Electric Measurement of Cultured Myoblast Oriented on
Scaffold with Micro-pattern
Haruka HINO, Shigehiro HASHIMOTO, Takuya TAMURA, Yusuke TAKAHASHI, Tetsuya SUGIMOTO
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/andToshitaka YASUDA
Bio-systems Engineering, Department of Electronic Engineering,Tokyo National College of Technology, Tokyo, Japan
ABSTRACTThe electric impedance of cultured myoblasts oriented on thescaffold with micro pattern has been measured between surface
electrodes in vitro. Several parallel lines of micro rectangularridges (0.001 mm height, 0.003 mm width, and 0.003 mm
interval) have been made between a pair of the surface titanium
electrodes at the surface of the scaffold of glass by thephotolithography technique. C2C12 (mouse myoblast cell line)
was cultured for 4 days on the micro-patterned scaffold in themedium of D-MEM (Dulbecco’s Modified Eagle Medium).
The electric impedance between electrodes was measured oncea day with the sinusoidal electric waves (frequency, 1 Hz < f < 1MHz; amplitude, ±0.1 V < V1 < ±5 V). The experimental
result shows that electric impedance has frequency
characteristic and that the impedance of the capacitancecomponent increases with proliferation of myoblasts. The
orientation of myoblasts is detected by the impedance between
the surface electrodes, when the orientation is varied by the
direction of micro ridges of the scaffold.
Keywords: Biomedical Engineering, Surface Electrode,
Micro-ridges, C2C12, Orientation and Electric Impedance.

biological tissue was measured in the previous study to
distinguish the kinds of tissue [5]. The biological membrane
has capacitance, and biological tissue has frequency
characteristic. The impedance depends on the orientation ofcells.
In the present study, the electric impedance of cultured
myoblast oriented on the scaffold with micro pattern has beenmeasured between surface electrodes in vitro.

2. METHODS
ScaffoldA scaffold of solid surface, which has both micro patterns andsurface electrodes, has been designed to measure electric
signals of oriented myoblast and myotubes (Fig. 1). Several
parallel lines of micro ridges have been made in the square areaof 1 mm × 1 mm at the surface of the scaffold. The height (H),the width (W), and the interval (I) of the rectangular ridge are
0.001 mm, 0.003 mm, and 0.003 mm, respectively (Fig. 2).
Two types of arrangements of lines are designed at the microridges: parallel (A in Fig. 1), and perpendicular (B in Fig. 1) tothe electric field.

1. INTRODUCTION
The cell culture technique has been developed and several
methodologies might clinically be applied to regenerative
medicine. C2C12 (mouse myoblast cell line) adheres to thescaffold, proliferates and differentiates to myotubes in vitro.
These behaviors of the cell depend on the micro property of thesurface. The effect of the surface property of the scaffold oncell culture has been studied in the previous studies [1].
Several micro-fabrication processes have been designed tocontrol adhesion of biological cells in vitro [2].
Photolithography technique enables machining for design of the
micro-pattern with the surface electrode [3]. The micro stripe
pattern on the scaffold plate is one of the effective methods tomake orientation of cells [4]. The electric impedance of theElectrode

Ld

B

Micro pattern
φ50

(A)

Fig. 1: Micro patterns and surface electrodes.

(B)

A

1 mm
1 mm

I

W
A
’

A

H
A’
Fig. 2: Dimension of micro pattern for scaffold.
Glass
OFPR800
Laser

Developing
Etching
RemoveFig. 3: Photolithography process for micro ridges.

The borosilicate glass (Tempax) disk was used for the base of
the scaffold in a photolithography process (Fig. 3). The
diameter and the thickness of the disk are 50 mm and 1.1 mm,
respectively. To remove micro particles on the surface of theglass, the oxygen (0.1 Pa, 30 cm3/min) plasma ashing wasapplied to the surface of the glass at 100 W for five minutes bya reactive ion etching system (FA-1, Samco Inc., Kyoto, Japan).
To improve the affinity between the glass and the photo-resist
material (OFPR-800), HMDS (hexamethyldisilazane: Tokyo
Chemical Industry Co., Ltd., Tokyo) was coated at 3000 rpm forthirty seconds with a spin coater (IH-DX2, Mikasa Co., Ltd.,Tokyo, Japan).
The positive photoresist material of
OFPR-800LB (Tokyo Ohka Kogyo Co., Ltd., Tokyo, Japan)
was coated at 3000 rpm for twenty seconds with the spin coater.
The photoresist was baked at the hotplate at 373 K for threeminutes. The pattern for the micro grooves was drawn on thedisk with a laser drawing system (DDB-201K-KH, Neoark
Corporation, Hachioji, Japan). To control the dimension of thepattern on the mold with the laser drawing system, theparameters were selected as follows: the voltage of 3.2 V, thevelocity of 0.14 mm/s, the acceleration of 0.34 mm/s2. The
pattern was baked at the hotplate at 373 K for three minutes.
The photo-resist was developed with tetra-methyl-ammoniumhydroxide (NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki,
Japan) for five minutes. The disk was rinsed two times withthe ultrapure water for one minute, 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 (0.1 Pa) at power of
100 W for five minutes using a reactive ion etching system(FA-1, Samco Inc., Kyoto): the oxygen plasma ashing. The
dimensions of the micro grooves of the pattern were measured
with a laser microscope (VK-X200, Keyence Corporation,
Osaka, Japan). The morphology along the transverse lines ofgrooves was traced (Fig. 9).
T-Shape Surface Electrodes
A couple of T-shaped surface electrode has been designed by
the micromachining technique (Fig. 1). The width of (B) the

electrode is 0.1 mm, the length of (L) each electrode is 1 mm,
and the distance between electrodes (d) is 1.2 mm. The
titanium film was used for the surface electrode on the glass.
The photomask B, which was made by the photomask A, wasused to make the pattern of the surface electrode. The
borosilicate glass (Tempax) disk (50 mm diameter, 1.1 mm
thickness) was used for the base of the photomask.
Photomask A
After plasma ashing, titanium (200 nm thickness) was depositedon the surface of the glass in the electron beam vapor deposition
apparatus (3.1×10－4 Pa, 0.5 nm/s, JBS-Z0501EVC JEOL Ltd.,Tokyo, Japan). To improve affinity between titanium andphotoresist material, HMDS was coated at 3000 rpm for thirty
seconds with a spin coater. The positive photoresist materialof OFPR-800LB was coated at 3000 rpm for twenty seconds
with the spin coater. The photoresist was baked at the hotplate
at 373 K for three minutes. The pattern was drawn on themask with a laser drawing system (DDB-201K-KH). To
control the dimension of the pattern on the mask with the laser
drawing system, the parameters were selected as follows: thevoltage of 3.2 V, the velocity of 0.14 mm/s, the acceleration of
0.34 mm/s2. The pattern was baked at the hotplate at 373 K
for three minutes. The photo-resist was developed withtetra-methyl-ammonium hydroxide (NMD-3) for five minutes.
The disk was rinsed, and dried. The surface of titanium with
OFPR-800LB was etched with the plasma gas using RIE-10NR(Samco International, Kyoto, Japan). For etching, the gas of
SF6 with Ar (50 cm3/min at 1013 hPa) was applied at 100 W at
4 Pa for nine minutes. OFPR-800LB was removed by acetone(Fig. 4).
Photomask B
After plasma ashing, titanium (200 nm thickness) was depositedon the surface of the glass in the electron beam vapor deposition
apparatus (3.1×10 － 4 Pa, 0.5 nm/s, JBS-Z0501EVC). The
negative photoresist material of high viscosity (SU-8 10: Micro
Chem Corp., MA, USA) was coated on the plate with the spincoater (at 1000 rpm for 10 s, and at 7000 rpm for 60 s). The
photoresist was baked in the oven at 368 K for five minutes.
The photoresist was baked at the hotplate at 368 K for threeminutes. The “photomask A” was mounted on the surface of
SU-8 10, and the photoresist was exposed to the UV lightthrough 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 five minutes. The photoresist wasdeveloped with SU-8 Developer (Micro Chem) 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 driedby the spin-dryer (Fig. 4).

Fig. 4: Photomask A (left), B (right): titanium on the glass.

Electrode with Micro Pattern
The surface electrode was made on the borosilicate glass(Tempax) disk with micro grooves, which was made the former
photolithography process (Fig. 5). After plasma ashing,
Titanium (200 nm thickness) was deposited on the surface ofthe glass. After HMDS coating, the positive photoresist
material of OFPR-800LB was coated at 3000 rpm for twenty
seconds with the spin coater. The photoresist was baked at the
hotplate at 373 K for three minutes. The “photomask B” wasmounted on the surface of OFPR-800LB, and the photoresist
was exposed to the UV light through the mask in the maskaligner (M-1S) at 15 mW/cm2 for 30 s. The photoresist was
baked at the hotplate at 373 K for three minutes. The
photo-resist was developed with tetra-methyl-ammoniumhydroxide (NMD-3) for five minutes. The disk was rinsed twotimes with the ultrapure water for one minute, and dried by the
spin-dryer. The titanium coated disk was etched with theplasma gas using RIE-10NR. For etching, the gas of SF6 withAr (50 cm3/min at 1013 hPa) was applied at 100 W at 4 Pa fornine minutes. OFPR-800LB was removed by acetone (Fig. 6).
PDMS Wall
The wall of culture dish is made of the donut-ring of
Polydimethylsiloxane (PDMS) (Fig. 6). After the borosilicate
glass (Tempax) disk (35 mm diameter) was enclosed with aperipheral wall of polyimide 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). The volume ratio of
PDMS to curing agent is ten to one. After degassing, PDMS
was poured on the glass disk. After degassing again, PDMS
was baked at 368 K for one hour in an oven (DX401, Yamato
Scientific Co., Ltd). The baked PDMS was exfoliated fromthe glass disk, and machined by the punch to make thedonut-ring. The outer diameter, the inner diameter and thethickness of the ring are 35 mm, 20 mm and 5 mm, respectively.
The donut-ring of PDMS was adhered on the disk with thesurface electrodes with extra past of PDMS. The lead wire
was connected to the surface electrode with crimp styleterminals.
Developing

Ti

OFPR 800
Etching
Mask

Exposure

Z
E
V1

R

V2

Fig. 7: Electric circuit.

Cell Culture
C2C12 (Mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) of the fifth passage was used for the cell
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 scaffold with the density of 5000 cells/cm2. Cells were
cultured in the incubator at 310 K with 5% CO2 for 4 days.
The medium were refreshed every two days. Cells wereobserved with an inverted phase-contrast microscope (IX71,
Olympus Co., Ltd., Tokyo) every 24 hours.
Electric Measurement
The sinusoidal electric waves (frequency, 1 Hz < f < 1 MHz;amplitude, ±0.1 V < V1 < ±5 V) were generated with an electric
stimulator (WS8102, Toyo Corporation, Japan; 1910 Function
Synthesizer, NF Corporation, Japan). The stimulator (E) wasconnected to the electrodes, and the sinusoidal electric waves
were introduced to the medium (Z) of cells (Fig. 7). An
electric resistance (R) of 10 kΩ is serially inserted between theelectrode and the stimulator. The electric voltages (V1, V2)
were monitored by an oscilloscope during measurement ofimpedance of the medium. V1 and V2 are the voltages between
the terminals of the electric stimulator, and of the resistance,
respectively.
Variation was made on the amount of medium to find the
optimum volume to measure stable electric signal between
electrodes.

3. RESULTS
RemovalFig. 5: Photolithography process for electrodes.

Fig. 6: Electrodes on glass (left), Culture dish with electrodes(right).

Fig. 8 shows the laser microscopic image of the micro patternon the scaffold. Measurement by the laser microscope showsthat the lines of ridges on the glass has the repetitive dimensionof 0.003 mm width, 0.003 mm interval, and 0.001 mm height(Fig. 9), which are close to the designed dimension. Fig. 10shows the relationship between the voltage and the volume of
the medium in the culture dish. The voltage V2 is proportional
to the current at the resistance, which depends on the impedance
between electrodes. As the volume of the media increases, theimpedance between electrodes decreases and the current
increases. In the following measurement, the enough volumeof the medium was maintained to stabilize the current between
electrodes. Fig. 11 exemplifies the tracings of the voltages (V1,and V2) during the electric measurement.

Fig. 12 exemplifies cells after 24 hours of culture on the micro
pattern A between electrodes. Each cell aligns to the lines ofridges (vertical in Fig. 12). Fig. 13 shows cells after 24 hoursof culture on the micro pattern B between electrodes. Eachcell aligns to the lines of ridges (horizontal in Fig. 13). Fig. 14
shows cells after 4 days of culture on the micro pattern between
electrodes. Cells proliferate to make confluent layer andorientation along the lines of ridges (vertical in Fig. 14a;horizontal in Fig. 14b). Fig. 15 shows the relation between the
impedance Z (between electrodes) and the frequency (f) (V1 =±1 V). The figure shows that the impedance is minimum at 10
kHz. The impedance varies at 10 Hz and 100 kHz during
cultivation for four days. Data in Fig. 15 are rearranged toshow the difference between data on the micro pattern A and B
in Fig. 16. Data are similar on day 1 (Fig. 16a). Data aredifferent each other between A and B at 10 Hz and at 100 kHz
on day 4 (Fig. 16b). At 10 Hz, the impedance on the micro
pattern B is higher than that on the micro pattern A.

Fig. 11: Tracings of V1 (upper ±5 V) and V2 (lower ±1 V): 250
Hz.

Fig. 12a: C2C12 after 24 hours of culture on micro pattern A.

Fig. 8: Laser microscopic image of micro pattern.
Height [μm]Fig. 12b: C2C12 after 24 hours of culture on micro pattern A
near electrode.

Transverse [μm]Fig. 9: Tracings of the micro pattern.
V2 [V]
Fig. 13a: C2C12 after 24 hours of culture on micro pattern B.

Volume [cm3]Fig. 10: V2 (V) (Fig. 7) vs. Volume of medium (cm3): circle (E= ±5 V), triangle (E = ±1 V).

Fig. 13b: C2C12 after 24 hours of culture on micro pattern B
near electrode.

Impedance (Z) [kΩ]

Fig. 14a: C2C12 after 4 days of culture on micro pattern A nearelectrode.

Frequency (f) [kHz]Fig. 16a: Impedance Z (kΩ) (cell culture) vs. frequency (kHz)on day 1: square, A; diagonal, B.
Impedance (Z) [kΩ]

Fig. 14b: C2C12 after 4 days of culture on micro pattern B nearelectrode.
Frequency (f) [kHz]Impedance (Z) [kΩ]

Fig. 16b: Impedance Z (kΩ) (cell culture) vs. frequency (kHz)on day 4: square, A; diagonal, B.
ElectrodeFrequency (f) [kHz]Fig. 15a: Impedance Z (kΩ) (cell culture on micro pattern A) vs.
frequency (kHz): diagonal, day 1; square, day 2; triangle, day 3;
circle, day 4.

Micro pattern

Cell
Fig. 17: Orientation of cells on micro pattern.

Impedance (Z) [kΩ]RC

Fig. 18: Serial connection between resistance (R) andcapacitance (C).

Frequency (f) [kHz]Fig. 15b: Impedance Z (kΩ) (cell culture on micro pattern B) vs.
frequency (kHz): diagonal, day 1; square, day 2; triangle, day 3;
circle, day 4.

4. DISCUSSION
To control the orientation of cultured cells in vitro, thephotolithography technique is effective to make micro
morphology on the surface of the scaffold [6, 7].

In the present study, the micro patterned scaffold has beensuccessfully manufactured between the surface titanium
electrodes on the glass by the photolithography technique.
Myoblasts have been able to be cultured on the micro patterned
scaffold. Orientation of myoblasts along the micro ridges has
been able to be observed by an inverted phase-contrast
microscope.
To detect the local electric impedance, several preparations ofelectrodes were designed in the previous studies [2-5, 8-11].
Those electrodes can be used for the electric stimulations on the
biological cells [2-4, 11-14]. The surface electrode of thepresent study can also be used for the electric stimulation on thecultured cells. The response of biological cells depends on the
direction of electric signal [12]. The electric stimulation

causes magnetic stimulation, simultaneously [15].
The variation of the resistance between electrodes for four daysof culture might indicate the variation of the organization of
myotubes in the present study. The impedance includes
capacitance component, which related to the membranes of thecell.
The orientation of cells might affect the capacitancecomponent (around 10 Hz) (Fig. 16b, Fig. 17). The impedance
also depends on the extracellular matrix [5].
In Fig. 11, V1 is delayed from V2, which indicates Z has
capacitance. Using the phase difference (θ) between V1 and V2,
the voltage between electrodes (V) is calculated by Eq. 1.
V 2 = V12 + V22 － 2 V1 V2 cos θ(1)When V1 = 5 V, V2 = 1 V, and θ is 0.94 rad, V = 4.5 V.
impedance Z is calculated by Eq. 2.

The

Z = V / (V2 / R)

(2)

When R = 10 kΩ, Z = 45 kΩ (the electric current: V2 / R = 0.1
mA). When the impedance Z consists of serial elements of thecapacitance C and the resistance R (Fig. 18), C is calculated by
Eq. 3 and Eq. 4.
Z 2 = R 2 + Zc2
Zc = 1 / (2πf C)

(3)
(4)

When the frequency f = 250 Hz, Zc = 43 kΩ, and thecapacitance C = 2 × 10－7 F

5. CONCLUSION
To measure the electric impedance of oriented cultured
myoblasts, the micro patterned scaffold has been manufactured
between the surface electrodes on the glass by thephotolithography technique. The experimental result showsthat electric impedance has the frequency characteristic between
1 Hz and 10 MHz and that the impedance of the capacitancecomponent increases with proliferation of myoblasts. The
orientation of myoblasts is detected by the impedance between
the surface electrodes, when the orientation is varied by the
direction of micro ridges 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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