Effect of Aspect Ratio of Checkered (Ichimatsu)
Convexo-concave Micro-pattern on Orientation of Cultured Cells
Kenta SUGIMOTO, Yusuke TAKAHASHI, Haruka HINO, Shigehiro HASHIMOTO
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACTThe effect of the pattern of micro ridges on orientation of thecell culture has been studied in vitro.
The checkered(Ichimatsu) convexo-concave pattern has been designed withmicro quadrangular prisms in the square area of 1 mm × 1 mm
on a disk of glass for a scaffold by the lithography technique.
Each prism has the following dimension: 0.01 mm length, and
0.005 mm (or 0.001 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 variation on the aspectratio of the top rectangular surface of the prism: 1, 1.25, and 2.
Three kinds of cells were used in the test: C2C12 (mouse
myoblast cell line), Neuro-2a (a mouse neural crest-derived cellline), and Hepa1-6 (mouse hepatoma cell line). Cells wereseeded on the micro pattern, and incubated for 21 days 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 myotubes 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 cell depend on the micro
morphology of the scaffold [1-10]. 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].
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 [11-13]. The
behavior of a cell depends on several factors: mechanical [14,15], electrical [12, 13, 16], and magnetic stimulations [17].
The effect of stimulations on the cell varies with the kind ofcells. Control methodology for proliferation, orientation anddifferentiation of cells would be applied to the regenerative
tissue technology.

In the present study, effects of the aspect ratio of the checkered
pattern of micro quadrangular prisms on orientation of the cell
culture have been studied in vitro.

2. METHODS
Micro Pattern
The “Ichimatsu” checkered convexo-concave pattern has been
designed with micro quadrangular prisms at the square area of 1
mm × 1 mm on a disk of glass for a scaffold by the lithography
technique (Fig. 1). Each prism has the following dimension.
The length of the top rectangular surface is 0.01 mm length.
Variation has been made on the width of the top square of theprism: 0.005 mm, 0.008 mm, and 0.01 mm. The variation of
the width makes the variation on the aspect ratio of the top
rectangular surface of the prism: 1, 1.25, and 2. The
arctangent of the each ratio is 45, 51, and 63 degree. Variation
has been made on the height of the prism using two kinds ofphotomasks: 0.005 mm by the mask A, and 0.001 mm by themask B.
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. The photomasks were made by two kinds of etchingprocess: wet and dry.

Fig. 1: Three kinds of checkered micro-pattern. Aspect ratioof the top rectangular surface of the prism: 1 (lower left), 1.25
(lower right), 2 (upper right), and flat (upper left).

Photomask A by Wet Etching
The borosilicate glass (Tempax) disk (35 mm diameter, 1mm
thickness) was used for the base of the photomask (Fig. 2).
After hydrophilization by the oxygen (30 cm3/min) plasma
ashing at 100 W for ten minutes by RIE (FA-1, Samco
International, Kyoto, Japan), chromium was deposited on thesurface with 300 nm thickness by the sputtering equipment of
L-210S-FH (Canon Anelva Corporation) for three minutes. To
improve affinity between chromium and photoresist material,
HMDS (hexamethyldisilazane: Tokyo Chemical Industry Co.,
Ltd., Tokyo) was coated on the disk at 3000 rpm for 30 s with aspin coater. The positive photoresist material of OFPR-800LB(Tokyo Ohka Kogyo Co., Ltd, Tokyo, Japan) was coated on thechromium at 5000 rpm for 30 s with the spin coater. The
photoresist was baked at the hotplate in two processes: at 338 K
for one minute, and 368 K for three minutes.

Fig. 3b: Photomask A after wet etching.
to right is 1.4 mm.

Dimension from left

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 wasdeveloped with tetra-methyl-ammonium hydroxide (NMD-3,
Tokyo Ohka Kogyo Co., Ltd., Kawasaki, Japan) for fiveminutes, rinsed with the distilled water, and dried by the
spin-dryer (SF-250, Japan Create Co., Ltd., Tokorozawa, Japan)(Fig. 3a).
The chromium coating was etched in etchant for chromium TW(Nihon Kagaku Sangyo Co., Ltd., Tokyo) for twenty minutes.
To remove the residual layer of OFPR-800LB, the mask wasdipped in acetone for two minutes, rinsed with the distilledwater, and dried by the spin-dryer (Fig. 3b).

Fig. 4: Photomask B by dry etching. Dimension from left toright is 0.7 mm.

Photomask B by Dry Etching
After hydrophilization by the oxygen plasma ashing by RIE,
titanium was deposited on the surface with 150 nm thickness on
the surface of the borosilicate glass by the sputtering equipment
for eight minutes. To improve affinity between titanium andphotoresist material, HMDS was coated.
The positive
photoresist material of OFPR-800LB was coated on the glass.
The checkered pattern was drawn on the mask with the laser
drawing system.
To control the dimension of the pattern on the photomask withthe laser drawing system, the parameters were selected asfollows: the voltage of 2.3 V, the velocity of 0.08 mm/s, theacceleration of 0.5 mm/s2.

Fig. 2: Photomask by photolithography.

The photo-resist was developed with tetra-methyl-ammoniumhydroxide. The glass was etched with the plasma gas using areactive 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 five minutes (Fig. 4).
To remove the residual layer of OFPR-800LB, the mask wasdipped in acetone for two minutes, rinsed with the distilledwater, and dried by the spin-dryer.

Fig. 3a: Photomask A before etching.
right is 0.7 mm.

Dimension from left to

Scaffold with Micro Pattern
The borosilicate glass (Tempax) disk (35 mm diameter, 1mm
thickness) was used for the base for micro quadrangular prisms,
after cleaning and hydrophilization. The negative photoresist
material of high viscosity (SU8: Micro Chem Corp., MA, USA)was coated on the glass at 3000 rpm (7000 rpm on B) for one
minute with a spin coater. The photoresist baked in the ovenat 368 K for five minutes. The photomask was adhered on thesurface of SU8-10, and the photoresist was exposed to the UV
light through the mask in the mask aligner (M-1S, Mikasa Co.
Ltd., Japan) for 250s (8 s on mask B) (Fig. 5A). The
photoresist was developed with SU8 developer (Nippon Kayaku
Co., Ltd, Tokyo, Japan) for six minutes to make micro prisms.
The glass with the micro pattern was rinsed with isopropyl
alcohol for two minutes, and dried by spin-dryer.
The dimension of the micro pattern of the mold was measured
with a laser microscope (VK-X200, Keyence Corporation,
Osaka, Japan) (Fig. 5B). The morphology along the transverselines of ridges was traced.
A disk of polydimethylsiloxane (PDMS), which has a donut
shape (35 mm outer diameter, 3 mm thickness) with a hole of
10 mm diameter (culture area of 3.1 cm2), was made for the
peripheral wall of the dish (Fig. 6).

Fig. 7: Tracings of contour of each myotube. Dimension fromleft to right is 0.7 mm.
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).
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).

Fig. 5A: Micro pattern by photolithography.

Cell Culture
Three kinds of cells were used in the test: C2C12 (mouse
myoblast cell line originated with cross-striated muscle of C3Hmouse), Neuro-2a (a mouse neural crest-derived cell line), andHepa1-6 (mouse hepatoma cell line of C57L mouse).
Cells were seeded on the micro pattern at the density of 1000
cells/cm2 (2000 cells/cm2 for pattern B), and incubated (< 21days) in the Dulbecco’s Modified Eagle Medium. The
medium contains 10% fetal bovine serum and 1% penicillin/
streptomycin. In the case of Neuro-2a, Retinoic Acid wasadded to the medium to induce differentiation.
The
concentration of Retinoic Acid in the medium is adjusted to 20
μM.

Fig. 5B: Tracing of scaffold by photomask A (by laser
microscope).

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 medium was changed every two days. The
cells were continuously observed with the phase contrast
microscope (IX71, Olympus, Tokyo) during the cell culture.
On the microscopic image, the outline of each cell was traced,
and the contour of each cell was approximated to ellipsoid (Fig.
7). The angle between the longitudinal axis of each cell andthe shorter side of the square of the checkered pattern wasmeasured.

3. RESULTS

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

Table 1 shows the dimension of height measured by the laser
microscope (Fig. 5B). At the manufactured micro pattern A,
there are slits between the micro quadrangular prisms, because
rectangles are bridged together at the photomask A (Fig. 3b).
At the manufactured micro pattern B, on the other hand, thereare bridges between the micro quadrangular prisms, because
each rectangle is separated island at the photomask B (Fig. 4).
Table 1: Dimension of the height of the prism, used in each testof three kinds of cells: micro pattern with mask A and B.
Cell

mm
Pattern A

Neuro-2a

0.005

C2C12

0.005

Hepa1-6

Pattern B
0.001
0.002

Neuro-2a extends the neurite along the slit between the micro
quadrangular prisms (Fig. 8). The proliferation of Neuro-2a
decreases at the micro pattern. The checkered pattern does notaffect orientation of Hepa1-6 (Fig. 9).

Fig. 9a: Hepa1-6 cultured on micro pattern B for 7 days.
Dimension from left to right is 1 mm.

On the checkered convexo-concave micro pattern, myoblasts(Figs. 10a & 11a) were differentiated to myotubes (Figs. 10b &11b). At the micro pattern A, the myotubes tend to makeorientation along the diagonal line of slit between the micro
quadrangular prisms (Fig. 10c). At the micro pattern B, on theother hand, the myotubes tend to make along the longitudinal
direction of the rectangular top surface of the micro
quadrangular prisms (Fig. 11b).
Fig. 12 shows the angle between the longitudinal axis of eachmyotube and the shorter side of the square of the checkered
pattern measured at the image of Fig. 10c.
Data are arranged in ascending order in Fig. 12a. Data will
make flat section at the large frequency. At the random
frequency, data will form a simple rising straight line. Everygroup of data at each micro pattern shows the character of theinverse “S”, which correspond to high frequency around themean value.

Fig. 9b: Hepa1-6 cultured on micro pattern B for 9 days.
Dimension from left to right is 1 mm.

Fig. 12b shows the relation between the mean longitudinal axis
direction of myotube and the angle of the diagonal line of eachrectangle. The angle of zero degree indicates direction toshorter side of the rectangle of checkered pattern. Fig. 12bshows that the angle of the myotube is proportional to that ofdiagonal line of the micro rectangle.
Fig. 10a: C2C12 cultured on micro pattern A for 1 day.
Dimension from left to right is 2 mm.

Fig. 8: Neuro-2a cultured on micro pattern A for 6 days.
Dimension from left to right is 0.9 mm.

Fig. 10b: C2C12 cultured on micro pattern A for 12 days.
Dimension from left to right is 2 mm.

Angle of myotube [degree]

Angle of diagonal line [degree]

Fig. 10c: C2C12 cultured on micro pattern A for 20 days.
Dimension from left to right is 2 mm.

Fig. 12b: Mean longitudinal axis direction of myotube vs.
diagonal line of each square. Angle of zero degree indicatesdirection to shorter side of the square of checkered pattern.

Fig. 13a: Orientation of cell on checkered pattern A.
Fig. 11a: C2C12 cultured on micro pattern B for 1 day.
Dimension from left to right is 2 mm.

Fig. 13b: Orientation of cell on checkered pattern B.

4. DISCUSSION

Fig. 11b: C2C12 cultured on micro pattern B for 10 days.
Dimension from left to right is 2 mm.
Angle [degree]

On the manufactured pattern A, the micro slits are left between
the micro quadrangular prisms (Fig. 13a). Myotubes mightmake orientation along the slit on checkered pattern A. On the
manufactured pattern B, on the other hand, the micro
quadrangular prisms are connected together (Fig. 13b). The
myotube 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 [1].
The height of the micro quadrangular prism is higher at thepattern A than that at the pattern B. The height of the pattern
B might have not enough height to make orientation of cells.

Fig. 12a: Angle of longitudinal axis of myotube: aspect ratio 1(circle), 1.25 (triangle), 2 (rhombus), and flat (square)

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 with the height between 0.00015 mm and 0.0025 mm.

The height (H) of the micro ridge is selected to be 0.001 mm <H < 0.005 mm in the present study.
The orientation in the surrounding area might follow that of therectangular pattern. The orientation of myotubes developed atday 20 of culture (Fig. 10c) after day 12 of culture (Fig. 10b).
Myotube has tendency to make orientation following directionof neighbor myotube [11].
On the Ichimatu pattern of 0.005 mm height, observation ofcells is not easy because of the light scattering through themicro pattern.
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 [13, 16]. The previous studies show that a mechanicalfield, on the other hand, governs behavior of cells. The shear
flow governs the orientation of endothelial cells [11, 14]. Toostrong mechanical stimulation damages cells. The moderatemechanical stimulation, on the other hand, might accelerate
differentiation of cells [15]. The mechanical stimulation candecrease proliferation of cells [15]. The mechanical stress also
exfoliates several cells [14], 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
Effects of the pattern of micro ridges on orientation of the cell
culture have been studied in vitro. The checkered (Ichimatsu)
convexo-concave pattern has been designed with micro
quadrangular prisms in the square area of 1 mm × 1 mm on adisk of glass for a scaffold by the lithography technique. Eachprism has the following dimension: 0.01 mm length, and 0.005
mm (or 0.001 mm) height. Variation has been made on thewidth of the prism: 0.005 mm, 0.008 mm, and 0.01 mm.
Three kinds of cells were used in the test: C2C12 (mouse
myoblast cell line), Neuro-2a (a mouse neural crest-derived cellline), and Hepa1-6 (mouse hepatoma cell line).
The
experimental results show that the orientation of myotubes canbe 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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