Effect of Extremely Low Frequency Magnetic Field
on Cultured Skeletal Myoblasts
Shigehiro Hashimoto, Wataru Ikeda, Yusuke Morita, Shuichi Mochizuki, Kenichi Yamasaki,
Hideo Kondo, Koichi Imoto, Yutaka Ishimoto, Jun Takase
Biomedical Engineering, Osaka Institute of Technology, Osaka, 535-8585, Japan
hasimoto@elc.oit.ac.jp
andHajime Otani, Hiroji Imamura, Toshiji Iwasaka
Cardiovascular Center, Kansai Medical University, Moriguchi, JapanandToshiya Fujisato
Department of Regenerative Medicine and Tissue Engineering,
the Advanced Medical Engineering Center, National Cardiovascular Center, Suita, JapanABSTRUCT

1. INTRODUCTION

The effect of a magnetic field at an extremely
low frequency on proliferation and orientationof cultured skeletal myoblasts has beenstudied in vitro. Skeletal muscle cells wereisolated from a hind-limb quadriceps. The
minced sample from a skeletal muscle wasrinsed with a phosphate buffer solution,
processed with an enzymatic digestion,
centrifuged at 250 G, cultured in the medium
for one hour, and filtered. The isolated cells
were collected and seeded onto a dish coatedwith collagen. The culture dishes were placedparallel to the uniform alternating magnetic
field, which was generated with themanufactured solenoid coil. The result showsthat the cultured skeletal myoblasts tend toalign to the direction of the magnetic field at
the extremely low frequency.

Cell culture technique has been progressed [1],and Myoblasts have been clinically applied toischaemic cardiomyopathy [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 [6-8].
The present experimental system has been
designed to evaluate the effect of magnetic
field on skeletal myoblasts in vitro.
Reconstructed model in cultured muscle cells
would be applied to investigation on muscle
control in the biomedical engineering research
field. The control methodology for orientationof cells might be applied to regenerated muscle
tissue to achieve its effective electro
mechanical function. In the present study,
the effect of a magnetic field at an extremely
low frequency on proliferation and orientationof cultured skeletal myoblasts has beenstudied in vitro.

Keywords: Bio-measurement, Magnetic field,
Skeletal
Myoblasts,Proliferation,Arrangement

2. METHODS
Experimental Equipment
Theexperimentalequipment
wasmanufactured
togeneratealternating
magnetic fields at an extremely low frequency.
An alternating magnetic field (60 Hz, 1 mT – 4mT) was generated through a solenoid withalternating voltage source of 6 V – 18 V. The
solenoid consists of a cupper wire of 0.6 mm
diameter coiled at 1800 turns aroundpolymethyl-methacrylate cylinder of 85 mm
diameter.
The generated magnetic flux
density is measured with a gauss-meter. Four
dishes (radius, 35 mm) were placed in thecenter part of the cylinder so that the culture
plane of the dish is parallel to the magneticfield (Fig. 1).
Another dish for control test was enclosed withthe flexible sheet of micro-grain metal to shield
the experimental space from the surrounding
magnetic field.
In the experiment, both the magnetic flux
density and frequency were controlled with theoscillator. The wave-forms of input voltage
and output current are measured with anoscilloscope.

Cell Isolation and Culture
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.
Figure 2 shows the experimental protocol.
Adult rat skeletal muscle cells were isolated
from a hind-limb quadriceps. The muscle in
the femoral region was removed, and placed ina sterilized tube with a medium. The muscle
was moved onto a dish with a saline solution
and minced into pieces smaller than three
cubic millimeter with the forceps scissors.
The sample was collected into a centrifugationtube, rinsed with a phosphate buffer solution(without calcium chloride and magnesiumchloride) three times. An enzymatic digestion
was processed for ten minutes shaking at 37
degrees centigrade with four milliliter of 0.25
percent trypsin and with an additional
phosphate buffer of eight milliliter. After theenzymatic process was stopped with anadditional medium solution of four milliliter,
the top layer of sample was collected andcooled in ice. After centrifugation at 250 G atroom temperature, the clear layer at the top
was discarded and medium of two milliliter
was added.

TransformerABCDE

Solenoid coilHGF

Fig. 2: Experimental protocol. A: muscle
extraction, B: mince, C: trypsin process, D:cooling, E: centrifugation, F: incubation, G:
filtration, H: cultivation.
Fig. 1: Experimental equipment.

The cells were dispersed with a pipette andexpanded on a dish of 90 mm diameter,
cultured in the medium of 15 milliliter at 37
degrees centigrade with carbon dioxide of five
percent in an incubator. After cultivation for
one hour, the suspension with cells wascollected under gentle stirring with a pipette.
After the suspension passed through the filterof 40 micrometer holes, the isolated cells werecollected and seeded onto a dish of 35 mm
diameter coated with collagen at a density of
500 thousand cells per milliliter. The density
was adjusted with a hemocyte-meter.
Cell Culture in Magnetic Field
Porcine skeletal myoblasts were collected froma femoral muscle. A piece of muscle tissue
was extracted from the femoral muscle, minced,
treated with trypsin, centrifuged, and filtered.

Figure 3 shows rat-cells observed with amicroscope after cultivation for one hour. Thecentrifugation was processed at room
temperature, because fat makes several
colloidal
massivestructures
atlow
temperature and interrupted collection ofisolated cells (Fig. 3 (a)). The filter with holesof 40 micrometer was effective to isolate
muscle cells (Fig. 3 (b)). The number offibroblasts in the suspension decreases after
cultivation for first one hour, because
fibroblasts adhere faster than the muscle cells.
Collagen coated on the dish promotes adhesionof muscle cells. Experimental results showthat important factors, which govern isolationof muscle cells, are filtration and temperatureduring centrifugation.
Figure 4 shows isolated skeletal myoblasts.

To isolate myoblasts, cells were cultured on a
100 mm dish coated with collagen in an
incubator through four generations andidentified with immunological stain techniqueof Desmin, which marks typical protein inmuscle cells.
The cells from the fifth generation of cultured
colony were used for the test.
The
myoblasts were cultured on dishes coated withcollagen in each magnetic condition for threedays.
The results were evaluated about
multiplication rate of cells, and thearrangement of cells.

Fig. 3 (a): Cells after one hour cultivation.

The population of cells in the certain area inthe dish was counted every twelve hours witha phase contrast microscope to observe
proliferation. The acute angle between themagnetic field and the longitudinal axes ofmyoblast was also measured on each cell toevaluate the effect of magnetic field onorientation of cells. The plate, which has fiveholes of 1 mm diameter, was attached on thebottom of the dish to trace the same area.
1 mm

3. RESULTS AND DISCUSSION
The intensity of surrounding magnetic fields
sufficiently decreased with the flexible sheet ofmetal.

Fig. 3 (b): Cells after one hour cultivation(filter of 0.04 mm).

Experimental systems had been designed toimprove magnetic conditions to study the effectof magnetic field on cell culture [6-8]. The
uniform alternating magnetic field wascontrolled in the present experimental device
with the solenoid coil and with the flexible
sheet of micro-grain metal.
4. CONCLUSION

control
2 mT
3 mT
1 mT

806040200122448
Time (h)72

Fig. 5: Proliferation in magnetic field.

1201008060
40
200

1 mT
2 mT
3 mT
4 mT
Fig. 6: Number of cells after 72 hours cultivation.

controlAngle(degree)

The experimental protocol about isolation ofmuscle cells and devices would be effective to
study the effect of magnetic field on cultured
muscle cells in vitro. The result shows thatthe cultured skeletal myoblasts tend to align tothe direction of the magnetic field at theextremely low frequency.

Number of cells

The angles distribute randomly in the cells
shielded from magnetic field, where they
distribute at smaller angles in the cells
exposed to the parallel magnetic field (Fig. 7).
The result shows that the cultured skeletal
myoblasts tend to align to the direction of themagnetic field at the extremely low frequency.

100

N u m b e r o f c e lls (% )The results show that population of cells
increases exponentially with time (Fig. 5)
except in the magnetic field of 4 mT (Fig. 6).

2 mT

3 mT

1 mT

90
80
70
60
50
40
30
20
100
24

48
72
Time(h)Fig. 7: Angles between cell and magnetic field.

5. ACKNOWLEDGMENT
0.01mmFig. 4: Isolated myoblasts.

This work was supported by a Grant in Aid for
Academic Frontier from the Japanese Ministryof Education, Culture, Sports, Science andTechnology: “ Application of Cultured Muscle
Cells on Medical Engineering“6. REFERENCES[1] Yasuda S.I., Sugiura S., “A Novel Method toStudy Contraction Chracteristics of a Single
Cardiac Myocyte Using Carbon Fibers”, AmJ Physiol Heart Circ Physiol, Vol. 281(3),2001, pp. H1442-1446.
[2] A.A. Hagege, C. Carrion, P. Menasche, et al,“Viability and Differentiation of Autologous
Skeletal Myoblast Grafts in Ischaemic
Cardiomyopathy”, Lancet, Vol. 361, 2003, pp.
491-492.
[3] Dawn M. Pedrotty, Jennifer Koh, Bryce H.
Davis, Doris A. Taylor, Patrick Wolf, andLaura E. Niklason, “Engineering Skeletal
Myoblasts: Roles of Three-Dimensional
Culture and Electrical Stimulation”, Am J
Physiol Heart Circ Physiol, Vol. 288, 2005,
pp. H1620-1626.
[4] C.D. McCaig, P.J. Dover, “Factors
Influencing Perpendicular Elongation ofEmbryonic Frog Muscle Cells in a Small
Applied Electric Field”, J. Cell Sci, Vol. 98,
1991, pp. 497-506.
[5] K. Kanda, T. Matsuda, ”MechanicalStress-InducedOrientation
andUltrastructural Change of Smooth Muscle
Cells Cultured in Three-Dimensional
Collagen Lattices”, Cell Transplant, Vol. 3,1994, pp. 481-492.

[6]

Shigehiro Hashimoto, Hidekazu Tsuji,
Chiaki Miyamoto, Atsushi Yamanaka,
Yoshiaki Hirano, Naoki Ogawa, Tomohiro
Sahara, Hajime Otani, Hiroji Imamura,
Yusuke Morita, “Effect of magnetic field at
low frequency on cells”, Proc. International

Federation for Medical and BiologicalEngineering, Vol.3, No.2, 2002, pp.

1402-1403.
[7] Chiaki Miyamoto, Shigehiro Hashimoto,
Yusuke Morita, Hidekazu Tsuji, Atsushi
Yamanaka, Takayuki Sekiyama, TomohiroSahara,
Kenichi Yamasaki, Teruyuki
Yamanari, Yuta Morioka, Yue Wu, Hajime
Otani, Hiroji Imamura, “Effect of magnetic
field at low frequency on cells arrangement”,

Proc. 7th World Multiconference onSystemics Cybernetics and Informatics,

Vol.8, 2003, pp. 62-66.
[8] Yuta Morioka, Shigehiro Hashimoto, Chiaki
Miyamoto,
YusukeMorita,Takayuki
Sekiyama, Tomohiro Sahara, Kenichi
Yamasaki, Hideo Kondo, Teruyuki Yamanari,
Yoshiaki Hirano, Hajime Otani, HirojiImamura, “Effect of Magnetic Field onCells”, Proc. 8th World Multiconference onSystemics Cybernetics and Informatics,Vol.7, 2004, pp. 177-182.