Effect of Electric Stimulation on Differentiation andHypertrophy of Fat Precursor Cells
Haruka HINO, Hiroaki NAKAJIMA, Shigehiro HASHIMOTO, Nobuyuki WAKURI,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/andToshitaka YASUDA
Bio-systems Engineering, Department of Electronic Engineering,Tokyo National College of Technology, Tokyo, Japan

ABSTRACTAn effect of electric stimulation with pulses on differentiationand hypertrophy of fat precursor cells has been investigated invitro. 3T3-L1 (mouse fat precursor cells) was seeded at theconcentration of 1000 cells per cm2, and cultured in Dulbecco’s
Modified Eagle Medium with 10 percent of fetal bovine serum.
Differentiation of cells was induced with insulin. An electric
stimulator was used to generate electric pulses with period of 1s, pulse width of 0.0025 s, and current amplitude of 0.03 A.
The electric pulses were applied to the medium through theelectrode for thirty minutes per day. The cells were observedwith an inverted phase contrast microscope for 24 days. At theend of the culture, the tissue was observed with the microscope,
after the tissue was stained with oil red O. The experimentshows that both differentiation and growth of 3T3-L1 aredelayed with electric pulses.
Keywords: Biomedical Engineering, 3T3-L1, Electric Pulses,
Cell Growth and Differentiation.

1. INTRODUCTION
Excess adipose tissue cause several disease in human body.
Many methods have been investigated to control adipose tissue.
Several methods include stimulation of electric pulses.
Electric pulses are applied to electrodes, which are attached on
the skin of human body. Electro-acupuncture might improvemetabolic disturbances [1].

appropriate conditions, 3T3-L1 differentiates into an
adipocyte-like phenotype. When cells enter a resting state,
they accumulate triglyceride and convert to adipose cells [4, 5].
Insulin has been used to induce 3T3-L1 to an adipocyte-likephenotype [6].
In the present study, the effect of electric stimulation withpulses on differentiation and hypertrophy of fat precursor cells
has been investigated in vitro.

2. METHODSElectrodeA dish of 35 mm diameter was used for the cell culture. A cap
with a couple of electrodes is made of Polydimethylsiloxane(PDMS) (Fig. 1). The electrodes are made of platinum wire of
0.2 mm diameter. The electrodes are mounted in the cap ofPDMS (Fig. 2). The electrodes are dipped into the medium,
when the cap is set on the dish.
A silicon wafer (Type N, Matsuzaki Seisakusyo, Co., Ltd.,Tokyo, Japan) was used for a mold for the upper wall of the cap.
The diameter of the wafer is 50 mm.

(d)(c)

Cell culture technique has been developed and several
methodologies might clinically be applied to regenerative
medicine [2, 3].
3T3-L1 was used for cell culture in the experiment. 3T3-L1 isa cell line derived from cells of mouse 3T3. The cells have afibroblast-like morphology. They fill the intercellular space inthe tissue, when some parenchymal cells disappear. Under

Fig. 1: Cap with a couple of electrodes (blue lines).
Dimension in mm. (c) and (d) show the position forobservation of cells (Figs. 11-15).

(V1), and the other signal is the voltage (V2) between terminalsof the resistance (r). The electric current (I) is calculated by
the following equation.
I = V2 / r(1)

Cell Culture
3T3-L1 (a cell line derived from cells of mouse 3T3) of the fifth
passage was used for the cell culture. D-MEM (Dulbecco’s
Modified Eagle Medium) containing 10% decomplemented
FBS (Fetal Bovine Serum) and 1% penicillin/ streptomycin(P/S) was used for the medium. The medium was refreshed
every two days during cell culture. The cells were seeded on
the dish with the density of 1000 cells/cm3. A hemocytometer(Burker-Turk) was used to count number of cells. Cells werecultured in the incubator at 310 K with 5% CO2 for 24 days.
The protocols of electric stimulation are classified into threegroups: A, B, and C.
Fig. 2: Cap of PDMS with a couple of electrodes of Platinum
wire. Top plate of cap (a), peripheral wall of cap (b), electrodeof platinum wire (c), electrodes are fixed on cap (d).
0.0025 s

10 V

1s
Fig. 3: Electric pulse.

After the wafer was enclosed with a peripheral wall ofpolyimide, 3 cm3 of PDMS (Dow Corning Corporation, MI,USA) was poured with the curing agent on the wafer (Fig. 2(a)).
PDMS was baked at 373 K for one hour in an oven. The baseof dish (35 mm diameter) with the peripheral wall of polyimide
was placed at the center of the disk of PDMS (Fig. 2(b)).
PDMS of 7 cm3 was poured into the donut canal between thewalls of polyimide to make the peripheral wall of the cap, andbaked at 333 K for 30 minutes in the oven. An electrode isformed with a platinum wire (Fig. 2(c)). A pair of electrodes
was placed on the inside of the cap with the counter position.
PDMS of 1 cm3 was poured to cover the root of electrode wire
for fixation, and baked at 393 K for one hour in an oven (Fig.
2(d)).
Electric stimulation
The electric pulses (period 1 s, amplitude 10 V, duration 0.0025s) were generated with an electric stimulator (SEN5201, Nihon
Kohden Corporation, Japan) (Fig. 3). The stimulator wasconnected to the electrodes, and the pulses (E) were applied tocells through the medium (Z) for thirty minutes per day. An
electric resistance of 30 ohm (r) is serially inserted between theelectrode and the stimulator (Fig. 4). The electric signals (V1,V2) were monitored by an oscilloscope during electric
stimulation to cultured cells (Fig. 8).
The interval between electric stimulations is 23 hours and a half.
One of the signals is the voltage between terminals of stimulator

In group A, the electric stimulation was applied after 24 hours
from cell seeding. In group B, the electric stimulation started
on the second day after confluent state of the cells. Both ingroup A and in group B, the electric stimulation wassubsequently applied every day (Figs. 5&6). In group C, cells
are cultured without electric stimulation. In every group,insulin was applied to the medium for differentiation on thesecond day after confluent state of the cells. The insulin of0.01 mg is dissolved in the medium (D-MEM+FBS+P/S) of 1
cm3.
Cells were observed with an inverted phase-contrast microscope(IX71, Olympus Co., Ltd., Tokyo) every day. For comparison,
typical figures not only of the center part (Fig. 1(c)) but also ofthe peripheral part (Fig. 1(d)) on the bottom of the dish are
exemplified in Figs. 11-15. For quantitative evaluation ofdifferentiation, the summation area of the rounded cell is
calculated by number of pixels in the contrasted microscopic
image (Fig. 7).
For chemical confirmation of differentiation, the cells on thebottom of the dish were stained with Oil Red O (C26H24N4O,
1-(2,5-dimethyl-4-(2-5-dimethylphenyl)
phenyldiazenyl)
azonapthalen-2-ol) at the end of the experiment.
IErZV2V1Fig. 4: Electric circuit: E, pulse generator; Z, medium; r,resistance; I, current; V, voltage.

Fig. 7: Measurement of area of lipocyte.

Fig. 5: Electric stimulation for thirty minutes per day.

Fig. 8: Voltage tracings of electric pulse.

Fig. 6: Cell culture dish with electrodes in incubator.
Fig. 9: Cells on day 2. Group C (left), and group A (right).
Dimension from left to right is 1 mm in each section.
3. RESULTS
Fig. 8 shows the electric signals monitored by an oscilloscopeduring electric stimulation to cultured cells. At the display ofoscilloscope (Fig. 8), the voltage tracings show that the voltage(V2 in Fig. 3) between terminals of resistance (r in Fig. 4) of 30
ohm is 0.8 V. These values make the electric current of 0.03 A
calculated by Eq. 1.
Fig. 9 shows cells on the second day of culture. The rightfigure (Fig 9(b)) shows cells exposed to electric pulses for thirty
minutes. Cells adhere to the bottom of the dish, and extend toshow the linear shape. Some floating cells show the sphericalshapes.

Fig. 10: Cells on day 6. Group C (left), and group A (right).
Dimension from left to right is 1 mm in each section.

Fig. 10 shows cells on the sixth day of culture. Althoughseveral vacancies are left among cells on the bottom of the dishin the group A with electric stimulation (Fig. 10(b), the cells
proliferate to confluent state without electric stimulation (Fig.
10(a)).
The cells proliferated to confluent state on the sixth day ofculture in the group C without electric stimulation so that themedium was changed to that with insulin on the eighth day ofculture in every group. The protocol of group B also started
on the eighth day of culture.
Fig. 11 shows cells on the eighth day of culture. The figure
shows several flat cells, which are exemplified with the circles
in Fig. 11.
Fig. 12 shows cells on the twelfth day of culture. The figure
shows several spherical cells, which include lipid droplets,
which are exemplified with the circles.
Fig. 13 shows cells on the 22nd day of culture. The figure also
shows several spherical cells, which include lipid droplets,
which are exemplified with the circles.

Fig. 12: Cells on day 12. Group C (a), group A (b) and group
B (c: center, d: peripheral (Fig. 1)). Dimension from left toright is 0.5 mm in each section.

On the 24th day, cell culture was stopped and the cells on thebottom of the dish were stained (Fig. 14).
The number of lipid droplet is maximum in the group C without
electric stimulation. The data of area of the rounded cell are
distinguished by the contrasted microscopic image, anddistributed between 0.00012 mm2 and 0.0057 mm2.
Fig. 15 shows relation between the areas (S [mm2]) of roundedcell and the culture days. Each figure shows maximum (A),mean (B) and minimum (C) area, respectively. In each figure,data of circle belongs to the group C, data of square belongs to
the group A, and data of triangle and cross belong to the group
B. Data of triangle belongs to the position of c in Fig.1 anddata of cross belongs to the position of d in Fig 1. The area
extends earlier without electric stimulation than with electric
stimulation (day 11 in Fig. 15). The growth of rounded cell
tends to decrease under electric stimulation.

Fig. 11: Cells on day 8. Group C (a), group A (b) and group B(c: center, d: peripheral (Fig. 1)). Dimension from left to rightis 0.5 mm in each section.

Fig. 13: Cells on day 22. Group C (a), group A (b) and group
B (c: center, d: peripheral (Fig. 1)). Dimension from left toright is 0.5 mm in each section.

Fig. 14: Cells stained with Oil Red O on 24th day. Group C(a), group A (b) and group B (c: center, d: peripheral (Fig. 1)).
Dimension from left to right is 0.5 mm in each section.

4. DISCUSSION

S, mm2The behavior of a biological cell depends on several factors:
electrical [7-10], magnetic [11], and mechanical factors [12-15].
The microfluidic system has been fabricated to study thebehavior of cells in vitro [16-19]. The pattern of the surface
has been applied to study the surface effect of adhesion of cells[20-21]. Electric stimulation has been applied to human bodyin many cases. Some of them are applied to clinical treatment[1].

T, dayFig. 15A: Maximum area (S [mm2]) of rounded cell vs. culturedays. Group C (circle), group A (square) and group B (c:triangle, d: cross (Fig. 1)).
S, mm2In the present study, the voltage of the electric pulse is selected
lower than the voltage, which generates electrolysis of themedium. The medium does not show any chemical changes
like bubble generation. The color the medium is maintained,
which indicates the constant value of pH.
3T3-L1 is a cell line derived from cells of 3T3 mouse [4-6].
3T3-L1 cells have a fibroblast-like morphology. The cells
differentiate into an adipocyte-like phenotype under appropriate
conditions. After differentiation, the cell become sphere,
which includes lipid droplets. The morphological change fromlinear to flat is used as prelude for differentiation.
Oil red O (C26H24N4O) is fat-soluble diazo dye. It is alysochrome used for staining of neutral triglycerides and lipids
on frozen sections and some lipoproteins on paraffin sections.
To evaluate the effect of electric stimulation on differentiationof 3T3-L1, electric stimulation is applied to 3T3-L1 before cells
proliferate to the confluent state in Group A in the present study.
Both number and growth of adipocyte-like cell tend to decrease
with exposure to electric pulses.

T, dayThe tendency is almost the same regardless the position fromthe electrode on the culture plate, which shows the uniform
electric field on the plate in the present study.

2Fig. 15B: Mean area (S [mm ]) of rounded cell vs. culture days.
Group C (circle), group A (square) and group B (c: triangle, d:cross (Fig. 1)).

S, mm2

5. CONCLUSION
An effect of electric stimulation with pulses on differentiationand hypertrophy of fat precursor cells has been investigated invitro. The experiment shows that both differentiation andgrowth of 3T3-L1 are delayed with electric pulses.

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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T, dayFig. 15C: Minimum area (S [mm2]) of rounded cell vs. culturedays. Group C (circle), group A (square) and group B (c:triangle, d: cross (Fig. 1)).

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