Culture of Myoblast on Micro Coil Spring with Electric Pulses
Yusuke TAKAHASHI, Kenta NODA, Shigehiro HASHIMOTO, Yuto YARIMIZU, 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/
ABSTRACT

2. METHODS

Myoblasts have been cultured on a micro coil spring to control
proliferation and differentiation on the scaffold with electricpulses in vitro. A micro coil spring made of the titanium wireof 0.085 mm was used for the scaffold for the cell culture.
The coil spring has the dimension as follows: 0.65 mm diameter,
0.15 mm pitch, 5 mm length. C2C12 (mouse myoblast cellline) was seeded and cultured in Dulbecco’s Modified Eagle
Medium with 10 percent of fetal bovine serum. The electricpulses were applied to the coil spring for thirty minutes per day:the period of 1 s, the pulse width of 0.001 s, and the current
amplitude of 0.02 A. The cells around the coil spring wereobserved with an inverted phase contrast microscope during thecell culture. The morphology of cells was observed with ascanning electric microscope at the end of the experiment.
The experimental results show that the micro coil spring oftitanium can be the scaffold of myoblasts and that electric
stimulation might control proliferation and differentiation ofmyoblasts.

Micro Coil Spring
A micro coil spring (Hi-Lex Corp., Takarazuka, Japan) made of
the titanium wire of 0.085 mm diameter was used for thescaffold for the cell culture (Fig. 1). The coil spring has thedimension as follows: 0.65 mm diameter, 0.15 mm pitch, 5 mm
length.

Keywords: Biomedical Engineering, C2C12, Micro CoilSpring, Electric Pulses, Cell Culture and Differentiation.

1. INTRODUCTION
The cell culture technique has been developed and several
methodologies have been clinically applied to regenerative
medicine [1].
The acceleration technique for orientation and proliferation ofcells has been studied to make a biological tissue in vivo or invitro [2-5]. Control methodology for behavior of cells would
be applied to regenerative tissue technology: orientation,
proliferation and differentiation.
The effect of the surface of the scaffold on cell culture has beenstudied in the previous studies [6-9]. Several factors, which
control adhesion of biological cells, have been studied in vitro.
Myoblast has been experimentally used to make a bio-actuatorin vitro [10-17]. Several methods have been applied to control
the bio-actuator: the electric stimulation, the electrode, and thescaffold.
In the present study, myoblasts have been cultured on a micro
coil spring to control proliferation and differentiation on thescaffold with electric pulses.

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. 2A).
The lead wires of the both ends of the spring were fixed on therim of the PDMS ring with the conductive tape to keep the
position of the micro spring in the medium (Fig. 2B). Bothends of the titanium coil spring were connected to the platinum
wire with the conductive paste (Dotite, D-550, Fujikura Kasei
Co., Ltd., Tokyo, Japan) to introduce electric pulses.
Cell Culture
The tenth passage of C2C12 (Mouse myoblast cell line
originated with cross-striated muscle of C3H mouse) was
seeded in the area inside of the PDMS ring on the culture dishat the concentration of 10000 cells per cm2.
The cells were cultured with the D-MEM (Dulbecco’s Modified
Eagle Medium) in the incubator for 30 days at 310 K with 5%of carbon dioxide gas (Fig. 3). The medium contains FBS(decomplemented fetal bovine serum) with the volume percent
of 10. The medium also contains penicillin and streptomycin
with the volume percent of one. The medium was changed
every two days. The whole body of the micro coil spring wasdipped in the culture medium during cell culture.
The cells were divided into three groups: A, B and C. In thegroup A, cells were cultured without electric pulses. In thegroup B, cells were cultured with electric pulses. The electricpulses are applied for thirty minutes per day after cell culture
for 24 hours for adhesion to the scaffold. The cultured cells
around the coil spring were observed in the groups A and B.
In the group C, cells were cultured on the bottom of the culture
dish without the coil spring to check the timing ofdifferentiation from myoblasts to myotubes.
The electric stimulation was stopped on the seventh day. The
coil spring was moved on the ninth day into the well without
collagen coating and cells were cultured successively till the
31st day.

stimulator (Fig. 4). The voltages (V1 and V2) were monitoredby an oscilloscope during application of electric pulse to thecoil spring. The electric current I (Fig. 4), which flows
through Z, is calculated by Eq. 1.
I = V2 / r(1)

Temperature around the coil spring was measured by an
infrared thermography (i5, FLIR Systems, Inc., Tokyo, Japan)
during electrical stimulation.
Observation
The cells around the coil spring were observed with an inverted
phase-contrast microscope (IX71, Olympus Co., Ltd., Tokyo)
every day. In the group of electric stimulation, the cells wereobserved before and after electric stimulation for thirty minutes.
Fig. 1: Scanning electron-microscopic image of the micro coilspring. The bar shows 0.2 mm.

The morphology of the surface of the micro coil spring wasobserved by a scanning electron microscope (SEM,
JSM6360LA, JEOL Ltd., Tokyo, Japan).
In the group C, Giemsa stain solution was applied to distinguishnuclei of myotubes on the twelfth day of culture.

Fig. 2A: Micro coil spring placed in the space of PDMS ring
contained in polystyrene dish, and connected to platinum wire.
Conductive paste

Polystyrene dishFig. 3: Cell culture on micro coil spring with electric pulses.
I

PDMS ring
Platinum wireE

Micro coilrFig. 2B: Micro coil spring placed in space of PDMS ring
contained in polystyrene dish, and connected to platinum wire(cross section).

Z

V2Electric Stimulation
The electric pulse E (period 1 s, amplitude 1 V, width 0.001 s)
was generated with an electric stimulator (SEN5201, Nihon
Kohden Corporation, Tokyo, Japan). The stimulator wasconnected to the coil Z, and the pulses were applied for thirty
minutes per day for one week. An electric resistance r of 10
ohm is serially inserted between the coil spring and theV1

Fig. 4: Electric circuit for application of electric pulses (E) tocoil spring (Z).

3. RESULTS(A)

Fig. 5 shows electric signal tracings during application ofelectric pulses through the coil spring. The signal 1 in Fig. 5
shows the electric pulse E (period of 1 s, amplitude of 1 V,
width of 0.001 s). The voltage between the ends of resistanceof 10 ohm is 0.2 V, which makes current of 0.02 A (signal 2 inFig. 5).
Fig. 6 shows temperature around the coil spring before and after
application of electric pulses for thirty minutes. Fig. 6B showsthat the temperature at the surface of the coil spring is 30.9
degree centigrade after application of electric pulses for thirty
minutes. The heating effect of the electric pulses on thesurface of the coil spring is small.
Fig. 7 exemplifies cells after cultivation for 24 hours around thecoil spring. The figure shows that cells adhere both on the coilspring and on the bottom of the culture dish, even after
application of the electric pulses for thirty minutes (Fig. 7B).

(B)

Fig. 8 shows that cells proliferate to make a confluent state inthree days of culture without the coil spring.
Fig. 9 exemplifies cells after cultivation for seven days aroundthe coil spring. The figure shows that the number of cells does
not increase much around the coil spring both without (Fig. 9A)
and with (Fig. 9B) the electric pulses.
In the group C, myoblasts differentiated to myotubes in elevendays of culture (Fig. 10). The myotubes with multiple nuclei
are observed by Giemsa stain (Fig. 11).
Fig. 12 exemplifies cells after cultivation for thirty days aroundthe coil spring. The cells on the bottom of the dish start todifferentiate into myotubes.
Fig. 13 shows SEM image of the coil at the 31st day of culture.
The figure shows that cells extend to make bridges between
springs over the pitch of turns both without and with electricpulses. Myoblasts adhere around the coil spring of titanium,
proliferate, extend to make bridges between wires over the pitchof turns, and make cylindrical layers around the coil.

Fig. 5: Electric signals during application of electric pulses.

Fig. 6: Thermography around coil spring before (A) and after(B) electric pulse application for 30 min. The value shows
temperature at the center. The coil spring is located at thecenter.

Fig. 7A: C2C12 cultured for 24 hours around coil spring
without electric pulse application. Dimension from left toright is 2 mm.

Fig. 7B: C2C12 after electric pulse application for 30 min on
the second day of culture. Dimension from left to right is 2
mm.

Fig. 8: C2C12 cultured for three days without coil spring.
Dimension from left to right is 2 mm.

Fig. 9A: C2C12 cultured for seven days around coil spring
without electric pulse application. Dimension from left toright is 2 mm.

Fig. 9B: C2C12 cultured for seven days around coil spring withelectric pulse application. Dimension from left to right is 2
mm.

Fig. 10: C2C12 cultured for eleven days without coil spring.
Dimension from left to right is 2 mm.

Fig. 11: C2C12 stained after culture for twelve days without
coil spring. Dimension from left to right is 1 mm.

Fig. 12A: C2C12 cultured for thirty days around coil spring
without electric pulse application. Dimension from left toright is 2 mm.

Fig. 13B: C2C12 cultured for thirty days on coil spring withelectric pulse application. Dimension from left to right is 1
mm.

4. DISCUSSION
The cell culture technique was applied to make a bio-actuatorcombined with micro-machine technique in the previous studies[11, 12]. In the previous studies, the several kinds ofacceleration technique to make orientation of cells were tried invitro: with the shear flow [2, 3], with the gravitational force [4],with the nanofiber [7], or with the morphology of the surface [6,8].
The micro coil spring gives a good scaffold for cell culture.
The spiral morphology of the coil spring might make the spiralorientation of myotubes [15]. 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.
Fig. 12B: C2C12 cultured for thirty days around coil springwith electric pulse application. Dimension from left to right is
2 mm.

The coil spring deforms in proportion to the force. The force
generated in the muscle tissue cultured on the coil spring might
be estimated by the displacement of the coil spring [15].
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 [18].
Titanium is one of the materials, which has been used forbiological application [19, 20]. 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 of titanium. The cells are also able todifferentiate into myotubes around the coil spring [15].

Fig. 13A: C2C12 cultured for thirty days on coil spring without
electric pulse application. Dimension from left to right is 1
mm.

The number of cells around the coil spring increases, after the
ninth day of culture. Under the electric stimulation of pulses,
the proliferation of cells might decrease [14].
Because the micro coil spring was slightly apart from thebottom of the culture dish, cells did not migrate from thebottom of the dish to the coil spring. It is not easy to control
the density of cells on the coil spring at the cell seeding. At
the beginning of the culture, cells might easily migrate to thesurface of the coil spring, when the coil spring is touching on
the bottom of the dish. The coil spring is located closer to thebottom of the well, after the ninth day of culture in the present
study.
The amount of the medium around the coil spring is more easily
maintained in the wells than in the ring of PDMS, which might
be one of the reasons why cells proliferate faster after the ninth
day of culture in the present study.
The cells on the coil spring are hard to be observed by the
optical microscope, because of the direction of the transmitted
light through the three dimensional cell culture (Figs. 7, 9, 12).
The extracellular matrix, which covers around the cells (Fig.
13), also makes the microscopic observation of cells difficult.

5. CONCLUSION
Myoblasts have been cultured on a micro coil spring to control
proliferation and differentiation on the scaffold with electricpulses: the period of 1 s, the pulse width of 0.001 s, the current
amplitude of 0.02 A, and thirty minutes per day. The
experimental result shows that electric pulses tend to decrease
the proliferation and differentiation of cells.

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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