusions This paper presents a visualization of mechanosensory properties in live MG-63 cells using microfluidics technology and an ICED device fabricated using soft lithography. First, a micro cell chip containing a microchannel array was fabricated to apply compressive stimulation to the cells, and intracellular calcium levels were measured. The cells were found to be sensitive to the external mechanical compressive stress. Under steady compressive stimulation, the calcium levels varied cyclically. Next, an ICED device with a seven-segment display similar to those used in electrical
engineering was fabricated and successfully used to demonstrate the visualization of mechanosensory properties in cells. Acknowledgments This research was supported by the Basic Science Research Program of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2009-0076333). References [1] C.R. Jacobs, C.E. Yellowley, B.R. Davis, Z. Zhou, J.M. Cimbalan , H.J. Donahue, J. Biomech. 31 (1998) 969–976. [2] A.M. Sorkin, K.C. Dee, M.L. Knothe Tate, Am. J. Physiol. Cell Physiol. 287 (2004) C1527–C1536. [3] E.H. Frank, M.S. Jin, A.M. Loening, M.E. Levenston, A.J. Grodzinsky, J. Biomech. 33 (2000) 1523–1527. [4] W.Y. Sim, S.W. Park, S.H. Park, B.H. Min, S.R. Park, S.S. Yang, Lab Chip 7 (2007) 1775–1782. [5] X.E. Guo, E. Takai, X.J. Jiang, Q. Xu, G.M. Whitesides, J.T. Yardley, C.T. Hung, E.M. Chow, T. Hantschel, K.D. Costa, Mol. Cell Biomech. 3 (3) (2006) 95–107. [6] S.W. Donahue, H.J. Donahue, C.R. Jacobs, J. Biomech. 36 (2003) 35–43. [7] Y. Xia, G.M. Whitesides, Annu. Rev. Mater. Sci. 28 (1998) 153–184. [8] S.M. Hong, S.H. Kim, J.H. Kim, H.I. Hwang, J. Phys: Conf. Ser. 34 (2006) 656–666. [9] H.T. Kim, O.C. Jeong, Microelectron. Eng. 88 (2011) 2281–2285. [10] J.H. Jeon, T.K. Kim, S.H. Park, J.W. Shin, O.C. Jeong, Proc. Micro Total Anal. Syst., Cheju, Korea, 2009. pp. 1156–1158. [11] M.J. Berridge, P. Lipp, M.D. Bootman, Nat. Rev. Mol. Cell Biol. (2000) 11–21. [12] M. Zayzafoon, J. Cell. Biochem. (2006) 56–70. [13] O.C. Jeong, S. Konishi, J. Micromech. Microeng. 18 (2008) 085017. [14] H.T. Kim, J.K. Kim, O.C. Jeong, Jpn. J. Appl. Phys. 50 (2011) 06GL04.