Effect of Electric Field on Myocytes in Vitro
Ryuuhei UEMURA, Shigehiro HASHIMOTO, Yuki KATAYAMA
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 an electric field on proliferation of cultured muscle
cells has been studied in vitro. Two kinds of myocytes wereexposed to electric stimulation: C2C12 (Mouse myoblast cellline originated with cross-striated muscle of C3H mouse) andL6 (rat skeletal muscle cell). The cells were suspended inDulbecco’s Modified Eagle’s Medium. The suspension wasexposed to the electric field between two electrodes made ofplatinum wire of 0.2 mm diameter at 25 degrees Celsius for fiveminutes a day. The electric pulses at a period of one second
with a pulse width of one millisecond were generated with afunction generator. Variation was made on amplitude of pulse
between 10 mV and 100 mV. After electric stimulation forfive minutes, cells were cultivated for one day in an incubator.
For comparative study, a part of the suspension was poured intothe same kind of dish without exposure to the electric field, andincubated. After incubation, the cells were observed with aninverted phase-contrast microscope, and the number of cells
was counted. The experimental results show that proliferation
decelerates with the amplitude of electric pulses lower than 0.1
V.
Keywords: Biomedical Engineering, Muscle Cells, CellCulture, Electric Field and Proliferation

1. INTRODUCTION
Behavior of biological cells depends on various environmental
factors, such as electric [1-3], magnetic [4] and mechanicalfields [5, 6]. Electric pulses are generated with ion movement
through membrane in vivo. The muscle tissue is controlledwith electric stimulation under nerve system in vivo [7]. The
electric stimulation has also been applied to a body in somemedical treatments for rehabilitation.

a flat bottom without coating of collagen.
diameter of each well is 35 mm.

The internal

The electrodes are made of platinum wire of 0.2 mm diameter,
and attached on a polypropylene plate. When the plate covers
the wells, a couple of electrodes in the rim position of each wellgenerate an electric field in the medium. The wire has asquare form in order to avoid concentration of the electric
current, which might induce electrolysis. The cover plate has
six square holes, through which the medium is observed during
electric stimulation (Fig. 1).
Electric pulses of a period of one second with a pulse width of
one millisecond were generated with a function generator(DF1906, NF Co. Ltd., Yokohama, Japan) (Fig. 2). Variation
was made on amplitude of pulse: 10 mV, 50 mV and 100 mV.
Cell Culture
Two kind of muscle cells were used in the experiment: C2C12(Mouse myoblast cell line originated with cross-striated muscleof C3H mouse) and L6 (rat skeletal muscle cell). Muscle cells
were suspended in Dulbecco’s Modified Eagle’s Medium(D-MEM). Fetal bovine serum (FBS) was added to themedium with the volume rate in 10 percent of FBS and 90
percent of D-MEM. The 4 mL of suspension including forty
thousands of cells was poured into each well.
To keep the temperature of 37 degrees Celsius and to keep thecarbon dioxide content of five percent, an incubator was usedfor cultivation of cells. Fig. 3 shows the procedure of theexperiment. After the cells were cultured in an incubator for
three hours, electric pulses were applied for five minutes atroom temperature. After additional 21 hours cultivation in an
incubator, electric pulses were applied again for five minutes.
The successive combined process of incubation for 24 hours
and electric stimulation for five minutes was repeated two moretimes.

Cell culture technique has been progressed and myoblasts havebeen clinically applied to ischaemic cardiomyopathy in the fieldof regenerative medicine.
Acceleration technique forproliferation of cells has been studied to make muscle tissue invivo and in vitro [8, 9]. Control methodology for proliferationand differentiation of cells would be applied to regenerative
tissue technology.
In the present study, the effect of electric stimulation on
proliferation of cultured muscle cells has been studied in vitro.

2. METHODSElectric Field
The cells were cultured in a culture plate, which has six wells of

Fig. 1: Six pairs of electrodes and wells for cell culture.

WidthPeriod

Fig. 2: Electric pulse.
03482472 hourFig. 4: Number of C2C12 after cultivation for three hours.

Stimulate
Count
Incubate
Incubate
Stimulate Stimulate
Count
Incubate
Incubate
Incubate
Stimulate
Stimulate Stimulate
Incubate

Incubate

Incubate

Count

Incubate

Fig. 3: Experimental Procedure.
At every time before electric stimulation for five minutes, thenumber of cells was counted in each well one by one. Whenthe number of cells was counted, cells were exfoliated from thebottom of the well with trypsin treatment. The exfoliated cells
were introduced into a counting chamber of 0.1 mm depth.
Fig. 5: Number of L6 after cultivation for three hours.
3. RESULTS
Figs. 4 and 5 show the number of C2C12 and of L6 in each wellafter cultivation for three hours, respectively. The number ofcells scatters between thirty thousand and fifty thousand, whichshow the error range of the following data.

4. DISCUSSION
The previous study shows that electric stimulation enhances
differentiation of muscle cells [1]. Another study shows
mechanical stimulation improves tissue-engineered humanskeletal muscle [5].

Figs. 6 and 7 show the number of C2C12 and of L6 in each wellduring cultivation for nine days. The column shows the meanvalue of four data, and the bar shows the range between themaximum value and the minimum value. Although thenumber of cells saturates in a week, the number of cells
increases during five days in a sub-confluent state. The effectof electric field on proliferation of myocytes was investigatedbefore sub-confluent state.

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 cell [10]. An alternating
magnetic field might affect on adhesive molecules on the cellmembrane.

Figs. 8-10 exemplify C2C12 under electric stimulation of 0.1 V.
Figs. 11-13 exemplify L6 under electric stimulation of 0.1 V.
The figures show that cells proliferate to a sub-confluent state inthree days of culture (Fig 10 and Fig. 13).

The muscle tissue is daily exposed to the field of electric pulses,
which are signals for control of contraction. It serves a double
purpose when the electric fields control regeneration of thetissue.

Fig. 14 shows the number of C2C12 tracings for three days withelectric stimulation. Fig. 15 shows the number of L6 tracings
for three days with electric stimulation. The column shows themean value of four data, and the bar shows the range between
the maximum value and the minimum value. The results showthat proliferation decelerates with the amplitude (< 100 mV) ofelectric pulses both in C2C12 and in L6.

The present study shows that cells can proliferate after fiveminutes of electric stimulation. The experimental results showthat proliferation decelerates with the amplitude of electricpulses. The results also show that deceleration of C2C12proliferation is small with the pulse amplitude of 10 mV.

Frontier from the Japanese Ministry of Education, Culture,Sports and Technology.

REFERENCES

Fig. 6: Number of C2C12 tracings for nine days.

Fig. 7: Number of L6 tracings for nine days.

n=4.

n=4.

5. CONCLUSION
The effect of an electric field on proliferation of cultured
myocytes has been studied in vitro. The experimental results
show that proliferation decelerates with the amplitude (< 100mV) of electric pulses.

6. ACKNOWLEDGMENT
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Fig. 8: C2C12 after three hours cultivation.
shows 0.1mm.

The barFig. 9: C2C12 after 48 hours cultivation (electricstimulation of 0.1 V). The bar shows 0.1mm.

Fig. 10: C2C12 after 72 hours cultivation (electricstimulation of 0.1 V). The bar shows 0.1mm.

Fig. 11: L6 after three hours cultivation.
0.1mm.

The bar shows

Fig. 12: L6 after 48 hours cultivation (electricstimulation of 0.1 V). The bar shows 0.1mm.

Fig. 13: L6 after 72 hours cultivation (electricstimulation of 0.1 V). The bar shows 0.1mm.

Fig. 15: Number of L6 tracings for three days.
Fig. 14: Number of C2C12 tracings for three days.

n=4.

n=4.