Design of Comb-shaped Surface Electrode to Measure Signal
from Tissue Cultured with Electric Stimulation
Shigehiro HASHIMOTO, Haruka HINO, Yusuke TAKAHASHI, Akira HIRAOKA
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACTThe comb-shaped surface electrode has been designed tomeasure the signal from the tissue cultured with electric
stimulation. The pattern of the thin film of the surface
electrode was made of indium tin oxide (ITO) coated on theglass by the photolithography technique. C2C12 (mouse
myoblast cell line) was cultured for 13 days on the patterned
scaffold. The electric pulses (period 1 s, amplitude 10 V,
duration 0.002 s) were applied for 10 minutes per day. The
experimental results show that myoblasts make orientation
along the comb-shaped surface electrode, which can beobserved through the transparent surface electrode by the
optical microscope. The change of the electric resistance ofthe cultured tissue is able to be detected between electrodes ofthe experimental system in vitro.
Keywords: Biomedical Engineering, Surface Electrode, C2C12,
Electric Pulse and ITO.

The membrane of the biological cell keeps difference of theelectric potential between inside and outside. The operation of
the ion channel makes change of the potential, which makes
electric pulses. The electric pulses are detected, when theelectrodes put on the biological tissue [11, 12].
Indium tin oxide (ITO) is an inorganic compound of indium
oxide (In2O3) and tin oxide (SnO2). Because the transmittancein the region of the visible light is high, the thin film of ITO iscolorless and transparent. ITO is one of the useful materials,
which is applied to the transparent electrode [13].
The response of biological cells might depend on the directionof electric signal [14]. Myotube contracts synchronously withthe electric pulse, when electric pulse is applied on the medium
through electrodes in vitro [15].
In the present study, the comb-shaped surface electrode of ITO
has been designed to measure the signal from the muscle tissue
cultured with the electric stimulation in vitro.

1. INTRODUCTION
2. METHODS
The cell culture technique has been developed and several
methodologies might clinically be applied to regenerative
medicine [1-4]. C2C12 (mouse myoblast cell line) adheres tothe scaffold, proliferates and differentiates to myotubes in vitro[5]. These behaviors of the cell depend on the micro propertyof the surface [6].
The effect of the surface property of the scaffold on cell culture
has been studied in the previous studies [7].
Severalmicro-fabrication processes have been designed to control
adhesion of biological cells in vitro [8, 9]. Photolithography
technique enables machining for design of the surface electrode.
The muscle tissue is exposed to electric pulses in vivo. The
movement is also controlled with the electric pulses. The
biological systems have ability to optimize themselves to theirenvironment. The optimum electric stimulation has a potential
to accelerate differentiation of myoblast and growth of themuscle tissue, which might contribute to the regenerative
medicine.
The electric impedance of the biological tissue was measured in
the previous study to distinguish the kinds of tissue [10]. The
impedance depends on the orientation of cells.

Comb-shaped Surface Electrode
A comb-shaped surface electrode has been designed by the
micromachining technique (Fig. 1). The comb-shaped pattern
has been selected for the following reasons: to make orientationof cells, to make oriented stimulation of electric pulses, and tomeasure oriented signal from cells.
The indium tin oxide (ITO) film was used for the surface
electrode [13]. A thin film of ITO is formed on the surface ofa glass disk with the comb-shaped micro pattern (line of 0.1 mm
width).
Photomask
The borosilicate glass (Tempax) disk was used for the base of
the photomask (Fig. 1b). After hydrophilization by the oxygen
plasma ashing for five minutes at 100 W by RIE (FA-1, Samco
International, Kyoto, Japan), titanium was coated on the surface
with 100 nm thickness in the sputtering system (L-332S-FH,
Canon Anelva Corporation, Kawasaki, Japan). The disk washydrophilized by the oxygen plasma ashing for ten minutes at
100 W by RIE again.
To improve affinity between titanium and photoresist material,
HMDS (hexamethyldisilazane: Tokyo Chemical Industry Co.,

Ltd., Tokyo) was coated on the disk with a spin coater. The
positive photoresist material of OFPR-800LB (Tokyo Ohka
Kogyo Co., Ltd, Tokyo, Japan) was coated on the disk at 5000
rpm for 20 s with the spin coater. The photoresist was bakedin the oven at 368 K for three minutes.
The pattern was drawn on the mask with a laser drawing system(DDB-201K-KH, Neoark Corporation, Hachioji, Japan). To
control the dimension of the pattern on the mask with the laser
drawing system, the parameters were selected as follows: thevoltage of 3.5 V, the velocity of 0.14 mm/s, the acceleration of
0.34 mm/s2. The pattern was baked on the heated plate at 393
K for five minutes. The photoresist was developed withtetra-methyl-ammonium hydroxide (NMD-3, Tokyo Ohka
Kogyo Co., Ltd., Kawasaki, Japan) for 3 minutes (Fig. 2). The
disk was rinsed by the ultrapure water for one minute, and driedby the spin-dryer.
The titanium coating disk was etched with the plasma gas using
RIE-10NR (Samco International, Kyoto, Japan). For etching,
the gas of SF6 with Ar was applied for six minutes.
OFPR-800LB was removed by acetone (Fig. 1).

Scaffold with Micro Pattern
The borosilicate glass (Tempax) disk (50 mm diameter, 1 mm
thickness) was used for the base of the scaffold. Afterhydrophilization by the oxygen plasma ashing for five minutes
at 100 W by RIE, the positive photo-resist material of
OFPR-800 was coated on the glass with the spin coater (Fig. 3).
The photo-resist was baked at 373 K for three minutes.
The surface of titanium coating mask was adhered on thesurface of OFPR-800, and the photoresist was exposed to the
UV light through the photomask in the single sided maskaligner (M-1S, Mikasa Co. Ltd., Japan) at 10 V for 30 s. The
photoresist was baked at 393 K for five minutes.
The photoresist was developed with tetra-methyl-ammoniumhydroxide (NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki,
Japan). The disk was rinsed by the ultrapure water.
ITO was coated on the surface with 0.08 mm thickness (1 mm
width × 30 mm length) in the electron beam vapor deposition
apparatus (JBS-Z0501EVC, JEOL Ltd., Japan).
After lift-off the ITO on OFPR-800LB by removal with acetone,
the pattern of ITO was left on the glass disk (Fig. 4a). The
disk was dried by the spin-dryer.
For the reference electrode, Ag/AgCl paste was baked at 393 K
for five minutes (Fig. 4b). The lead wire is soldered on theeach end of the film to introduce the electric current.
The dimension of the micro pattern was measured with a laser
microscope (VK-X200, Keyence Corporation, Osaka, Japan).
The height along the cross sectional line of the electrode pattern
was traced.

Fig. 1a: Micro pattern of mask for comb-shaped surface
electrode. Dimension from left to right is 1.3 mm.

Glass

Developing

Deposition

Exposure

Lift-off

Fig. 3: Photolithography process for surface electrode.
Fig. 1b: Photomask of titanium on the glass.

Fig. 2: Micro pattern of mask developed on the glass.
Dimension from left to right is 1.3 mm.

Fig. 4a: Comb-shaped surface electrode.
to right is 1.3 mm.

Dimension from left

The cells were seeded on the scaffold with the density of 10000
cells/cm2. Cells were cultured in the incubator at 310 K with
5% CO2 for 13 days. The medium were refreshed every twodays. Cells were observed with an inverted phase-contrast
microscope (IX71, Olympus Co., Ltd., Tokyo) once a day.
The electric potential was traced at the electrode of ITO during
the pulse application once a day (Fig. 7). During themeasurement, the culture dish was kept at room temperature outof the incubator.

Fig. 4b: Surface electrodes on glass.

The differentiation from myoblasts to myotubes was confirmed
by contractive movement of myotubes during electric pulse
application to the medium once a day, after cultivation for sevendays. The cyclic electric pulses (amplitude of 10 V, durationof 0.003 s) at the period between 0.1 s and 2 s were applied tothe medium through electrodes of titanium wire.

3. RESULTS
Fig. 8 shows the dimension of the micro pattern of the surface
electrode. Measurement by the laser microscope shows thatthe comb-shaped micro pattern of surface ITO film has thedimension of 0.07 mm width, and 0.0014 mm thickness.
Fig. 5: PDMS ring-wall and cap with electrode wire.

PDMS Wall and Cap
A donut-ring of polydimethylsiloxane (PDMS, Dow Corning
Corporation, MI, USA) is adhered with the affinity of surfaces
to make the peripheral walls for the cell culture on the Tempax
glass disk. The outer diameter and the inner diameter of thering are 25 mm, and 10 mm, respectively (Fig. 5).
A pair of electrodes of titanium wire (0.5 mm diameter) for
electric stimulation to observe synchronous contraction of
myotubes are put through the cap of PDMS.

Fig. 9 shows the contact angle on the film of ITO (A) and on
the base of the glass (B). The contact angle is smaller on theglass (0.12 rad) than on the ITO film (0.16 rad).
Fig. 10 shows C2C12 around the micro pattern of surface
electrode.
On the thirteenth day of culture, C2C12
differentiated to myotubes, and the myotubes orient along thelongitudinal direction of the comb-shaped surface electrode.
The electric resistance r between the surface electrodes iscalculated as 9 Ω from Eq. 1, when the electric resistance of 51
Ω is inserted for R (Fig. 6).

Both on the ITO film and on the base of the glass, the purewater 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 10 V, duration 0.002s) were generated with an electric stimulator (SEN5201, Nihon
Kohden Corporation, Japan). The stimulator was connected to
the ITO film, and the pulses were introduced to the scaffold ofcells for ten minutes per day (Fig. 6). An electric resistance R
of 51 Ω is serially inserted between the ITO film and thestimulator. The electric signals (V1, V2) were monitored by an
oscilloscope during electric stimulation to the ITO film. V1
and V2 are the voltages between the terminals of resistance, andof electric stimulator, respectively. The electric stimulation
was applied to the scaffold after 24 hours of culture foradhesion of the cells to the scaffold.
Cell Culture
C2C12 (Mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) of the ninth passage was used for thecell culture with electric stimulation. D-MEM (Dulbecco’s
Modified Eagle Medium) containing 10% FBS (Fetal Bovine
Serum) and 1% penicillin/ streptomycin was used for the
medium in first two days. The medium was changed to
D-MEM containing 2% HS (Horse Serum) on the third day ofcultivation.

Fig. 6: Electric circuit.

Fig. 7: Measurement of electric signal on stage of microscope(left) between electrodes (right).

r = (V2 – V1) / (V1 / R)

(1)

In Eq 1, V1 is voltage between the terminals of resistance of R,and (V2 – V1) is voltage between the terminals of resistance of r.

The cells are easily observed with the microscope through thetransparent thin film of ITO on the glass surface (Figs. 10-12).
The cells adhere, and proliferate both on glass and on the ITO
film.

Figs. 11a and 12a show microscopic image of C2C12 on thesixth day and the tenth day of culture, respectively. Figs. 11b
and 12b show the voltage monitored during application ofelectric pulses on the ITO film on the sixth day and the tenthday of culture, respectively. The parameters of voltage
between the terminals of electric stimulator are 1 s of period
and 0.002 s of duration, respectively. The amplitudes of V2 in
Fig. 11b and 12b are 1.8 V and 2.3 V, respectively. The
amplitudes of V1 in Fig. 11b and 12b are 1.0 V and 1.5 V,
respectively. The ratio of V1/V2 increases from 0.56 to 0.65,
which corresponds to decrease of the resistance between
electrodes for four days of culture.
C2C12 of the same lot was cultured in the culture dish coatedwith collagen for the control test. The myotube shows
contraction synchronous to the electric pulse stimulation after
cultivation for eight days (Fig. 13).
The synchronous
contraction was not able to be observed, on the other hand, at
myotubes cultured on the micro patterned scaffold.

Fig. 10a: C2C12 on the second day of culture.

Fig. 10b: C2C12 on the thirteenth day of culture.

Fig. 8: Dimension of micro pattern measured with laser
microscope.

Fig. 11a: C2C12 on the sixth day of culture.
left to right is 1 mm.

Dimension from

Fig. 9a: Contact angle on the film of ITO.

Fig. 9b: Contact angle on the base of the glass.

Fig. 11b: Tracings of V1 (yellow, middle) and V2 (purple,
higher) on the sixth day of culture.

was frequently observed on the culture dish of polystyrene in
the previous study [16].
The decrease of the resistance between electrodes for four daysof culture might indicate organization of myotubes.
Impedance also depends on the extracellular matrix [17]. The
electric current of 0.03 A and electric resistance of 9 Ω makes
0.008 W of the electric power consumption between theelectrodes of ITO film during the stimulation of pulse.
Because the electric power consumption is very small and theelectric current is pulsatile (short duration of pulse), the ITO
film is not heated a lot by the electric current [13].
Fig. 12a: C2C12 on the tenth day of culture.
left to right is 1 mm.

Dimension from

The behavior of cells depends on several kinds of environment:
electric field [18-25], magnetic field [26], mechanical force
field [27-31]. The microfluidic system has been applied tosort biological cells [26], and to trap biological cells [6]. The
system also used to study local environment around the cultured
cell. The micro pattern of the surface has been applied to
study the surface effect of adhesion of cells [8].

5. CONCLUSION

Fig. 12b: Tracings of V1 (yellow, middle) and V2 (purple,
higher) on the tenth day of culture.

To measure the signal from the tissue cultured with electric
stimulation, the comb-shaped surface electrode has been
designed and manufactured with indium tin oxide (ITO) coatedon the glass. C2C12 (mouse myoblast cell line) was cultured
for 13 days on the glass with intermittent stimulation of electricpulses. The experimental results show that myoblasts oriented
along the comb type of the surface electrode are observed
through the transparent surface electrode, and the change of theelectric resistance with the change the cells is detected between
electrodes in vitro.
The designed comb-shaped surface
electrode is effective to apply the asymmetric electric field on
the cell culture and to measure the electric signal from the cell
culture.

6. ACKNOWLEDGMENT

Fig. 13: C2C12 on the eighth day of culture.
left to right is 1 mm.

Dimension from

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

4. DISCUSSION

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ITO has been selected as the material for the film type of thesurface electrode [13] coated on the glass, because of thefollowing properties: high conductivity, optical transparency,
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hand, cannot be lift-off in the micro machining process to makethe comb-shaped surface electrode in the present study.

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