Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)

Tracings of Orientation of Cell on Scaffold with Micro Striped
Pattern after Stimulation of Vertical Excess Gravity
Takuya TAMURA, Shigehiro HASHIMOTO, Haruka HINO,
Tsubasa ITO, Yuji ENDO
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/and differentiation of cells would be applied to the regenerative
tissue technology [5-29].

ABSTRACTThe orientation of a single cell on the scaffold with the micro
striped pattern after stimulation of the vertical excess gravity
has been traced in vitro. The micro striped pattern (3 μm
width, 3 μm interval, and 0.2 μm < height < 0.4 μm) was madeon the PDMS (polydimethylsiloxane) plate of the scaffold by
the photolithography technique. C2C12 (mouse myoblast cellline) was used in the test. To apply the normal mechanicalforce field (< 100 G) to the cells adhered on the scaffold of theplate, the plate was set at the bottom of the culture dish in theswing rotor in a conventional centrifugal machine placed in an
incubator (310 K, 5% CO2). After the centrifugation forseveral (1, 2, or 5) hours, the behavior of each cell was traced at
the time lapse images for 12 hours. For the control study, cells
were cultured on the scaffold of PDMS with the same micro
pattern. The experiments show that the orientation along the
micro striped pattern is strengthened by the vertical excess
gravity and the effect is maintained for several hours after
stopping of the excess gravity.

In the present study, the effect of mechanical field onorientation and deformation of cells has been studied bycentrifuge in vitro, and the orientation of each cell on thescaffold with the micro striped pattern after stimulation of thevertical excess gravity has been traced in vitro.

2. METHODS
Micro Striped Pattern
The micro striped pattern has been made in three partial areas (1mm square each) on the PDMS (polydimethylsiloxane) plate of
the scaffold by photolithography technique (Fig. 1). Both thewidth (W) and the interval (I) of the rectangular ridges are 3 μm.
Variation has been made on the height (H) of the ridges. Eacharea has its own constant height: 0.2 μm, 0.3 μm, or 0.4 μm.
Mold for Micro Pattern
The borosilicate glass (Tempax) disk was used for the base of
the mold through micromachining process (Fig. 2). The
diameter and the thickness of the disk are 50 mm and 1.1 mm,
respectively. To remove micro particles on the surface of theglass, the oxygen (0.1 Pa, 30 cm3/min) plasma ashing wasapplied to the surface of the glass at 100 W for five minutes bya reactive ion etching system (FA-1, Samco Inc., Kyoto, Japan).

Keywords: Biomedical Engineering, Cell Culture, Excess
Gravity, Micro Pattern and C2C12.

1. INTRODUCTION
A biological cell shows passive and active behaviors in anenvironment.
The cell is sensitive to the mechanicalstimulation, and shows several responses: deformation, andmigration. While the shear stress deforms the cell, the cell
deforms by itself to adapt to the shear field. The cell has
compliance, and is deformed by force. The cell deforms, on
the other hand, to minimize the intra force. The cell is movedby the force. The cell moves in response to the force.

1 mm

1 mm

While the strong stimulation above the threshold damages thecell, the stimulation below the threshold might remain in thecell as a memory for the response in the next step. The muscle
tissue might decrease in the micro gravitational field [1-6].
The muscle tissue might increase, on the other hand, in thehypergravity [7-17]. The previous study shows that the excess
gravitational field thickens the myotubes in vitro [13].

W

I
H

Fig. 1: Dimension of micro pattern (at cross section, left) inthree areas on scaffold (right).

The acceleration technique for orientation, proliferation anddifferentiation of cells has been studied to make tissue in vivo orin vitro. Control methodology for orientation, proliferation150

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To improve affinity to photoresist material, HMDS
(hexamethyldisilazane: Tokyo Chemical Industry Co., Ltd.,Tokyo) was coated on the glass plate at 3000 rpm for 30 s witha spin coater. The positive photoresist material of OFPR-800(Tokyo Ohka Kogyo Co., Ltd, Tokyo, Japan) was coated on theglass with the spin coater (at 3000 rpm for 20 s). The
photoresist was baked in the oven (DX401, Yamato ScientificCo., Ltd) at 373 K for ninety seconds.

Developing

The pattern for the slit was drawn on the mold with a laser
drawing system (DDB-201K-KH, Neoark Corporation,
Hachioji, Japan). The pattern was baked in the oven at 373 K
for five minutes.

RemovalFig. 2: Photolithography process for mold.

The photoresist was developed with tetra-methyl-ammoniumhydroxide (NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki,
Japan) for two minute, rinsed with the ultrapure water for threeminutes twice, and dried by the spin-dryer (SF-250, Japan
Create Co., Ltd., Tokorozawa, Japan).

Excess gravity

C2C12

The glass plate with the photoresist material was etched withthe plasma gas using RIE-10NR (Samco International, Kyoto,
Japan). For etching, the gas of CF4 (30 cm3/min at 1013 hPa)
was applied at 100 W at 2 Pa. The duration time was varied
between four and nine minutes, according to the depth of the
micro groove. Each area was separately etched by covering
the other area by the glass plate.

PDMS
Fig. 3: Excess gravity applied to cell at micro pattern.
Cell
PDMS

To remove the residual OFPR-800LB on the surface of the glass,the oxygen (0.1 Pa, 30 cm3/min) plasma ashing was applied tothe surface of the glass at 100 W for five minutes by a reactive
ion etching system (FA-1).centrifuge

Excessgravity

After the mold of the glass disk was enclosed with a peripheral
wall of polyimide tape, PDMS (Sylgard 184 Silicone Elastomer
Base, Dow Corning Corp., MI, USA) was poured with thecuring agent (Sylgard 184 Silicone Elastomer Curing Agent,
Dow Corning Corp., MI, USA) on the mold. The volume ratioof PDMS to curing agent is ten to one. After degassing,
PDMS was baked at 338 K for one hour in an oven (DX401).
The baked plate of PDMS (1 mm thickness) was exfoliatedfrom the mold of the slide glass plate. The PDMS plate wascut to make a disk of 25 mm diameter, and stacked on thebottom of the culture dish (without coating of collagen) of 35
mm diameter. The surface of the PDMS plate was exposed tothe oxygen gas (0.1 Pa, 30 cm3/min) in the reactive ion etchingsystem (FA-1: oxygen plasma ashing, 50 W) for thirty seconds
just before cell culture.

Fig. 4: Swing rotor in centrifugal machine.

Fig. 5: Centrifugal machine (left) in incubator (right).

In the present study, the centrifugal acceleration (< 100 G: 1 G
is equal to the gravitational acceleration) is generated with thecentrifugal machine (CN-1040, Matsuuraseisakusyo. Ltd,Tokyo, Japan) (Fig. 5).
Asymmetrical position of the
micro-stripe areas are used for the marker to trace each cell
behavior (Fig. 1).

Hyper Gravitational Force Field
The hyper-gravitational force was applied vertically on thescaffold plane with the micro striped pattern by the centrifugal
force (Fig. 3). A swing rotor has been used to apply thevertical force to cultured cells (Fig. 4). The normal force atthe bottom surface of the culture dish was applied by thecentrifuge. The centrifugal force (Fc) is calculated by Eq. 1.
Fc= m r ω2

Cell Culture
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) was used in the experiment. C2C12 of
the passage between five and ten was cultured in D-MEM(Dulbecco’s Modified Eagle’s Medium) containing 10%
decomplemented FBS (fetal bovine serum) and 1% penicillin/
streptomycin. The cells were seeded on the micro pattern atthe density between 2000 cells/cm2 and 10000 cells/cm2.

(1)

In Eq. 1, m is mass, r is radius of the rotation, and ω is angularvelocity. In the gravitational field, gravitational force (Fg) is
calculated by Eq. 2, where g is gravitational acceleration.
Fg = m g

PDMS

(2)

After the cells were cultured in the incubator (CO2 5%, 310 K)

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Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)

for one hour in the static state, excess gravitational stimulation(50 G, or 100 G) was applied for several hours (1, 2, or 5 hours)by centrifuge in the incubator.

ab

To keep the content of carbon dioxide of 5 % at 310 K, the cells
were incubated in an incubator through the entire experimental
term including the term of exposure to the excess gravitational
field. Namely, the centrifugal machine was placed in the
incubator during the centrifugation (Fig. 5). In the control
group (normal force of 1 G), the cells were cultured without
centrifuge on the PDMS plate, which was placed in thepolystyrene dish (35 mm diameter) with collagen coating.

Fig. 6: Outline of each single cell is traced (A), andapproximated to ellipsoid (B).

Image Analysis
After stimulation of the excess gravity, cells were subsequently
cultured in the dish placed in the incubator combined with aninverted phase-contrast microscope (LCV110-SK, Olympus Co.,
Ltd., Tokyo). The behavior of each cell was analyzed on thetime lapse image captured every five minutes for 12 hours after
stimulation of the excess gravity. “Image J” was applied toanalyze the behavior of each cell. On the microscopic image,the outline of each cell was traced, and the contour of each cell
was approximated to ellipsoid (Fig. 6). As to the ellipsoid, thelength of the major axis (a), and the minor axis (b) were
measured. The ratio of axes is calculated as the shape index (S)by Eq. 3.
S=1–b/a

Ridge

Cell
θ

(3)

Fig. 7: Angle (θ) of major axis of cell related to longitudinal
direction of micro ridge.

At the circle, S = 0. As the ellipsoid is elongated, Sapproaches to unity. The number of cells (Ns), of which S ishigher than 0.5, was counted, and the shape index ratio (Rs) is
calculated by Eq. 4.
Rs = Ns / Nt

Fig. 9 shows the orientation ratio after stopping the excess
gravity of 100 G for 1 hour. The orientation ratio is higher inthe stimulation group than in the control group (without thecentrifuge).
Especially at the zero hour, the ratio is
approximately 80 % in the stimulation group, while the ratio iszero in the control group. The ratio scatters in the control
group during 12 hours after the stimulation of the gravity. Fig.
10 shows the shape index ratio after stopping the excess gravityof 100 G for 1 hour. The ratio is high in the stimulation groupat the zero hour. The high ratio is maintained even after 8hours. Fig. 11 shows the shape index ratio after stopping theexcess gravity of 100 G for five hours. The ratio is rather low
compared with Figs. 9 and 10. The cells are rounded after five
hours of the stimulation of the excess gravity of 100 G.

(4)

In Eq. 4, Nt is the total number of cells.
The cell, of which S is higher than 0.5, was selected, and theangle (θ) of the major axis of the cell was measured related to
the longitudinal direction of the micro ridge (Fig. 7). Whenthe major axis of the cell aligned to the longitudinal direction of
the micro ridge, the angle (θ) is defined as 90 degrees. The
number of the oriented cells (No) was counted as the cell, of
which the angle (θ) is between –75 degrees and +105 degrees.
In the area of each micro-pattern, the orientation ratio (Ro) was
calculated by Eq. 5.
Ro = No / Nt

Fig.12 exemplifies the tracings of the angle (θ) of the major axisof each cell related to the longitudinal direction of the microridge in the control group.
The value scatters, andasymptotically approaches to 90 degrees after 6 hour. Fig.13
exemplifies the tracings of the angle (θ) of the major axis ofeach cell related to the longitudinal direction of the micro ridgein the stimulation group of 100 G. After five hours of thestimulation of the excess gravity, the value asymptoticallyapproaches to 90 degrees. Each cell makes orientation along
the longitudinal direction of the ridge faster in the stimulationgroup than in the control group.

(5)

In Eq. 5, Nt is the total number of cells.

3. RESULTS
Fig. 8a shows the manufactured mold on the borosilicate glass
disk, which has micro pattern at the center part of the surface.
Fig. 8b exemplifies cells on the micro pattern, which hasvertical stripes in Fig. 8b.

Fig.14 exemplifies the tracings of the angle (θ) of the major axisof each cell related to the longitudinal direction of the microridge in the stimulation group: after two hours of thestimulation of the excess gravity of 50 G. Variation was madeon the height (H) of the ridge in each area: 0.4 μm (a), 0.3 μm(b), or 0.2 μm (c). The tendency for each cell to align to the

In Figs. 9-16, data at zero hour shows the value immediately
after stopping the stimulation of the centrifuge. In Figs. 9-11,
each bar shows the mean value of nine samples.

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Shape Index Ratio [%]longitudinal direction of the ridge decreases on the low ridge(Fig. 14c). Fig.15 exemplifies the tracings of the angle (θ) of
the major axis of each cell related to the longitudinal directionof the micro ridge in the control group of 1 G. Variation wasmade on the height (H) of the ridge in each area: 0.4 μm (a), 0.3
μm (b), or 0.2 μm (c). The tendency for each cell to align to
the longitudinal direction of the ridge decreases on the low
ridge (Fig. 15c). Fig.16 exemplifies the tracings of the angle(θ) of the major axis of each cell on the surface without the
micro pattern in the control group of 1 G. The angle ismeasured between 0 degree and 180 degree related to thevoluntary defined line of direction. The angle of each cell
varies at random with rotational migration of the cell on theplane surface.

100

80
60

Hyper Gravitational
Force group
Control group

40
20

0
0

1

2

3

4 5 6
Time [h]789Fig. 11: Shape index ratio (Rs [%]) after stopping excess
gravity (100 G for 5 hours).
180
150

Angle [degree]

120
90

60
30
0
0

2

4

6

8

10

Time [h]Fig. 12: Angle (θ) tracings (control, 1 G).
180

Angle [degree]

50 mm
Fig. 8a: Micro pattern (center) on surface of mold: diameter, 50
mm

150
120
90
60
30
0
0

2

4

6

8

10

Time [h]Direction of
micro stripes

Fig. 13: Angle (θ) tracings after stopping excess gravity (100 G
for 5 hours).
180

Angle [degree]150

0.2 mm

120
90
60
30
0
0

80

2

3

4
Time [h]

5

6

7

8

180

Control

60

150

40

Hyper gravitational
force

20
0

Shape Index
Ratio [%]

1

2

3

4
Time [h]

5

6

7

8

Fig. 14b: Angle (θ) tracings after stopping excess gravity (50 G
for 2 hours, 0.3 μm).
Angle [degree]

4Time
6 [h]860

0Control2

900Fig. 9: Orientation ratio (Ro [%]) after stopping excess gravity(100 G for 1 hour).
100
80
60
40
20
0

120

30

0 1 2 3 4 5 6 7 8 9 10 11 12Time [h]01Fig. 14a: Angle (θ) tracings after stopping excess gravity (50 G
for 2 hours, 0.4 μm).

100

Angle [degree]

Orientation Ratio [%]Fig. 8b: Cells on micro pattern (vertical stripes): dimension
from left to right is 1 mm.

10 12

180
150
120
90
60
30
0
0

Fig. 10: Shape index ratio (Rs [%]) after stopping excess
gravity (100 G for 1 hour).

1

2

3

4
Time [h]

5

6

7

8

Fig. 14c: Angle (θ) tracings after stopping excess gravity (50 G
for 2 hours, 0.2 μm).

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Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)

after stimulation.

180

Angle [degree]

150

Migration of a cell on the scaffold plate was observed inprevious studies [26]. The time lapse image of every ten
minutes is effective to trace the movement of each cell. The
orientation of the single cell depends on the morphology of thescaffold surface [24-29]. The height of the micro ridge of 0.4
μm is too law for C2C12 to make orientation at 1 G [29]. The
normal force by the excess gravity applied to the cell mightemphasize the orientation of the cell along to the striped micro
pattern. The variation of the height of micro pattern in threeareas realizes the simultaneous observation of the behavior ofeach cell (Figs. 14 & 15).

120
90
60
30

0
0

1

2

3

4
Time [h]

5

6

7

8

Fig. 15a: Angle (θ) tracings (control 1G, 0.4 μm).
Angle [degree]

180
150
120
90

During centrifugation, the direction of the force field tilts fromhorizontal direction because of the gravity (1 G) of the earth.
In the case of centrifuge of 50 G, the shift of the angle is 1
degree. The gravity in the fluid is reduced by the buoyancy.
The density of the cell of C2C12 is 1.065×103 kg m–3 measured
by the centrifugal method: each separator adjusted to various
density of the fluid of phthalate-ester is compared with thedensity of the cell in the glass capillary by centrifuge [30].
The density of medium of DMEM, on the other hand, is1.0055×103 kg m–3. The difference between two densities is0.0595×103 kg m–3. The net effect of the centrifugal force on
the cell in the medium is 6 G at centrifuge of 100 G.

60
30
0
0

1

2

3

4
Time [h]

5

6

7

8

7

8

Fig. 15b: Angle (θ) tracings (control 1G, 0.3 μm).
Angle [degree]

180
150
120
90
60
30
0
0

1

2

3

4
Time [h]565. CONCLUSION

Fig. 15c: Angle (θ) tracings (control 1G, 0.2 μm).

The orientation of a single cell on the scaffold with the micro
striped pattern after stimulation of the vertical excess gravity
has been traced in vitro. The micro striped pattern (3 μm
width, 3 μm interval, and 0.2 μm < height < 0.4 μm) was madeon the PDMS (polydimethylsiloxane) plate of the scaffold by
the photolithography technique. After the vertical force of theexcess gravity (< 100 G) was applied to C2C12 (mouse
myoblast cell line) for several hours by the swing rotor in aconventional centrifugal machine placed in an incubator, theorientation of each cell was traced at the time lapse images for
12 hours. The experiments show that the orientation along the
micro striped pattern is strengthened by the vertical excess
gravity and the tendency is maintained for several hours after
stopping of the excess gravity.

180

Angle [degree]

150
120
90
60

30
0
0

1

2

3

4

5

6
Time [h]

7

8

9

10

11

12

Fig. 16: Angle (θ) tracings on flat surface (control 1G).

4. DISCUSSION
To apply continuous uniform mechanical stimulation to the cells,
centrifugal force is used in the present study. The effect of
mechanical field on orientation and deformation of several
kinds of cells was studied using centrifuge in vitro in theprevious studies [5-17]. The differentiation of cells was
accelerated by the mechanical stimulation by centrifuge in theprevious study [11, 14]. The response of biological system tothe microgravity field has been studied at a space satellite.
The cell cycle might extend in the space [6]. C2C12
differentiation might be accelerated and the myotube mightalign to the direction perpendicular to the centrifugal field [8].

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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