Effect of Static Magnetic Field on Muscle Cells in Vitro
Yuma SAKATANI, Shigehiro HASHIMOTO, Junichi YORIKI,
Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, 535-8585, Japanhashimoto@bme.oit.ac.jp http://www.oit.ac.jp/bme/~hashimoto

ABSTRACTAn effect of a static magnetic field on proliferation and onorientation of cultured muscle cells has been studied in vitro.
An experimental system was manufactured to apply a staticmagnetic field to muscle cell culture. The system consists of aneodymium magnet bar, a culture dish of 52 mm internal
diameter contained in a transparent plastic chamber, and an
inverted phase-contrast microscope. Air including carbon
dioxide in five volume percent was introduced to the chamber
to maintain atmosphere. C2C12 (Mouse myoblast) cells weresuspended in Dulbecco’s Modified Eagle’s Medium with fetal
bovine serum. The suspension was poured into the plastic dishplaced on the stage of the microscope, where cells are exposedto the static magnetic field around the magnet bar at 29 degrees
Celsius. For a comparative study, a part of the suspension waspoured into the same kind of dish without exposure to themagnetic field. The number of cells, which adhered to thebottom of the culture dish, was traced according to the time(<120 min) during exposure to the magnetic field. The
experimental results show that the cells tend to tilt to thedirection of the static magnetic field of < 270 mT.

neodymium magnet bar, a culture dish of 52 mm internal
diameter contained in a transparent plastic chamber, and an
inverted phase-contrast microscope. The neodymium magnetbar has a rectangular dimension of 4 mm × 8 mm × 52mm. A
static magnetic field of < 270 mT was applied to cell culture
with neodymium magnet bar, which is attached under the outersurface of the bottom plate of a culture dish. Air including
carbon dioxide in five volume percent was introduced to thechamber to maintain atmosphere around the culture dish.
For comparative study, another similar experimental system wascomposed without magnet bar (Fig.1). The experiments of
two comparative conditions were performed simultaneouslywith two systems.
The magnetic flux density was measured with a gauss meter(Model 5080, F. W. Bell Inc., USA).

With magnet

Without magnet

Keywords: Biomedical Engineering, Muscle Cells, Cell
Culture, Magnetic Field, Proliferation and Orientation

1. INTRODUCTION
Behavior of biological cells depends on various environmental
factors, such as electric [1, 2], magnetic [3-9] and mechanicalfields [10].
Cell culture technique has been progressed and myoblasts havebeen clinically applied to ischaemic cardiomyopathy in the fieldof regenerative medicine.
Acceleration technique fororientation and proliferation of cells has been studied to makemuscle tissue in vivo and in vitro [1, 10]. The previous studies
show the experimental design to evaluate the effect ofalternating magnetic fields on cells [3-5].
Controlmethodology for adhesion and proliferation of cells would beapplied to regenerative tissue technology.
In the present study, the effect of static magnetic stimulation onorientation of cultured muscle cells has been studied in vitro.

2. METHODS
Magnetic Field
An experimental system was manufactured to apply a magnetic
field to muscle cell culture. The system consists of aFig. 1: Experimental system: with and without magnet.

Cell Culture
C2C12 (Mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) cells were suspended in Dulbecco’s
Modified Eagle’s Medium (D-MEM). Fetal bovine serum(FBS) was added to the medium with the volume rate in 10
percent of FBS and 90 percent of D-MEM. The density ofcells in the suspension was adjusted to 38 thousands cells/cm3.
The six milliliters of suspension was divided into two parts andeach part of three milliliters was poured into each polystyrene
dish of 52 mm internal diameter without collagen coating.
The cells were cultured for 13 hours in a conventional incubator,
where the partial pressure of carbon oxide and temperature werekept at 5 percent and 37 degrees Celsius, respectively.
For the successive 12 hours, one dish was set in the chamberwith the magnet bar, and the other dish was set in the chamber
without the magnet bar. A selected area near the magnetic bar
was observed with the microscope every four hours (Fig. 2).
In the test without magnetic field, the cells in a randomly
selected area were observed. The room temperature was kept
29 degrees Celsius during the test.

0.1 mmCellAngle

Magnetic field

Fig. 3: Cells cultured in the static magnetic field.
Fig. 3 exemplified cells cultured in the static magnetic field.
The arrow shows direction of the magnetic field (Fig.3). The
angle between the longitudinal axis of each cell and thedirection of the magnetic field was measured one by one.
In another test, cells were cultured for nine days in the medium
with HS (horse serum) to observe differentiation, after cultured
for three days in the medium with FBS. The cells wereobserved in three selected areas of different magnetic flux
density: 15-45 mT, 60-90 mT, and 270 mT (Fig. 4).

Culture dishFig. 4: Magnetic flux density in relation to distance from thecenterline on the inner surface of the dish bottom, of which themagnet bar is attached on the outer surface.

3. RESULTS
Fig. 4 shows the magnetic flux density in relation to thedistance from the line on the inner surface of the dish bottom, of
which the magnet bar is attached on the outer surface. The
results show that the maximum value of the magnetic flux
density is 270 mT on the line, and that the magnetic flux density
decreases rapidly as the distance increases.
Observed area

Fig. 2: Observed area is near the magnet bar in the culture
dish contained in the transparent chamber.

Fig. 5 shows distribution of angles as a function of exposure
time in the magnetic field. The figure shows that the angles
tend to decrease with time.

reFig. 4: Magnetic flux density in relation to distance fromthe centerline on the inner surface of the dish bottom, of
which the magnet bar is attached on the outer surface.

Fig. 5: Angles distribution as a function of exposure timein the magnetic field.
Figs. 6 exemplifies myotubes cultured for one day in the static
magnetic field. The four pictures show C2C12 after one dayof cultre in the medium with HS under each magnetic
conditions: control, 15-45 mT, 60-90 mT, and 270mT (from topto bottom). Fig. 7 exemplifies myotubes cultured for eightdays in the static magnetic field. The four pictures show
C2C12 after eight days of cultre in the medium with HS undereach magnetic conditions: control, 15-45 mT, 60-90 mT, and270mT (from top to bottom). The cells were differentiated tomyotubes regardless of the magnetic flux density.

4. DISCUSSION
The previous study shows that exposure to the static magnetic
field of 80 mT for five days accelerates accumulation of actin
and myosin in the differentiation process of L6 cells [7]. The
study also shows that the static magnetic field of 80 mT for
previous three days of differentiation affects on alignment ofcells. Another previous study shows that the static magnetic
field of 30 mT stimulates migration of cells.
The previous study shows that magnetic fields acceleratedifferentiation of cells.
The result in the previous study shows that the temperature
increases form 25 degrees Celsius to 29 degrees Celsius for one
hour exposure to the magnetic field and saturates at 29 degrees
Celsius after one hour exposure [5]. When electric current
applies to a coil, the coil generates not only magnetic field, butalso thermal effect. The temperature of control study wasadjusted to the same value as that of magnetic field study, sothat the effect of magnetic field can be distinguished from thethermal effect.
The previous study shows that electric stimulation enhances
differentiation of muscle cells [1, 2]. Another study shows
mechanical stimulation improves tissue-engineered humanskeletal muscle [10].
Several factors might govern adhesion of biological cells. The
previous study shows that electric stimulation can restrict
adhesion of muscle cells [2]. Another study shows anelectromagnetic field affects on the cellular collagen [6]. AnFig. 6: C2C12 after one day of cultre in the medium with
HS under each magnetic conditions: control, 15-45 mT,
60-90 mT, and 270mT. The bar shows 0.1 mm.

alternating magnetic field might affect on adhesive moleculeson the cell membrane.
In the previous study, an alternating magnetic field of 1-4 mT ata period of 0.017 s was applied to the muscle cells, which
tended to align to the direction of the magnetic field [3]. The
slightly higher alternating magnetic field of 13 mT at a periodof 0.01 s was applied to muscle cells in the present study, tocompare results to that of the previous study. Distinguishingthermal effects, the effect of magnetic field is evaluated clearerin the present experimental system than in the previous one.
The previous study shows that adhesion of muscle cells to thebottom of the culture dish is accelerated with alternatingmagnetic fields.

5. CONCLUSION
The effect of a static magnetic field on orientation of cultured
muscle cells to the culture plate has been studied in vitro. The
experimental results show that proliferation of muscle cells is
decelerated and the cells tend to tilt with a static magnetic fieldof < 270 mT.

6. ACKNOWLEDGMENT
This work was supported by a Grant-in-Aid for Academic
Frontier from the Japanese Ministry of Education, Culture,
Sports and Technology.

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