Culture of Myoblast on Conductive Film with Electric Pulses
Kenta NODA, Yusuke TAKAHASHI, Shigehiro HASHIMOTO, Haruka HINO
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
shashimoto@cc.kogakuin.ac.jp http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACTThe effect of stimulation of electric pulses through the scaffold
on proliferation and on differentiation of myoblasts has beenstudied in vitro. A transparent thin film of indium tin oxide(ITO) coated on the glass was used for the conductive scaffold
for the cell culture. C2C12 (mouse myoblast cell line
originated with cross-striated muscle of C3H mouse) was
cultured for 22 days on the film. Electric pulses (period 1 s,amplitude 1 V (0.0005 A), duration 0.001 s) were applied on thefilm for thirty minutes per day. During culture, the cells wereobserved with an inverted phase contrast microscope every day.
The experimental results in 21 days of culture without electricpulses show that myoblast differentiate to myotube on ITO,
although myoblasts do not differentiate to myotube on glass.
On the scaffold with electric pulses in 21 days, myoblasts
proliferate to confluent state, although they do not differentiate
to myotubes.
Keywords: Biomedical Engineering, C2C12, Electric Pulse andITO.

1. INTRODUCTION
Cell culture technique has been developed and several
methodologies might clinically be applied to regenerative
medicine [1]. C2C12 (mouse myoblast cell line) adheres tothe scaffold, proliferates and differentiates to myotubes in vitro[2]. These behaviors of the cell depend on the micro propertyof the surface.
The effect of the surface of the scaffold on cell culture has beenstudied in the previous studies [3]. Several micro-fabrication
processes have been designed to control adhesion of biologicalcells in vitro [4].
The muscle tissue is exposed to electric pulses in the biologicalbody. The movement is also controlled with the electric pulses.
The biological systems have ability to optimize themselves totheir environment. The optimum electric stimulation has apotential to control growth of the muscle tissue, which might
contribute to the regenerative medicine.
Indium tin oxide (ITO) is an inorganic compound of indium
oxide (In2O3) and tin oxide (SnO2). Because the transmittancein the visible light region is high, it is colorless and transparent
in thin films. ITO is one of the useful materials, which isapplied to the transparent electrode.

In the present study, the effect of stimulation of electric pulses
through the scaffold of conductive film of ITO on proliferationand on differentiation of myoblasts has been studied in vitro.

2. METHODS
Conductive Scaffold
The indium tin oxide (ITO) film was used for the conductive
scaffold for the cell culture. A transparent thin film of ITO isformed on the surface of a glass disk (Fig. 1). The thickness
and the width of the film are 30 nm and 40 mm, respectively.
The diameter and the thickness of the disk are 100 mm and 1
mm, respectively. The lead wire is soldered on the each end of
the film to introduce the electric current.
Three small donut-rings of polydimethylsiloxane (PDMS, Dow
Corning Corporation, MI, USA) are adhered with the affinity ofsurfaces to make the peripheral walls for the cell culture (Fig. 2).
The outer diameter, the inner diameter and the thickness of thering are 32 mm, 10 mm and 5 mm, respectively. The locationsof the rings are on the ITO film, on the glass, and on theboundary area between the ITO film and the glass, respectively.
The glass disk was placed in the dish of 100 mm diameter (Fig.
3).
Both on the ITO film and on the base of the glass, the purified
water contact angles were measured by the contact angle
analyzer (Phoenix-300, Meiwafosis Co., Ltd., Tokyo, Japan).
Electric Pulses
The electric pulses (period 1 s, amplitude 1 V, duration 0.001 s)
were generated with an electric stimulator (SEN5201, NihonKohden Corporation, Japan). The stimulator was connected tothe ITO film, and the pulses were introduced to the scaffold ofcells for thirty minutes per day (Fig. 4). An electric resistanceof 10 ohm is serially inserted between the ITO film and thestimulator (Fig. 5). The electric signals (V1, V2) weremonitored by an oscilloscope during electric stimulation to theITO film. V1 and V2 are the voltages between the terminals ofelectric stimulator, and between terminals of resistance,respectively. The electric stimulation was applied to thescaffold after adhesion of the cells by 24 hours of culture.
The temperature at the surface of the ITO film was measured byan infrared thermography (i5, FLIR Systems, Inc., Tokyo,Japan), before and after the introduction of electric pulses in theincubator.

GlassLead wire

Fig. 4: Electric pulses introduced to scaffold of cells inincubator.
IIndium Tin Oxide

Fig. 1: ITO film on the disk of glass.

ErZ

V2

V1

Fig. 5: Electric circuit during introduction of electric pulses (E)to scaffold (Z) of cells.

Polydimethylsiloxane

Fig. 2: PDMS rings are attached on the ITO and glass.

Cell Culture
C2C12 (Mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) of the ninth passage was used for thecell culture without electric stimulation, and C2C12 of the
fourth passage was used for the cell culture with electricstimulation. D-MEM (Dulbecco’s Modified Eagle Medium)
containing 10% FBS (Fetal Bovine Serum) and 1% penicillin/streptomycin was used for the medium. The cells were seededon the scaffold with the density of 1000 cells/cm2. Cells werecultured in the incubator at 310 K with 5% CO2 for 21 days
(with electric pulses for 30 minutes per day). The medium
were refreshed every two days. Cells were observed with aninverted phase-contrast microscope (IX71, Olympus Co., Ltd.,Tokyo) every day.

3. RESULTS
Fig. 6 shows the contact angle on the film of ITO (A) and on
the base of the glass (B). The contact angle is smaller on filmof ITO (61 degree) than on the glass (65 degree).

Fig. 3: Glass with ITO film in the dish of 100 mm diameter.

Fig. 7 shows the voltage monitored during introduction ofelectric pulses to the ITO film. The parameters of voltage
between the terminals of electric stimulator are 1 s of period, 1V of amplitude, and 0.001 s of duration, respectively. The

parameters of the voltage between terminals of resistance of 10
ohm are 1 s of period, 0.005 V of amplitude, and 0.001 s ofduration. The 0.005 V of amplitude and 10 ohm of resistancemakes 0.0005 A of current.
Fig. 8 shows the distribution of temperature around the ITO
film on the glass, before (A) and after (B) introduction ofelectric pulses. The temperature at the center of ITO film iselevated from 33 degree centigrade to 36 degree centigrade in
the incubator. The local temperature at the film is same as thatat the glass during the introduction of electric pulses for one
hour in the incubator. The three red rings in Fig. 8 correspondto the ring of PDMS, which has the rate of thermal emissivity
different from the glass.
The cells are easily observed with the microscope through thetransparent thin film of ITO on the glass surface (Figs. 9-11).
The Cells adhere, extend pseudo, and proliferate both onpolystyrene and on the ITO film.
Fig. 9 exemplifies cells on 11th day of culture without
stimulation of electric pulses. Fig. 10 exemplifies cells on
21st day of culture without stimulation of electric pulses.
C2C12 proliferated to the confluent state on the eighth day ofculture on ITO, although C2C12 proliferated to the confluent
state on the sixth day of culture on polystyrene dish.
Myoblasts differentiated into myotubes on the 21st day ofculture on ITO (Fig. 10B), although myoblasts differentiatedinto myotubes on the 11th day of culture on polystyrene dish(Fig. 9A).

Fig. 7A: Electric pulse and voltage between terminals ofresistance. Scale: Ordinate, 0.5 V (Channel 1) and 0.005 V(Channel 2); abscissa, 0.001 s.

Fig. 11 exemplifies cells on the 22nd day of culture withintermittent stimulation of electric pulses. C2C12 proliferated
to the confluent state on the 15th day of culture on ITO withintermittent stimulation of electric pulses. Myoblasts do notdifferentiate into myotubes in 22 days of culture on ITO (Fig.
11B). Myoblasts do not proliferate to the confluent state in 22days of culture on the glass (Fig. 11D).

Fig. 7B: Electric pulse and voltage between terminals ofresistance. Scale: Ordinate, 1 V (Channel 1) and 0.2 V(Channel 2); abscissa, 0.2 s.
Fig. 6A: Contact angle on ITO film.
4. DISCUSSION
The electric current was low so that the temperature at thesurface of metal coated on the glass was kept around 310 K
during the cell culture. From the current of 0.0005 A, and thevoltage of 1 V, the resistance of the ITO film is estimated to be2000 S-1. The estimation makes of 0.0005 W of the electricpower consumption at the ITO film during the pulse. Because
the electric power consumption is very small and the electric
current is pulsatile (short duration of pulse), the ITO film is notheated by the electric current.
Fig. 6B: Contact angle on glass.

Fig. 9A: Myotubes on polystyrene dish on 11th day of culture
without stimulation of electric pulses. Dimension from left toright is 1 mm.

Fig. 8A: Temperature around ITO film before introduction ofelectric pulses. Temperature at the center of the film is 33.4
degree centigrade.

Fig. 9B: Myoblasts on ITO film on 11th day of culture without
stimulation of electric pulses.

Fig. 8B: Temperature around ITO film after introduction ofelectric pulses for one hour. Temperature at the center of thefilm is 36.1 degree centigrade.

Fig. 10A: Myotubes on polystyrene dish on 21st day of culture
without stimulation of electric pulses.

Fig. 10B: Myotubes on ITO film on 21st day of culture without
stimulation of electric pulses.

Fig. 11B: Cells on ITO film on 22nd day of culture withstimulation of electric pulses.

Fig. 10C: Cells on boundary area between ITO film and glasson 21st day of culture without stimulation of electric pulses.

Fig. 11C: Cells on boundary area between ITO film and glasson 22nd day of culture with stimulation of electric pulses.

Fig. 10D: Cells on glass on 21st day of culture withoutstimulation of electric pulses.

Fig. 11D: Cells on glass on 22nd day of culture withstimulation of electric pulses.

To inhibit the migration of cells on the glass of the culture disk,
the area on the glass was divided by the ring of PDMS. Thecells were cultured in each area within the ring.
The transparency of ITO film is good enough to observe thebehavior of cells on the film. C2C12 is able to be cultured anddifferentiated on the ITO film.
The contact angle on the gold film deposited on PDMS depends
on the process of deposition in the previous study [5]. The
contact angle governs behavior of cells on the scaffold:adhesion, proliferation, and differentiation. The surface ofITO is more hydrophilic than the surface of glass in the present
study. Proliferation of C2C12 tends to be faster on the ITO
film than on the glass.
The behavior of cells depends on several kinds of environment:
electric field [2], magnetic field [6], gravitational force [7], andshear flow [8, 9]. The microfluidic system has been applied tosort biological cells [10, 11], and to trap biological cells [12, 13].
The system also used to study local environment around thecultured cell. The micro pattern of the surface has been
applied to study the surface effect of adhesion of cells [4, 14,
15].
The moderate electric stimulation might accelerate
differentiation of myoblasts into myotubes [2]. In the present
study, myoblasts do not differentiate into myotubes on ITO withelectric pulses in 21 days, although they differentiate into
myotubes on ITO in 21 days without electric pulses.

5. CONCLUSION
The effect of stimulation of electric pulses through the scaffoldof conductive film on proliferation and on differentiation ofmyoblasts has been studied in vitro. A transparent thin film ofindium tin oxide (ITO) coated on the glass was used for
conductive scaffold for the cell culture. C2C12 (mousemyoblast cell line) was cultured for 22 days on the film.
Electric pulses (period 1 s, amplitude 1 V (0.0005 A), duration0.001 s) were applied on the film for thirty minutes per day.
The experimental results show that myoblasts do notdifferentiate into myotubes on ITO with electric pulses in 22days, although they differentiate into myotubes on ITO in 21
days without electric pulses.

6. ACKNOWLEDGMENT
This work was supported by a Grant-in-Aid for Strategic
Research Foundation at Private Universities from the JapaneseMinistry of Education, Culture, Sports and Technology.

REFERENCES[1] T. Shimizu, M. Yamato, A. Kikuchi and T. Okano, “Cell

Sheet Engineering for Myocardial Tissue Reconstruction”,
Biomaterials, Vol. 24, No. 13, 2003, pp. 2309-2316.
[2] S. Hashimoto, F. Sato, R. Uemura and A. Nakajima, “Effectof Pulsatile Electric Field on Cultured Muscle Cells in Vitro”,
Journal of Systemics Cybernetics and Informatics, Vol.
10, No. 1, 2012, pp. 1-6.
[3] H. Hino, S. Hashimoto and F. Sato, “Effect of Micro Ridges
on Orientation of Cultured Cell”, Journal of Systemics
Cybernetics and Informatics, Vol. 12, No. 3, 2014, pp.
47-53.
[4] C.S. Chen, M. Mrksich, S. Huang, G.M. Whitesides and D.E.
Ingber, “Geometric Control of Cell Life and Death”, Science,Vol. 276, No. 5317, 1997, pp. 1425-1428.
[5] K. Noda, S. Hashimoto, T. Yasuda and H. Fujie, “Culture ofMyoblast on Gold Film Sputtered on Polydimethylsiloxane
Disk”, Proc. 18th World Multi-Conference on Systemics
Cybernetics and Informatics, Vol. 2, 2014, pp. 150-155.
[6] S. Hashimoto and K. Tachibana, “Effect of Magnetic Fieldon Adhesion of Muscle Cells to Culture Plate”, Journal ofSystemics Cybernetics and Informatics, Vol. 11, No. 4,
2013, pp. 7-12.
[7] S. Hashimoto, H. Hino and T. Iwagawa, “Effect of Excess
Gravitational Force on Cultured Myotubes in Vitro”,
Journal of Systemics Cybernetics and Informatics, Vol.
11, No. 3, 2013, pp. 50-57.
[8] S. Hashimoto and M. Okada, “Orientation of Cells Culturedin Vortex Flow with Swinging Plate in Vitro”, Journal ofSystemics Cybernetics and Informatics, Vol. 9, No. 3,
2011, pp. 1-7.
[9] S. Hashimoto, F. Sato, H. Hino, Hiromichi Fujie, Haruka
Iwata and Yuma Sakatani, “Responses of Cells to Flow inVitro”, Journal of Systemics Cybernetics and Informatics,Vol. 11, No. 5, 2013, pp. 20-27.
[10] S. Hashimoto, T. Horie, F. Sato, T. Yasuda and H. Fujie,“Behavior of Cells through Micro Slit”, Proc. 17th World
Multi-Conference on Systemics Cybernetics andInformatics, Vol. 1, 2013, pp. 7-12.
[11] S. Hashimoto, A. Mizoi, H. Hino, K. Noda, K. Kitagawa
and T. Yasuda, “Behavior of Cell Passing through MicroSlit”, Proc. 18th World Multi-Conference on Systemics
Cybernetics and Informatics, Vol. 2, 2014, pp. 126-131.
[12] S. Hashimoto, R. Nomoto, S. Shimegi, F. Sato, T. Yasuda
and H. Fujie, “Micro Trap for Flowing Cell”, Proc. 17th
World Multi-Conference on Systemics Cybernetics andInformatics, Vol. 1, 2013, pp. 1-6.
[13] S. Hashimoto, Y. Takahashi, H. Hino, R. Nomoto and T.
Yasuda, “Micro Hole for Trapping Flowing Cell”, Proc.
18th World Multi-Conference on Systemics Cybernetics
and Informatics, Vol. 2, 2014, pp. 114-119.
[14] T. Okano, N. Yamada, M. Okuhara, H. Sakai and Y. Sakurai,“Mechanism of Cell Detachment from TemperatureModulated, Hydrophilic-Hydrophobic Polymer Surfaces”,
Biomaterials, Vol. 16, No. 4, 1995, pp. 297-303.
[15] T. Okano, S. Nishiyama, I. Shinohara, T. Akaike, Y. Sakurai,
K. Kataoka and T. Tsuruta, “Effect of Hydrophilic andHydrophobic Microdomains on Mode of Interaction between
Block Polymer and Blood Platelets”, Journal of Biomedical
Materials Research, Vol. 15, No. 3, 1981, pp. 393-402.