Effect of Ultrasonic Vibration on Proliferation of Cultured Cell
Hiroaki NAKAJIMA, Haruka HINO, Shigehiro HASHIMOTO, Yusuke TAKAHASHI
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
proliferation of cells has been studied in vitro. To apply thevibration on the cells, a piezoelectric element was attached on
the outside surface of the bottom of the culture plate of six wells.
The piezoelectric element was vibrated by sinusoidal alternating
voltage at 1.0 MHz generated by a function generator. Threekinds of cells were used in the experiment: C2C12 (mousemyoblast cell), L929 (fibroblast connective tissue of mouse),
Hepa1-6 (mouse hepatoma cell). After incubation for 24 hours,
cells were exposed to the ultrasonic vibration intermittently forthree days: for thirty minutes per day. At the end of theexperiment, the number of cells was counted by colorimetricmethod with the microplate photometer. The experimental
results show that proliferation of cells is enhanced with mildvibration, and that the optimum vibration depends on the kindof cells.
Keywords: Biomedical Engineering, C2C12, L929, Hepa1-6,
Cell Culture and Vibration.

1. INTRODUCTION
Ultrasonic vibration has been applied to human body in several
cases: measurements of deep structure of body, and lithotrity [1,2].

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 [3, 5-7].
In the present study, the effect of ultrasonic vibration on
proliferation of cultured cells 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 28
mm diameter and 1 MHz resonance frequency, is used forvibrator (Fig. 1). A plate of six wells with a flat bottom of 35
mm internal diameter (Falcon) was used for the cell culture.
The area of the bottom of each well for the cell culture is 9.6
cm2. A polydimethylsiloxane (PDMS) disk, which contains
the piezoelectric element, is attached on the outside surface of
one of the bottom of the well (Fig. 2). The corner well isselected for attachment, so that the distance from thepiezoelectric element varies at each well. The wells forcontrol group are located at the separated plate (Fig. 3).

The cell culture technique has been developed, and cells havebeen cultured in the controlled environment. The effect ofvibration on cell culture was studied in previous studies [3].
In most cases, the vibration with low frequency was applied tocell culture: shaking the scaffold, or vibrating the scaffold at theaudible 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 [3-7]. Control methodology for
proliferation and differentiation of cells would be applied to theregenerative tissue technology.

Fig. 1: Piezoelectric element.

135PC (Oscilloscope)Probe
6-well multi-plate

246Function generator

Piezoelectric element
Fig. 2: Position of each well in the culture plate. Piezoelectricelement is attached on the back of the well “number one”.

7Fig. 4: Diagram of measurement of vibration in the medium.

8Fig. 3: Wells in the separated plate for control group (No. 8 &9).

The contact between the disk and the bottom of the plate is kept
by affinity between them without adhesive. To keep acoustic
contact between the outside surface of the bottom of the dishand the piezoelectric element, a porous sheet filled with water issandwiched between them.
The piezoelectric element was vibrated by sinusoidal alternating
current at 1.0 MHz generated by a function generator(PM8572A, Tabor Electronics Ltd.). The amplitude (peak topeak) of the sinusoidal voltage (Vp-p) was adjusted to 16 V.
During incubation, the sinusoidal voltage is transmitted to thepiezoelectric element from the function generator placedoutside of the incubator (Fig. 7).
Measurement of Vibration
Before the test for cell culture, the vibration in the medium wasmeasured with a piezo probe (Figs. 4&5). The piezo electricelement attached on the bottom of the culture dish is vibrated bya function generator (the inverse piezoelectric effect). The
vibration propagates through the phosphate buffer solution of 2
cm3 in a well to a piezo probe, which is dipped in the center ofthe solution in the well. The piezo probe translates thevibration to the electric oscillation (the piezoelectric effect).
The oscillation is monitored by an oscilloscope.

Fig. 5: Measurement of vibration in the medium.

Calibration of cell Count
Cells were pre-cultured in a polystyrene dish, and exfoliatedfrom the bottom of each well with trypsin. The number ofcells is counted with Burker-Turk hemocytometer under a phase
contrast microscope (IX71, Olympus, Tokyo). The suspension
at the density of 500000 cells/cm3 was prepared for three kindsof cells.
Before the suspension of 0.1 cm3 was added into one of thewells, the medium of 0.1 cm3 was poured into every well of the96-wells-plate.
D-MEM (Dulbecco’s Modified EagleMedium) containing 10% FBS (fetal bovine serum, afterdecomplementation) and 1% penicillin/ streptomycin was used
for the medium. After mixing the suspension with the medium
in a well, the suspension of 0.1 cm3 in the well was collected
and poured into the medium of the next well for mixing. The
repetitive process makes dilution doubled of the density of cells
in the suspension of successive wells.
Dojindo’s Kit-8 (CCK-8), which includes highly water-solubletetrazolium salt (WST-8), was added to every well, andincubated for several hours: 120 minutes for Hepa1-6, and 150
minutes for C2C12 and for L929. The intensity of absorbanceat the wave length of 450 nm was measured with a microplate
photometer (Multiskan FC Microplate Photometer, Thermo
Fisher Scientific, Inc.) (Fig. 6).

Fig. 6: Mounted samples in microplate photometer.

Fig. 7: Piezoelectric element in incubator is vibrated byfunction generator.

Cell Culture
Three kinds of cells were used in the test: C2C12 (mousemyoblast cell line originated with cross-striated muscle of C3Hmouse), L929 (fibroblast connective tissue of C3H mouse), andHepa1-6 (mouse hepatoma cell line of C57L mouse).
D-MEM (Dulbecco’s Modified Eagle Medium) containing 10%
FBS (fetal bovine serum, after decomplementation) and 1%
penicillin/ streptomycin was used for the medium. The
medium of 2 cm3 in the well was refreshed every two days.
Cells were cultured at 310 K with 5 % of CO2 in an incubator
throughout the test including exposure to the vibration.
In the case of C2C12, fourth passage was used. Cells wereseeded on the bottom of each well at the density of 1000
cells/cm2. After incubation for 24 hours, cells were exposed tothe ultrasonic vibration intermittently for three days: for thirty
minutes per day. After the last exposure to the vibration for
thirty minutes, cells were incubated for another 24 hours. At
the end of every test, the cells were observed by a phase
contrast microscope (IX71, Olympus, Tokyo), and exfoliatedfrom the bottom of each well with trypsin. Each suspension ofcells was centrifuged, and the supernatant was discarded.
After the medium of 3 cm3 was mixed, the suspension of 1 cm3
was collected. WST-8 of 0.1 cm3 was added to the suspension.
After incubation for 150 minutes, the number of cells at eachwell was counted by colorimetric method with the microplate
photometer.
In the case of L929, tenth passage was used. Cells wereseeded on the bottom of each well at the density of 1000
cells/cm2. After incubation for 24 hours, cells were exposed tothe ultrasonic vibration intermittently for three days: for thirty
minutes per day. After the last exposure to the vibration for
thirty minutes, cells were incubated for 24 hours. At the endof the test, cells were exfoliated from the bottom of each wellwith trypsin. Each suspension of cells was centrifuged, andthe supernatant was discarded. After the medium of 3 cm3 wasmixed, the suspension of 1 cm3 was collected, and WST-8 of
0.1 cm3 was added. After incubation for 150 minutes, thenumber of cells at each well was counted by colorimetricmethod with the microplate photometer.

Fig. 8: Cells in wells exposed to vibration in incubator.

In the case of Hepa1-6, sixth passage was used. Cells wereseeded on the bottom of each well at the density of 2000
cells/cm2. After incubation for 24 hours, cells were exposed tothe ultrasonic vibration intermittently for three days: for thirty
minutes per day. After the last exposure to the vibration for
thirty minutes, cells were incubated for 24 hours. At the endof the test, cells were exfoliated from the bottom of each wellwith trypsin. Each suspension of cells was centrifuged, andthe supernatant was discarded. After the medium of 3 cm3 wasmixed, the suspension of 1 cm3 was collected, and WST-8 of
0.1 cm3 was added. After incubation for 120 minutes, thenumber of cells at each well was counted by colorimetricmethod with the microplate photometer.
During the exposure to the vibration for thirty minutes, the cells
were cultured in the separated compact incubator (Figs. 7&8).
After the exposure for thirty minutes, cells were returned to theoriginal incubator, and cultured for the rest of the day. In thecontrol group, cells were continuously cultured in the originalincubator.

3. RESULTS
Table 1 shows the results of the measurement of vibration in themedium at the center of each well. The distance is measured
by a scale between the centers of the wells. The number of thewell corresponds to that of Fig. 2. The level of -20 dB means
one tenth by attenuation. The well number one is the origin in
Table 1. The result shows that the damping of the vibration isproportional to the distance from the piezoelectric element.

Table 1: Vibration in the medium in each well.
Well
123456Distance [mm]03939557889

Voltage [mV]
3.88
0.245
0.195
0.1550.0489
0.0388

Level [dB]0-24-26-28-38-40

In Figs. 9-11, relation between the absorbance of light at thewave length of 450 nm and the density of cells is shown forsuspension of three kinds of cells, respectively.
Theabsorbance is proportional to the density of cells. The
approximated line is used to calculate the number of the cells
from the absorbance of the light.

Fig. 10: Relation between absorbance and the number of L929
in a well of microplate.

Figs. 12-14 show the cells observed at the end of the experiment.
The dimension of left to right is 2 mm. The rounded particles
are floating cells. C2C12 proliferates to the confluent manner
even in the vibration group.
Figs. 15-17 show the number of cells in each well. Eachcolumn shows mean value of five samples from each well andthe vertical bar shows the range between maximum andminimum data of the five samples. The number of wellcorrespond to that of Fig. 2 and Fig. 3.

Fig. 11: Relation between absorbance and the number of
Hepa1-6 in a well of microplate.

Fig. 15 shows that C2C12 proliferates higher than 15000 cells,
which corresponds to the confluent state in every well in fourdays (Fig. 12). The number of cells of 9600 corresponds tothe same number of seeding.
Fig. 16 shows that the number of L929 increases in the well
distant from the source of vibration (number one), although thenumber of L929 is very small in the well at the source ofvibration (Fig. 13(B)).
The number of cells of 9600
corresponds to the same number of seeding.

Fig. 9: Relation between absorbance and the number of C2C12
in a well of microplate.

Fig. 17 shows that vibration does not affect the number of
Hepa1-6. The number of cells of 19200 corresponds to thesame number of seeding.

(A)

(B)

(B)

Fig. 14: Hepa1-6 at the end of test in well: control (A), No. 1(B).

Fig. 12: C2C12 at the end of test in well: control (A), No. 1 (B).
(A)

Fig. 15: Number of C2C12 in each well.
(B)

Fig. 13: L929 at the end of test in well: control (A), No. 1 (B).
(A)

Fig. 16: Number of L929 in each well.

enhance the proliferation of cells.
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, Vp-p is 16 V,
although micro vibration might have local stirring effect.
The effect of low-frequency ultrasound on neuronal activity wasstudied in the previous study [1]. Low-intensity ultrasoundtreatment might increase mass transport, and enhance C2C12proliferation, metabolic activity, and differentiation of cells [2].

5. CONCLUSION

Fig. 17: Number of Hepa1-6 in each well.

4. DISCUSSION
Dojindo’s highly water-soluble tetrazolium salt (WST-8) is
reduced by dehydrogenase activities in cells to give ayellow-color formazan dye. The formazan dye has themaximum point of absorption at the wave length of 450 nm.
The dye is soluble in the media of the cell culture. The
formazan dye is generated by the activities of dehydrogenases
in cells, so that the amount of the formazan dye might beproportional to the number of living cells. The calibration line
is convenient to be used for calculation of the number of cells in
the present study, because the absorbance was proportional tothe density of cells of three kinds: C2C12, L929, and Hepa1-6.
Fig. 12 shows that C2C12 proliferates to the confluent state inevery well. In the previous study, the mild vibration tends toaccelerate the proliferation of C2C12 [3]. In the present
experiment, the number of cells in vibration group is almost
equal to that of control group, and cells proliferate to confluent
state in four days. Most of cells of C2C12 are not exfoliatedin the present study, although several cells of C2C12 areexfoliated during exposure to the vibration in thirty minutes inthe previous study. The difference of dimension of the dishmight make variation of the flow in the culture dish, whichgoverns the exfoliation of cells.
The proliferation of L929 is not so fast as C2C12 in the presentstudy. Fig. 16 shows that the proliferation of L929 increases
with mild vibration (No. 2-6 in Fig. 16), although the strong
vibration (in the well number one) inhibits proliferation of L929.
Fig. 17 shows that vibration does not affect proliferation ofHepa1-6, although cells do not proliferate to the confluent state.
The numbers of cells are small in the group of L929 andHepa1-6 including control tests. The small number of cells
might be results of exfoliation of cells, or of little adhesion ofcells. Although the culture condition might not be good forproliferation in the present experiments, the numbers of cells
are larger in the mild vibration group than in control group.
The experimental results show that the mild vibration might

The effect of mechanical stimulation of vibration at 1.0 MHz on
proliferation of cells has been studied in vitro. Three kinds ofcells were used in the experiment: C2C12 (mouse myoblastcell), L929 (fibroblast connective tissue of mouse), Hepa1-6(mouse hepatoma cell). The experimental results show thatproliferation of cells is enhanced with mild vibration, and thatthe optimum vibration depends on the kind of cells.

6. ACKNOWLEDGMENT
This work was supported by a Grant-in-Aid for Strategic
Research Foundation at Private Universities from the Japanese
Ministry of Education, Culture, Sports and Technology.

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