Culture of Myoblast on Gold Film Sputteredon Polydimethylsiloxane Disk
Kenta NODA, Shigehiro HASHIMOTO
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/andToshitaka YASUDA
Bio-systems Engineering, Department of Electronic Engineering,Tokyo National College of Technology, Tokyo, Japan
and
Hiromichi FUJIE
Human Mechatronic Systems, Faculty of System Design,
Tokyo Metropolitan University, Tokyo, Japan

ABSTRACTA transparent thin gold film has been designed to culture
myoblasts on the surface. The thin gold film was formed on
the surface of the polydimethylsiloxane disk by vacuum
deposition. The thickness of the film is 30 nm estimated withthe current during sputtering. C2C12 (mouse myoblast cellline originated with cross-striated muscle of C3H mouse) was
cultured for one week on the film. The proliferation of cells
was observed with an inverted phase contrast microscope everyday. The experimental results show that the proliferation of
the cell on the gold film is able to be observed through thetransparent gold thin film with the microscope. The watercontact angle shows hydrophilic surface of the gold film, which
accelerate adhesion and proliferation of cells.
Keywords: Biomedical Engineering, C2C12, Film Electrode,Gold, Sputtering and Polydimethylsiloxane.

1. INTRODUCTION
Cell culture technique has been developed and several
methodologies might clinically be applied to regenerative
medicine [1]. C2C12 (mouse myoblast cell line originatedwith cross-striated muscle of C3H mouse) adheres to thescaffold, proliferates and differentiates to myotubes in vitro [2].
These behaviors of the cell depend on the micro property of thesurface.

The photolithography technique enables design of microstructure of the solid surface for the scaffold of cell culture.
The contact angle is one of the parameters for the physicalproperty of the surface. When the water contact angle in the
air is smaller than 1.6 rad on a surface, the surface ishydrophilic. The biological cell adheres to the hydrophilic
surface. The oxygen plasma ashing is one of the procedures tomake the surface hydrophilic. Polydimethylsiloxane (PDMS)
is used frequently in the photolithography process. The
surface of PDMS is hydrophobic. The surface of the metal, on
the other hand, is hydrophilic.
The effect of the surface of the scaffold on cell culture has beenstudied in the previous studies [3-6].
Several microfabrication processes have been designed to control adhesion of
biological cells in vitro [4-6].
In the present study, gold has been sputtered to make atransparent thin film of gold on polydimethylsiloxane disk, andthe behavior of myoblast on the surface has been observed
microscopically.
2. METHODS
Film ElectrodeA transparent thin gold film has been designed to culture
myoblasts on the film. The thin gold film is formed on thesurface of the polydimethylsiloxane (PDMS) disk
A silicon wafer (Type N, Matsuzaki Seisakusyo, Co., Ltd.,

Tokyo, Japan) is used for a surface mold for the disk. The
diameter and the thickness of the wafer are 50 mm and 0.30 mm,
respectively. The surface of the wafer was coated with 0.001
mm thickness of Parylene (Specialty Coating Systems, Inc., IN,
USA). After the wafer is enclosed with a peripheral wall ofpolyimide, PDMS (Dow Corning Corporation, MI, USA) is
poured with the curing agent on the wafer. After being
degased, PDMS is baked at 383 K for one hour in an oven.
The diameter of PDMS disks is 51 mm. The thicknesses of
the two kinds of PDMS disks are 2 mm and 5 mm.
To trace the area of the film, a mask of aluminum disk (52 mm
diameter, 0.2 mm thickness) has been manufactured (Fig. 1).
The mask has a pair of void of 15 mm square at the counterends for gold deposition. Both surfaces of the disk of PDMS
and of the mask of aluminum were cleaned in the etchingsystem (FA-1, Samco Inc., Kyoto), before the sputtering
process.

Fig. 1: Mask of aluminum for deposition.

The thin gold film was formed on the surface of the PDMS diskof 2 mm thickness by vacuum deposition in the sputtering
equipment (SC-70AT, Sanyu-Electron Co., Ltd., Tokyo, Japan)(Fig. 2). The thickness of the deposited film on the PDMS
disk is controlled with the electric current of the equipment: 30
nm with 300 mA in the present study.
Another PDMS disk of 5 mm thickness, which has a hole of 32
mm diameter, is attached on the PDMS disk with electrode to
make a rim along the circle (Figs. 3&4). Two PDMS disks are
fixed each other with PDMS as adhesive by the following
process. The liquid of PDMS was painted on the surface ofthe two disks. Two disks were attached together, degassed,and baked at 383 K.

Fig. 2: Deposited gold film on the surface of PDMS.

After sterilized in the autoclave, the assembled disks are
exposed to the oxygen gas in a reactive ion etching system(FA-1, Samco Inc., Kyoto) to be characterized as hydrophilic(oxygen plasma ashing). The disks are placed in polystyrene
dish of 70 mm diameter, and used as the culture dish (Fig. 5).
Contact Angle
For comparison, gold was deposited on the half of the surface ofPDMS disk (Fig. 6). Variation was made on the process ofelectric current at spattering: 300 mA, 200 mA followed by 100
mA, and 100 mA followed by 200 mA. Water contact angles
were measured by the contact angle analyzer (Phoenix-300,
Meiwafosis Co., Ltd., Tokyo, Japan).
The surface of the disks was exposed to the oxygen gas in areactive ion etching system (FA-1, Samco Inc., Kyoto) to be
characterized as hydrophilic (oxygen plasma ashing). The
contact angles after five minutes from exposure were comparedwith that before exposure.
Cell Culture
C2C12 (Mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) of thirteenth passage was used for thecell culture. D-MEM (Dulbecco’s Modified Eagle Medium)
containing 10% FBS (Fetal Bovine Serum) and 1% penicillin/
streptomycin was used for the medium. The cells were seededon the dish of PDMS with the density of 1000 cells/cm3. Cells
were cultured in the incubator at 310 K with 5% CO2 for one
week. The medium were refreshed every two days. Cells
were observed with the inverted phase-contrast microscope(IX71, Olympus Co., Ltd., Tokyo) every day.

Fig. 3: Design of culture dish of PDMS. Cross-section at line of
A-A’ (bottom).

Fig. 4: Culture dish of PDMS.

Fig. 7: Contact angle (1.7 rad) on the surface of PDMS before
oxygen plasma ashing.
Fig. 5: Culture dish of PDMS placed in polystyrene dish of 70
mm diameter.

Fig. 8: Contact angle (1.1 rad) on the surface of gold sputtered(300mA) area on PDMS before oxygen plasma ashing.

Fig. 6: Gold was deposited on the half of the surface of PDMS
disk (upper half in the figure) by three kinds of sputtering
process. 300 mA (left), 200 mA followed by 100 mA (middle),
and 100 mA followed by 200 mA (right).

3. RESULTS
The color of 100 mA followed by 200 mA is darker than that of
300 mA (Fig. 6), which shows that the thickness of thedeposited gold film might increase by sputtering two times.
Figs. 7-11 show the water contact angle on the surface ofPDMS and gold in the air. Fig. 7 shows the water contact
angle bigger than 1.6 rad, which reveals that the surface ofPDMS is hydrophobic. Fig. 8 shows the angle smaller than
1.6 rad, which reveals that the surface of the gold film ishydrophilic.

Fig. 9: Contact angle (0.45 rad) on the surface of PDMS after
oxygen plasma ashing.

The water contact angle decreases after sputtering, so that theoxygen plasma ashing was effective for the surface to behydrophilic (Figs. 9&10).
Fig. 11 shows smaller angle than Fig. 8, which shows theprocess of the spattering two times is better to make thehydrophilic surface.

Fig. 10: Contact angle (0.38 rad) on the surface of gold
sputtered area on PDMS after oxygen plasma ashing.

Fig. 11: Contact angle (0.80 rad) on the surface of gold
sputtered (100mA & 200 mA) area on PDMS before oxygen
plasma ashing.

Fig. 14: C2C12 on PDMS after 6 days.
to right is 1 mm.

Fig. 12: C2C12 on PDMS after 24 hours.
to right is 1 mm.

Dimension from left

Dimension from left

Fig. 15: C2C12 on gold after 6 days. Dimension from left toright is 1 mm.

Fig. 13: C2C12 on gold film after 24 hours. Dimension fromleft to right is 1 mm.

The cells are easily observed with the microscope through thetransparent thin film of gold on the PDMS surface (Figs. 13, 15,16). The Cells adhere, extend pseudo, and proliferate both on
PDMS and on gold film (Figs. 12-15). Proliferation of C2C12
tends to be faster on the gold film than on the PDMS even after
oxygen plasma ashing.

Fig. 16: More cells adhere in the gold film area (right half) on
the surface of PDMS. Dimension from left to right is 1 mm.

4. DISCUSSION
The behavior of a biological cell depends on several factors:
electrical [7], magnetic [8], and mechanical factors [9-12].
The gold film has a potential to be used as an electrode tocontrol the electrical factors.
The electrical resistance (R) between the ends of the film is
calculated by Eq. 1.
R = k L / (d b)(1)

In Eq. 1, k is resistivity [S-1 m], L is length [m], d is thickness[m], and b is width [m].
The resistivity of gold is 2.21 × 10-8 m/S at 293.15 K. The
electrical resistance (R) between the ends of the film isestimated 2 S-1, when k = 2.21 × 10-8 m/S, L = 15 mm, d =0.00001 mm, and b = 15 mm.
To avoid cells get into the space between the PDMS disk andthe culture dish, the rim was formed around the PDMS disk,
and the suspension of the cells was poured on the PDMS disk.
The contact angle on the gold film deposited on PDMS depends
on the process of deposition.
Several factors might govern the adhesiveness of the cell: themicromorphology of the deposited gold film, and the chemical
characteristics of the surface. The contact angle is one of theparameters for the physical property of the surface.
Proliferation of cells also might depend on these factors.
The photolithography technique has been applied to fabricate
the micro channel [13]. The microfluidic system has been
applied to sort biological cells [14, 15], and to trap biologicalcells [16, 17].
The system also used to study local
environment around the cultured cell [3, 6]. The micro patternof the surface has been applied to study the surface effect ofadhesion of cells [4, 5, 18-20].
The micro-fabrication technique has also been applied to design
microfluidic systems in vitro [21-24]. The technique will also
be applied to handle cells in diagnostics in vitro. The
difference of the affinity between the scaffold and the cell might
control adhesion of cells in vitro. The methodology wasdeveloped to estimate adhesiveness of myoblast [25]. The
microstructure was designed to capture biological cells [26].

5. CONCLUSION
A transparent thin gold film on the surface of thepolydimethylsiloxane disk has been designed to culture
myoblasts on the surface. The experimental results show thatthe proliferation of the cell on the gold film is able to beobserved through the transparent gold thin film with themicroscope. Proliferation of myoblast tends to be faster on thegold film than on the PDMS even after oxygen plasma ashing.

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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