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

Design of Scaffold with Taper-Striped Pattern to Observe
Durotactic Migration of Cell
Shigehiro HASHIMOTO, Kenta SUGIMOTO, Yusuke TAKAHASHI
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/
ABSTRACT

2. METHODS

The scaffold with the taper-striped pattern has been designed toobserve durotactic migration of a biological cell. The surfaceof the scaffold with the micro taper-striped (0.05 mm < width <0.2 mm) pattern was made by the photolithography technique.
The variation of hardness of the surface of the band has been
made of the materials alternately: polydimethylsiloxane(PDMS), and epoxy based negative photoresist material (SU-8).
C2C12 (mouse myoblast) was seeded on the micro pattern, andincubated for 48 hours in the medium. The projected area of
each cell was traced in each band at the microscopic images,
and the total area was compared between bands of two kinds ofmaterials. The experimental result shows that each cell tends
to migrate to the area of the band of SU-8. The experimental
system is effective to investigate durotaxis of cells in vitro.

Micro Hybrid Triangular Striped Pattern
The hybrid taper-striped pattern of scaffold has been designed
on a plate of polydimethylsiloxane (PDMS) for a flat scaffold
by the photolithography technique. A part of each band has
gradual reduction at the width: from 0.2 mm to 0.1 mm, or 0.15
mm to 0.05 mm. The surface of the scaffold consists of bands
made of two alternate materials: PDMS, and SU-8 (epoxy based
negative photoresist material). The pattern was controlled by aphotomask. The surface of the photomask was observed by
the laser microscope (VK-X200, Keyence Corporation, Osaka,
Japan) (Fig. 1).
Mold
The borosilicate glass (Tempax) disk is used for the base of themold (Fig. 2). The diameter and the thickness of the disk are
35 mm and 1.1 mm, respectively. The surface of the glass disk
was cleaned by the oxygen (0.1 Pa, 30 cm3/min) plasma ashing(100 W, for five minutes) in the reactive ion etching system(FA-1, Samco Inc., Kyoto, Japan).

Keywords: Biomedical Engineering, C2C12, Durotaxis andPhotolithography.

1. INTRODUCTION
The negative photoresist material of high viscosity (SU-8 10:
Micro Chem Corp., MA, USA) was coated on the disk by thespin coater (at 1000 rpm for 1 min). The photoresist was
baked in the oven (DX401, Yamato Scientific Co., Ltd) at 368
K for five minutes.

After adhesion on the scaffold, a biological cell migrates on thescaffold. The cell migration might govern the subsequentorientation of cells. Control methodology for orientation ofcells would be applied to the regenerative medicine.
The migration depend on several factors: the micro morphologyof the scaffold [1-8], the vibration [8, 9], the shear flow field[10-14], and the gravitational field [15, 16].
The cellmigration might also be guided by the rigidity gradients on thescaffold (durotaxis) [17-29].
The photolithography technique is available to make the micro
patterns on the scaffold of the cell culture. The surface of thescaffold with the micro stripe pattern of alternative bands(polydimethylsiloxane (PDMS) and epoxy based negative
photoresist material (SU-8)) was made by the photolithography
technique to observe durotaxis in the previous study [17]. At
the parallel stripe pattern, migration to the longitudinal directionof the stripe has no relation to durotaxis. At the band of thegradually decreased width, on the other hand, the longitudinal
direction of the stripe has asymmetry.

0.2 mm

In the present study, the scaffold with the triangular striped
pattern has been designed to observe durotactic migration of abiological cell in vitro.

Fig. 1: Laser microscopic image of photomask for taper-stripe
pattern. Dimension from left to right is 1.5 mm.

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

Cell Culture
C2C12 of the passage 6 was cultured in D-MEM (Dulbecco’s
Modified Eagle Medium) containing 10% FBS (fetal bovine
serum) and 1% penicillin/ streptomycin.

Exposure

Mask

In each test, cells were seeded on the micro pattern of the disk
placed in the culture dish at the density of 2000 cells/cm2. The
culture dish was kept in the incubator to maintain both thetemperature of 310 K and the carbon dioxide partial pressure of
5 percent for 48 hours.

SU-8

Micro
pattern

Glass
Developing

The migration tendency of each cell was checked by the timelaps image of the phase contrast microscope during the cell
culture. Figs. 7a, 8a, and 9a show microscopic images of cells.
On the microscopic image, the contour of each cell was traced,
and the projected area of each cell (Ak) was calculated. At thetwo dimensional image, the total area of cells was evaluated ateach band of the micro pattern by the occupation ratio of thearea (R).

Fig. 2: Photolithography process for mold.

The photomask with the taper-stripe patterns was mounted on
the surface of SU-8 10, and the photoresist was exposed to the
UV light through the mask in the mask aligner (M-1S, Mikasa
Co. Ltd., Japan) at 15 mW/cm2 for 20 s. The photoresist was
baked in the oven at 368 K for ten minutes. The photoresist
was developed with SU-8 developer (Micro Chem Corp., MA,
USA) for five minutes. The glass surface with the micro
pattern was rinsed with IPA (2-propanol, Wako Pure Chemical
Industries, Ltd.) for one minute, and pure water for one minute.
The surface was dried by the spin-dryer (SF-250, Japan Create
Co., Ltd., Tokorozawa, Japan).

R = 100 (Σ Ak) / S(1)

In Eq. 1, S is the total scaffold area of each kind of band in eachsegment of the microscopic image. R becomes 100 % at theconfluent state in each kind of band. When the cell across theborder between the bands, the area is divided into two parts at
the line of the border. Each partial area is added to each band,
respectively.

The morphology of the surface of the mold was measured
across the bands by the stylus of the contact profilometer(Dektak XT-E, Bruker Corporation) (Fig. 4).

3. RESULTS
Fig. 3 shows the phase contrast microscopic image of thescaffold. The taper-stripe pattern of SU-8 (wider on the lefthand) and PDMS (wider on the right hand) have beensuccessfully made on the surface of the scaffold for the cell
culture.

Scaffold with Taper-stripe Micro Pattern
After the mold was enclosed with a peripheral wall ofpolyimide tape, 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) on the mold. The
volume ratio of PDMS to the curing agent is ten to one.
PDMS was baked at 373 K for thirty minutes in an oven.

Fig. 4 and Fig. 5 show the tracings of the surface of the moldmeasured by the stylus of the contact profilometer. Fig. 4bshows the tracing measured by the stylus at the line (Fig. 4a) on
the surface of the mold. The tracing shows the height of 0.015
mm, and the pitch of 0.25 mm with the narrower top width.

The baked disk of PDMS (3 mm thickness) was exfoliated fromthe mold, and punched to make a disk (diameter of 25 mm) withthe micro pattern. The disk was exposed to the oxygen gas ina reactive ion etching system (FA-1) to be characterized ashydrophilic (oxygen plasma ashing).
On the micro pattern of the disk, another disk without micro
pattern, which has two holes, was attached. SU-8 2 wasintroduced by the capillary effect into the space, which is kept
by the micro pattern, between two disks through the hole at thedisk with no micro pattern [17]. After the disks with SU-8 2
was baked at 393 K for five hours in the oven, the PDMS disk
with no micro pattern was exfoliated, and discarded.

PDMS
SU-8

The pattern of the surface of the scaffold was observed by thephase contrast microscope (IX71, Olympus, Tokyo) (Fig. 3).
Before the cell cultivation, the surface of the culture plate washydrophilized by the oxygen (30 cm3/min, 0.1 Pa) plasma
ashing for thirty seconds at 50 W by the reactive ion etchingsystem (FA-1).

Fig. 3: Phase contrast microscopic image of hybrid taper-stripe
pattern of SU-8 (0.05 mm – 0.15 mm) and PDMS (0.1 mm –0.2 mm) for scaffold of cell culture. Dimension from left toright is 2.2 mm.

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

Fig. 5b shows the tracing measured by the stylus at the line (Fig.
5a) on surface of the mold. The tracing shows the height of
0.015 mm, and the pitch of 0.25 mm with the wider top width.
The height of 0.15 mm corresponds to the thickness of SU-8
layer on the surface of the scaffold.

Fig. 8a shows C2C12 cultivated on the micro pattern for 48
hours. The contour of each cell is traced to calculate the total
area of cells on each band. Fig. 8b shows the occupation ratioof the area (R) of C2C12 cultivated on micro pattern for 48
hours. The ratio is higher at SU-8 than at PDMS.

Fig. 6 shows the tracings measured by the stylus across the
stripe on the scaffold surface with indentation load of 3×10－5 N.
The tracings show the step of 0.001 mm, and the pitch of 0.25mm.

Height: 0.015 mm

Fig. 7a shows C2C12 cultivated on the micro pattern for 24hours. The contour of each cell is traced to calculate the total
area of cells on each band: SU-8 (yellow), and PDMS (red).
Fig. 7b shows the occupation ratio of the area (R) of C2C12
cultured on micro pattern for 24 hours. The ratio is higher at
SU-8 than at PDMS.

Pitch: 0.25 mm

Fig. 5b: Tracing (height, μm; distance, mm) measured by stylus
at the line (Fig. 5a) on surface of the mold.

Height: 0.001 mm

Stylus

Pitch: 0.25 mm

Fig. 4a: Measurement of morphology of surface of mold acrossbands by stylus (left).

Fig. 6: Tracing (height [μm] vs. distance [mm]) measured bystylus across stripe on scaffold surface with indentation load of
3×10－5 N.

Height: 0.015 mm

Pitch: 0.25 mm
PDMS
SU-8

Fig. 4b: Tracing measured by stylus at the line (Fig. 4a) on
surface of mold.

0.2 mmStylusFig. 7a: C2C12 cultivated on micro pattern for 24 hours.
Dimension from left to right is 2.2 mm. Trace of contour ofeach cell to calculate total area of cells on each band.

Fig. 5a: Measurement of morphology of surface of mold acrossbands by stylus (left).

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

Fig. 9a shows C2C12 cultivated on the micro pattern for 24hours, and the contour of each cell is traced to calculate the total
area of cells on left and right sides. Fig. 9b shows the total
area of C2C12 cultivated on micro pattern for 24 hours. The
total area is wider at the left side than at the right side. The
ratios of SU-8 area at left and right sides are 48 % and 32 %,respectfully.

R [%]Fig. 10 shows the migration of C2C12 at every three hours for
24 hours after seeding. At the initial position, five cells are
selected on each band. Migration tends to be active in thewide band of SU-8.

R [%]Fig. 8b: Occupation ratio of area (R) of C2C12 cultivated on
micro pattern for 48 hours: SU-8 (left), PDMS (right).

Fig. 7b: Occupation ratio of area (R) of C2C12 cultivated on
micro pattern for 24 hours: SU-8 (left), PDMS (right).
SU-8
PDMS
PDMS

0.2 mmSU-8

Fig. 9a: C2C12 cultivated on micro pattern for 24 hours.
Dimension from left to right is 2.2 mm. Trace of contour ofeach cell to calculate total area of cells on each side (left(yellow) or right (green)).
2

[μm ]

0.2 mmFig. 8a: C2C12 cultivated on micro pattern for 48 hours.
Dimension from left to right is 2.2 mm. Trace of contour ofeach cell to calculate total area of cells on eachband.

Fig. 9b: Total area of C2C12 cultivated on micro pattern for 24hours: left side, right side (Fig. 9a).

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Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)the surface of the band has been made of the materials
alternately: polydimethylsiloxane (PDMS), and epoxy based
negative photoresist material (SU-8). The experimental resultshows that each cell tends to migrate to the area of the bandwith SU-8 in 24 hours. The experimental system is effective
to investigate durotaxis of cells in vitro.

μm

SU-8

PDMS
6. ACKNOWLEDGMENT

SU-8
0.5 mm

0.5 mmThis 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.

μm

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Fig. 10: Tracings of centroid of each C2C12 at every threehours for 24 hours: unit, μm.

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5. CONCLUSION
The flat scaffold with the taper-striped pattern has been
designed to observe durotactic migration of a biological cell.
The surface of the scaffold with the micro taper-striped (0.05mm < width < 0.2 mm) pattern has successfully been made by
the photolithography technique. The variation of hardness of100

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