Effect of Aspect Ratio of Checkered Convexo-concave
Micro-pattern on Orientation of Cultured Single Cell
Kenta SUGIMOTO, Yusuke TAKAHASHI, Shigehiro HASHIMOTO, Haruka HINO
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACTThe effect of the aspect ratio of checkered convexo-concave
micro-pattern on orientation of the cultured single cell has beenstudied in vitro. The checkered convexo-concave pattern has
been designed with micro quadrangular prisms in the square
area of 1 mm × 1 mm on a disk of glass for a scaffold by thephotolithography technique. Each prism has the following
dimension: 0.01 mm length, and 0.0007 mm height. Variation
has been made on the width of the prism: 0.005 mm, 0.008 mm,
and 0.01 mm. The variation of the width makes the variationon the aspect ratio of the top rectangular surface of the prism: 1,
1.25, and 2. C2C12 (mouse myoblast cell line) were seededon the micro pattern, and incubated for 24 hours in theDulbecco’s Modified Eagle Medium containing 10% fetal
bovine serum and 1% penicillin/ streptomycin. The cells wereobserved with a phase contrast microscope. The experimental
results show that the orientation of myoblast can be controlled
by the aspect ratio of the checkered micro convexo-concave
pattern of the surface of the scaffold.
Keywords: Biomedical Engineering, Cell Culture, Checkered
Micro pattern and Orientation.

1. INTRODUCTION
A biological cell adheres, migrates, rotates, and deforms on thescaffold. These behaviors of the cell depend on the micro
morphology of the scaffold [1-13]. The cell might be sensitive
to the morphology of the similar dimension to itself at thescaffold. The photolithography technique is available to makethe micro patterns on the scaffold of the cell culture. The
previous study showed that the orientation of myoblast depends
on the height of the micro ridges [1].
In most of the previous studies, cells were forced to makeorientation along the space between walls. In the case ofparallel micro bands, cells just trace the direction of lines. To
investigate the sensitivity of a biological cell against thedimension, variation has been made on the aspect ratio ofcheckered micro pattern of the scaffold [2].
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. The behavior of acell depends on several factors: mechanical [14-19], electrical[20-22], and magnetic stimulations [23]. The effect ofstimulations on the cell varies with the kind of cells. Control
methodology for proliferation, orientation and differentiation ofcells would be applied to the regenerative tissue technology.
In the present study, the effect of the aspect ratio of checkered
convexo-concave micro-pattern on orientation of the cultured
single cell has been studied in vitro.

2. METHODS
Micro Pattern
The checkered convexo-concave pattern has been designed withmicro quadrangular prisms at the nine square areas (1 mm × 1
mm each) on a disk of glass for a scaffold by the lithography
technique (Fig. 1). Each prism has the following dimension.
The height of the prism is 0.0007 mm. The length of the top
rectangular surface is 0.01 mm length. Variation has been
made on the width of the top square of the prism: 0.005 mm,
0.008 mm, and 0.01 mm. The variation of the width makes
the variation on the aspect ratio R of the top rectangular surfaceof the prism: R = 1, R = 1.25, and R = 2. The arctangent of theeach ratio R is 45, 51, and 63 degree, respectively.
Each pattern is drawn in the square area of 0.5 mm × 0.5 mm,
which is the quarter part of the square area of 1.0 mm × 1.0 mm.
The square area is surrounded by a smooth surface without
pattern as control. Photomasks were used to trace the micro
checkered pattern on the mold.

500

500

Control
area

5

10
10

10

5 μm
area
8

10 μm
area

10

8 μm
area
[μm]

Fig. 1: Three kinds of checkered micro-pattern: aspect ratio of
top rectangular surface of prism: 1 (lower left), 1.25 (lower
right), 2 (upper right), and flat (upper left): mirrored pattern in
Fig. 3, 5, 7, 8.

Photomask
The borosilicate glass (Tempax) disk (35 mm diameter, 1mm
thickness) was used for the base of the photomask (Fig. 2).
After the surface of the glass disk was cleaned by the oxygen(0.1 Pa, 30 cm3/min) plasma ashing (100 W, for ten minutes) in
the reactive ion etching system (FA-1, Samco Inc., Kyoto,
Japan), titanium was deposited on the surface with 150 nm
thickness by the sputtering equipment of L-210S-FH (Canon
Anelva Corporation) for eight minutes. The surface of theglass disk was cleaned again by the oxygen plasma ashing forten minutes in the reactive ion etching system (FA-1). To
improve affinity between titanium and photoresist material,
HMDS (hexamethyldisilazane: Tokyo Ohka Kogyo Co., Ltd.,
Kawasaki, Japan) was coated on the disk at 3000 rpm for 30 swith a spin coater. The positive photoresist material of
OFPR-800 (Tokyo Ohka Kogyo Co., Ltd, Kawasaki, Japan) was
coated on the titanium at 7000 rpm for 30 s with the spin coater.
The photoresist was baked in the oven (DX401, Yamato
Scientific Co., Ltd) at 368 K for three minutes.
The checkered pattern was drawn on the mask with a laser
drawing system (DDB-201K-KH, Neoark Corporation,
Hachioji, Japan). To control the dimension of the pattern on
the photomask with the laser drawing system, the parameters
were selected as follows: the voltage of 2.3 V, the velocity of
0.08 mm/s, the acceleration of 0.5 mm/s2. The photoresist was
baked again in the oven at 368 K for ten minutes. The
photoresist was developed with tetra-methyl-ammoniumhydroxide (NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki,
Japan) for ten minutes, rinsed with the distilled water, and driedby the spin-dryer (SF-250, Japan Create Co., Ltd., Tokorozawa,
Japan).
Glass

Development

The titanium was etched with the plasma gas using a reactive
ion etching system (RIE-10NR, Samuco Inc., Kyoto, Japan).
For etching, the gas of SF6 (50 cm3/min at 1013 hPa) with Ar(50 cm3/min at 1013 hPa) was applied at 100 W at 4 Pa for fiveminutes. To remove the residual layer of OFPR-800LB, themask was dipped in acetone for two minutes, rinsed with thedistilled water, and dried by the spin-dryer (Fig. 3).
Mold
The borosilicate glass (Tempax) disk (35 mm diameter, 1mm
thickness) was used for the base of mold for micro quadrangularprisms, after cleaning and hydrophilization (Fig. 4). The
negative photoresist material of low viscosity (SU-8 2: Micro
Chem Corp., MA, USA) was coated on the glass at 7000 rpm
for one minute with the spin coater. The photoresist baked in
the oven at 368 K for five minutes.
The photomask was adhered on the surface of SU-8 2, and thephotoresist was exposed to the UV light through the photomask
in the mask aligner (M-1S, Mikasa Co. Ltd., Japan) for 8 s.
The photoresist baked in the oven at 368 K for ten minutes.
The photoresist was developed with SU-8 developer (Nippon
Kayaku Co., Ltd, Tokyo, Japan) for six minutes to make micro
prisms. The glass with the micro pattern was rinsed withisopropyl alcohol for one minutes, and dried by the spin-dryer(Fig. 5).
Nine unites of the micro patterns were made at the interval of 2mm at the bottom of the culture dish (Fig. 6).
Themorphology of the surface of the mold was measured by thestylus of the contact profilometer (Dektak XT-E, Bruker
Corporation) (Fig. 10).

Glass

Resist(OFPR-800)
Etching
SU-8
Laser

Coating

ResistRemoveFig. 2: Photomask by photolithography.

Exposure
Mask

Micro
patternFig. 3: Photomask after etching: dimension from left to right is
0.7 mm.

Fig. 4: Micro pattern on mold by photolithography.

The culture dish was kept in the incubator to maintain both thetemperature of 310 K and the carbon dioxide partial pressure of
5 percent. The cells were continuously observed with thephase contrast microscope (IX71, Olympus, Tokyo) during thecell culture (Fig. 7).
In order to improve the contrast of the image of the contour ofeach cell, the cells were fixed and dried after the incubation for
24 hours. Cells were fixed with 4% paraformaldehyde for 20
minutes. In order to dehydrate the sample, the ethanol
concentration was exchanged in the order of 50%, 70%, 80%,90%, 95%, 99.5% for 3 minutes each (Fig. 8).

Fig. 5: Mold of micro pattern by photomask.

On the microscopic image, the contour of each cell was traced,
and approximated to ellipsoid (Fig. 9). The angle (0 degree <θ < 90 degree) between the longitudinal axis of each ellipsoid
and the shorter side of the square of the checkered pattern wasmeasured. The angle of zero degree indicates direction toshorter side of the rectangle of the checkered pattern.

Fig. 6: Culture dish (35 mm diameter) with micro patterns.

Scaffold with Micro Pattern
After the disk of Tempax glass was enclosed with a peripheral
wall of polyimide, degassed 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). The volume ratio of
PDMS to curing agent is ten to one. PDMS was baked at 373
K for one hour in an oven (DX401, Yamato Scientific Co., Ltd).

Fig. 7: C2C12 cultured on micro pattern for 24 hours:
dimension from left to right is 2 mm.

The morphology of the surface of the scaffold was observed bya scanning electron microscope (SEM, JSM6380LD, JEOL Ltd.,Tokyo, Japan), after the cell culture (Fig. 11).
A disk of polydimethylsiloxane (PDMS), which has a donut
shape (35 mm outer diameter, 3 mm thickness) with a hole of
20 mm diameter (culture area of 3.1 cm2), was made for the
peripheral wall of the dish (Fig. 6).
The culture plate was exposed to the oxygen gas in a reactive
ion etching system (FA-1, Samco Inc., Kyoto) to be
characterized as hydrophilic (oxygen plasma ashing).
Cell Culture
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) was seeded on the micro pattern at thedensity of 2000 cells/cm2, and incubated (24 hours) in theDulbecco’s Modified Eagle Medium. The medium contains
10% fetal bovine serum and 1% penicillin/ streptomycin.

Fig. 8: C2C12 image after fixation of Fig.7: dimension fromleft to right is 2 mm.

θFig. 9: Contour of each cell was traced: dimension from left toright is 0.7 mm.

3. RESULTS

Fig. 11: SEM image of surface of scaffold with checkered
micro-pattern: dimension from left to right is 0.1 mm. C2C12
adheres to pattern.

Angle [degree]

Fig. 10 shows morphology of the surface of the mold. The
tracings shows the height of 0.0007 mm of the micro pattern on
the mold. Fig. 11 exemplifies SEM image of the surface of thescaffold. The figure shows the checkered convexo-concave
pattern with micro quadrangular prisms (the top of the square of
0.01 mm × 0.01 mm). C2C12 adheres to the pattern.
Fig. 12 shows the angle θ [degree] between longitudinal axis ofeach cell of ellipsoid and shorter side of square of checkered
pattern: R = 2 (Fig. 12a), R = 1.25 (Fig. 12b), R = 1 (Fig. 12c),
and control (Fig. 12d). Data are arranged in ascending orderin Fig. 12a. Data will make flat section at the large frequency.
Data make straight line in Fig. 12d, which shows random
distribution of angles. Data on a line of small inclination
around 60 degrees (Fig.12b) show the high probability
distribution around 60 degrees.
Fig. 13 shows the mean value at each area of R. Each bar
shows the standard deviation. The mean value of θ is 45
degree at the random distribution between 0 degree and 90
degree. The angle shows random distribution in the area of R
= 1 and control (without micro pattern). The mean value of θ
approaches to the value of the arctangent of R, in the area of R =2 and R = 1.25.

Cell number
Fig. 12a: Angle [degree] between longitudinal axis of each cellof ellipsoid and shorter side of square of checkered pattern: R =2 (88 cells).
Angle [degree]

0.0007 mm

Cell number

Fig. 10: Tracing (height [nm] vs. distance [mm]) measured bystylus on surface of mold.

Fig. 12b: Angle [degree] between longitudinal axis of each cellof ellipsoid and shorter side of square of checkered pattern: R =1.25 (121 cells).

The biological cell is sensitive to the property of the surface ofthe scaffold. The hydrophilic property of the scaffold on thescaffold in the present study is confirmed by the very small
contact angle.

Angle [degree]

In the previous studies, cells were cultured on various
morphological pattern of scaffold. In the conventional
preparation, the morphology of the scaffold surface can becontrolled with the dimension of sub-millimeter.
The
biological cell might be sensitive to the morphology of thescaffold surface at the dimension, which is smaller than the
diameter of the cell. In the present study, the dimension of
sub-micrometer at the morphology of the scaffold surface has
been controlled by the photolithography technique.
Cell number
Fig. 12c: Angle [degree] between longitudinal axis of each cellof ellipsoid and shorter side of square of checkered pattern: R =1 (93 cells).
Angle [degree]

The previous study shows that the orientation of myotubes canbe controlled by the aspect ratio of the checkered micro
convexo-concave pattern of the surface of the scaffold [2].
The contrast of the image of each cell has been improved bysample fixation. The contour of each cell can be easily traced
without deformation artifact of the cell (Figs. 7&8).
The myoblast might make orientation along the longitudinal
direction of the rectangular top surface of the micro
quadrangular prisms. C2C12 migrates and extends along thelongitudinal axis of the ridges [3].
On the pattern of 0.005 mm height, observation of cells was noteasy because of the light scattering through the micro pattern inthe previous study [2]. The height of the prism of 0.0007 mm
has been selected as the minimum height to make orientation ofa single cell in the previous study.

Cell number
Fig. 12d: Angle [degree] between longitudinal axis of each cellof ellipsoid and shorter side of square of checkered pattern:
control (without micro pattern) (110 cells).
Angle [degree]

The effect of the height of micro ridges on the orientation of
C2C12 was studied in the previous study [1].
Theexperimental results show that myoblasts adhere on the top of
the ridge and align to the longitudinal direction of the microridges higher than 0.0007 mm.
The orientation in the surrounding area might follow that of therectangular pattern.
Myotube has tendency to makeorientation following direction of neighbor myotube [16]. The
behavior of a cell depends on that of the neighbor cell. The
behavior of a single cell has been investigated in the present
study. The orientation of the single cell might accelerate
subsequent orientation of cells in the tissue.

Fig. 13: Angle [degree] between longitudinal axis of each cellof ellipsoid and shorter side of square of checkered pattern.
From left to right: R = 2, R = 1.25, R = 1, and control. Mean(column) ± SD (bar).

4. DISCUSSION
The control technique of orientation of cells might be applied tothe alternative system to the animal experiment for tissue
technology: making the engineered pseudo-environment, or thepartial biological tissue in vitro.

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 [20-22]. The previous studies show that a mechanicalfield, on the other hand, governs behavior of cells. The shear
flow governs the orientation of endothelial cells [14-18]. Toostrong mechanical stimulation damages cells. The moderatemechanical stimulation, on the other hand, might accelerate
differentiation of cells. The mechanical stimulation candecrease proliferation of cells. 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.

5. CONCLUSION
The checkered convexo-concave pattern has been designed, andthe effect of the aspect ratio of each micro quadrangular prism(0.01 mm length, and 0.0007 mm height) on orientation of thecultured single cell has been studied in vitro. The micro
pattern for the scaffold has been successfully made by thephotolithography technique. Variation has been made on thewidth of the prism: 0.005 mm, 0.008 mm, and 0.01 mm, whichmakes the variation on the aspect ratio of the top rectangularsurface of the prism: 1, 1.25, and 2. C2C12 (mouse myoblast
cell line) were seeded on the micro pattern, and incubated for 24hours. The experimental results show that the orientation ofmyoblast can be controlled by the aspect ratio of the checkered
micro convexo-concave pattern of the surface of the scaffold.

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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