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

Behavior of Cell Cultured on Micro Striped Pattern after
Stimulation of Excess Gravity
Takuya TAMURA, Haruka HINO, Shigehiro HASHIMOTO
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.

ABSTRACTThe behavior of a single cell cultured on the micro striped
pattern has been observed after stimulation of the excess gravity
in vitro. The lines of parallel micro quadrangular ridges (0.7μm height, 3 μm width, and 3 μm interval) are made on thesurface of the plate as the scaffold to make orientation of eachcell.
Variation is made about the angle between thelongitudinal direction of the ridge and the direction of theexcess gravity: 0 degree, 45 degrees, and 90 degrees. To apply
the excess gravitational force field (50 G, or 100 G) to the cells,
the plate was set in the tube in a conventional centrifugal
machine placed in an incubator. The contour of each cell at
the time lapse images was traced for 24 hours and approximatedto ellipse to analyze the angle between the longitudinal axis of
the cell and the direction of the excess gravity, after the excess
gravitational stimulation for 5 hours. The experiment showsthat the longitudinal axis of C2C12 (mouse myoblast cell line)
tends to align to the direction of the excess gravity, and returns
to the direction of longitudinal direction of micro pattern after
stopping of the excess gravity.

In the previous study, the longitudinal axis of C2C12 (mouse
myoblast cell line) tends to align to the direction of the excess
gravity, and turns to the perpendicular direction after stoppingof the excess gravity [3]. A single cell migrates at random on
the scaffold. The cell tends to align to the longitudinal
direction of the micro ridge line [8].
In the present study, the effect of mechanical field onorientation and deformation of a single cell on the micro striped
pattern has been studied by centrifuge in vitro, and the behaviorof each cell has been traced after stopping of the stimulation of
the excess gravity.

2. METHODS
Micro-pattern on Scaffold Plate
The micro striped pattern has been made in three partial
rectangular areas of 0.4 mm × 1.6 mm on the PDMS
(polydimethylsiloxane)
plate
of
the
scaffoldbyphotolithography technique. The height (H), the width (W),and the interval (I) of the quadrangular ridges are 0.7 μm, 3 μm,
and 3 μm, respectively (Fig. 1a). Variation has been made on
the angle (θ) between the longitudinal direction of the ridge andthe direction of centrifuge: 0, 45, 90 degrees (perpendicular)(Fig. 1b).

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

1. INTRODUCTION
A biological cell shows passive and active behaviors in anenvironment. While the shear stress deforms the cell, forexample, the cell deforms by itself to adapt to the shear field.
While the strong stimulation above the threshold damages thecell, the stimulation below the threshold remains in the cell as amemory for the response in the next step.

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).

A biological cell is exposed to the mechanical force field in vivo.
The cell is sensitive to the mechanical stimulation, and shows
several responses: deformation, and migration. 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 by itself. The muscle tissue
might decrease in the micro gravitational field [1]. The
muscle tissue might increase, on the other hand, in thehypergravity [2-7].
The previous study shows that thehyper-gravitational field thickens the myotubes in vitro [2].
The acceleration technique for orientation, proliferation anddifferentiation of cells has been studied to make tissue in vivo orin vitro. Control methodology for orientation, proliferation

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.

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

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.

W

I

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).

Fig. 1a: Dimension of micro pattern.
1.6 mmThe 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.

90 degrees

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).

45 degrees

Centrifuge

0.4 mmAfter 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 plate of 15 mm × 10 mm × 1 mm, and stacked on
the glass plate of 50 mm × 13 mm × 1 mm (Fig. 3). The
surface of the PDMS plate was exposed to the oxygen gas (0.1
Pa, 30 cm3/min) in the reactive ion etching system (FA-1:
oxygen plasma ashing, 50 W) for thirty seconds just before cell
culture.

0 degree

50 µm

Fig. 1b: Three kinds of micro pattern on PDMS plate.
1. Coating

5. Removal

2. Exposure

6. Transfer

3. Developing

Hyper-gravitational Force Field
The hyper-gravitational force was applied to cultured cells withthe centrifugal force. The culture plate is inserted in the tube,
which is contained in the rotor. The angle between the radial
direction of the rotation of the rotor and the axial direction of
the tube in the rotor is 60 degree. The glass plate was set sothat the culture surface is parallel to the radial direction of therotation. The centrifugal force is applied parallel to the culture
surface. The variation is made on the position of the glass
plate in the tube: at the middle of the tube (X), or at the bottomof the tube (Y). To stabilize the position of the glass plate in
the tube, Polydimethylsiloxane (PDMS) is filled in the tube tofill the vacancy below the glass plate in the group X. The
centrifugal force (Fc) is calculated by Eq. 1.
Fc = m r ω2

H

7. Exfoliation
8. Adhesion

4. Etching

PDMS

Glas
s

Fig. 2: Micro machining.

(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(2)

In the present study, the centrifugal acceleration of < 100 G (1G is equal to the gravitational acceleration) is generated withthe centrifugal machine (CN-1040, Matsuuraseisakusyo. Ltd,Tokyo, Japan).

Fig. 3: PDMS plate with micro pattern stacked on glass plate.

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

the centrifuge and the direction of the major axis of each cell
was measured at the microscopic image. When the major axis
is parallel to the direction of centrifuge, θ = 0. When the
major axis is perpendicular to the direction of centrifuge, θ = 90
degree.

3. RESULTS
Centrifuge

In Figs. 5-10, each datum point shows the value of each cell
before centrifuge (before), immediately after stopping
centrifuge (after), and every 6 hours after stopping centrifuge,
respectively.
Fig. 5 shows the direction of the major axis of each cell without
centrifuge (control study) on each micro pattern: 0 degree (Fig.
5a), 45 degrees (Fig. 5b), and 90 degrees (Fig. 5c), respectively.
Most of cells tend to tilt along the longitudinal direction of theridge during 24 hours of culture. Fig. 5a shows that data
concentrate at zero degree.
Fig. 5b shows that data
concentrate to 45 degrees. Fig. 5c shows that data concentrate
to 90 degrees.

Fig. 4: Plate position in tube: 50 G (left), and 100 G (right).

Cell Culture
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) was used in the experiment. C2C12 of
the passage four 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 glass plate at the density of 3000 cells/cm2.
After the cells were cultured for 12 hours in the static state,excess gravitational stimulation was applied for five hours.
The gravity of 50 G (r = 0.045 m, group X), and the gravity of
100 G (r = 0.09 m, group Y) were applied at ω = 104 rad/s.

Figs 6 & 7 show the angle between the direction of the major
axis of each cell and the tangential force (on the surface of thescaffold by centrifuge) at 50 G, and at 100 G, respectively. Fig.
6 shows the angle between the direction of centrifuge of 50 G
and the direction of the major axis of each cell on each micro
pattern: 0 degree (Fig. 6a), 45 degrees (Fig. 6b), and 90 degrees(Fig. 6c), respectively. Fig. 7 shows the angle between the
direction of centrifuge of 100 G and the direction of the major
axis of each cell on each micro pattern: 0 degree (Fig. 7a), 45
degrees (Fig. 7b), and 90 degrees (Fig. 7c), respectively. The
tendency of tilting of each cell to the direction of excess gravity
is kept for 12 hours after stopping centrifugation. The numberof cells, which do not align to the longitudinal direction ofridges, tends to decrease by the hyper-gravitational stimulation.
The tendency of the cell to align to the longitudinal direction ofridges is enhanced on the ridge perpendicular to the direction of
the excess gravity, after centrifuge (Figs. 6c & 7c).

To apply the excess gravity, the plate, on which cells adhered,was set in the medium in the tube. Several tubes with theglass plate were set in the rotor to cultivate cells of group X andY, simultaneously. To keep the content of carbon dioxide of
5 % at 310 K, the cells were incubated in an incubator throughthe entire experimental term including the term of exposure to
the hyper-gravitational field. Namely, the centrifugal machine
was placed in the incubator during the centrifugation. In thecontrol group, the cells were cultured without centrifuge on theplate, which was placed in the polystyrene dish.

Figs. 8-10 show the shape index (P). Fig. 8 shows the shapeindex of each cell without centrifuge (control study) on eachmicro pattern: 0 degree (Fig. 8a), 45 degrees (Fig. 8b), and 90
degrees (Fig. 8c), respectively. Fig. 9 shows the shape index
of each cell at 50 G on each micro pattern: 0 degree (Fig. 9a),45 degrees (Fig. 9b), and 90 degrees (Fig. 9c), respectively.
Fig. 10 shows the shape index of each cell at 100 G on eachmicro pattern: 0 degree (Fig. 10a), 45 degrees (Fig. 10b), and 90
degrees (Fig. 10c), respectively. The number of rounded cells(small value of P) on the ridges decreases immediately after
centrifugation (Fig. 10). Cells tend to elongate to the directionof centrifuge. The tendency is apparent, when the longitudinal
direction of the micro ridge is parallel to the direction ofcentrifuge (Fig. 10a).

Image Analysis
After stimulation of the excess gravity, the glass plate was
moved from the centrifugal tube to the culture dish. Cells
were subsequently cultured in the dish placed in the incubator
combined with an inverted phase-contrast microscope(LCV110-SK, Olympus Co., Ltd., Tokyo). The behavior ofeach cell was analyzed on the time lapse image captured every
five minutes for 24 hours after stimulation of the excess gravity.
“Image J” was applied to analyze 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.
On the ellipsoid, the length of the major axis (a), and the minor
axis (b) were measured. The ratio of axes is calculated as theshape index (P) by Eq. 3.
P=1 － b/a
At the circle, P = 0.
approaches to unity.

4. DISCUSSION(3)

The previous studies showed that a mechanical field governs
behavior of cells [2-14]. The effect of shear flow onorientation of cells depends on the kinds of cells [13].

As the ellipsoid becomes flat, P

The angle (0 degree < θ < 90 degrees) between the direction of146

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

DegreeDegreeBefore

After

6

12

18

24 hour

BeforeFig. 5a: Angle of C2C12: control, 0 degree.

After

After

12

18

24 hour

Degree61218

24 hour
BeforeFig. 5b: Angle of C2C12: control, 45 degrees.
DegreeBefore6

Fig. 6c: Angle of C2C12: 50 G, 90 degrees.

DegreeBefore

After

6

12

18

After

6

12

18

24 hourFig. 7a: Angle of C2C12: 100 G, 0 degree.
Degree24 hour
BeforeFig. 5c: Angle of C2C12: control, 90 degrees.

After

6

12

18

24 hourFig. 7b: Angle of C2C12: 100 G, 45 degrees.

DegreeDegreeBefore

After

6

12

18

24 hour
BeforeFig. 6a: Angle of C2C12: 50 G, 0 degree.

After

6

12

18

24 hourFig. 7c: Angle of C2C12: 100 G, 90 degrees.

DegreeBefore

After

P

6

12

18

Before24 hour

After

6

12

18

24 hourFig. 8a: Shape Index (P) of C2C12: control, 0 degree.

Fig. 6b: Angle of C2C12: 50 G, 45 degrees.

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

P

Before

P

After

6

12

18

24 hour

BeforeFig. 8b: Shape Index (P) of C2C12: control, 45 degrees.
P

After

6

12

18

24 hourFig. 10a: Shape Index (P) of C2C12: 100 G, 0 degree.
P

Before

After

6

12

18

24 hour
BeforeFig. 8c: Shape Index (P) of C2C12: control, 90 degrees.

6

12

18

24 hourFig. 10b: Shape Index (P) of C2C12: 100 G, 45 degrees.
P

P

Before

After

After

6

12

18

24 hourFig. 9a: Shape Index (P) of C2C12: 50 G, 0 degree.

Before

After

6

12

18

24 hourFig. 10c: Shape Index (P) of C2C12: 100 G, 90 degrees.

P

Although HUVEC (human umbilical vein endothelial cells)
orients along the stream lines, C2C12 tilts from the stream lines
to make myotubes. When mechanical stimulation is applied tothe scaffold, the whole stimulation cannot always be transmitted
to the cells. When the tension applied to a scaffold, thedeformation of the scaffold generates compression and shear in
the different direction simultaneously [10].
Before

After

6

12

18

24 hour

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 [4-6]. The response of biological system tothe microgravity field has been studied using a space satellite[1]. C2C12 differentiation might be accelerated and the
myotube might align to the direction perpendicular to thecentrifugal field [6].

Fig. 9b: Shape Index (P) of C2C12: 50 G, 45 degrees.
P

Before

After

6

12

18

The variation was made on the position (radius) of cell culturein the same rotor of the centrifuge, to test in the different
gravitational environment simultaneously. Cells passively
follow the direction in the strong tangential force field.
Immediately after the centrifugation, cell might start to show

24 hourFig. 9c: Shape Index (P) of C2C12: 50 G, 90 degrees.

148Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)active response to the mechanical stimulation. The mild
tangential force field induces the active reflection of cells to tilt
perpendicularly to decrease internal force of the cells. The cellshows adaptation against stimulation. The stimulation leaves
hysteresis in the cell. The hyper-gravitational stimulation
governs the behavior of the cell after stimulation. C2C12
made perpendicular orientation of myotubes to the flow
direction in the previous study [13].

REFERENCES[1] H.R. Fitts, R.D. Riley and J.J. Widrick, “Functional and
Structural Adaptations of Skeletal Muscle to Microgravity”,
Journal of Experimental Biology, Vol. 204, No. 18, 2001,
pp. 3201–3208.
[2] S. Hashimoto, H. Hino and T. Iwagawa, “Effect of Excess
Gravitational Force on Cultured Myotubes in Vitro”,
Journal of Systemics, Cybernetics and Informatics, Vol.
11, No. 3, 2013, pp. 50-57.
[3] T. Tamura, H. Hino, S. Hashimoto, H. Sugimoto and Y.
Takahashi, “Cell Behavior After Stimulation of Excess
Gravity”, Proc. 21st World Multi-Conference on
Systemics Cybernetics and Informatics, Vol. 2, 2017, pp.
263-268.
[4] H. Hino, H. Sugimoto, Y. Takahashi, S. Hashimoto and S.
Miura, “Behavior of Cells in Excess Gravitational Field:
Using Centrifuge”, Proc. 20th World Multi-Conference on
Systemics Cybernetics and Informatics, Vol. 2, 2016, pp.
123-128.
[5] H. Hino, H. Sato, S. Hashimoto and Y. Takahashi, “Effect ofExcess Gravitational Force and Electric Pulse Field on
Myoblast”, Proc. 19th World Multi-Conference on
Systemics Cybernetics and Informatics, Vol. 2, 2015, pp.
258-263.
[6] H. Hino, S. Hashimoto and T. Yasuda, “Effect of Centrifugal
Force
on
Cell
Culture”,
Proc.
18th World
Multi-Conference on Systemics Cybernetics andInformatics, Vol. 2, 2014, pp. 132-137.
[7] A. Tschopp and A. Cogoli, “Hypergravity Promotes Cell
Proliferation”, Experientia, Vol. 39, No. 12, 1983, pp.
1323-1329.
[8] H. Hino, S. Hashimoto and F. Sato, “Effect of Micro Ridges
on Orientation of Cultured Cell”, Journal of Systemics,
Cybernetics and Informatics, Vol. 12, No. 3, 2014, pp.
47-53.
[9] H. Hino, S. Hashimoto, Y. Shinozaki, H. Sugimoto and Y.
Takahashi, “Effect of Flow on Cultured Cell at Micro-patternof Ridge Lines”, Journal of Systemics, Cybernetics andInformatics, Vol. 15, No. 5, 2017, pp. 1-7.
[10] J.H.-C. Wang, G. Yang, Z. Li and W. Shen, “Fibroblast
Responses to Cyclic Mechanical Stretching Depend on Cell
Orientation to the Stretching Direction”, Journal of
Biomechanics, Vol. 37, 2004, pp. 573-576.
[11] P. Uttayarat, M. Chen, M. Li, F.D. Allen, R.J. Composto
and P.I. Lelkes, “Microtopography and Flow Modulate the
Direction of Endothelial Cell Migration”, Am. J. Physiol.
Heart Circ. Physiol., Vol. 294, 2008, pp. H1027-H1035.
[12] S. Hashimoto, F. Sato, H. Hino, H. Fujie, H. Iwata and Y.
Sakatani, “Responses of Cells to Flow in Vitro”, Journal of
Systemics Cybernetics and Informatics, Vol. 11, No. 5,
2013, pp. 20-27.
[13] S. Hashimoto and M. Okada, “Orientation of Cells
Cultured in Vortex Flow with Swinging Plate in Vitro”,
Journal of Systemics Cybernetics and Informatics, Vol. 9,
No. 3, 2011, pp. 1-7.
[14] H. Hino, S. Hashimoto, Y. Takahashi and M. Ochiai,“Effect of Shear Stress in Flow on Cultured Cell: Using
Rotating Disk at Microscope”, Journal of Systemics,
Cybernetics and Informatics, Vol. 14, No. 4, 2016, pp.
6-12.

The time lapse image of every ten minutes is effective to trace
the movement of each cell. The response of the cell depends
on the direction of the hyper-gravitational field. Each cellchanges its own direction during migration. The migration
depends on the morphology of the scaffold surface [11].
When a cell cannot keep adhesion under stimulation, the shapeindex (P) approaches to “0” to be a sphere.
During centrifugation, the direction of the force field slightly
tilts from horizontal direction (normal at the scaffold in thepresent study) because of the gravity (1 G) of the earth. In thecase of centrifuge of 5 G, the shift of the angle is 11 degrees.
The gravity in the fluid is reduced by the buoyancy. From thedifference of densities, the effect of the centrifugal force on thecell in the medium is estimated to be 6 G at centrifuge of 100 G(group Y in the present study) [3].
To align the direction of the single cell on the scaffold before
stimulation, the micro stripe pattern has been used in the present
study. The height of the ridge is designed as low as possible(0.7 μm: just higher than threshold [8]) to allow the effect of
centrifuge simultaneously. The change between before andafter stimulation can be easily observed with the micro striped
pattern on the scaffold.

5. CONCLUSION
The behavior of a cell cultured on the micro striped pattern has
been observed after stimulation of the excess gravity in vitro.
The lines of parallel micro quadrangular ridges are made on thesurface of the plate as the scaffold to make orientation of eachcell.
Variation is made about the angle between thelongitudinal direction of the ridge and the direction of theexcess gravity: 0 degree, 45 degrees, and 90 degrees. The
hyper- gravitational force field (50 G, or 100 G) was applied to
C2C12 (mouse myoblast cell line) adhered on the micro patternby a conventional centrifugal machine placed in an incubator.
The behavior of cells was traced at the time lapse images for 24hours, after the hyper-gravitational stimulation for 5 hours.
The longitudinal axis of C2C12 tends to align to the direction of
the excess gravity, and returns to the direction of longitudinal
direction of micro pattern after stopping of the excess gravity.
The present study shows that the hyper-gravitational stimulation
affects the behavior of cells after stimulation.

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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