Cell Behavior around Surface-Electrode with Electric Pulses
Yusuke TAKAHASHI, Atsushi MIZOI, Shigehiro HASHIMOTO, Haruka HINO, Kenta NODA
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACT
Behavior of biological cells around the surface-electrode withelectric pulse has been observed in vitro. A thin film
(thickness of 300 nm) of titanium coated on the glass was used
for the surface-electrode. Variation has been made on the tipof the pairs of surface electrodes: rectangular and triangular.
The distance between electrodes is 0.1 mm, or 0.3 mm. Twokinds of cells were seeded around the surface electrodes:
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse), or Hepa1-6 (mouse hepatoma cell lineof C57L mouse). After 2 minutes from seeding, electric pulses(period 0.00001 s, amplitude 10 V (0.0005 A), duration
0.000005 s) were applied to the film. The microscopic
observation on the experimental system shows movement ofcells between electrodes depends on the shape of tips.
Keywords: Biomedical Engineering, Dielectrophoresis,
Titanium, Surface Electrode, C2C12, Hepa1-6, and Electric
Pulse.

1. INTRODUCTION
Several methods have been applied to control the movement of
biological cell suspended in the medium in vitro: micro slit [1,2], flow [3, 4], gravitational field [5], electric field [6], magnetic
field [7], Van der Waals force, affinity of surface, and pressure.
These methods might contribute to several applications ofmanipulation of cells: arrangement of cells to make a tissue [8],sorting of cells, and measurement of cells.
Movement of a charged particle depends on the electric field.
The effect is applied to the electrophoresis device.

surface-electrode with electric pulse has been observed in vitro.

2. METHODS
Surface ElectrodeThe titanium film was used for the surface electrode. Titanium
was coated on the borosilicate glass (Tempax) disk (50 mm
diameter, 1mm thickness). The thickness of coating is 300 nm.
The width of the coating is 4 mm. The pattern of the coatingis controlled by the film type of mask.
To make variation on non-uniformity of the electric field,
variation has been made on the tip of the cathode of surface
electrodes: rectangle and triangle. The distance between
electrodes is 0.3 mm (Fig. 1A&B), or 0.1 mm (Fig. 1C).
Photomask for ElectrodeThe pattern of the coating is controlled by the film type of mask
(Fig. 2). A carbon disk (0.5 mm of thickness) was used for themask for the deposition of titanium surface electrode. Threekinds of hole was machined at the mask by the ultrashort pulse
laser (IFRIT, Cyber Laser Inc., Tokyo, Japan): rectangles of 5
mm × 20 mm (A); a rectangle and a pentagon (B); a rectangle
and a closer thin pentagon (C). The distances between surface
electrodes are 0.3 mm at A and B, and 0.1 mm at C.
Deposition of Titanium
Before the deposition of titanium, the surface of the glass washydrophilized by the oxygen (>0.1 Pa) plasma ashing for ten
minutes at 100 W by RIE (FA-1, Samco International, Kyoto,
Japan). Titanium was deposited on the surface of theborosilicate glass (Tempax) disk (50 mm diameter, 1.1 mm
thickness) in the electron beam vapor deposition apparatus(JBS-Z0501EVC JEOL Ltd., Tokyo, Japan). The thickness ofdeposition of titanium film is 300 nm.

When a particle is subjected to a non-uniform electric field, aforce is exerted even on a non-charged particle, because thepolarization generates in the particle. The phenomenon iscalled dielectrophoresis, which depends on the several
parameters: the electrical property of the particle, shape and sizeof the particle, the electrical property of the medium, andfrequency of the electric field [9].
The behavior of the cell depends on the micro morphology ofthe surface of the scaffold [10, 11]. The electric pulses affectto the behavior of the cell [12, 13]. The cell can be cultured on
the surface of the electrode [14, 15].
In the present study, behavior of biological cells around the

Rectangle,

Rectangle-triangle,

Rectangle-triangle

Fig. 1: Three kinds of surface electrode on glass.

After cleaning and hydrophilization, the negative photoresist
material (SU-8 10) was coated over the titanium surface
electrodes on the glass at 2000 rpm for one minute with the spincoater. The photoresist was baked in the oven at 368 K forseven minutes. The photomask 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 10 V for 30 s. The photoresist was baked in the ovenat 368 K for three minutes. The photoresist was developedwith Remover PG for twenty seconds. The glass surface withthe micro pattern was rinsed with acetone, ethanol, andultrapure water, and dried by the spin-dryer.

Fig. 2: Photomask (upper) for surface electrode (lower).

Photomask for Cover of PDMS
The borosilicate glass (Tempax) disk was used for the base of
the photomask. After hydrophilization by the oxygen plasma
ashing for ten minutes at 100 W by RIE, the negative
photoresist material of high viscosity (SU-8 10: Micro Chem
Corp., MA, USA) was coated on the disk with the spin coater.
The photoresist was baked in the oven. The pattern was drawn
on the mask (in the area of 1 mm × 1 mm) with a laser drawing
system (DDB-201K-KH, Neoark Corporation, Hachioji, Japan):the diameter of the circle is 0.03 mm, and the distance between
centers of the circle is 0.1 mm. The photoresist was baked in
the oven at 368 K for three minutes. The photoresist wasdeveloped with Remover PG (MicroChem Corp., MA, USA)for twenty seconds, and rinsed with the ultrapure water.
Surface Electrode Covered with Micro-pattern of SU-8 10
The surface electrode of titanium covered with SU-8 10 filmwith micro holes (0.03 mm diameter) was made by thefollowing process (Fig. 3).

PDMS Ring
The wall of culture dish was made of the donut-ring of
Polydimethylsiloxane (PDMS) (Fig. 4). PDMS (Sylgard 184
Silicone Elastomer Base, Dow Corning Corp., MI, USA) was
mixed 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 373 K for one hour in an oven (DX401,
Yamato Scientific Co., Ltd). The baked PDMS was machinedby the punch to make the donut-ring. The outer diameter, theinner diameter and the thickness of the ring are 32 mm, 10 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.
Electric Pulses
The electric pulses (period 0.00001 s, amplitude 10 V, duration
0.000005 s) were generated with an electric stimulator (1910
FUNCTION SYNTHESIZER, NF Corporation, Japan). The
stimulator was connected to the titanium film, and the pulses
were introduced to the medium of cells. An electric resistanceof 1000 ohm is serially inserted between the electrode and thestimulator. The electric signal (V) is monitored by an
oscilloscope during electric pulse application between thetitanium surface electrodes. V is the voltage between theterminals of the resistance.

Surface electrodeof titanium
Glass
Donut-ring of polydimethylsiloxane
Fig. 3: Surface electrode covered with SU-8 with micro-holes.
Fig. 4: PDMS rings are attached on surface electrodes.

Most of cells adhere to the scaffold of the plate in two minutes.
Between the electrodes with 0.3 mm distance, a few cells
moved to electrode (anode). Any movement of cells wasobserved between electrodes with 0.1 mm distance.
The electrical measurement at the electric circuit shows that the
conductance of the film is 0.002 S. Cells show vibrationsynchronous to the electric pulse, which is applied between theinsulated electrodes with SU-8 10 coating. The focus on thecell adhered on the surface is shifted, which shows exfoliationof the cell.
Fig. 5: Electric circuit during introduction of electric pulses (E)to scaffold (Z) of cells.

Fig. 6a: C2C12 between electrodes A immediately after electric
stimulation.
Fig. 5: Observation of cells between electrodes with an inverted
phase-contrast microscope.

A Crimp terminal was used to make connection between thesurface electrode and the lead wire.
Cell Culture
Two kinds of cells were used in the test: C2C12 (Mouse
myoblast cell line originated with cross-striated muscle of C3Hmouse), and Hepa1-6 (mouse hepatoma cell line of C57L
mouse).
D-MEM (Dulbecco’s Modified Eagle Medium)
containing 10% FBS (Fetal Bovine Serum) and 1% penicillin/
streptomycin was used for the medium. The cells weresuspended in the medium and poured on the surface electrode.
The cells were rest for two minutes, before stimulation byelectric pulses.

Fig. 6b: C2C12 between electrodes A at 210 s.

Behavior of cells was observed with an inverted phase-contrast
microscope (IX71, Olympus Co., Ltd., Tokyo).

3. RESULTS
Figs. 6-9 exemplify cells during the experiment.
Themovement of cells is able to be microscopically observed
through the transparent scaffold between the surface electrodes.
The cells are not able to be observed by the optical microscopeon the other hand, through the surface electrode of titanium: theblack part in Figs. 6-9.
A micro bubble has been generated by electrolysis adjacent to
the electrode. While the electric pulses applied to theelectrode, the bubble vibrates synchronous with the electricpulse.

Fig. 7a: C2C12 between electrodes B immediately after
stimulation.

Fig. 7b: C2C12 between electrodes B at 210 s.

Fig. 8a: C2C12 between electrodes C, immediately after
stimulation.

Fig. 9a: Hepa1-6 between electrodes C immediately after
electric stimulation.

Fig. 9b: Hepa1-6 between electrodes C at 270 s

Fig. 10: Hepa1-6, 10 min
Fig. 8b: C2C12 between electrodes C at 210 s.

Fig. 11a: ITO, Hepa1-6 without stimulation.

Fig. 11d: ITO, electrode, C2C12 with stimulation.

Fig. 11 shows the movement in the x-y plane of five cells traced
every minute at the time-lapse image. The origin corresponds
to the starting point of each cell. The cells move together tothe same direction as flow of the medium without electricpulses (Fig. 11a & c). The cells, on the other hand, change thedirection to move, during application of electric pulses (Fig. 11
b & d).

4. DISCUSSION
To maximize the effect of dielectrophoresis, the values (10 V,
and 100 kHz (period of 0.00001 s)) are selected for theamplitude and frequency of the rectangular wave as the
maximum value at the instrument (the electric stimulator) in thepresent study [9].
Fig. 11b: ITO, Hepa1-6 with stimulation.

When the film electrode of titanium is covered with SU-8, thecells show the vibrating movement in the medium. The cells,on the other hand, do not show the vibrating movement in themedium near the film of electrode without SU-8. The electric
voltage signal is modified from the rectangular wave, when thefilm of electrode is dipped in the medium without the cover of
SU-8.
Although the contact area between medium and electrode waslimited by the film of SU-8 with micro holes, the film iseffective to protect the titanium film on the glass fromexfoliation during electric stimulation. The behavior of cells isable to be observed through transparent film. The holes play
the role of marker, which is convenient to trace the movementof cells.
The electric resistance (R) of the titanium is calculated by Eq. 1.
R = (ρ l) / A

Fig. 11c: ITO, C2C12 without stimulation.

(1)

In Eq. 1, A is cross sectional area [m2], ρ is resistivity [Ω m],and l is length [m].
l = 0.04 [m]A = 0.004× (300 × 10-9) [m2]
ρ = 55×10-8 [Ω・m]The electric resistance is calculated as 18 Ω.
The electric pulses applied to the medium are confirmed by thesynchronized vibration of cells and bubbles in the present study.
Two minutes is enough for cells to adhere on the scaffold ofplate.
Titanium is one of the materials which has been implanted invivo. In the previous study, myoblast was cultured on the
micro coil spring of titanium [13].
In the previous study, dielectrophoresis has been tried to applyon the biological cell manipulation technology [9]. The micro
grooves were used for trapping of flowing cells in the previous
study [11]. The movement of cells also depends on the flow of
the medium. Cells do not fall into the micro holes at film
during the observation in the present study.
The movement of cells between surface electrodes depends onthe morphology of surface electrodes (the angle of the tip),which relates to non-uniformity of electric field.
Dielectrophoresis of cell depends on asymmetric figure ofelectrodes.
The higher slope of electric field with
non-uniformity might be necessary to move cells around theelectrode.
The effect of the surface of the scaffold on cell culture wasstudied in the previous studies [10]. Several micro-fabrication
processes have been designed to control adhesion of biologicalcells in vitro [14].

5. CONCLUSION
Behavior of biological cells around the surface-electrode withelectric pulse has been observed in vitro. A thin film
(thickness of 300 nm) of titanium coated on the glass was used
for the surface-electrode. Variation has been made on the tipof the pairs of surface electrodes: rectangular and triangular.
The distance between electrodes is 0.1 mm, or 0.3 mm.
C2C12 moves to the anode at the potential difference of 10 V
between asymmetric electrodes with short distance.

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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