Environmental Design of Muscle Cell Culture
for Micro-actuator
Shigehiro Hashimoto, Hiroshi Tsutsui, Shuichi Mochizuki, Masahiko Yoshiura,
Sadahito Uto, Kenzo Akazawa, Mieko Ohsuga, Hiroyuki Kobayashi, Toshikazu Kawai,
Kenichi Yamazaki, Hideo Kondo, Koichi Imoto, Jun Takase, Masahide Okada
Dept. Biomedical Engineering, Osaka Institute of Technology, Osaka, 535-8585, Japanhashimoto@bme.oit.ac.jp
http://www.oit.ac.jp/bme/
andHajime Otani
Cardiovascular Center, Kansai Medical University, Moriguchi, JapanandToshia Fujisato
Department of Regenerative Medicine and Tissue Engineering,
the Advanced Medical Engineering Center, National Cardiovascular Center, Suita, JapanandKiyoshi Yoshinaka
National Institute of Advanced Industrial Science and Technology, Life Science and Technology,
Institute for Human Science and Biomedical Engineering, Surgical Assist Technology Group,Tsukuba, Japan
ABSTRUCT
An interdisciplinary research project on“Application of cultured muscle cells on
medical engineering” has been proposed
between engineering and medicine to beapplied to actuators. In the present study,
environment of muscle cell culture has been
designed to accelerate cells proliferation,differentiation, and arrangement to be applied
to micro-fabricated scaffolds with magnetic
and electric fields in vitro. In the magnetic
fields test, isolated muscle cells from ahind-limb quadriceps of an adult miniature pig

were cultured in the controlled magnetic field
for 72 hours. The culture dishes were placedparallel to the uniform alternating magnetic
field, which was generated by a manufactured
solenoid coil with a cooling water circulation
system. In the electric stimulation test, L6
rat’s myoblasts were cultured and electricallystimulated between two electrodes of platinum
wire with 4 V rectangular pulse waves for ten
minutes every two days for one week. On theother hand, cell culture scaffolds have been
designedandfabricatedwithastereo-lithography system.
The designed
environment has possibility to accelerate cells

proliferation, differentiation, and arrangement
to be applied to micro-fabricated scaffolds.
Keywords: Biomeasurement, Cell-Culture,Muscle Cell, Environment, Micro-Actuator,
Magnetic Field, Electric Stimulation
1. INTRODUCTION
An interdisciplinary research project on“Application of cultured muscle cells on
medical engineering” has been proposed
between engineering and medicine to beapplied to actuators.
Cell culture technique has been progressed [1],and myoblasts have been clinically applied toischaemic caridiomyopathy in the field ofregenerative medicine [2].
Electrical andmechanical stimulation to cell culture havebeen investigated in the previous studies tocontrol orientation of cells [3-5]. The previous
study shows the experimental design toevaluate the effect magnetic field on cells[6-10].
The engineering on robotics has progressed, on
the other hand, with biomimetic studies, which
has been applied to control methodology inmultidisciplinary fields. A lot of simulation
models have been proposed in the biomedical
engineering field [11].

2. METHODS
Magnetic field test
An alternating magnetic field between 2 mT
and 3 mT for 72 hours is generated around acoil with alternating electric current.
To
generate a magnetic field, a solenoid coil of1800 turns was manufactured. The coil wasenclosed with the flexible sheet of micro-grain
metal to shield the experimental space fromthe surrounding magnetic field (Fig. 1).
Cooling water of 33 degrees Celsius wascirculated around the coil in the incubator tocontrol temperature in the culture dish duringexposure to the magnetic field (Fig. 2). In theexperiment, both the magnetic flux densityand frequency were controlled with theoscillator in sinusoidal wave of 60 Hz. The
wave-forms of input voltage and output
current are measured with an oscilloscope.
The culture medium consists of Eagle’s MEM
medium (including kanamycin), 10 percent offetal bovine serum, fibrobrast growth factor,
and was sterilized by an autoclave. Adult
miniature pig skeletal muscle cells wereisolated from a hind-limb quadriceps [8, 9].
The muscle in the femoral region was removed,

Micromachining technique has also been
progressed with optic technology and been
tried to be applied to the biomedical
engineering field.
Biological muscles control smooth movements,and have high efficiency to generate powerwith its light structure, which might bridge toan ideal actuator.
In the present study, environment of muscle
cell culture for micro-actuator has been
designed to accelerate cells proliferation,differentiation, and arrangement to be applied
to micro-fabricated scaffolds with magneticand electric fields in vitro.
Fig. 1: Experimental system for magnetic field.

Fig. 2: Cooling system of cell culture during exposure to magnetic field.

10 mm

0.05 mmFig. 3. L6 rat’s myoblast.

and placed in a sterilized tube with a medium.
The muscle was moved onto a dish with asaline solution and minced into pieces with theforceps scissors. After an enzymatic digestion
was processed with trypsin, the suspension
was filtered and collected cells were seeded
onto a dish of 35 mm diameter coated withcollagen at a density of 50 thousand cells permilliliter. The density was adjusted with aFig. 4: Electric stimulation apparatus.

hemocyte-meter. Dulbecco’s Modified Eagle’s
Medium with 10 % of fetal bovine serum andwith 0.02 mg/mL of fibroblast growth
factor-basic was used for the culture medium.
The experiment with magnetic field wasperformed with cells between sixth and tenthTemperature of medium [℃]60
Control

Cooling

50403020100410Time [h]Fig. 6: Cooling effect.

Fig. 5: Stereo-lithography system. Acrytic plasticlayers are accumulated one by one.

Cell Numbers [×104 cells/cm3]

1614Control

2 [mT]

024

3 [mT]

8 [mT]

1210864204872

Time (hour)generation during the successive subculture.
The pig’s skeletal myoblasts were cultured ondishes coated with collagen in each magnetic
condition. The results were evaluated about
multiplication rate of cells, and thearrangement of cells.
Electric stimulation testL6 rat’s myoblasts (Fig. 3) cultured on the dishwithout collagen coating were used in theexperimentforelectricstimulation.
Dulbecco’s Modified Eagle’s Medium with 10 %of fetal bovine serum was used for the culture
medium. Platinum wires of 0.1 mm diameter
were used for electrodes (Fig. 4). After cells
proliferated into confluent on the culture dish,cells were electrically stimulated with 4 V
rectangular pulse waves of 1.0 ms at thefrequency of 0.5 pulse per second for ten
minutes every two days for one week.
Micromachining
Cell culture scaffolds have been designed andFig. 7: Proliferation in magnetic field.

fabricated with a stereo-lithography system(Fig. 5). The scaffolds consist of collagenbridges and acrylic plastic anchors.
3. RESULTS AND DISCUSSION
Temperature in the culture dish was
successfully controlled around 37 degrees
Celsius during exposure to the magnetic fieldsof 3 mT with the aid of circulation of thecooling water (Fig. 6).
The results show that magnetic field does notaffect on cells proliferation (Fig 7), and thatcells tend to tilt to the direction of themagnetic field [10]. Cultured cells formed amass in several days after confluent state inmonolayer (Fig. 8).

10 mmFig. 8: Mass formed after confluent state.

1 ㎜

0.050 mm

1 ㎜
Fig.
10:Scaffold
fabricatedwithstereo-lithography system; design (upper), anchor(center), with collagen bridge (lower).

0.05 mmBAFig. 9: L6 rat’s myoblasts proliferated, arranged(upper), fused and formed myotubes (lower A &B).

Myotubes were formed in 19 days fromconfluent layer formation of myoblasts underthe electric stimulation (Fig. 9). Myoblasts
fusion and myotubes formation might beaccelerated with the electric stimulation.
The scaffold was successfully fabricated with astereo-lithography system (Fig. 10).
Reconstructed model in cultured muscle cells
would be applied to investigation on muscle
control in the biomedical engineering researchfield.

4. CONCLUSION
Environment of muscle cell culture formicro-actuators has been designed withmagnetic and electric fields in vitro. The
designed environment has possibility toaccelerate cells proliferation, differentiation,
andarrangement
tobeappliedtomicro-fabricated scaffolds.
5. ACKNOWLEDGMENT
This work was supported by a Grant-in-Aid for
Academic Frontier from the Japanese Ministryof Education, Culture, Sports, Science andTechnology.
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