Electric Stimulation for Acceleration of Cultivation of Myoblaston Micro Titanium Coil Spring
Yusuke TAKAHASHI, Shigehiro HASHIMOTO, Haruka HINO, Tomokazu TAKEDA
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACTThe electric stimulation has been applied on myoblasts toaccelerate cultivation on a micro coil spring in vitro. A micro
coil spring made of the titanium wire of 0.085 mm was used forthe scaffold for the cell culture. The coil spring has thedimension as follows: 0.65 mm diameter, 0.15 mm pitch, 5 mm
length. For the counter electrode, the titanium film (100 nm
thickness) was coated on the glass. C2C12 (mouse myoblast
cell line) was seeded and cultured in Dulbecco’s Modified
Eagle Medium with 10 percent of fetal bovine serum. The
electric pulses were applied between the coil spring and thetitanium film for thirty minutes per day: the period of 1 s, thepulse width of 0.001 s, and the voltage amplitude of 0.5 V (thecurrent amplitude of 0.5 mA). The cells around the coil springwere observed with an inverted phase contrast microscope forten days. The experimental results show that the movement ofcell depends on the local electric field, and that the electric
stimulation tends to accelerate differentiation of myoblasts tomyotubes around the micro coil spring.
Keywords: Biomedical Engineering, Micro Coil Spring,Electric Pulses, C2C12, Movement, and Differentiation.

1. INTRODUCTION
Biological cells are exposed to electric fields in vivo. The cells
are sensitive to the electric stimulation. In the previous study,
the cells showed accelerated adhesion to the scaffold by electric
stimulation [1]. Cell culture technique has been progressed
recently. Acceleration technique for making tissue from cells in
vitro might be applied to regenerative medicine [2]. Micromachining technique enables the design of the electrode aroundcell culture. The movement of cell depends on the local
electric field [3, 4]. Dielectrophoresis is a phenomenon, in
which a cell moves by the local electric field distortion [5, 6].

spring. The force can be calculated from the deformation of
the spring [7]. The balance between forces by myotubes andby the spring can form a micro actuator. In the present study,
the electric pulses have been applied to myoblast to accelerateadhesion of cells to the scaffold of the titanium micro coilspring.

2. METHODS
Micro Coil Spring
A micro coil spring (Hi-Lex Corp., Takarazuka, Japan) made of
the pure titanium (JIS class 2) wire of 0.085 mm was used forthe scaffold. The coil spring has the dimension as follows:
0.65 mm diameter, 0.15 mm pitch, 5 mm length.
Titanium Surface ElectrodeThe titanium film was machined for the surface electrode (Fig.
1). Before the coating of titanium, the surface of the glass washydrophilized by the oxygen (> 0.1 Pa) plasma ashing for ten
minutes at 100 W by RIE (FA-1, Samco International, Kyoto,
Japan). Titanium was coated on the surface of the borosilicate
glass (Tempax) disk (50 mm diameter, 1.1 mm thickness) or
slide glass in the electron beam vapor deposition apparatus(JBS-Z0501EVC JEOL Ltd., Tokyo, Japan). The thickness of
coating is 100 nm.
The pattern of the coating is controlled by the film type of mask.
A carbon disk (0.5 mm of thickness) was used for the mask forthe deposition of titanium surface electrode (Fig. 2). The
rectangular hole (5 mm × 20 mm) was machined at the mask by
the ultrashort pulse laser (IFRIT, Cyber Laser Inc., Tokyo,
Japan).

In the previous study, myoblasts were cultured on a micro coilspring in vitro [7, 8]. In the previous experiment, the coilspring was inserted in the electric circuit and the electric current
flowed along the coil spring [8]. If the electric field iseffective to accelerate adhesion of cells to the micro spring,
additional electrodes should be equipped around the spring toapply electric pulses.
If the myotubes cultured on the micro coil spring, the force
generated among the tissue might make deformation of theFig. 1: Surface electrode of titanium on glass.

Fig. 2: Carbon disk of mask for pattern of surface electrode.
Titaniumtitanium electrode: the naked titanium surface electrode (Fig. 1),
and the titanium surface electrode covered with the negative
photoresist material of high viscosity (SU-8 10: Micro Chem
Corp., MA, USA) (Fig. 5). The glass slide (25 mm × 75 mm ×1 mm) coated with titanium was hydrophilized by the oxygen(>0.1 Pa) plasma ashing for ten minutes at 100 W by RIE.
SU-8 10 was coated on the surface at 5000 rpm for one minutewith a spin coater. After baked in the oven at 373 K for six
minutes, the photoresist was exposed to the ultraviolet light inthe mask aligner (M-1S, Mikasa Co. Ltd., Japan) at 10 V for
250 s. The photoresist was baked in the oven at 373 K for ten
minutes. The photoresist was developed with SU8 developer(Nippon Kayaku Co., Ltd, Tokyo, Japan) for five minutes, andrinsed with the ultrapure water.

Micro Coil Spring

PDMS
Titanium
Titanium wire
Fig. 3a: Design of electrodes of titanium film and spring.
Fig. 4a: Design of electrodes of covered titanium film and wire.

Fig. 3b: Manufactured electrodes of titanium film and spring.
Fig. 4b: Manufactured electrodes of covered titanium film andwire.
PDMS Ring
To control the scattering of the cell, the space around the coilspring is limited within a center hole (diameter of 10 mm) of thepolydimethylsiloxane (PDMS) ring, which is placed on thebottom of the dish (Fig. 3).
The wall of culture dish was made of the ring of
Polydimethylsiloxane (PDMS) (Fig. 3). PDMS (Sylgard 184
Silicone Elastomer Base, Dow Corning Corp., MI, USA) was
mixed with the curing agent (Sylgard 184 Silicone Elastomer
Curing Agent, Dow Corning Corp., MI, USA). The volume
ratio of PDMS to curing agent is ten to one. After degassing,
6 cm3 of PDMS was poured on the glass disk. After degassing
again, PDMS was baked at 373 K for one hour in an oven
(DX401, Yamato Scientific Co., Ltd). The baked PDMS wasmachined by the punch to make the ring: the internal diameteris 20 mm for the micro coil spring, or 10 mm for the titanium
wire.

PDMS
Titanium
Titanium wire

Fig. 5a: Design of electrodes of titanium film and wire.

The micro coil spring was fixed at the ring (Fig. 3). A
titanium wire of 0.5 mm diameter was used to compare with thetitanium coil spring (Fig. 4).
Titanium Surface Electrode Covered with SU-8 10
For the comparison, variation was made on the surface of theFig. 5b: Manufactured electrodes of titanium film and wire.

Titanium
PDMS

Fig. 9: Circuit for electric stimulation.
Fig. 6: Design of electrodes of titanium films.

Micro coil spring

PDMS
Titanium

PDMS
Fig. 7a: Design of spring between electrodes of titanium film.

Four kinds of setting of couple of electrodes were designed: thesurface film and the surface film (Fig. 6), the surface film andthe coil spring (Fig. 3), the surface film and the titanium wire
(Fig. 4), the surface film coated with SU8-10 and the wire (Fig.
5).
In the setting A, the coil spring is placed in the middle position
between two surface electrodes (Fig. 7). In the setting B, thecoil spring is placed as one of the electrode: the counterelectrode is the surface electrode (Fig. 8). The distance
between electrodes is 5 mm.
Electric Stimulation
The electric pulse E was generated with an electric stimulator(SEN5201, Nihon Kohden Corporation, Tokyo, Japan). The
stimulator was connected to the electrodes (Fig. 9). An electric
resistance r of 1 kΩ is serially inserted between the micro coilspring and the stimulator. The voltages (V) were monitored byan oscilloscope during application of electric pulse to the coilspring.
The electric current I, which flows through Z(medium), is calculated by Eq. 1.
I=V/r

Fig. 7b: Scaffold of spring between electrodes of titanium film
placed in dish.
Micro coil spring

PDMS
Titanium

Slide glass
Fig. 8a: Design of electrodes of titanium film and spring.

Fig. 8b: Manufactured electrodes of titanium film and spring.

(1)

In Eq. 1, V is the voltage between terminals of the electric
resistance.
Cell
C2C12 (Mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) was seeded in the area inside of the
PDMS ring.
The cells were cultured in the D-MEM(Dulbecco’s Modified Eagle Medium). The medium contains
FBS (decomplemented fetal bovine serum) with the volume
percent of ten. The medium also contains penicillin andstreptomycin with the volume percent of one. The surface
electrodes and the micro coil spring were dipped in the medium.
Cell Movement
At the first trial, C2C12 was seeded in the area inside of the
PDMS ring at the concentration of 1000 cells per cm2. The
electric pulses were applied between the surface electrode andthe micro coil spring: the period of 0.00001 s, the pulse widthof 0.000005 s, and the voltage amplitude of 1 V. The cells
were observed with an inverted phase-contrast microscope(IX71, Olympus Co., Ltd., Tokyo) for eight hours. The
time-lapse microscopic image was recorded every five minutes.
At the second trial, C2C12 was seeded at the concentration of
10000 cells per cm2. The electric pulses were applied between
the surface electrode and the micro coil spring: the period of
0.00001 s, the pulse width of 0.000005 s, and the voltage
amplitude of 10 V. The time-lapse microscopic image wasrecorded every thirty seconds for thirty minutes, and every two
minutes for six hours.
At the third trial, C2C12 was seeded at the concentration of
10000 cells per cm2. The electric pulses were applied between
the surface electrode covered with SU8-10 and the titanium
wire: the period of 0.00001 s, the pulse width of 0.000005 s,
and the voltage amplitude of 10 V.
The time-lapse
microscopic image was recorded every thirty seconds for thirty
minutes.
At the fourth trial, C2C12 was seeded at the concentration of
10000 cells per cm2. The electric pulses were applied between
the surface electrode and the titanium wire: the period of
0.00001 s, the pulse width of 0.000005 s, and the voltage
amplitude of 10 V. The time-lapse microscopic image wasrecorded every thirty seconds for thirty minutes.

Fig. 12a: C2C12 near surface electrode and wire before electric
stimulation. Dimension from left to right is 0.9 mm.

During the microscopic observation, the temperature and thecontent of the carbon dioxide gas were maintained at 310 K and
5%, respectively.
Cell Culture
C2C12 was seeded in the area inside of the PDMS ring at theconcentration of 10000 cells per cm2. After cultivation of 24hours, the electric pulses were applied to the medium for thirty
minutes per day (Fig. 10): the period of 1 s, the pulse width of
0.001 s, and the voltage amplitude of 0.5 V (the current
amplitude of 0.5 mA). The cells around the coil spring wereobserved with the inverted phase contrast microscope for ten
days. The cell culture experiments are divided into twogroups: using the setting A (Fig. 7), or B (Fig. 8).
At the end of the culture, the morphology of the surface of the
micro coil spring was observed by a scanning electron
microscope (SEM, JSM6360LA, JEOL Ltd., Tokyo, Japan).

Fig. 12b: C2C12 near surface electrode and wire electric
stimulation for 8 min. Dimension from left to right is 0.9 mm.

3. RESULTS
The results of cell movement with the electrode of the micro
coil spring show the following movement of cell. The cells
show vibration under electric stimulation between the surface
electrode covered with SU8-10 and the titanium wire (Fig. 11).
The cells approach to the titanium wire under electric
stimulation between the surface electrode and the titanium wire.
Fig. 12b shows the cells out of focus because of the movement.
In the successive cultivation, cells differentiate into myotubes inten days, and cells make tissue layer around the micro coilspring in sixteen days (Fig. 13).
Cells survive undercontinuous application of cyclic electric pulses for six hours(Fig. 14).
Fig. 15 shows the tracing of the electric pulse which is applied
between the electrodes: amplitude of 0.5 V, and width of 0.001
s.

Fig. 10: Cell culture with electric stimulation.

Fig. 11: C2C12 near covered surface electrode and wire in 2
min. Dimension from left to right is 0.9 mm.

Although the image of cells on the coil spring is not very clear,several cells adhere on the coil spring in several days of culture:
7 days on the coil spring between the surface electrodes, and 5days on the coil spring as one of the electrodes (Fig. 16),
respectively.

Fig. 13: Cell culture on coil spring on day 16.
from left to right is 0.9 mm.

Dimension

The microscopic image shows that myoblast cultured on theglass differentiates into myotubes. Between two surface
electrodes, myotubes are observed in 10 days of culture (Fig.
16b). Between the surface electrode and the coil spring
electrode, on the other hand, myotubes are observed in 6 days
(Fig. 16a).
Fig. 17 exemplifies SEM image of the coil spring at the end of
the cell culture. The figure shows that the cells and theextracellular matrix make bridges between pitch of the spring.

Fig. 14: C2C12 near coil spring under electric stimulation for 6hours. Dimension from left to right is 0.9 mm.

4. DISCUSSION
Electrolysis does not occur in the present experiment. To
prevent the medium from electrolysis, electric pulses areapplied to the medium.
The movement synchronous to the electric pulse applied
between the electrodes was observed at the cells between
electrodes. The movement of the cell might depend on the
distance between electrodes. The movement of cell is notobserved between electrodes with the distance of 5 mm, butobserved between electrodes with the distance of 3 mm in thepresent experiment.

Fig. 15: Voltage at resistance during application of electricpulse between film and spring.

Fig. 16a: Cell culture on day 6; surface electrode (left), spring
electrode (right). Dimension from left to right is 0.9 mm,
each.

Fig. 16b: Cell culture on day 10; surface electrode (left), spring
electrode (right). Dimension from left to right is 0.9 mm,each.

Most of cells adhere and differentiate to myotubes not on the
micro coil spring, but on the glass plate in the present study.
In the previous study, more cells adhered to the micro coilspring on the culture dish [7, 8]. The behavior of cells might
depend on affinity between cell and the scaffold. The
differentiation of myoblast seems to be accelerated at the setting
B in the present study, in which the coil spring is placed as oneof the electrode.
The electric stimulation has been applied to human body in theclinical treatment [9]. The cell culture technique was applied
to make a bio-actuator combined with micro-machine techniquein the previous studies [10, 11]. In the previous studies, theseveral kinds of acceleration technique to make orientation ofcells were tried in vitro: with the shear flow [12, 13], with thegravitational force [14], with the electric field [1, 15], with themagnetic field [16], or with the micro pattern of the surface [1719].
The micro coil spring gives a good scaffold for cell culture.
The spiral morphology of the coil spring might make the spiralorientation of myotubes [8]. When cells make tissue, a spacefor supplying medium is necessary around cells. A coil spring
has a spiral space along the wire: the interval of 0.085 mm
between the wires. The space might give a path for the
medium to approach to the cells. The coil spring deforms inproportion to the force. The force generated in the muscle
tissue cultured on the coil spring might be estimated by thedisplacement of the coil spring [7].
The movement of cultured myotubes is able to be controlledwith electric pulses supplied to the medium. The laser system
has been applied to measure the cyclic movement in the
biological system [20].

0.05 mmFig. 17: SEM image of cells on coil spring.

Titanium is one of the materials, which has been used forbiological application [21, 22]. Titanium has been implanted
to human body as a strut of valves, a root of teeth, pins inorthopedic treatment, and a part of joint. C2C12 is able toadhere and proliferate on the surface of the micro coil spring oftitanium. The cells are also able to differentiate into myotubes
around the coil spring [7, 8].
Under the electric stimulation of pulses, the proliferation ofcells might decrease [1]. The electric pulses, on the other hand,might control the migration to the micro coil spring, andaccelerate differentiation to myotubes.

5. CONCLUSION
The electric stimulation has been applied on myoblasts toaccelerate cultivation on a micro coil spring in vitro. A micro
coil spring made of the titanium wire of 0.085 mm was used forthe scaffold for the cell culture. The electric pulses wereapplied between the coil spring and the titanium film coated on
the glass for thirty minutes per day: the period of 1 s, the pulse
width of 0.001 s, and the voltage amplitude of 0.5 V. The
experimental results show that the movement of cell depends on
the local electric field, and that the electric stimulation tends toaccelerate differentiation of myoblasts to myotubes around themicro coil spring.

6. ACKNOWLEDGMENT
Authors thank to Hi-Lex Corp. for supply of the micro coilspring. This work was supported by a Grant-in-Aid for
Strategic Research Foundation at Private Universities from theJapanese Ministry of Education, Culture, Sports andTechnology.

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