Effect of Magnetic Field on Adhesionof Muscle Cells to Culture Plate
Junichi YORIKI, Shigehiro HASHIMOTO, Keiji TACHIBANA, Masahide OKADA,Shuichi MOCHIZUKI, Toshia FUJISATO
Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, 535-8585, Japanhashimoto@bme.oit.ac.jp http://www.oit.ac.jp/bme/~hashimotoandHajime OTANI
Cardiovascular Center, Kansai Medical University, Moriguchi, JapanABSTRACT

2. METHODSThe effect of a magnetic field on adhesion of cultured muscle
cells to the culture plate has been studied in vitro. An
experimental system was manufactured to apply a magnetic
field to muscle cell culture. The system consists of a couple ofsolenoid coils, a culture dish of 52 mm internal diameter, and an
inverted phase-contrast microscope.
The solenoid coilgenerates the alternating magnetic field of 13 mT of theeffective value at a period of 0.01 s with the electric current of
the rectangular pulses. C2C12 (Mouse myoblast cell line
originated with cross-striated muscle of C3H mouse) cells weresuspended in Dulbecco’s Modified Eagle’s Medium. The
suspension was poured into the plastic dish for culture placed on
the stage of the microscope, where is exposed to the magnetic
field between the solenoid coils at 29 degrees Celsius. For
comparative study, a part of the suspension was poured into thesame kind of dish without exposure to the magnetic field at 29
degrees Celsius. The number of cells, which adhered to thebottom of the culture dish, was traced according to the time(<130 min) during exposure to the alternating magnetic field.
The experimental results show that adhesion is accelerated withalternating magnetic field of 13 mT.

Magnetic Field
The experimental system consists of a couple of solenoid coils,
a culture dish of 52 mm internal diameter, and an inverted
phase-contrast microscope (Fig. 1). The magnetic field isapplied to the culture dish with the solenoid coil. The solenoid
coil was manufactured with cupper wire of 0.5 mm diameter(Fig. 1). The wire is coiled 1000 turns around a plastic
cylinder solid, which has a diameter of 10 mm and a length of500 mm.

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

The magnetic flux density was measured with a gauss meter(Model 5080, F. W. Bell Inc., USA). The alternating magnetic
field of 13 mT of effective value at a period of 0.01 second isgenerated with alternating electric current, which is measured at
the observation point in culture dish. Alternating electric
power, which have 0.01 second period, are generated withBipolar DC Power (BP4610, NF Co. Ltd., Yokohama, Japan).

DishCoil

1. INTRODUCTION
Behavior of biological cells depends on various environmental
factors, such as electric, magnetic and mechanical fields.

Microscope

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, 2]. The previous studies
show the experimental design to evaluate the effect of magnetic
fields on cells [3, 4]. Control methodology for adhesion andproliferation of cells would be applied to regenerative tissue
technology.
Bipolar DC PowerIn the present study, the effect of electric stimulation onadhesion of cultured muscle cells has been studied in vitro.

Fig. 1: Experimental system.

CoilDishHeater

electric heaters with a thermo-regulator (TF4-10, Keyence Co.
Ltd., Osaka, Japan) (Fig. 2). The distribution of temperature at
the surface of the water in the culture dish placed on the heater
without the magnetic field was measured with an infra-red
thermo camera, while the heater was controlled to 29 degrees
Celsius.

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 three
milliliters of suspension, which was kept at 29 degrees Celsius
in the water bath before test, was poured into the polystyrene
dish of 52 mm internal diameter without collagen coating in 2
million cells/cm2.
Ten sectors of 4 mm square in the culture dish were randomly
selected to trace the behavior of the muscle cells in the fixedarea under the microscopic observation. The time until thefirst adhesion of cell was measured in each sector. The
numbers of cells, which adhered on the bottom in each sector ofthe dish during continuous exposure to the magnetic field, werecounted at 15, 30, 45, 60, 75, 90, 105, 120 minutes, respectively.

Microscope

Thermo-regulator

Magnetic flux density [mT]Fig. 2: Control study with thermo-regulator.

At the end of exposure to the magnetic field for 130 minutes,
five sectors of 4 mm square were selected independent ofobserved sectors for 120 minutes. The numbers of cells, which
adhered on the bottom in each sector of the dish, were counted
after exposure to the magnetic field for 130 minutes,
respectively.

252015105The ratio (R) of number of adhered cells to number of total cells
is calculated by Eq. 1.

0020406080Distance [mm]Fig. 3: Magnetic flux density in relation to distance fromthe right end of the left coil.

The culture dish is placed between two coils so that theirlongitudinal symmetry lines are duplicated each other. The
distance between two coils is 70 mm to leave space for theculture dish.
Temperature
The temperature of the liquid in the culture dish duringexperiment was adjusted to the same level (29 degrees Celsius)
between two conditions: with and without magnetic field.
Distilled water of three milliliter was contained in the culture
dish of 52 mm internal diameter to measure distribution oftemperature. The distribution of temperature at the surface of
the water in the culture dish between two coils during exposure
to the magnetic field was measured with an infra-red thermo
camera (Neo Thermo, TVS-700, NEC Avio Infrared
Technologies Co. Ltd., Tokyo, Japan). The temperature wastraced for 160 minutes from the start of the magnetic field
exposure, while the room temperature was maintained at 25
degrees Celsius.
In the control study, no magnetic stimulation was applied to themedium after cell seeding. To adjust the temperature without
exposure to the magnetic field same as that with exposure to themagnetic field, control dish was placed between the plate type

R=A/T

(1)

In Eq.1, A is the number of the adhered cells and T is thenumber of total cells in the sector.

3. RESULTS
Fig. 3 shows the magnetic flux density in relation to the distancefrom the right end of the left coil. The distance of 35 mm
corresponds to the middle point between two coils in Fig. 3.
The observation point is the distance of 15 mm, where themagnetic flux density is 13 mT.
Fig. 4 exemplifies the distribution of temperature at the surfaceof the water in the culture dish between two coils duringexposure to the magnetic field, where the upper figure shows
the distribution of temperature after exposure for thirty minutes
and the lower figure shows that for sixty minutes. The uniform
temperature area in the middle of the figure corresponds to thesurface of the water in the dish of 52 mm diameter (Figs. 3, 4).
The horizontal distance is 86 mm in Figs. 3 and 4. The figure
shows area of higher temperature at the coil (left and right in Fig.
3). The black filled circle marks the point of measure for Fig.
5. The point was selected as a typical point for observation ofcells in the dish.
Fig. 5 exemplifies the distribution of temperature at the surfaceof the water in the culture dish placed between the heaters.
The black filled circle marks the point of measure for Fig. 6.
The high temperature area around the culture dish corresponds
to the surface of the heater.

Temperature [deg C]

30292827262524050100150200

T ime [min]Fig. 6: Tracings of temperature at marked point of Fig. 4.

30

Fig. 4: Distribution of temperature between coils. Upper,
30 min; Lower, 60 min. Distance between left and right is
87 mm in the figure.

Temperature [deg C]

292827262524050

100150200

T ime [min]Fig. 7: Tracings of temperature at marked point of Fig. 5.
Fig. 6 exemplifies tracings of temperature at the surface of thewater in the culture dish (Fig. 3) for 160 minutes from the startof the magnetic field exposure, while the room temperature wasmaintained at 25 degrees Celsius. The result shows that thetemperature increases form 25 degrees Celsius to 29 degrees
Celsius for one hour exposure to the magnetic field andsaturates at 29 degrees Celsius after one hour exposure.
Fig. 7 exemplifies tracings of temperature at the surface of thewater in the culture dish (Fig. 4) between the heaters for 160
minutes from the start of the heating, while the room
temperature was maintained at 25 degrees Celsius. The resultshows that the temperature increases form 25 degrees Celsius to
29 degrees Celsius for one hour heating and saturates at 29
degrees Celsius after one hour heating. The results show thatthe arranged heater works well to adjust the condition of thetemperature in the liquid in the culture dish to the same level asthat exposure to the magnetic field.
Fig. 8(a) exemplifies muscle cells in the medium duringexposure to the magnetic fields with comparison to control
study (Fig. 8(b)). The floating cells are rounded, whileadhered cells are deformed. With the focus of their fringe, theadhered cells are able to be distinguished from floating cells.
Fig. 5: Distribution of temperature between heaters.
Upper, 30 min; Lower, 60 min. Distance between left andright is 87 mm in the figure.

Fig. 9 shows the time for the first cell adhesion. The test wasrepeated 13 times, and each result is displayed at each case in
Fig. 9. In the most of cases, the time is shorter in magnetic
field than in control. The experimental results show that the12001000
Time [s]8006004002000051015CaseFig. 9: Time for the first cell adhesion. Circle, withmagnetic field; triangle, control.

Fig. 8(a): Muscle cells exposed to magnetic field for 30min (upper) and for 60 min (lower). Distance between
left to right is 0.8 mm in the figure.

Adhesion ratio [％]

10080
60
40
2001530456075time [min]90105 120

15304560 75 90
time [min]105 120

Adhesion ratio [％]1008060
40
200Fig. 10: Ratio of cells adhesion in relation to exposure
time to the magnetic field (upper), and that of control
study.
Fig. 8(b): Muscle cells without exposure to magnetic
field for 30 min (upper) and for 60 min (lower).
Distance between left to right is 0.8 mm in the figure.

earlier with the magnetic field than without magnetic field.
The ratio of cells adhesion saturates for 2 hours exposure to themagnetic field (Fig. 10). Each bar shows the range between
the maximum and minimum values in Fig. 10.

cells tend to adhere faster with the magnetic field than withoutmagnetic field.

Fig. 11 shows the ratio of cells adhesion after exposure to themagnetic field for 130 minutes. The test was repeated 13 times,and each result is displayed at each case in Fig. 11. The ratioof cells adhesion was counted at five sectors in the area marked
in Figs. 4&5 in each case. In the most of cases, the ratio ishigher in magnetic field than in control. The results show thatFig. 10 shows the ratio of cells adhesion in relation to exposure
time to the magnetic field. The results show that the ratio ofadhesion increases with culture time and that the ratio increases

%

thermal effects, the effect of magnetic field is evaluated clearer
in the present experimental system than in the previous one.

1009080

The present study shows that adhesion of muscle cells to thebottom of the culture dish is accelerated with alternatingmagnetic fields.

70605040

5. CONCLUSION

302010M agnet

Control

0The effect of a magnetic field on adhesion of cultured muscle
cells to the culture plate has been studied in vitro. The
experimental results show that adhesion is accelerated withalternating magnetic field of 13 mT.

1 2 3 4 5 6 7 8 9 10 11 12 13Case

6. ACKNOWLEDGMENT

Fig. 11: Ratio of cells (%) adhesion after exposure to themagnetic field for 130 minutes.

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

the ratio of adhesion for 130 minutes is higher with themagnetic field than without magnetic field.

REFERENCES

The experimental results show that adhesion is accelerated withalternating magnetic fields.

4. DISCUSSION

The length of the cylinder is enhanced to 500 mm for thediameter of 10 mm, because the larger ratio between length anddiameter approximates uniform magnetic field between twosolenoid coils.
When electric current applies to a coil, the coil generates not
only magnetic field, but also thermal effect. The temperatureof control study was adjusted to the same value as that ofmagnetic field study, so that the effect of magnetic field can be
distinguished from the thermal effect.
The previous study shows that electric stimulation enhances
differentiation of muscle cells [1, 5]. Another study shows
mechanical stimulation improves tissue-engineered humanskeletal muscle [2].
Several factors might govern adhesion of biological cells. The
previous study shows that electric stimulation can restrict
adhesion of muscle cells [5]. Another study shows anelectromagnetic field affects on the cellular collagen [6]. Analternating magnetic field might affect on adhesive moleculeson the cell membrane.
In the previous study, an alternating magnetic field of 1-4 mT at
a period of 0.017 s was applied to the muscle cells, which
tended to align to the direction of the magnetic field [3]. The
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