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

Design of Surface Electrode for Measurement of Electric
Impedance of Arrangement of Cells Oriented on Micro Striped
Pattern
Shigehiro HASHIMOTO, Haruka HINO, Toshihiro YAMAMOTO,
Takuya TAMURA, Kazuyuki ABE
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/of the scaffold for the cell culture in vitro. The micro stripe
pattern on the scaffold plate is one of the effective methods tomake orientation of cells [14]. In the present study, the surface
electrode has been designed for measurement of the electric
impedance of the group of biological cells oriented on the micro
striped pattern in vitro.

ABSTRACTThe surface electrode has been designed for measurement of theelectric impedance of biological cells oriented on the micro
striped pattern in vitro. Several parallel lines of micro
rectangular grooves (1 μm depth, 3 μm width, and 3 μm
interval) have been made between a pair of the surface titanium
electrodes at the surface of the scaffold of glass by thephotolithography technique. Two types of arrangements oflines are designed at the micro ridges: parallel, andperpendicular to the electric line of force. Variation has also
been made on the shape of surface electrodes (thickness; 0.2μm): I-shape and T-shape.
Two kinds of cells werealternatively cultured on the micro-patterned scaffold: C2C12(mouse myoblast cell line), and 3T3-L1 (mouse fat precursor
cells).
The electric impedance between electrodes wasmeasured for 24 hours with the sinusoidal electric waves(frequency, 1 kHz < f < 1 MHz; amplitude, V = ±0.05 V,). The
experiment shows that the impedance is higher at thearrangement of myoblasts oriented in perpendicular position
than that in parallel position to the electric line of force, and thatthe impedance increases with the number of adhered cells.

2. METHODS
ScaffoldA scaffold of solid surface, which has both micro patterns andsurface electrodes, has been designed to measure electric
signals of oriented cells. Several parallel lines of micro
grooves have been made in the square area of 1 mm × 1 mm at
the surface of the scaffold. The depth (H), the width (W), andthe interval (I) of the rectangular grooves are 1 μm, 3 μm, and 3
μm, respectively. Two types of arrangements of lines aredesigned at the micro ridges: parallel, and perpendicular to theelectric line of force (Fig. 1).
Three types of electrodes has been designed: T-shape, I-shape,
and asymmetric shape (Fig. 2). In T-shape, the width (B1) of
the electrode is 0.1 mm, the length (L1) of each electrode is 1
mm, and the distance between electrodes (d1) is 1.2 mm (Fig.
2a). In I-shape, the width (B2) of the electrode is 0.1 mm, thelength of (L2) each electrode is 0.1 mm, and the distance
between electrodes (d2) is 1.4 mm (Fig. 2a).

Keywords: Biomedical Engineering, Electric Impedance,
Micro-machining, Surface Electrode, C2C12, 3T3-L1 andOrientation.

1. INTRODUCTION
In previous studies the orientation of cells in the tissue was tried
to be controlled by several methods. The orientation of cells
in the tissue also was tried to be measured by several methods.
The electric impedance is one of the method to measure theorientation of cells in the tissue [1-13].
The electric
impedance of the biological tissue was measured in the previous
study to distinguish the kinds of tissue [11]. The biologicalmembrane has capacitance, and biological tissue has frequency
characteristic.
The electric impedance depends on theorientation of cells.

Electrode
Pattern

The surface morphology might govern the orientation of cells.
Several kinds of the surface micro morphology were applied tothe scaffold in the previous studies [14]. The photolithography
technique enables making the micro morphology on the surface

Fig. 1: Micro pattern between electrodes: parallel (left),
perpendicular (right).

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the glass and the photo-resist material.
The positive
photoresist material of OFPR-800 (Tokyo Ohka Kogyo Co.,
Ltd., Tokyo, Japan) was coated at 3000 rpm for thirty seconds
with the spin coater. The photoresist was baked at the hotplate
at 373 K for ninety seconds.

0.1 mm

0.1 mm
0.1 mmInsulation
1 mm
1.2 mmThe photomask of the shape of the electrodes (Fig. 2) was
mounted on the surface of OFPR-800, and the photoresist was
exposed to the UV light through the mask in the mask aligner(M-1S, Mikasa Co. Ltd., Japan). The photoresist was baked in
the oven (DX401, Yamato Scientific Co., Ltd) at 373 K for
three minutes.
The photoresist was developed withtetra-methyl-ammonium hydroxide (NMD-3, Tokyo Ohka
Kogyo Co., Ltd., Kawasaki, Japan) for one minute. The disk
was rinsed with the ultrapure water, and dried by the spin-dryer(SF-250, Japan Create Co., Ltd., Tokorozawa, Japan). The
titanium was etched with the plasma gas using a reactive ion
etching system (RIE-10NR, Samuco Inc., Kyoto, Japan). For
etching, the gas of SF6 (30 cm3/min at 1013 hPa) with Ar (30cm3/min at 1013 hPa) was applied at 100 W for nine minutes.

1.4 mm1 mm
0.1 mm
0.1 mm

0.1 mmFig. 2a: Surface electrode of T-shape (left) and I-shape (right).
1.2 mm2 mm

1 mm

To insulate the residual area of the surface electrode, thetitanium was covered with the coating of the negative
photoresist material (SU-8: Micro Chem Corp., MA, USA).
SU-8 was coated with the spin coater (at 3000 rpm for 20 s).
The photoresist was baked in the oven at 373 K for threeminutes. The coating area was exposed to the UV lightthrough the mask (Fig. 3) in the mask aligner. The photoresist
was baked in the oven at 393 K for five minutes. The
photoresist was developed with SU-8 Developer (Micro Chem
Corp., MA, USA).
The surface was rinsed with IPA(2-propanol, Wako Pure Chemical Industries, Ltd.) and theultrapure water.

2 mm
0.1 mmFig. 2b: Dimension of asymmetric surface electrodes.

PDMS Wall
The wall of culture dish is made of the donut-ring of
Polydimethylsiloxane (PDMS) (Fig. 4a).
After theborosilicate glass (Tempax) disk (35 mm diameter) was
enclosed with a peripheral wall of polyimide tape, PDMS
(Sylgard 184 Silicone Elastomer Base, Dow Corning Corp., MI,
USA) was poured into the dish 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 toone. After degassing, PDMS was baked at 373 K for one hourin an oven. The baked PDMS was machined by the punch to
make the donut-ring.

10 mm

Fig. 3: Photomask for SU-8: dimension 50 mm (left to right).

In the asymmetric shape, the distance between electrodes (d3) is
1.2 mm. At the left electrode, the width (B3) of the electrode is
2 mm, and the length of (L3) the electrode is 2 mm. At theright electrode, the width (B4) of the electrode is 0.1 mm, andthe length of (L4) the electrode is 1 mm (Fig. 2b).
The borosilicate glass (Tempax) disk was used for the base of
the scaffold through micromachining process. The diameterand 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 by a reactive ion etchingsystem (FA-1, Samco Inc., Kyoto, Japan).

50 mm

50 mm

Electrode with Micro Pattern
After machining the micro striped grooves [1], Titanium of 0.2
μm thickness was deposited on the surface of the glass in theelectron beam vapor deposition apparatus (JBS-Z0501EVC
JEOL Ltd., Tokyo, Japan).
Fig. 4a: A pair of devices fixed on plate to be placed on stage of
microscope (left), asymmetry (middle), T-shape (right).

After plasma ashing, HMDS (hexamethyldisilazane: Tokyo
Chemical Industry Co., Ltd., Tokyo) was coated at 3000 rpm forthirty seconds with a spin coater to improve the affinity between

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electric current (I) (Eq. 1). The voltage is calculated by thedifference between V2 and V1. The electric current (I) is
calculated V1 divided by R.

E

R

Z=V/I

(1)

D
3. RESULTS
V1
Fig 5 exemplifies cells on the micro pattern (parallel) between
electrodes.
Most of cells make orientation along thelongitudinal direction of the micro striped pattern. Figs. 6-11
show the relationship between the impedance between
electrodes and the frequency of the sinusoidal electric voltage
load. The impedance is the lowest at 0.1 MHz.

V2
Fig. 4b: Electric circuit.

Fig. 6a shows the impedance of medium without cells between
three kinds of electrodes related to frequency: long dashed line
(I-shape), dotted line (T-shape), solid line (asymmetry). The
impedance is the highest between the electrodes of I-shape, andthe lowest between the electrodes of asymmetric shape. Fig.
6b shows the impedance between three kinds of electrodes
immediately after seeding of cells (C2C12). Fig. 6c shows the
impedance between three kinds of electrodes at 24 hours after
seeding of cells (C2C12). The impedance tends to increase
and the difference between three kinds of electrodes tends todecrease. Fig. 7 shows the impedance of medium with C2C12
on two kinds of micro pattern between T-shape electrodes.
The impedance with the perpendicular micro stripe pattern ishigher than that with the parallel one.

0.5 mmFig. 5: Cells on micro pattern (parallel) between electrodes.

The donut-ring of PDMS was adhered on the disk with thesurface electrodes with extra paste of PDMS. The lead wire
was connected to the surface electrode with the crimp styleterminals and with the conductive paste (Dotite, Fujikura Kasei
Co., Ltd., Tokyo) (Fig. 4a). A pair of devices enables
simultaneous tests of two kinds of the setting of the devices.

Fig. 8a shows the impedance between T-shape electrodes
immediately after seeding of 3T3-L1 at 5×103 cell/cm2. Fig.
8b shows the impedance between T-shape electrodes at 24 hours
after seeding of 3T3-L1. The impedance with the parallel
micro stripe pattern is higher than that with the perpendicular
one at 24 hours. Fig. 9a shows the impedance between
T-shape electrodes immediately after seeding of 3T3-L1 at
5×104 cell/cm2. Fig. 9b shows the impedance between T-shapeelectrodes at 24 hours after seeding of 3T3-L1.
The
impedance is higher at 5×104 cell/cm2 than at 5×103 cell/cm2.
The impedance with the parallel micro stripe pattern is higher
than that with the perpendicular one.

Cell Culture
Two kinds of cells were used in the experiment: C2C12 (Mouse
myoblast cell line originated with cross-striated muscle of C3Hmouse; passage between fifth and tenth), and 3T3-L1 (mouse
fat precursor cells, a cell line derived from cells of mouse 3T3;
passage between third and sixth). D-MEM (Dulbecco’s
Modified Eagle Medium) containing 10% FBS (Fetal Bovine
Serum) and 1% penicillin/ streptomycin was used for the
medium. After the oxygen plasma ashing by a reactive ion
etching system (FA-1), cells were seeded on the scaffold withthe density of 5×103 cells/cm2 (or 5×104 cells/cm2). Cells were
cultured in the incubator at 310 K with 5% CO2. Cells wereobserved with an inverted phase-contrast microscope (IX71,
Olympus Co., Ltd., Tokyo) for 24 hours.

Fig. 10a shows the impedance between T-shape electrodes
immediately after seeding of C2C12 at 5×103 cell/cm2. Fig.
10b shows the impedance between T-shape electrodes at 24hours after seeding of C2C12. The impedance with theperpendicular micro stripe pattern tends to be higher than thatwith the parallel one at 24 hours. Fig. 11a shows the
impedance between T-shape electrodes immediately after
seeding of C2C12 at 5×104 cell/cm2. Fig. 11b shows the
impedance between T-shape electrodes at 24 hours after seedingof C2C12. The impedance is higher at 5×104 cell/cm2 than at
5×103 cell/cm2. The impedance with the perpendicular micro
stripe pattern is higher than that with the parallel one.

Electric Measurement
The sinusoidal electric waves (frequency, 1 kHz < f < 1 MHz;amplitude, V = ±0.05 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 (D) of cells. An electric
resistance (R) of 10 kΩ is serially inserted between theelectrode and the stimulator (Fig. 4b). The electric voltages(V1, V2) were monitored by an oscilloscope during measurementof impedance of the medium. V1 and V2 are the voltages
between the terminals of the resistance, and of the electric
stimulator, respectively. The impedance of the specimen (Z) is
calculated by the voltage (V) between electrodes divided by

4. DISCUSSION
The impedance increases with the distance between electrodes.
The impedance also increases with decrease of the crosssectional area of electric flow path between electrodes. The
results of the present experiments of three kinds of electrodes
are consistent to the law of physics.

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Impedance [Ω]Impedance [Ω]1000000

インピーダンス [Ω]インピーダンス [Ω]100000

10000

100000

10000

1000
1k

10k

100k

1000

1M

1k

周波数
[Hz][Hz]
Frequency

100k

1M

Frequency[Hz]
周波数 [Hz]

Fig. 6a: Impedance of medium between three kinds ofelectrodes without cells related to frequency: long dashed line
(I-shape), dotted line (T-shape), solid line (asymmetry).

Fig. 7: Impedance of medium with C2C12 on two kinds ofmicro pattern at 24 h between T-shape electrodes related tofrequency: circle (perpendicular), triangle (parallel).
Impedance [Ω]Impedance [Ω]1000001000000

I-shape
I字電極

インピーダンス [Ω]インピーダンス [Ω]

10k

T字電極
T-shape
100000異面積電極
Asymmetry

10000

10000

1000
1000

1k
1k

10k

100k

1M

100k

1M

周波数 [Hz][Hz]
Frequency

周波数 [Hz]
Frequency[Hz]

Fig. 8a: Impedance of 3T3-L1 (5×103 cell/cm2) at 0 h on two
kinds of micro pattern between T-shape electrodes related tofrequency: long dashed line (parallel), dotted line
(perpendicular) in Figs. 8-11.

Fig. 6b: Impedance of medium with C2C12 at 0 h between
three kinds of electrodes related to frequency: long dashed line
(I-shape), dotted line (T-shape), solid line (asymmetry).
Impedance [Ω]Impedance [Ω]100000インピーダンス [Ω]1000000

インピーダンス [Ω]10k100000

10000

1000
1k

10k

100k

10000

1000

1M

1k

周波数 [Hz][Hz]
Frequency

10k100k
周波数 [Hz]
Frequency[Hz]

1M

Fig. 8b: Impedance of 3T3-L1 (5×103 cell/cm2) at 24 h on two
kinds of micro pattern between T-shape electrodes related tofrequency.

Fig. 6c: Impedance of medium with C2C12 at 24 h between
three kinds of electrodes related to frequency: long dashed line
(I-shape), dotted line (T-shape), solid line (asymmetry).

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Impedance [Ω]1000000(a)

インピーダンス [Ω]インピーダンス [Ω]100000

Impedance [Ω]100000

10000

10000

1000

1000
1k

10k

100k

1k

1M

(b)

Impedance [Ω]

1M

(a)

1000000

インピーダンス [Ω]インピーダンス [Ω]100kFig. 10b: Impedance of C2C12 (5×103 cell/cm2) at 24 h
between T-shape electrodes related to frequency:

Impedance [Ω]10000010k

周波数 [Hz]
Frequency

周波数 [Hz]
Frequency
[Hz]

10000

100000

1000
1k

10k

100k

1M

周波数 [Hz]
Frequency
[Hz]

10000

1000
1k

Fig. 9: Impedance of 3T3-L1 (5×104 cell/cm2) at 0 h (upper) 24
h (lower) on two kinds of micro pattern between T-shapeelectrodes related to frequency.
Impedance [Ω]

10k

100k

1M

周波数[Hz][Hz]
Frequency

Impedance [Ω]1000000(a)

インピーダンス [Ω]1000000
インピーダンス [Ω](b)100000(b)

100000

10000

10000

1000

1000
1k

10k

100k

1k

1M

10k

100k

1M

周波数[Hz][Hz]
Frequency

Frequency
周波数 [Hz]
Fig. 10a: Impedance of C2C12 (5×103 cell/cm2) at 0 h on two
kinds of micro pattern between T-shape electrodes related tofrequency.

Fig. 11: Impedance of C2C12 (5×104 cell/cm2) at 0 h (upper) 24
h (lower) on two kinds of micro pattern between T-shapeelectrodes related to frequency.

In the previous study, impedance was measured at the frequency
between 1 Hz and 1 MHz. The range of the frequency
between 10 kHz and 100 kHz is selected for comparison ofimpedance of the colony of cells in the present study, because
impedance depended on the orientation of cells in the range ofthe frequency in the previous study [1].

The number of cells of C2C12 was not the same as that of
3T3-L1 in the present study. As the number of cells adheredon the scaffold between the electrodes increases, the impedance
between the electrodes might increase. The mean size of
3T3-L1 is smaller than that of C2C12, which might affect thevalue of impedance. C2C12 is differentiated into myotubes tomake fusion between cells. The fusion might be decrease theimpedance between electrodes, especially at the parallel

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orientation of cells. The sinusoidal electric impedance signal
between 10 kHz and 0.1 MHz is sensitive to the condition of thecolony of cells, which might be related to the structure of eachcell. To control the orientation of cultured cells in vitro, thephotolithography technique is effective to make optimal size of
micro morphology on the surface of the scaffold [1, 14]. 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 grooves
has been able to be observed by an inverted phase-contrast
microscope. To detect the local electric impedance, several
preparations of electrodes were designed in the previous studies[2, 11, 15]. Those electrodes can be used for the electric
stimulations on the biological cells [2, 10]. The surface
electrode of the present study can also be used for the electric
stimulation on the cultured cells. The response of biologicalcells depends on the direction of electric signal. The electric
stimulation causes magnetic stimulation, simultaneously. The
impedance includes capacitance component, which related tothe membranes of the cell [1].

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Types Using Electric Cell–Substrate Impedance Sensing”,
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[6] L. Vistejnova, J. Dvorakova, M. Hasova, T. Muthny, V.
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5. CONCLUSION
The surface electrode has been designed for measurement of theelectric impedance of the group of biological cells oriented on
the micro striped pattern in vitro. Several parallel lines of
micro rectangular grooves (0.001 mm depth, 0.003 mm width,and 0.003 mm interval) have been made between a pair of thesurface titanium electrodes (I-shape and T-shape) at the surfaceof the scaffold of glass by the photolithography technique.
Two kinds of cells were alternatively cultured on the
micro-patterned scaffold: C2C12 (mouse myoblast cell line),and 3T3-L1 (mouse fat precursor cells).
The electric
impedance between electrodes was measured for 24 hours withthe sinusoidal electric waves (frequency, 1 kHz < f < 1 MHz;amplitude, V = ±0.05 V,). The experiment shows that the
impedance is higher at the arrangement of myoblasts oriented inperpendicular position than that in parallel position to theelectric line of force, and that the impedance increases as thenumber of adhered cells. The designed surface electrodes candetect the arrangement of the orientation 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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