Effect of Micro Ridges on Cell Culture
Yoshinori YAGUCHI, Shigehiro HASHIMOTO, Takahiro MINEZAKI, Haruka HINO
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/andHiromichi FUJIE
Human Mechatronic Systems, Faculty of System Design,
Tokyo Metropolitan University, Tokyo, Japan

ABSTRACT

2. METHODSThe effect of micro ridges on the cell culture has been studied invitro. Several patterns of micro ridges have been fabricated ona transparent polydimethylsiloxane disk with the photo
lithography technique: a single line or ten parallel lines of microridges (0.001 mm height, 0.003 mm width). C2C12 (mousemyoblast cell line originated with cross-striated muscle of C3Hmouse) was cultured on the disk for one week and was observedwith an inverted phase contrast microscope. The experimental
results show that cells align to the direction of micro ridges, andthat the differentiation of cells can be accelerated with microridges.

Micro Ridges
Three patterns of micro ridges have been made on a disk oftransparent polydimethylsiloxane (PDMS): a single line or ten
parallel lines of micro ridges. The height and the width of theridge are around 0.001 mm and 0.003 mm, respectively.
Variation is made on the interval of the parallel lines of theridges: 0.002 mm and 0.004 mm.

Keywords: Biomedical Engineering, Cell Culture, C2C12,
Lithography, Micro Ridge and Polydimethylsiloxane.

1. INTRODUCTION
Biological cells respond to various environmental factors, suchas electric [1], magnetic [2] and mechanical [3, 4] fields.

A silicon wafer (Type P, Matsuzaki Seisakusyo, Co., Ltd.,Tokyo, Japan) is used for a surface mold for the disk (Fig. 1).
The diameter and the thickness of the wafer are 50 mm and 0.30mm, respectively.
The surface of the wafer was cleaned three times: with theacetone for five minutes in an ultrasonic cleaner, with theisopropyl alcohol for five minutes in an ultrasonic cleaner, andwith the ultrapure water for ten minutes. Then, the wafer wasdried on the hot plate (AHP-300, Asahi-rika, Chiba, Japan) at
383 K for 10 minutes, and exposed to the oxygen gas in areactive ion etching system (FA-1, Samco Inc., Kyoto) to be
characterized as hydrophilic (the oxygen plasma ashing).

Cell culture technique has been developed and several
methodologies have been clinically applied to regenerative
medicine. The acceleration technique for orientation andproliferation of cells has been studied to make a biologicaltissue in vivo or in vitro [1-3]. Control methodology forbehavior of cells would be applied to regenerative tissue
technology: orientation, proliferation and differentiation [5].
The morphology of the surface of the scaffold might affect theorientation of cells and might govern organization of cells.
Several methods have been applied to make orientation of cell
culture in the previous studies: fibers and grooves [6-8].
The photo lithography technique is effective to make micro
patterns on the cell culture plate. In the present study, theeffect of micro ridges on the cell culture has been studied withthe photo lithography technique in vitro.

Fig. 1: Silicon wafer (diameter: 50 mm).

Fig. 2: Silicon wafer coated with photo-resist material.

The photo-resist material of OFPR-800 (Tokyo Ohka KogyoCo., Ltd, Tokyo, Japan) was coated on the wafer with 0.002 mmthick at 5000 rpm with a spin coater (Fig. 2). The photo-resist
was baked on the heated plate at 338 K for 3 minutes.

Fig. 3: Degassing of PDMS on mold of silicon wafer withmicro pattern.

The pattern of lines was drawn on the wafer with a laser
drawing system (DDB-201K-KH, Neoark Corporation,Hachioji, Japan). To control the dimension of the lines of themold with the laser drawing system, the parameters wereselected as follows: the voltage of 3.5 V, the velocity of 3 mm/s,
the acceleration of 0.5 mm/s2.
The photo-resist was developed with tetra-methyl-ammoniumhydroxide (NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki,Japan) for several minutes. The wafer was rinsed with thedistilled water, and dried on the heated plate. To decrease theremaining stress and to increase the adhesiveness of the coating,
the wafer was baked at 393 K for 3 minutes.
The wafer was etched with the plasma gas using the Si DeepRIE System (MUC-21 ASE-SRE, Sumitomo Precision Products
Co., Ltd., Amagasaki, Japan) to make lines of the micro groove.
The switching mode between C4F8 gas and SF6 gas was appliedon the disk.
The residual photo-resist was exfoliated in the separating
solution (Hakuri 105, Tokyo Ohka Kogyo Co., Ltd, Kawasaki,Japan). The wafer was dipped in the isopropyl alcohol, before
rinsed with the distilled water. Then, the wafer with grooves
was dried on the hot plate, and used for the concave mold to
make micro ridges in the following process.
The dimension of the micro groove was measured with a laser
microscope (VK-9510, Keyence Corporation, Osaka, Japan).
The surface of the wafer with micro pattern was coated with0.001 mm thickness of parylene in the parylene coater(PDS-2010, Speciality Coating Systems, Indianapolis).
After the wafer was enclosed with a peripheral wall ofpolyimide, PDMS (Sylgard 184 Silicone Elastomer Base, DowCorning Corporation) was poured with the curing agent (DowCorning Corporation) on the wafer. The volume ratio between
PDMS and the curing agent is ten to one. After degassing (Fig.
3), PDMS was baked at 383 K for one hour in an oven (DX401,Yamato Scientific Co., Ltd) (Fig. 4).

Fig. 4: PDMS baked at 383 K for one hour.

The baked disk of PDMS is exfoliated from the mold, andsterilized in an autoclave. The disk was exposed to the oxygengas in a reactive ion etching system (FA-1, Samco Inc., Kyoto)
to be characterized as hydrophilic (oxygen plasma ashing), justbefore the cell culture.
Cell Culture
The culture dish, which consists of 6 cylindrical wells of 35 mm
diameter, was used for cell culture (Fig. 5). The PDMS disk,
which has micro ridges on the upper surface, was placed in thebottom of each well. C2C12 (mouse myoblast cell line
originated with cross-striated muscle of C3H mouse) was usedin the experiment. Cells were seeded on the culture plate withD-MEM (Dulbecco’s Modified Eagle Medium) at density of 1.5
× 103 cells per cm2. Fetal bovine serum (FBS) was added tothe medium with the volume rate of 10 percent. Cells werecultured in the incubator for one week.
The cells near the micro ridges were observed with an inverted
phase-contrast microscope (IX71, Olympus Co., Ltd., Tokyo)
day by day.

3. RESULTS
Figs. 6-9 show the results of the measurement of the micro
grooves of the manufactured mold of A, B, C and D,respectively. The dimension of micro grooves measured withthe micro scope is summarized in Table 1.
Table 1. Dimension of micro grooves of mold
Mold

Width (W)[mm]

Interval (I)[mm]

Depth (D)[mm]

A

0.0034B

0.00370.00160.0003

C

0.00380.00170.0013D

0.00410.00310.00110.0002

Figs. 10-13 show cells near the single micro ridge of “A” after
one, two, three and seven days cultivation, respectively. The
figure shows that cells adhere over the ridge.
Figs. 14-17 show cells near the micro ridges of “B” after
cultivation for one, two, three and seven days, respectively.
Figs.18-21 show cells near the micro ridges of “D” after
cultivation for one, two, three and seven days, respectively.
The figure shows orientation of cells along the ridges. Thefigure also shows that some cells are differentiated to myotubes
near the ridges.
The experimental results show that cells align to the direction of
micro ridges, and that the differentiation of cells can beaccelerated with micro ridges. The tendency is accelerated on
the multiple parallel ridges.

Fig. 6: Dimension of the single groove (A) measured with alaser microscope.

Fig. 5: Culture dish with 6 cylindrical wells.
Fig. 7: Dimension of the micro groove (B) measured with alaser microscope.

Fig. 10: Cell near the single ridge (one day).

Fig. 8: Dimension of the micro groove (C) measured with alaser microscope.

Fig. 11: Cell near the single ridge (two days).

Fig. 12: Cell near the single ridge (three days).

Fig. 9: Dimension of the micro groove (D) measured with alaser microscope.

Fig. 13: Cell near the single ridge (seven days).
from left to right is 1.0 mm.

Dimension

Fig. 14: Cell near the multiple ridges (B) (one day).

Fig. 18: Cell near multiple ridges (D) (one day).

Fig. 15: Cell near the multiple ridges (B) (two days).

Fig. 19: Cell near multiple ridges (D) (two days).

Fig. 16: Cell near the multiple ridges (B) (three days).

Fig. 20: Cell near the multiple ridges (D) (three days).

Fig. 17: Cell near the multiple ridges (B) (seven days).
Dimension from left to right is 1.0 mm.

Fig. 21: Cell near the multiple ridges (D) (seven days).
Dimension from left to right is 1.0 mm.

Cell

Cell Cell
RidgeGroove
Fig. 22: Orientation of cell in groove.

CellRidge
DW IFig. 23: Orientation of cell on multiple ridges (W, I, D: see
Table 1).

4. DISCUSSION
Several morphologies were applied to cell culture in theprevious studies: a micro capillary of glass and a groove ofsub-millimeter. In these experiments, the cells orient along thewall (Fig. 22).
The biological cells, on the other hand, might sense micro
morphology of the surface smaller than their own dimension[6-9]. The effect of the curvature of grooves of micro meterorder on the behavior of cell was studied in the previous study[10]. Morphology of nanometer order of the surface might
affect behavior of cells [11, 12]. In the present study, theeffect of smaller dimension of morphology on the orientation ofcells has been studied.
Control methodology for orientation and proliferation of cells
has a potential to be applied to a bio-actuator [13].
The experimental results show that cells move and elongate
according to the ridge. Although the single ridge is notenough to make orientation of cells, multiple ridges affect thecells orientation (Fig. 23). The surface morphology of
micrometer affects cells behavior. The differentiation of cells
can be accelerated with micro ridges.

5. CONCLUSION
Responses of cells to micro ridges have been studied in vitro.
Several patterns of micro ridges have been fabricated on atransparent polydimethylsiloxane disk with photo lithography
technique.
The experimental results show that cells align tothe direction of micro ridges, and that the differentiation of cells
can be accelerated with micro ridges.

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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