Effect of Ultrasonic Vibration on Culture of Myoblast
Hiroaki NAKAJIMA, Shigehiro HASHIMOTO
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
shashimoto@cc.kogakuin.ac.jp http://www.mech.kogakuin.ac.jp/labs/bio/andToshitaka YASUDA
Bio-systems Engineering, Department of Electronic Engineering,Tokyo National College of Technology, Tokyo, Japan

ABSTRACTThe effect of mechanical stimulation of vibration on cultured
cells has been studied in vitro. To apply the vibration on thecultured cells, a piezoelectric element was attached on theoutside surface of the bottom of the culture dish. The
piezoelectric element was vibrated by sinusoidal alternating
voltage (Vp-p < 16 V) at 1.0 MHz generated by a function
generator. C2C12 (mouse myoblast cell line originated withcross-striated muscle of C3H mouse) was used in the test.
Cells were seeded on the polystyrene bottom of the dish at thedensity of 1000 cells/cm2. After adhesion of cells in 24 hour,cells are exposed to the ultrasonic vibration in two manners:
intermittently (for thirty minutes a day) or continuously. The
cells were observed with a phase contrast microscope every day
during the cell culture. The experimental results show that
C2C12 proliferates and differentiates even under continuousvibration.
Keywords: Biomedical Engineering, C2C12, Cell Culture andVibration.

The mechanical stress is one of the interested points in theenvironment of cells, because they receive mechanical force invivo. The mechanical stress on cells might induce various
responses: deformation, migration, proliferation, anddifferentiation. Several methods have been designed to apply
the mechanical stress to cells [4-12].
In the present study, the effect of ultrasonic vibration on cultureof myoblast has been studied in vitro.

2. METHODS
Experimental System
Mechanical vibration was applied to cultured cells with thefollowing experimental system.
A piezoelectric element(1Z28D-SYX, Fuji ceramics Corporation, Tokyo), which has 28mm diameter and 1 MHz resonance frequency, is used forvibrator (Fig. 1). A polystyrene dish of 60 mm of internal
diameter (IWAKI 3010-060-MYP) was used for cell culture.
A polydimethylsiloxane (PDMS) disk, which contains apiezoelectric element, is attached on the outside surface of thebottom of the dish (Fig. 2).

1. INTRODUCTION
Ultrasonic vibration has been applied to human body in several
cases: measurements of deep structure of body, and lithotrity [1,2].
Cell culture technique has been developed, and cells have been
cultured in controlled environment. Effect of vibration on cell
culture was studied in previous studies. In most cases, thevibration with low frequency was applied to cell culture:shaking the scaffold, or vibrating the scaffold at audible
frequency.
Several methodologies have been clinically applied toregenerative medicine.
The acceleration technique forproliferation and differentiation of cells has been studied tomake tissue in vivo or in vitro [3-11]. Control methodology
for proliferation and differentiation of cells would be applied to
the regenerative tissue technology.

Fig. 1: PDMS disk contains piezoelectric element.
the drop of water reveal vibration.

Waves on

The contact between the disk and the dish is kept by affinity
between them without adhesiveness. To keep acoustic contact
between the outside surface of the bottom of the dish and thepiezoelectric element, water is filled between them.
The piezoelectric element was vibrated by sinusoidal alternating
current at 1.0 MHz generated by a function generator(PM8572A, Tabor Electronics Ltd.) (Fig. 3). Variation wasmade on the amplitude (peak to peak) of the sinusoidal voltage(Vp-p): between 3V and 16 V (Fig. 4).
The surface of the medium was observed macroscopically,
while the ultrasonic vibration is applied during cell culture.
Cell Culture
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) was used in the test. In the case ofintermittent vibration test, sixth passage of C2C12 was used.
In the case of continuous vibration test, fifth passage of C2C12
was used. Cells were seeded on the polystyrene bottom of thedish at the density of 1000 cells/cm2. Cells are cultured at 310
K with 5 % of CO2 in an incubator. After adhesion of cells in
24 hour, cells are exposed to the ultrasonic vibration in two
manners: intermittently (for thirty minutes a day) orcontinuously.
C2C12 was cultured with the D-MEM (Dulbecco’s Modified
Eagle’s Medium) containing 10% FBS (fetal bovine serum,after decomplementation). The medium containing penicillin
and streptomycin was refreshed every two days.

Fig. 3: Electric circuit: oscilloscope (left), piezoelectric element(middle), function generator (right).

Vp-p

The culture dish with the piezoelectric element was kept in theincubator to maintain both the temperature of 310 K and thecarbon dioxide partial pressure of 5 percent (Fig. 5).
Sinusoidal voltage is transmitted to the piezoelectric elementfrom the function generator placed outside of the incubator (Fig.
6).
The cells were observed with a phase contrast microscope(IX71, Olympus, Tokyo) every day during the cell culture for 5days. On the outer surface of the bottom of the dish, a line of
0.2 mm width or a circle of 1 mm diameter is marked to trace
the same area for the cell culture. When the number of cells iscounted with Burker-Turk hemocytometer, cells are exfoliatedwith trypsin, and stained with trypan blue (GIBCO) to eliminate
dead cells.

Fig. 4: Voltage tracings of 1 MHz with function generator.

The electric pulses were applied to the cells through themedium at the end of the culture to confirm differentiation of
C2C12 to myotubes.

Fig. 5: Cell culture dish with piezoelectric element in incubator.

Fig. 2: Piezoelectric element attached on the bottom of the dish.

(A)

(B)

(C)

Fig. 6: Sinusoidal voltage is transmitted to the piezoelectricelement from function generator placed outside of the incubator.

3. RESULTS
The observation of the surface of the medium shows nomacroscopic vibration during cell culture in the present
experiment (Fig. 7B). Two lines of lights are reflected on thesurface in Fig. 7. The surface of the medium shows several
slightly convex parts, while the ultrasonic vibration is applied
during cell culture with the condition in the present study. At
the intensity of the vibration, the electric current is estimated to
0.1 A. The value is calculated from the voltage between theends of the resistance (Vp-p = 12 V), when the resistance of 51
S-1 is connected in series of the piezoelectric element.

(D)

To select the density of seeding, C2C12 was cultured withoutvibration (Figs. 8 & 9). Fig. 8 shows that C2C12 proliferates
to confluent state in three days, when C2C12 seeded in thedensity of 5000 cells/cm2. Fig. 9 shows, on the other hand,that C2C12 proliferates to sub-confluent state in three days,
when C2C12 seeded in the density of 1000 cells/cm2. For thedensity of seeding, 1000 cells/cm2 is selected in the following
cell culture.

Fig. 7: Surface of medium before vibration (A) and during
vibration at 6 V (B), 10 V (C), 16 V (D).

The experimental results show no significant effect ofcontinuous vibration at 10 kHz for four days on proliferation ofC2C12 (Figs. 10-12). The frequency of 10 kHz makes audible
sound, which confirms continuous vibration at the piezoelectricelement. Some cells of C2C12 show morphological change
after vibration: extended pseudopodia, flake of pseudopodia,
decrease of cytoplasm, and exfoliation of cells (Fig. 13).
Figs. 14-17 exemplify C2C12 cultured under intermittent
vibration at 1 MHz for 30 minutes a day compared with control
study. Fig. 18 shows the number of cells in relation toamplitude of the sinusoidal voltage of vibration at 1 MHz. In
every cases, the number of cells increases more than 10 times in5 days. The number of cells is standardized with that ofcontrol in Fig. 18. The proliferation of C2C12 tends toaccelerate with the ultrasonic vibration of Vp-p lower than 13 V.

Fig. 8: C2C12 after 3 days after seeding of 5000 cells/cm2
without vibration. Dimension from left to right is 2 mm.

Figs. 19-21 show C2C12 culture under continuous vibrationcompared with control study. Even under continuous vibration,C2C12 differentiate to myotubes, which shows repetitive
contraction with stimulation of electric pulses.

Fig. 9: C2C12 after 3 days after seeding of 1000 cells/cm2
without vibration. Dimension from left to right is 2 mm.

Fig. 12: C2C12 after 5 days after seeding of 1000 cells/cm2
without vibration. Dimension from left to right is 2 mm.

Fig. 10: C2C12 after 1 days after seeding of 1000 cells/cm2
without vibration. Dimension from left to right is 2 mm.

Fig. 13: C2C12 after vibration of 16 V for 30 min.

Fig. 14: C2C12 before vibration.
2Fig. 11: C2C12 after 3 days after seeding of 1000 cells/cm
without vibration. Dimension from left to right is 2 mm.

4. DISCUSSION
When C2C12 is seeded with the density of 5000 cells/cm2, cells
proliferate to confluent density in 3 days, which is too short toobserve the effect of vibration to proliferation of cells. That is
the reason why the density of 1000 cells/cm2 is selected in thepresent experiment.

When the voltage, which is applied to the probe, increases, thesurface of the medium becomes convex and vibrates (Vp-p > 16V). The prominent vibration might generate macroscopic flow,which has stirring effect. In the present sturdy, variation has
been made on Vp-p in the range smaller than 16 V, although
micro vibration might have local stirring effect.

Fig. 15: C2C12 after vibration of 3 V for 30 min.

Fig. 18: Relation between number of cells and Vp-p after 5days of culture. Datum at 0 V shows datum without vibration.

Fig. 16: C2C12 after 4 days without vibration.
Fig. 19: C2C12 cultured for 48 hours under continuousvibration. Diameter of the circle is 1 mm. Circle marked on
the outside of the bottom has 1 mm diameter to trace the same
area of culture.

Fig. 17: C2C12 after 24 hours after three days’ vibration of 3 V
for 30 minutes a day.

Fig. 20: C2C12 cultured for 11 days without vibration.
Dimension from left to right is 2 mm.
Vibration makes agitation in the liquid. Vibration at low
frequency makes flow in the liquid. Vibration with highenergy destroys structure. The surface of the medium does notvibrate macroscopically during cell culture in the presentexperiment. The surface of the medium shows slight convex,
while the ultrasonic vibration is applied during cell culture withthe condition in the present study (Fig. 7).

Both acceleration of proliferation and orientation of cells areimportant targets in the research field of regenerative medicineon the cultured biological tissue. The previous study showsthat electrical stimulation enhances differentiation of muscle
cells [3]. Another study shows that mechanical stimulation
improves a tissue-engineered human skeletal muscle [4].

Research Foundation at Private Universities from the Japanese
Ministry of Education, Culture, Sports and Technology.

REFERENCES

Fig. 21: C2C12 cultured for 11 days under continuous vibration.
Dimension from left to right is 2 mm.

The previous studies show that a mechanical field, on the other
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the orientation of the smooth muscle cells in the biologicaltissue [5]. The direction of the mechanical field affects
fibroblasts [7].
Too strong mechanical stimulation damages cells.
The
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The mechanicalstimulation can decrease proliferation of cells [8]. The
mechanical stress also exfoliates several cells, which makes
vacancy around the adhesive cell. The differentiation might be
optimization of cells to the changing environment. The
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make myotubes (Fig. 17).
The effect of low-frequency ultrasound on neuronal activity wasstudied in the previous study [1]. Low-intensity ultrasoundtreatment might increase mass transport, and enhance C2C12cell proliferation, metabolic activity, and differentiation of cells[2]. The wave length of ultrasound at frequency of 1 MHz
through water is 1 mm, when the velocity of ultrasound throughwater is 1 km/s. The wave length is near the dimension of
aggregation of cell of 0.01 mm.

5. CONCLUSION
Effect of ultrasonic vibration on culture of myoblast has beenstudied in vitro. To apply the vibration on the cultured cells, apiezoelectric element was attached on the outside surface of thebottom of the culture dish. The experimental results show that
C2C12 proliferates and differentiates even under continuousvibration.

6. ACKNOWLEDGMENT
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