Behavior of Cell on Vibrating Micro Ridges
Haruka HINO, Shigehiro HASHIMOTO, Shoma NISHINO, Hiroaki NAKAJIMA, Yusuke TAKAHASHI,
Hiromi SUGIMOTO
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/
ABSTRACTThe effect of micro ridges on cells cultured at vibrating scaffold
has been studied in vitro. Several parallel lines of micro ridges
have been made on a disk of transparent polydimethylsiloxane
for a scaffold. To apply the vibration on the cultured cells, apiezoelectric element was attached on the outside surface of thebottom of the scaffold. The piezoelectric element was vibrated
by sinusoidal alternating voltage (Vp-p < 16 V) at 1.0 MHz
generated by a function generator. Four kinds of cells wereused in the test: L929 (fibroblast connective tissue of C3Hmouse), Hepa1-6 (mouse hepatoma), C2C12 (mouse myoblast),
3T3-L1 (mouse fat precursor cells). Cells were seeded on the
micro pattern at the density of 2000 cells/cm2 in the medium
containing 10% FBS (fetal bovine serum) and 1% penicillin/
streptomycin. After adhesion of cells in several hours, cells
are exposed to the ultrasonic vibration for several hours. The
cells were observed with a phase contrast microscope. The
experimental results show that the cells adhere, deform andmigrate on the scaffold with micro pattern regardless of theultrasonic vibration. The effects of the vibration and the micro
pattern depend on the kind of cells.
Keywords: Biomedical Engineering, Cell Culture, Micropattern and Vibration.

1. INTRODUCTION
A biological cell adheres, migrates, rotates, and deforms on thescaffold. These behaviors of cell depend on the morphologyof the scaffold [1-6]. The previous study show that theorientation of myoblast depends on the height of the microridges [1].
The ultrasonic vibration has been applied to human body inseveral cases: measurements, and lithotrity [7-9]. Cell culture
technique has been developed, and cells have been cultured incontrolled environment. Effect of vibration on cell culture wasstudied in previous studies [10-14]. The moderate ultrasonicvibration accelerated proliferation of myoblasts in the previous
experiment.
The cells might be exfoliated from theultrasonically vibrating scaffold. The exfoliation depends on
affinity between the cell and the scaffold. Specific characterof interaction between the cell and the scaffold might beenhanced by vibration. The interaction might depend on thesurface micro morphology. The behavior might depend on thekind of cells. The technology can be applied to sorting ofcells.

Several methodologies have been clinically applied toregenerative medicine.
The acceleration technique forproliferation, orientation and differentiation of cells has beenstudied to make tissue in vivo or in vitro [10, 11, 13, 15].
Control methodology for proliferation and differentiation ofcells would be applied to the regenerative tissue technology.
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, orientation,and differentiation. Several methods have been designed toapply the mechanical stress to cells [16-18].
In the present study, the effect of micro ridges on cells cultured
at vibrating scaffold has been studied in vitro.

2. METHODS
Micro Pattern
Several parallel lines of micro ridges have been made at thecenter on a disk of transparent polydimethylsiloxane (PDMS).
The height (H), the interval (I), and the length (L) of therectangular ridge (Fig. 1) are around 0.001 mm, 0.003 mm, and
0.5 mm, respectively. Variation has been made on the width(W) of the ridge: 0.001 mm, 0.003 mm, and 0.005 mm. Eachpattern is drawn in the square area of 0.5 mm × 0.5 mm, whichis the quarter part of the square area of 1.0 mm × 1.0 mm.
One of the quarter areas has a smooth surface, and has no ridges(Figs. 8-11).
A silicon wafer (CZ, Type N, Matsuzaki Seisakusyo, Co., Ltd.,Tokyo, Japan) was used for a surface mold for the disk in aphoto lithography process [1]. The diameter and the thicknessof the wafer are 50 mm and 0.30 mm, respectively. The
surface of the wafer was cleaned by a spin-drier (SF-250, Japan
Create Co., Ltd., Tokorozawa, Japan). To improve the affinity
between the wafer and the photo-resist material (OFPR-800),hydrophobic treatment was applied to the wafer.
Hexamethyldisilazane (HMDS) was coated at 3000 rpm forthirty seconds with a spin coater (IH-DX2, Mikasa Co., Ltd.,Tokyo, Japan). The photo-resist material of OFPR-800 (Tokyo
Ohka Kogyo Co., Ltd., Tokyo, Japan) of 0.0017 mm thick wascoated on the wafer at 7000 rpm (for twenty seconds) and at
3500 rpm (subsequently for five seconds) with the spin coater.
The photo-resist was baked on a heated plate at 373 K for
ninety seconds. The pattern for the micro grooves was drawn
on the wafer with a laser drawing system (DDB-201K-KH,
Neoark Corporation, Hachioji, Japan). To save the 0.001 mm

thickness of parylene coating, the dimensions of the width of
the three kinds of grooves on the mold are 0.003 mm, 0.005 mm,
and 0.007 mm, respectively (Fig. 1). To control the dimensionof the pattern on the mold with the laser drawing system, theparameters were selected as follows: the voltage of 3.25 V, thevelocity of 0.15 mm/s, the acceleration of 0.375 mm/s2. The
pattern was baked on the heated plate at 393 K for five minutes.
The photo-resist was developed with tetra-methyl-ammoniumhydroxide (NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki,
Japan) for 60 seconds. The wafer was rinsed two times withthe distilled water for three minutes, and dried by the spin-drier.
To increase the adhesiveness of the coating, the wafer was
baked at 393 K for five minutes. The wafer was etched withthe plasma gas using a reactive ion etching system (RIE-10NR,
Samuco Inc., Kyoto, Japan) to make lines of the micro groovesof 0.001 mm depth. The gas of sulfur hexafluoride (10 cm3
min-1) with argon (20 cm3 min-1) and with oxygen (10 cm3
min-1) was applied on the wafer at 15 Pa for three minutes. To
exfoliate the residual photo-resist material from the surface, thewafer was exposed to the oxygen gas of 30 milliliter per minuteat power of 100 W for five minutes using a compact etcher(FA-1, Samco Inc., Kyoto): (the oxygen plasma ashing).
The dimensions of the three kinds of the micro grooves of themold were measured with a laser microscope (VK-X200,
Keyence Corporation, Osaka, Japan). The morphology along
the transverse lines of ridges was traced. The morphology ofthe micro grooves was observed by a scanning electron
microscope (SEM, JSM6360LA, JEOL Ltd., Tokyo, Japan).
The surface of the wafer with micro pattern was coated with
0.001 mm thickness of parylene in a parylene coater (PDS-2010,
Speciality Coating Systems, Indianapolis, USA) (Fig. 1).
After the wafer was enclosed with a peripheral wall ofpolyimide, PDMS (Sylgard 184 Silicone Elastomer Base, Dow
Corning Corp., MI, USA) was poured with the curing agent(Sylgard 184 Silicone Elastomer Curing Agent, Dow Corning
Corp., MI, USA) on the wafer. The volume ratio of PDMS tocuring agent is ten to one. After degassing, PDMS was baked
at 383 K for one hour in an oven (DX401, Yamato ScientificCo., Ltd). The baked disk of PDMS of 0.25 mm thickness was
exfoliated from the mold, and sterilized in an autoclave. The
disk with the micro ridges was used for the bottom of the dish.
Another disk of PDMS, which has a donut shape (50 mm outer
diameter, 5.5 mm thickness) with a hole of 33 mm diameter,
was made for the peripheral wall of the dish (Fig. 2). These
two disks are contacted with the affinity between them, andmake a culture area of 8.5 cm3 at the bottom. The cap for thedish of PDMS was made of PDMS. The dimension of thediameter and the thickness are 50 mm and the 1 mm,
respectively. The dish of PDMS was exposed to the oxygengas for one minute in a reactive ion etching system (FA-1,
Samco Inc., Kyoto) to be characterized as hydrophilic (oxygen
plasma ashing). The dish was preserved in the ultrapure water
to keep the hydrophilic property of the surface, before the cell
culture.
The contact angles [1] were measured between the disk of
PDMS and the medium (D-MEM) at 298 K by the contact angle
analyzer (Phoenix-300, Meiwafosis Co., Ltd., Tokyo, Japan),before and after the oxygen plasma ashing.
Experimental System
The mechanical vibration was applied to cultured cells with thefollowing experimental system. A donut shape (2 mm thick,
20 mm outer diameter, and 10 mm inner diameter) of apiezoelectric element (1Z10x20W-SYX(C-21), Fuji Ceramics
Corporation, Tokyo, Japan), which has the resonance frequencyof 1 MHz, is used for a vibrator. After the piezoelectricelement was placed on the silicon wafer, PDMS was poured
with the curing agent over the element to make the holder of theelement. The piezoelectric element mounted in the holder(total thickness of 2.2 mm) of PDMS was attached on theoutside of the bottom of the dish of PDMS (Figs. 2&3). The
contact between the holder and the dish is kept by affinity
between them without adhesive. To keep acoustic contact
between the outside surface of the bottom of the dish of PDMS
and the piezoelectric element, Vaseline is filled between them.
The micro pattern is located at the center of donut ring ofpiezoelectric element to be observed by the microscope.
Measurement of Vibration
The vibration in the medium was measured with a piezo probe[19]. The piezo electric element attached on the bottom of theculture dish is vibrated by a function generator (inverse
piezoelectric effect). The vibration propagates through themedium in the dish to a piezo probe, which is dipped in thecenter of the medium in a well. The piezo probe translates thevibration to the electric oscillation (piezoelectric effect). The
oscillation was monitored by an oscilloscope.
Cell Culture
Four kinds of cells were used in the test. C2C12 (mouse
myoblast cell line originated with cross-striated muscle of C3Hmouse), 3T3-L1 (mouse fat precursor cells, cell line of 3T3mouse), Hepa1-6 (mouse hepatoma cell line of C57L mouse),
L929 (fibroblast connective tissue of C3H mouse). The
passage between the fourth and the eighth of cell line was usedin the experiment with vibration (Table 1). The passage
between the third and the eighth of cell line was used in theexperiment without vibration (control).
PDMS
W

I
0.001

H

H
Parylene

0.001
W+0.002

Mold
I－0.002
Fig. 1: Dimension for parylene coating on mold for microridges on PDMS. Unit of numbers is mm.

MediumCell
Vibration
PDMS

Piezo

Micro patternFig. 2: Cell culture on vibrating scaffold.

Table 2 shows the mean values of the dimensions of the threekinds of the grooves on the mold, as the result of themeasurement with the laser microscope.
The scanning electron microscope images of the lines of ridges
are exemplified in Fig. 5. Fig. 5 shows the border of threekinds of ridges.
Fig. 6 shows the contact angle of D-MEM on PDMS. The
angle is 92 degree before the oxygen plasma ashing, and 3.7
degree after the oxygen plasma ashing. The property of thesurface changed from hydrophobic to hydrophilic by the oxygen
plasma ashing.
Fig. 3: Cell on the vibrating scaffold placed in chamber of
incubator at microscope.

Table 2: Dimension of the groove on the mold.
Group

Table 1: Passage of cell line.
Cell

ControlVibration

C2C12

8

6

3T3-L1

7

8

Hepa1-6

3

4

L929

6

5

EMEM (Eagle's Minimum Essential Medium) containing 10%
FBS (decomplemented fetal bovine serum) and 1% penicillin/
streptomycin was used for the culture medium of L929.
D-MEM (Dulbecco’s Modified Eagle’s Medium) containing
10% FBS (decomplemented fetal bovine serum) and 1%
penicillin/ streptomycin was used for the culture medium of
C2C12, 3T3-L1, and Hepa1-6.

Depth

mm
Width

IntervalA

0.00120.001

0.0028B

0.00130.001

0.0049C

0.00130.001

0.0069Fig. 4: Surface morphology of mold measured by laser
microscope.

Cells were seeded on the dish of PDMS at the density of 20
cells/mm2. The culture dish with the piezoelectric element
was kept in the small chamber of the incubator placed on thestage of the microscope to maintain both the temperature of 310
K and the carbon dioxide partial pressure of 5 percent (Fig. 3).
The cells were continuously observed with the phase contrast
microscope (IX71, Olympus, Tokyo) during the cell culture.
After the culture term for adhesion of cells to the scaffold withthe micro pattern, the vibration was continuously applied to thescaffold with micro pattern. The term before vibration was
one hour for Hepa1-6. The term before vibration was threehours for 3T3-L1, C2C12, and L929. After the start ofvibration, the behavior of cells was successively recorded withthe time-lapse microscopic pictures: pseudo extension,
migration, exfoliation, and re-adhesion. The interval of thetime-lapse was selected to be several minutes: between two andfive minutes. The exceptional interval longer than ten minutes
was selected for the long time recording for 24 hours.

Fig. 5: Scanning electron microscope image of the lines ofgrooves on mold before parylene coating. Perspective view.

3. RESULTS
The tracing of the surface morphology across the lines of ridges
on the mold measured by the laser microscope is exemplified inFig. 4. The tracing shows the height of 0.0013 mm, width of
0.0010 mm, and interval of 0.0049 mm.

Fig. 6: Contact angle of D-MEM on PDMS before (upper) andafter (lower) oxygen plasma ashing.

The vibration in the medium measured with the piezo probe is
exemplified in Fig. 7. The wave pattern shows the frequencyof 1 MHz.
The behavior of cells on the vibrating ridges is exemplified in
Figs. 8-11. The longitudinal direction of ridge is vertical.
The upper left square area of 0.5 mm edge has a smooth surface,and has no ridges. The width of ridges in each quarter area
among 1.0 mm edge is 0.001 mm (upper right), 0.003 mm(lower right), and 0.005 mm (lower left), respectively.
Dimension from left to right is 1.3 mm in Figs 8-11.
The behavior of C2C12 on the vibrating ridges is exemplified inFig. 8. The cells keep adhesion on the top of the ridge andmigrate along the longitudinal direction of the ridges, althoughsome cells exfoliate from the smooth surface without ridges.
Migration is faster at the ridges of the narrow ridges (0.001 mm
and 0.003 mm width) than at the ridges of the wide ridges(0.005 mm width).
Vibration accelerates migration andextension of cells, especially on the ridges of 0.001 mm width.
The repetitive elongation along the longitudinal direction of theridges is observed on the vibrating scaffold. The elongation
occurs most frequently on the ridges of 0.003 mm width.

Fig. 8: C2C12 cultured for 3 hour with vibration.

Fig. 9 shows the behavior of 3T3-L1 on the ridge. The
number of cells adhered on the scaffold is smaller than that of
the other kinds of cells. Cells adhere faster on the smoothsurface than on the ridges. Cells extend pseudo to every
direction regardless to the longitudinal direction of the ridges.
On the ridges of 0.001 mm and of 0.003 mm width, cells makeorientation along the longitudinal direction of the ridges in 24
hours of incubation, after the stop of the vibration for threehours.
Fig. 10 shows the behavior of Hepa1-6 on the ridge. The cellhas round shape, so that it is difficult to distinguish deformation
by adhesion. A few cells migrate along the longitudinal
direction of the ridges of 0.001 mm and 0.003 mm without
vibration. The vibration was started, when the cells extend
pseudo. The number of adhesion of cell decreases on thewider ridges with vibration. The cell repeats extension andshrinkage of pseudo on the vibrating scaffold regardless of thelongitudinal direction of ridges.
Vibration decelerates
migration of cells. A few cells make orientation along thelongitudinal direction of the ridges in 24 hours of vibration.
The exfoliated cells do not re-adhere to the scaffold during
vibration. Cells make orientation along the longitudinal
direction of the ridges of 0.001 mm width in 24 hours of
incubation, after the stop of the vibration for 24 hours.

Fig. 9: 3T3-L1 cultured for 3hour with vibration.

Fig. 10: Hepa1-6 cultured for 24 hour with vibration.
show oriented cells.

Fig. 7: Vibration in the medium. From left to right is 0.1 s.

CirclesFig. 11 shows the behavior of L929 on the ridge. The cells
adhere to the scaffold in one hour. The cells slightly extend to
the longitudinal direction of the ridges, but do not migrate along
the ridges. Cells adhere to the scaffold more stable than theother kinds of cells, so that every cell keeps adhesion at thesame place for three hours under vibration.
Fig. 12
exemplifies migration of C2C12 on the ridges of 0.003 mm
width for three hours with vibration. Data shows the position

of every ten minutes from original position (origin). The
coordinates of y and x are longitudinal and perpendicular axisof the ridge, respectively. C2C12 migrates along the ridge.
Fig. 13 shows migration of cells on the ridges of 0.003 mm
width for three hours with vibration. Data shows the ratio ofΔy per Δx, where Δy and Δx are migration for every ten minutes.
The ratio higher than one corresponds to the migration along theridge. The ratio is high at C2C12. Table 3 shows the numberof cells of four kinds oriented along the longitudinal direction of
micro ridge, and adhered on the scaffold at each timing. The
number was counted in each square area of 0.5 mm edge.
When the angle between the longitudinal axis of the cell and thelongitudinal axis of the ridge is smaller than 0.17 rad, the cell iscounted as the oriented cell.

Table 3: Number of cells.
Adhesion.
C2C12

Flat

vib: vibration.

Orientation/

Width of ridge [mm]
0.001
0.003
0.005

3h

0/3

1/7

2/9

0/8

+3 h vib

0/4

3/6

2/9

5/7

3T3-L1

Flat

Width of ridge [mm]
0.001
0.003
0.005

3h

0/4

0/4

0/9

0/5

+3 h vib

0/5

0/3

1/5

0/5

+24 h

2/8

5/8

3/7

3/12

Hepa1-6

Flat

Width of ridge [mm]
0.001
0.003
0.005

1h

0/35

0/33

0/13

0/14

+24 h vib

0/19

1/26

0/20

1/35

+24 h

0/14

5/14

0/23

0/18

L929

Flat

Width of ridge [mm]
0.001
0.003
0.005

3h

0/5

0/5

0/9

0/14

+3 h vib

0/4

0/5

1/10

0/12

Fig. 11: L929 cultured for 3 hour with vibration.
4. DISCUSSION

Longitudinal y [μm]Perpendicular x [μm]Fig. 12: Migration of C2C12 on 0.003 mm width for 3 hourwith vibration.
Ratio Δy / Δx

The effect of the height of micro ridges on the orientation of
C2C12 was studied in the previous study. The experimental
results show that myoblasts adhere on the top of the ridge andalign to the longitudinal direction of the micro ridges with theheight between 0.00015 mm and 0.0025 mm [1]. The heightof the micro ridge is selected to be 0.001 mm in the present
study. The time-lapse is convenient not only to catch themoment of the behavior of the cell, but also to observe themovement of the cell. The migration of the cell is able to be
traced, because the micro ridges play a role as markers. In thepresent methodology of lithography technique, the dimension oflines of rectangular ridges smaller than 0.001 mm is difficult tocontrol. The dimensions of the lines of rectangular ridges ofthe present study are selected to save the thickness of parylene
coating.
When the voltage, which is applied to the probe, increases, thesurface of the medium becomes convex and vibrates (Vp-p > 16V) [11]. In the present sturdy, Vp-p is 16 V, although micro
vibration might have local stirring effect. Vibration makes
agitation in the liquid. Vibration at low frequency makes flowin the liquid. Vibration with high energy destroys structure.
The surface of the medium is not vibrating macroscopicallyduring cell culture in the present experiment.

C2C12

3T3-L1

L929

Fig. 13: Direction of migration of C2C12 on 0.003 mm widthfor 3 hour with vibration.

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 [15]. The previous studies show that a mechanical field,

on the other hand, governs behavior of cells. The shear flow
governs the orientation of endothelial cells [17, 18]. Toostrong mechanical stimulation damages cells. The moderatemechanical stimulation, on the other hand, might accelerate
differentiation of cells [16]. The mechanical stimulation candecrease proliferation of cells [16]. The mechanical stress also
exfoliates several cells, which makes vacancy around theadhered cell. The differentiation might be optimization ofcells to the changing environment. The mechanical stress canaccelerate differentiation of C2C12 into myotubes.
Low-intensity ultrasonic treatment might increase masstransport, and enhance C2C12 cell proliferation, metabolic
activity, and differentiation of cells [10]. The wave length ofultrasonic vibration at frequency of 1 MHz through water is 1
mm, when the velocity of ultrasonic vibration through water is
1 km/s. The wave length is near the dimension of aggregationof cell. Vibration accelerates adhesion of C2C12 on the microridges, although vibration decelerates adhesion of 3T3-L1 on
the micro ridges. C2C12 migrates and extends along thelongitudinal axis of the ridges, although 3T3-L1 migrates
regardless of the direction of the ridges.

5. CONCLUSION
The effect of micro ridges on cells cultured at vibrating scaffold
has been studied in vitro. Four kinds of cells were used in the
test: C2C12 (mouse myoblast), 3T3-L1 (mouse fat precursor
cells), Hepa1-6 (mouse hepatoma), L929 (fibroblast connective
tissue of mouse). The experimental results show that the cells
adhere, deform and migrate on the scaffold with micro patternregardless of the ultrasonic vibration. The effects of thevibration and the micro pattern depend 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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