Effect of Surface Morphology of Scaffold with Lines of Micro
Ridges on Deformation of Cells
Hiromi SUGIMOTO, Haruka HINO, Yusuke TAKAHASHI, 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 micro ridges of the scaffold on deformation ofcells has been studied in vitro. Several parallel lines of microridges have been made on a disk of transparent
polydimethylsiloxane. The height, the width, and the length of
the rectangular ridge are 0.001 mm, 0.003 mm, and 0.5 mm,
respectively. Variation has been made on the interval of theridge: 0.001 mm, 0.003 mm, 0.005 mm and 0.007 mm. Fivekinds of cells were used in the test. C2C12 (mouse myoblast
cell line), L929 (fibroblast connective tissue of mouse),
Neuro-2a (a mouse neural cell line), 3T3-L1 (mouse fat
precursor cells), and HUVEC (human umbilical vein
endothelial cells). Cells were seeded on the micro pattern atthe density of 2000 cells/cm2 and cultured for 24 hours. The
cells were observed with a microscope. The experimental
results show that C2C12 lengthen along the line of ridge of
0.003 mm interval. The effects of the micro pattern depend on
the kind of cells.
Keywords: Biomedical Engineering, Cell Culture, Micro
Ridges, Photolithography, and Deformation.

1. INTRODUCTION
Multi-factors govern organization of cells in vivo. Because
relations among the factors have not been analyzed, it is noteasy to design simulator in vitro. Most of trials of regenerative
medicine were arranged in situ,
A biological cell adheres, migrates, rotates, and deforms on thescaffold. These behaviors of cell depend on the micro patternof the scaffold [1-6]. The previous study showed that theorientation of myoblast depends on the height of the microridges [1].
Cell culture technique has been developed, and cells have been
cultured in controlled environment [1-21]. The behavior of thecell might depend on the surface micro morphology. The
dependency might depend on the kind of cells.
The
technology can be applied to sorting of cells.
The acceleration technique for proliferation, orientation [1, 14,20] and differentiation [3, 7] of cells has been studied to maketissue in vivo or in vitro. Several methods have been designed
to stimulate cells [17-21].

In the present study, the effect of the surface morphology withlines of micro ridges of the scaffold on deformation of cells has
been studied in vitro.

2. METHODS
Micro Pattern
Several parallel lines of micro ridges have been made at thecenter on a disk of transparent polydimethylsiloxane (PDMS)(Fig. 1). The height (H), the width (W), and the length (L) of
the rectangular ridge (Fig. 1) are 0.001 mm, 0.003 mm, and 0.5mm, respectively. Variation has been made on the interval (I)of the ridge: 0.001 mm, 0.003 mm, 0.005 mm and 0.007 mm.
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 withoutridges as control.
The borosilicate glass (Tempax) disk was used for a surface
mold for the disk in a photo lithography process (Fig. 2). The
diameter and the thickness of the disk are 50 mm and 1.1 mm,
respectively. The surface of the glass was cleaned by the
spin-dryer (SF-250, Japan Create Co., Ltd., Tokorozawa, Japan).
To improve the affinity between the glass and the photo-resist
material (OFPR-800), hydrophobic treatment was applied to theglass by the oxygen (30 cm3/min) plasma ashing at 100 W forten minutes by a compact etcher (FA-1, Samco Inc., Kyoto).
Hexamethyldisilazane (HMDS) was coated at 3000 rpm forthirty seconds with a spin coater (IH-DX2, Mikasa Co., Ltd.,Tokyo, Japan).
The photoresist material of OFPR-800LB (Tokyo Ohka Kogyo
Co., Ltd., Tokyo, Japan) of 0.0017 mm thick was coated on theglass at 3500 rpm for five seconds with the spin coater. The
photoresist was baked in the oven at 373 K for ninety seconds.
The pattern for the micro grooves was drawn on the disk with alaser drawing system (DDB-201K-KH, Neoark Corporation,
Hachioji, Japan). To control the dimension of the pattern on
the mold with the laser drawing system, the parameters wereselected as follows: the voltage of 3.2 V, the velocity of 0.151
mm/s, the acceleration of 0.350 mm/s2. The pattern was bakedin the oven at 393 K for five minutes.
The photo-resist was developed with tetra-methyl-ammoniumhydroxide (NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki,
Japan) for 60 seconds. The disk was rinsed two times with theultrapure water for three minutes, and dried by the spin-dryer.

The glass was etched with the plasma gas using a reactive ion
etching system (RIE-10NR, Samuco Inc., Kyoto, Japan) tomake lines of the micro grooves of 0.001 mm depth. For
etching, the gas of CF4 (30 cm3/min at 1013 hPa) was applied at
100 W at 2 Pa for thirty minutes. To exfoliate the residual
photo-resist material from the surface, the disk was exposed tothe oxygen gas of 30 milliliter per minute at power of 100 W forten minutes using a compact etcher (FA-1, Samco Inc., Kyoto):(the oxygen plasma ashing).

Fig. 4: Micro ridges of PDMS. Interval I = 0.001 mm (right),
0.003 mm (upper), 0.005 mm (left), 0.007 mm (lower).
Dimension from left to right is 0.1 mm.

The dimensions of the four kinds of the micro grooves of themold were measured with a laser microscope (VK-X200,
Keyence Corporation, Osaka, Japan). The morphology along
the transverse lines of grooves was traced (Fig. 3).
After the mold was enclosed with a peripheral wall ofpolyimide tape, 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) on the mold. The volume ratio of
PDMS to curing agent is ten to one. After degassing, PDMS
was baked at 373 K for one hour in an oven (DX401, Yamato
Scientific Co., Ltd). The baked disk of PDMS was exfoliatedfrom the mold. The disk with the micro ridges was used as thebottom of the culture dish.
W

Fig. 5: Tracings of micro ridges of PDMS. Interval I = 0.001
mm (upper right), 0.003 mm (upper left), 0.005 mm (lower left),
0.007 mm (lower right).

L

H

I
Fig. 1a: Parallel lines of micro ridges.

Fig. 6: Disk with micro pattern, wall and cap of PDMS (filledwith medium) contained in polystyrene dish (left), placed inincubator on microscope (right).

The morphology of the micro ridges for scaffold was observed
by a scanning electron microscope (SEM, JSM6360LA, JEOL
Ltd., Tokyo, Japan) (Fig. 4). The dimensions of the four kindsof the micro ridges of PDMS were measured with a laser
microscope. The morphology along the transverse lines ofridges was traced (Fig. 5).

Fig. 2: Mold of micro grooves on glass plate. Interval I =0.001 mm (upper left), 0.003 mm (upper right), 0.005 mm(lower right), 0.007 mm (lower left). Dimension from left toright is 1.5 mm (left), 0.1 mm (right).

Fig. 3: Tracings of mold of micro grooves on glass plate.
Interval I = 0.001 mm (upper left), 0.003 mm (upper right),
0.005 mm (lower right), 0.007 mm (lower left).

Another disk of PDMS, which has a donut shape (50 mm outer
diameter, 5 mm thickness) with a hole of 33 mm diameter, wasmade for the peripheral wall of the dish (Fig. 6). These two
disks are contacted with the affinity between them, and make aculture area of 8.5 cm3 at the bottom. The cap for the dish of
PDMS was made of PDMS. The dimension of the diameterand the thickness are 50 mm and the 0.5 mm, respectively.
The donut shape wall and the cap are for multiple use bysterilization in the autoclave.
The culture dish of PDMS was exposed to the oxygen gas of 30
milliliter per minute at power of 50 W for one minute in acompact etcher (FA-1, Samco Inc., Kyoto) to be characterized
as hydrophilic (oxygen plasma ashing).
The dish waspreserved in the ultrapure water to keep the hydrophilic
property of the surface, before the cell culture. The contact
angles were measured between the disk of PDMS and theultrapure water at 298 K by the contact angle analyzer(Phoenix-300, Meiwafosis Co., Ltd., Tokyo, Japan), before andafter the oxygen plasma ashing (Fig. 7).

microscope. After fixation of cells by immersion for twenty
minutes in 4% paraformaldehyde solution, cells were
dehydrated by ethanol. Then, the sample was dried at criticalpoint by JCPD-5 (JEOL Ltd., Japan).

Fig. 7a: Contact angle of water on smooth surface (left), on
micro pattern (middle and right), before oxygen plasma ashing.
Perpendicular (left and middle) and parallel (right) view to linesof ridge.

Fig. 7b: Contact angle of water on PDMS, after oxygen plasma
ashing.

Cell Culture
Five kinds of cells were used in the test. C2C12 (mouse
myoblast cell line originated with cross-striated muscle of C3Hmouse), L929 (fibroblast connective tissue of C3H mouse),
Neuro-2a (a mouse neural crest-derived cell line), 3T3-L1(mouse fat precursor cells, cell line of 3T3 mouse), andHUVEC (human umbilical vein endothelial cells).
The
passage between the fourth and the eighth of cell line was usedin the experiment.
D-MEM (Dulbecco’s Modified Eagle’s Medium) containing
10% FBS (decomplemented fetal bovine serum) and 1%
penicillin/ streptomycin (GIBCO, Life Technologies Japan Ltd.,Tokyo, Japan) was used for the culture medium of C2C12, and
3T3-L1. HUVEC was cultured in EBM-2 (Endothelial Cell
Basal Medium, Lonza) containing EGM-2 (Endothelial Cell
Growth Medium, Lonza) and 2% decomplemented FBS (fetal
bovine serum).
Cells were seeded on the dish of PDMS at the density of 2000
cells/cm2. The culture dish was kept in the small chamber ofthe incubator placed on the stage of the microscope to maintainboth the temperature of 310 K and the carbon dioxide partialpressure of 5 percent. The cells were continuously observedwith the phase contrast microscope (IX71, Olympus, Tokyo)
during the cell culture (Fig. 6).
The behavior of cells was successively recorded with thetime-lapse microscopic pictures: deformation, pseudo extension,
migration, exfoliation, re-adhesion, and proliferation. Theinterval of the time-lapse was selected to be two minutes for 24hours. On the microscopic image, the outline of each cell was
traced, and the contour of each cell was approximated toellipsoid (by Image J). The length of the major axis (A), theminor axis (B), and the area (S) were measured. The ratio ofaxes is calculated as the shape index (X) by Eq. 1. X
approaches unity at rounded cells, and zero at elongated cells.
The extension is calculated as the extension ratio (E) by Eq. 2.
X=B/A
E = A / S 1/2

(1)
(2)

At the end of the experiment, the micro morphology of the cellwith the micro ridges was also observed by a scanning electron

3. RESULTS
The tracing of the surface morphology across the lines ofgrooves on the mold measured by the laser microscope is
exemplified in Fig. 3. The tracing shows the height of 0.0009
mm, and width of 0.0028 mm. The tracing shows also thevariation on the interval: 0.0010 mm, 0.0032 mm 0.0052 mm,
and 0.0072 mm. The scanning electron microscope images ofthe lines of ridges are exemplified in Fig. 4. Fig. 4 shows theborder of four kinds of ridges. The tracing of the surface
morphology across the lines of ridges of PDMS measured by
the laser microscope is exemplified in Fig. 5. The tracingshows the height of 0.0009 mm, and width of 0.0025 mm.
The tracing shows also the variation on the interval: 0.0012 mm,
0.0033 mm 0.0052 mm, and 0.0074 mm. Fig. 7a shows thecontact angle of water on PDMS before oxygen plasma ashing.
The angle on the micro pattern in the direction perpendicular tothe line of the ridges is 2.3 rad, which is bigger than 2.0 degree
on the flat surface. The angle is 1.6 rad, on the other hand, on
the micro pattern in the longitudinal direction of the line of theridges. Fig. 7b shows the contact angle of 0.07 rad, after
oxygen plasma ashing. The property of the surface changed
from hydrophobic to hydrophilic by the oxygen plasma ashing.
Fig. 8 shows C2C12. Most of cells extend along the lines ofridges. Fig. 9 shows L929. L929 bridges between ridges.
Fig. 10 shows Neuro-2a. The most of cell are rounded on the
micro pattern, although several cells extend the neurite on thesmooth surface. Fig. 11 shows 3T3-L1. Most of cells extend
along the lines of ridges, although cell extends to random
direction on the smooth surface. Fig. 12 shows HUVEC.
HUVEC is rounded on the micro pattern at SEM image,although extension of cell observed at time lapse image. Fig.
13 shows the extension ratio E (Eq. 2) on the micro pattern.
C2C12 extends at the ridges of 0.001 mm< I < 0.004 mm. The
extension ratios E of L929, Neuro-2a, 3T3-L1, and HUVEC do
not depend on ridges. Fig. 14 shows the shape index (X). X
of C2C12 decreases on the ridge of I = 0.001 mm. The shapeindex of L929, Neuro-2a, 3T3-L1, and HUVEC do not dependon ridges.

Fig. 8a: C2C12 immediately after seeding (left) and cultured
for 24 hours (right): interval I = 0.001 mm (upper right), 0.003
mm (upper left), 0.005 mm (lower left), 0.007 mm (lower right).
Dimension from left to right is 0.13 mm, each.

Fig. 8b: SEM image of C2C12 cultured on ridges for 24 hours:
Interval I = 0.001 mm (A), 0.003 mm (B), 0.005 mm (C), 0.007
mm (D), smooth (E).

Fig. 9a: L929 immediately after seeding (left) and cultured for
24 hours (right): interval I = 0.001 mm (upper right), 0.003 mm(upper left), 0.005 mm (lower left), 0.007 mm (lower right),
dimension from left to right is 0.13 mm, each.
A

C

B

Fig. 10b: SEM image of Neuro-2a cultured on ridges for 24hours: Interval I = 0.001 mm (A), 0.003 mm (B), 0.005 mm (C),0.007 mm (D), smooth (E).

Fig. 11a: 3T3-L1 immediately after seeding (left) and cultured
for 24 hours (right): interval I = 0.001 mm (upper right), 0.003
mm (upper left), 0.005 mm (lower left), 0.007 mm (lower right).
Dimension from left to right is 0.13 mm, each.
A

B

D

E

C

D

Fig. 9b: SEM image of L929 cultured on ridges for 24 hours:
Interval I = 0.001 mm (A), 0.003 mm (B), 0.005 mm (C), 0.007
mm (D).

Fig. 10a: Neuro-2a immediately after seeding (left) andcultured for 24 hours (right): interval I = 0.001 mm (upper
right), 0.003 mm (upper left), 0.005 mm (lower left), 0.007 mm(lower right). Dimension from left to right is 0.13 mm, each.

Fig. 11b: SEM image of 3T3-L1 cultured on ridges for 24hours: Interval I = 0.001 mm (A), 0.003 mm (B), 0.005 mm (C),0.007 mm (D), smooth (E).

Fig. 12a: HUVEC immediately after seeding (left) and cultured
for 24 hours (right): interval I = 0.001 mm (upper right), 0.003
mm (upper left), 0.005 mm (lower left), 0.007 mm (lower right).
Dimension from left to right is 0.13 mm, each.

X

Fig. 12b: SEM image of HUVEC cultured on ridges for 24hours: Interval I = 0.001 mm (A), 0.003 mm (B), 0.005 mm (C),0.007 mm (D), smooth (E).
E

I [μm]
Fig. 14a: Shape index (X) vs. interval (I) [μm]: C2C12.
X

I [μm]
I [μm]
Fig. 13a: Extension ratio (E) vs. interval (I) [μm]: C2C12.
E

Fig. 14b: Shape index (X) vs. interval (I) [μm]: L929.
X

I [μm]
I [μm]
Fig. 13b: Extension ratio (E) vs. interval (I) [μm]: L929.

Fig. 14c: Shape index (X) vs. interval (I) [μm]: Neuro-2a.
XE

I [μm]

I [μm]

Fig. 14d: Shape index (X) vs. interval (I) [μm]: 3T3-L1.
X

Fig. 13c: Extension ratio (E) vs. interval (I) [μm]: Neuro-2a.
E

I [μm]
Fig. 14e: Shape index (X) vs. interval (I) [μm]: HUVEC.
I [μm]
Fig. 13d: Extension ratio (E) vs. interval (I) [μm]: 3T3-L1.
E

I [μm]
Fig. 13e: Extension ratio (E) vs. interval (I) [μm]: HUVEC.

4. DISCUSSION
The dimension of 0.003 mm is selected for the width of theridge, because the value is effective to make orientation of
C2C12 in the previous study [2]. The dimension of 0.001 mm
is selected for the height of the ridge, because the value between
0.0005 mm and 0.002 mm is effective to make orientation of
C2C12 [1]. The dimension of interval of the ridge is selected
smaller value compared to the diameter of cells in the present
study. The height of the ridge is 0.001 mm, which is low
enough for a cell to get over. The conventional way uses
pattern with the higher wall to control orientation of cells in
vitro. If the surface micro morphology is effective to control
the orientation of cells, the variation of methodology would be
extended.

The behavior of a cell might depend on the adhesion of the cellto the scaffold. The adhesive property varies with thematerials of the scaffold [7]. In the present study, the surfaceof PDMS is modified with oxygen ashing. Although thediameter of the droplet of distilled water is bigger than theinterval of the lines of the ridges, the contact angle shows
asymmetrical properties of the surface with ridges (Fig. 7a).
Asymmetry on the hydrophilic property might govern theextension of the cell.
Some parts of the ridge are destroyed near the cell at SEM
image (Figs. 8-12). The part might be destroyed by the cell, orby the treatment for microscopic observation.

5. CONCLUSION
The effect of micro ridges of the scaffold on deformation ofcells has been studied in vitro. Several parallel lines of microridges have been made on a disk of transparent
polydimethylsiloxane. The height, the width, and the length of
the rectangular ridge are 0.001 mm, 0.003 mm, and 0.5 mm,
respectively. Variation has been made on the interval of theridge: 0.001 mm, 0.003 mm, 0.005 mm and 0.007 mm. Fivekinds of cells were used in the test. C2C12 (mouse myoblast
cell line), L929 (fibroblast connective tissue of mouse),
Neuro-2a (a mouse neural cell line), 3T3-L1 (mouse fat
precursor cells), and HUVEC (human umbilical vein
endothelial cells). The experimental results show that C2C12
lengthen along the line of ridge of 0.003 mm interval. The
effects of the micro pattern depend on the kind of cells.

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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