Effect of Electric Field Direction on Contractile Movement ofLongitudinally Oriented Myotubes Cultured in Vitro
Shigehiro HASHIMOTO, Ayumi NAKAGAWA, Kana NAKAMURA, Masahide OKADA,Shuichi MOCHIZUKI, Toshia FUJISATO, Sadahito UTO, Hiroyuki TONAMI
Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, 535-8585, Japanhashimoto@bme.oit.ac.jp http://www.oit.ac.jp/bme/~hashimotoandTetsuji YAMAOKA
Department of Biomedical Engineering, Research Institute of National Cardiovascular Center,Suita, Japan
biological cells depends on various environmental factors, suchas electric, magnetic and mechanical fields.

ABSTRACTThe effect of electric field direction on contraction of
longitudinally oriented myotubes has been studied in vitro.
The oriented myotubes were made by the cultivation in thevortex flow around a silicone rubber disk in the culture dishswung by a rotating tilted plate at 30 revolutions per minute.
C2C12 (Mouse myoblast cell line) cells are cultured in theDulbecco’s Modified Eagle’s Medium for seven days. The
horse serum was added to differentiate the muscle cells tomyotuybes. After the cultivation, periodical electric pulses (1ms pulse width with a period of 0.1 s) were applied to themedium with a pair of electrodes of platinum wire of 0.2 mm
diameter. The myotubes were tetanized, when the electricpulses were applied. The distance between traceable points ona myotube in longitudinal direction was measured during themicroscopic observation. Variation was made on the angle
between the longitudinal direction of myotube and the directionof the electric field. The experimental results show that theamplitude of contraction is maximized when the direction of theelectric field is parallel to the longitudinal direction of myotubes,
and that the rate increases when the myotubes are oriented
parallel each other.

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 [2-5]. Reconstructed
myotubes have a potential to be applied to a micro actuator.
Orientation of myotubes might affect on their controllability
with an electric stimulation.
Several effects of electric stimulation have been reported on
biological cells. The previous study shows that electric
stimulation enhances differentiation of muscle cells [1].
Another study shows mechanical stimulation improves
tissue-engineered human skeletal muscle [4]. The previous
study also shows that muscle cells can adhere and proliferate
under electric stimulation with periodical pulses, and thatadhesion of muscle cells can be controlled with the amplitude ofpulse [5].
In the present study, the effect of electric field direction oncontraction of longitudinally oriented myotubes has beenstudied in vitro.

Keywords: Biomedical Engineering, Muscle Cells, Cell Culture,
Electric Field, Tetanic Contraction, Vortex Flow and Orientation
2. METHODS
1. INTRODUCTION
Electrical stimulation has been applied in rehabilitation
medicine. The effect of electrical stimulation on muscle cells
was studied in previous study in vitro [1]. Behavior ofFig. 1: Cultivation in the vortex flow.

Cell Culture in Vortex Flow
The oriented myotubes were made by the cultivation in thevortex flow [6] (Fig. 1). A silicone rubber disk (22 mmdiameter, 3 mm thick) was attached on the bottom at the centerof a plastic culture dish of 35 mm internal diameter. The

Rotation of tilted disk generates swing motion in incubator.

Glass capillary

8 mm

Platinum wire
3 mm
5 mm
Fig. 2: Electrodes.
silicone rubber disk is stuck on the bottom of the dish withaffinity between their surfaces without bond.

Longitudinal
direction
MyotubeA polystyrene culture dish was used in the first group, while atemperature-responsive polymer (Poly-N-isopropylacrylamide)[7] coated dish was used in the second group.
The culture dish was placed on the plate, which tilted by 0.1 radto the horizontal plane. The plate rotated to generate a swing
motion of 30 revolutions per minute (WAVE-SI, Taitec, Co.,Ltd., Koshigaya). The motion produces a vortex flow in themedium in the dish. C2C12 (Mouse myoblast cell line
originated with cross-striated muscle of C3H mouse) cells weresuspended in the Dulbecco’s Modified Eagle’s Medium. The
suspension of 2 mL was poured into the dish and cultured in an
incubator for seven days, while the plate was continuously
rotating to make a steady vortex flow around the silicone rubber
disk in the medium (Fig. 1).
Fetal bovine serum (FBS) was added to the medium with thevolume ratio in 10 percent of FBS and 90 percent of D-MEM.
The medium was exchanged every two days. Since the second
day of culture, FBS was changed to the horse serum (HS) todifferentiate the muscle cells to myotuybes. HS was addedwith the volume ratio in 7 percent of HS and 93 pecent ofD-MEM.
Control cells were cultured and differentiated in the dish, which
was placed in the incubator without exposure to the flow.
Electric Stimulation
After the cultivation, periodical electric pulses were applied tothe medium to generate contraction of myotubes at 27 degrees
Celsius.
A block of electrodes was manufactured. The block consists ofa pair of electrodes of platinum wire of 0.2 mm diameter and aplastic block of supporter (Fig. 2). The wire was inserted
through a glass capillary, which passes through the block to be
fixed at the position. The distance is 3 mm between twoelectrodes of rectangle of 5 mm width in opposed position eachother. The electrodes of a couple of parallel platinum wires
generate a uniform electric field between them.

Electricfield
90 deg-60 deg

60 deg-30 deg

30 deg0 deg

Fig. 3: Electrodes rotate on the cap of culture dish toselect angle to myotube.

The block was placed on the cap with a rectangular hole (10 mmwidth × 12 mm) of the culture dish, to be able to be rotated for
adjusting its direction. The electrode of wire was dipped with
2 mm depth, and positioned above 2 mm from the bottom of theculture dish. Several radial lines are marked on the cap to keep
the direction of electrode (Fig. 3). The electrodes dipped in themedium, and the periodical electric pulses were introduced.
The rectangular repetitive pulses, which have a pulse width of 1
ms with a period of 0.1 s and amplitude of 40 V, were generated
by the electric stimulator (SEM-4201, Nihon-Koden Co., Ltd.,Tokyo) (Fig. 4).
Contraction Measurement
The myotubes were tetanized, when the electric pulses wereapplied. The culture dish was took down from the rotatingplate in the incubator at 37 degrees Celsius, and placed on theinverted microscope. The dish was placed on the heater tomaintain temperature at 27 degrees Celsius in the room
temperature of 26 degrees Celsius (Fig. 5). The distribution of
the temperature around the culture dish was measured by an
infrared thermography. The movement of myotubes wasobserved at 27 degrees Celsius, while the electric pulses wereapplied.
The distance between traceable points on the myotube inOrientation

Fig. 6: Oriented myotubes by vortex flow.
between left to right is 0.8 mm in the figure.

Fig. 4: Myotube contraction measurement system.

Distance38.3℃
36.4
34.6

Electrode

32.7
30.9
29.0

DishDish27.2
25.3
23.5

HeaterFig. 7: Distribution of temperature around the culture dish.

Fig. 5: Dish on the heater plate for temperature control.

longitudinal direction was measured during the microscopic
observation (Fig. 8). The distance after tetanic contraction iscompared to that before tetanic contraction. The traceable
points were selected in the myotube, where bigger contraction
occurs. Variation was made on the angle between thelongitudinal direction of myotube and the electric field direction(Fig. 3).

Distance between
two points

Measurement was applied to three conditions of myotubes: theculture on the polystyrene dish in the vortex flow, the culture on
the temperature-responsive polymer coating dish in vortex flow,
and the culture on the polystyrene dish without flow.
Fig. 8: Distance between traceable points on the myotube.
Distance between left to right is 0.8 mm in the figure.

The contraction ratio (R) is calculated by Eq. 1.
R = (L0 – L1) / L0

(1)

In Eq. 1, L0 is the distance before tetanic, and L1 is the distanceafter tetanic.

3. RESULTS
The muscle cells proliferated to the sub-confluent state in one
day of incubation. The oriented myotubes beyond the area of 1
mm square were observed around the silicone disk in seven days

with flow (Fig. 6), where directions of myotubes were random
in the culture without flow.
Fig. 7 exemplifies the distribution of the temperature around theculture dish on the heater plate measured by an infrared
thermography, which shows the temperature around the culture
dish was 27 degrees Celsius.
Fig.8 exemplifies traceable points on the myotube to measure
the contraction ratio.

The experimental result shows that contraction occurs even at
90 degree, when myotubes are lifted up on the temperaturesensitive polymer coating form the bottom of the dish. The
contraction might occur combined with that of adjacent
myotubes, which are not perpendicular to the electric field.

0.14

Contraction ratio

0.12
0.1

The temperature sensitive polymer Poly-N-isopropylacrylamide
is hydrophilic below 32 degrees Celsius [7]. The polymer
reversibly changes conformation in the water. When themedium is cooled down from 37 degrees Celsius to 20 degrees
Celsius, the cultured cells are exfoliated from thepolymer-coated bottom of the dish. Temperature of medium
was kept at 27 degrees Celsius to keep lifted condition ofmyotubes in the culture dish in the present experiment.

0.08
0.06
0.04
0.02
0-100

-50

050

100Angle [deg]Fig. 9: Relation between contraction ratio and angle:
square, control; triangle, flow on temperature-responsive
polymer; circle, flow on polystylene.

The myotubes might shrink a little and the tension in myotubes
might be released, when they are lifted with the temperaturesensitive polymer.
The relaxation might decrease thecontraction ratio in the test. The lift, on the other hand, might
help contraction of myotubes even in the electric field
perpendicular to the longitudinal direction of the myotubes.

5. CONCLUSION

Fig. 9 shows the relation between contraction ratio and the angle.
The angle of “zero” corresponds to the direction of the electricfield parallel to the longitudinal direction of myotube, and thatof “90” corresponds to the direction of the electric field
perpendicular to the longitudinal direction of myotube,respectively. The data of square show the contraction ratio of
myotubes cultured without flow. The data of triangle show thecontraction ratio of myotubes cultured with flow on thetemperature-responsive polymer coating dish, and the data ofcircle show that on the polystyrene dish, respectively.
The experimental results show that the amplitude of tetanic
contraction is maximized when the direction of electric field isparallel to the longitudinal direction of myotubes, and that theratio increases when the myotubes are oriented parallel eachother on the polystyrene dish.

The effect of electric field direction on contraction of
longitudinally oriented myotubes has been studied in vitro.
The experimental results show that the amplitude of repetitive
contraction is maximized when the direction of electric field isparallel to the longitudinal direction of myotubes, and that theratio increases when the myotubes are highly oriented.

6. ACKNOWLEDGMENT

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

REFERENCES

The cuture layer including myotubes on the temperature-sensitive-polymer-coating slightly lifted up from the bottom ofthe dish at 28 degrees Celsius. The experimental result showsthat contraction ratio of myotubes on the temperature sensitive
polymer decreases compared with those on the polystylene.
The contraction occurs even at 90 degree, when myotubes lifton the temperature-responsive polymer coating dish (Fig. 9).

4. DISCUSSION

The present study shows that the amplitude of contraction ismaximized when the direction of electric field is parallel to thelongitudinal direction of myotubes. The electric potential
difference across the cell membrane might be maximized when
the direction of electric field is parallel to the longitudinal
direction of myotubes.
The periodical electric pulses were applied to the culture
medium in the present study. The previous study shows thatmyotube repeats contraction and relaxation synchronized withthe periodical electric pulses, and that myotube is tetanized withthe electric pulses at period of 0.1 s [9].
Contraction is observed in the second layer from the bottom
layer. Contraction in the bottom layer cannot be observed,
because the myotubes might be strictly attached to thepolystyrene surface of the dish in the bottom layer.

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