Effect of Excess Gravitational Force and Electric Pulse Field on
Myoblast
Haruka HINO, Hiroki SATO, Shigehiro HASHIMOTO, 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/
ABSTRACTThe effects of mechanical and electric stimulations on cell
culture have been studied in vitro. C2C12 (mouse myoblast
cell line originated with cross-striated muscle of C3H mouse)
was cultured in Dulbecco’s Modified Eagle’s Medium with 10
percent of fetal bovine serum on a polystyrene dish for 12 days,
while the mechanical force field or the electric field isintermittently applied, alternatively. To apply the mechanicalforce field to the cells on the bottom of the dish, the dish wasset on a swing-rotor. The rotor was placed in a conventional
centrifugal machine, to set the surface of the culture plate in theperpendicular position to the centrifugal force. Throughelectrodes of platinum wire, the electric pulse (width of 0.0025
s, period of 0.5 s) was applied to the medium, where cells were
cultured. The experiment shows following results: both theexcess gravitational force and the electric pulse fields decelerate
proliferation of myoblasts, and accelerate differentiation ofmyoblasts, and mild gravitational force field is effective toaccelerate differentiation of myoblast.

The biological cells receive gravitational force on the earth.
An astronaut needs exercise before standing, when he comesback from the space station. The characteristic of muscle
tissue might change in the space station, where the tissue does
not receive gravitational force [3, 4].
The electromagnetic stress is another factor in the environmentof cells [18-23]. The muscle tissue is exposed to electricpulses in the biological body.
The movement is also
controlled with the electric pulses. The biological systems
have ability to optimize themselves to their environment. The
electric stimulation has also been applied to a body in somemedical treatments for rehabilitation. The optimum electric
stimulation has a potential to control growth of the muscle
tissue, which might contribute to regenerative medicine.
In the present study, the effects of mechanical and electric
stimulations on cell culture have been studied in vitro.

2. METHODS
Keywords: Biomedical Engineering, Cell Culture, C2C12,
Excess Gravitational Field and Electric Field.

1. INTRODUCTION
The acceleration technique for orientation, proliferation anddifferentiation of cells has been studied to make tissue in vivo orin vitro [1-23].
Control methodology for orientation,
proliferation and differentiation of cells would be applied to theregenerative tissue technology.
The mechanical stress is one of the interested factors in theenvironment of cells, because they receive mechanical force invivo. The mechanical stress on cells might induce various
responses: deformation, migration, proliferation, anddifferentiation. Several methods have been designed to apply
the mechanical stress to cells [1-17].
A transmission point of the stress to a specimen is important.
In many studies, the stress is applied to a scaffold [8, 10, 11].
When fixation between the cell and the scaffold is not enough,the stress is not transmitted to the cell. A mechanical field, on
the other hand, can be used to apply a continuous stress to a
specimen [1-6, 12-17]. The specimen fixed at a position
receives the shear stress in the mechanical field.
Thegravitational field is one of the mechanical fields [1-6].

Excess Gravitational Force Field
The excess gravitational force was applied to cultured cells withthe centrifugal force. A swing rotor has been manufactured toapply the vertical force to cultured cells (Figs. 1-3). The rotor
consists of a fixing bracket, an arm, a shaft and a dish. The
fixing bracket and the shaft are made of the steel (SS400).
The arm and the dish are made of aluminum alloy (A7075).
When the rotor swings, the culture dish swings. During therotation, the centrifugal force acts perpendicularly to the bottom
plane of the culture dish. The bottom of the dish has a line on
the outside of the culture surface to mark the observation point.
The dish was set on the swing rotor in the centrifugal machine(CN-1040, Matsuuraseisakusyo. Ltd, Tokyo, Japan).
The rotor (turning radius of 8.5 cm) rotates with the constant
speed of 1025 revolutions per minute, which makes the excess
gravitational force of 100 G at the surface of the culture plate.
The centrifugal machine was placed in an incubator to keep thepartial pressure of bicarbonate of 5 % at 310 K. Ethanol isfilled in the counter dish to make balance the mass
(approximately 0.12 kg) between two dishes on the both ends ofthe arm of the rotor.

through a couple of electrodes of platinum wire, which wasdipped into the medium of the culture dish (Fig. 6). A
resistance of thirty ohms (r) is serially connected in the electric
circuit (Fig. 7). The electric signal was monitored with an
oscilloscope. From the monitored potential difference of 5mV (V2) between the terminals of the resistance, the electric
current is calculated as 0.17 mA (I).
Cell Culture
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) of the passage four was used in the test.
D-MEM (Dulbecco’s Modified Eagle’s Medium) containing
10% decomplemented FBS (fetal bovine serum) and 1%
penicillin/ streptomycin was used for the medium.
The cells were seeded on the culture dish of 35 mm diameter atthe density of 2000 cells/cm2. After the cells were cultured in
the incubator for 24 hours without stimulation, the cells were
exposed to the excess gravitational field and to the electric pulse
field.

Fig. 1: Dimension (mm) of designed swing rotor.

Fig. 2: Manufactured swing rotor in centrifugal machine.

Fig. 4: Electrodes on cap.

Cell

Centrifugal force

Force

Force

Electrodes
Fig. 5: Dimension (mm) of electrodes on cap.

Fig. 3: Normal force on cell culture plane during centrifuge.
Platinum Wire
Electric Field
To apply electric pulse to culture cells, a pair of electrode has
been made. The electrode is made of a platinum wire of 0.2
mm diameter. To keep the position of the electrode, the wire isfixed on a cap made of polydimethylsiloxane (PDMS). To
decrease concentration of electric current around the wire, thewire is bended to make a square of 15 mm × 10 mm (Figs. 4&5).
The distance between two electrodes is 30 mm.
The electric pulse (interval of 0.5 s, amplitude of 0.1 V, durationof 0.0025 s) was generated by an electric stimulator (SEN5201,
Nihon Kohden Corporation, Japan). The pulses were applied

Cap of PDMS

Cells

Fig. 6: Electrodes on cap of culture dish.

In every group, C2C12 proliferates to the confluent state anddifferentiates into myotubes. In the group C, proliferation ofcells to the confluent state delays one day compared with theother groups.

I

E
r
Z

V2

V1

Fig. 7: Electric circuit: E, pulse generator; Z, medium; r,
resistance; I, current; V, voltage.

Several cells exfoliate from the bottom of the dish and are
floating in the medium in group A and B (Figs. 9-13).
The maximum value of the width of the myotube (bars in Figs.
14&15) in each group (A, B, C, D) on the twelfth day is 0.096
mm, 0.103 mm, 0.087 mm, 0.089 mm, respectively (Figs.
13-15). The value is highest in the group B.
In every group, differentiation into myotube is confirmed by theelectric stimulation under the microscopic observation. The
repetitive cyclic contraction of the myotube is observed on theeleventh day of culture, when the electric pulses of theamplitude higher than 5 V are applied to the medium. The
number of the contractive myotubes is small in the group C.
Several myotubes exfoliate from the bottom of the dish, when
they repeat cyclic contraction.

The cell culture tests for 12 days were divided into four groups:
excess gravity for one hour per day and electric pulse for one
hour per day (A), excess gravity for two hours per day (B),electric pulse for two hours per day (C), and control without any
stimulation (D) (Figs. 8-15).
The medium was refreshed every two days. The dish wasplaced in an incubator through the entire experimental term
including the term of stimulation to keep the partial pressure ofbicarbonate of 5 % at 310 K.
Morphological Study
Density and morphology of cells was observed with an inverted
phase-contrast microscope (IX71, Olympus, Tokyo) every 24
hours during the test. During the microscopic observation, theculture dish with cells was taken out from the incubator. On
the eleventh day, the width of each myotube was gauged, andthe maximum value was compared among the groups. The
data in the position, where the multiple myotubes aresuperimposed, were not counted for the maximum value.

Fig. 8: C2C12 on the first day before stimulation.
from left to right is 1 mm in each section.

Dimension

Electric Stimulation to Confirm Differentiation
At the end of tests, myotubes were stimulated with electricpulses to confirm differentiation of C2C12. The electric pulse(interval of 0.5 s, amplitude of 10 V, duration 0.003 s) was
generated by an electric stimulator (SEN5201, Nihon Kohden
Corporation, Japan). The pulses were applied through acouple of electrodes of platinum wire of 0.2 mm diameter,
which was dipped into the medium of the culture dish (Fig. 6).
The movement of the myotubes was observed with the inverted
phase-contrast microscope.

3. RESULTS
Centrifugal force is normal direction in the following figures(Figs. 8-15). Figs 8-13 show cells of each group: A (upper
right), B (lower left), C (lower right), and D (upper left). The
experiments show the following results.

Fig. 9: C2C12 on the second day (extends pseudopod).
Dimension from left to right is 1 mm in each section.

Fig. 10: C2C12 on the fourth day.
right is 1 mm in each section.

Dimension from left toFig. 13: Myotubes on the twelfth day. Dimension from left toright is 1 mm in each section.

Fig. 11: C2C12 on the sixth day (confluent). Dimension fromleft to right is 1 mm in each section.

Fig. 14: Width of myotubes on the twelfth day in group A.
Dimension from left to right is 1 mm. Bars show width of
myotube.

Fig. 12: Myotubes on the eighth day. Dimension from left toright is 1 mm in each section.

Fig. 15: Maximum width of myotube on the twelfth day ingroup C. Dimension from left to right is 1 mm. Bar shows
width of myotube.

4. DISCUSSION
Although the serum in the medium is usually decreased toinduce differentiation from myoblasts to myotubes in vitro, thecontents of the medium were not changed throughout thepresent study. The myoblasts tends to differentiate into
myotubes, when the cells proliferate to the confluent state. In
the most of the cell culture experiments, contents of the medium
are changed to that for differentiation such as the medium
includes horse serum [1].
Several electric pulses are generated in the biological tissue.
The electric pulses might have multiple effects on biologicalactivities [18-23].
The electric pulse might decelerate
proliferation of cells. The electric stimulation might accelerate
differentiation of myoblast to myotubes. Because of themultiple effects, myoblast does not show maximal
differentiation in the present study.
To apply electric
stimulation on myoblasts for differentiation, the optimum
intensity should be chosen: the frequency, the amplitude, andthe exposure time.
The excess gravitational force field, on the other hand, shows
maximal effect on differentiation of myoblast in the present
study. The mechanical stimulation might have more direct
relation to differentiation of myoblast than electric stimulation,
because the muscle operates against forces.
The culture dish without treatment was used in the present study,
to emphasize mechanical and electrical effects.
The response of biological system to the microgravity field has
been studied using a space satellite. The cell cycle might
extend in the space [3]. C2C12 differentiation might beaccelerated and the myotube might align to the direction
perpendicular to the centrifugal field [2].
When mechanical stimulation is applied to the scaffold, thewhole stimulation cannot always be transmitted to the cells.
When the tension applied to a scaffold, the deformation of thescaffold generates compression and shear in the different
direction simultaneously [8, 10, 11]. To apply continuous
uniform mechanical stimulation to the cells, centrifugal force isused in the present study [1].
Both acceleration of proliferation and orientation of cells areimportant targets in the research field of regenerative medicineon the cultured biological tissue. The previous study showedthat the behavior of cells depends on the electric [18-22] andmagnetic stimulation [23].
Another study shows thatmechanical stimulation improves a tissue-engineered skeletal
muscle [7]. The results of the study will contribute toacceleration technique in regenerative medicine
The previous studies showed that a mechanical field governs
behavior of cells [1-17].
The shear flow governs theorientation of endothelial cells [15-17]. The shear stressaffects the orientation of the smooth muscle cells in the
biological tissue [8]. The direction of the mechanical field
affects fibroblasts [10]. The effect of shear flow on orientationof cells depends on the kinds of cells [12]. Although HUVEC(human umbilical vein endothelial cells) orients along thestream lines, C2C12 tilts from the stream lines to makemyotubes. The previous study showed orientation of cells
perpendicular to the stretch direction [9].

Too strong mechanical stimulation damages cells.
The
moderate mechanical stimulation, on the other hand, might
accelerate differentiation of cells [1].
The mechanicalstimulation can decrease proliferation of cells [1]. The
mechanical stress also exfoliates several cells, which makes
vacancy around the adhesive cell. The differentiation might be
optimization of cells to the changing environment. The
mechanical stress can accelerate differentiation of C2C12 to
make myotubes [1].

5. CONCLUSION
Effects of intermittent application of the excess gravitational
force and the electric pulse fields on differentiation of myoblast
have been studied in vitro. To apply the excess gravitational
force field on cells, the cell culture dish was set in the
manufactured swing rotor set in a conventional centrifugal
machine. To apply the electric pulse field on cells, a pair ofelectrodes was dipped in the medium. The experiment shows
following results: both the excess gravitational force and theelectric pulse fields decelerate proliferation of myoblasts, andaccelerate differentiation of myoblasts, and mild gravitational
force field is effective to accelerate differentiation of myoblast.

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.

REFERENCES[1] S. Hashimoto, H. Hino and T. Iwagawa, “Effect of Excess
Gravitational Force on Cultured Myotubes in Vitro”,
Journal of Systemics, Cybernetics and Informatics, Vol.
11, No. 3, 2013, pp. 50-57.
[2] H. Hino, S. Hashimoto and T. Yasuda, “Effect of Centrifugal
Forceon
Cell Culture”,
Proc. 18th World
Multi-Conference on Systemics Cybernetics andInformatics, Vol. 2, 2014, pp. 132-137.
[3] T.B. Damm, A. Franco-Obregón and M. Egli, “Gravitational
Force Modulates G2/M Phase Exit in Mechanically
Unloaded Myoblasts”, Cell Cycle, Vol. 12, No. 18, 2013, pp.
3001-3012.
[4] T.B. Damm, I. Walther, S.L. Wueest, J. Sekler and M. Egli,“Cell Cultivation Under Different Gravitational Loads Usinga Novel Random Positioning Incubator”, Biotechnology
and Bioengineering, Vol. 111, No. 6, 2014, pp. 1180-1190.
[5] G. Ciofani, L. Ricotti, J. Rigosa, A. Menciassi, V. Mattoli
and M. Monici, “Hypergravity effects on myoblast
proliferation and differentiation,” Journal of Bioscienceand Bioengineering, Vol. 113, No. 2, 2012, pp. 258-261.
[6] G.G. Genchi, F. Cialdai, M. Monici, B. Mazzolai, V. Mattoli
and G. Ciofani, “Hypergravity Stimulation Enhances PC12
Neuron-Like Cell Differentiation”, BioMed ResearchInternational, Vol. 2015, 2015, Hindawi. 748121, pp. 1-10.
[7] Y. Akiyama, R. Terada, M. Hashimoto, T. Hoshino, Y.
Furukawa and K. Morishima, “Rod-shaped Tissue
Engineered Skeletal Muscle with Artificial Anchors to
Utilize as a Bio-Actuator”, Journal of Biomechanical
Science and Engineering, Vol. 5, No. 3, 2010, pp. 236-244.
[8] K. Nakagawa, N. Morishima and T. Matsumoto, “Effect ofThree-Dimensional Culture and Cyclic Stretch Stimulation
on Expression of Contractile Protein in Freshly Isolated Rat
Aortic Smooth Muscle Cells”, Journal of Biomechanical
Science and Engineering, Vol. 4, No. 2, 2009, pp. 286-297.
[9] L. Terracio, B. Miller and T. Borg, “Effects of Cyclic
Mechanical Stimulation of the Cellular Components of theHeart: in Vitro”, In Vitro Cellular & Developmental
Biology, Vol. 24, No. 1, 1988, pp. 53-58.
[10] J.H.-C. Wang, G. Yang, Z. Li and W. Shen, “Fibroblast
Responses to Cyclic Mechanical Stretching Depend on Cell
Orientation to the Stretching Direction”, Journal of
Biomechanics, Vol. 37, 2004, pp. 573-576.
[11] K. Sato, S. Kamada and K. Minami, “Development ofMicrostretching Device to Evaluate Cell Membrane Strain
Field around Sensing Point of Mechanical Stimuli”,
International Journal of Mechanical Sciences, Vol. 52, No.
2, 2010, pp. 251-256.
[12] S. Hashimoto and M. Okada, “Orientation of Cells
Cultured in Vortex Flow with Swinging Plate in Vitro”,
Journal of Systemics Cybernetics and Informatics, Vol. 9,
No. 3, 2011, pp. 1-7.
[13] S. Hashimoto, F. Sato, H. Hino, H. Fujie, H. Iwata and Y.
Sakatani, “Responses of Cells to Flow in Vitro”, Journal of
Systemics Cybernetics and Informatics, Vol. 11, No. 5,
2013, pp. 20-27.
[14] Y. Hayamizu, T. Hyakutake, K. Matsuura, S. Yanase, S.
Morita, S. Ohtsuka and T. Gonda, “Behavior of Motile
Sperm in Taylor-Couette Flow: Effect of Shear Stress on the
Behavior of Motile Sperm,” Open Journal of Fluid
Dynamics, Vol. 3, No. 2A, 2013, pp. 9-13.
[15] Y. Sugaya, N. Sakamoto, T. Ohashi and M. Sato,“Elongation and Random Orientation of Bovine Endothelial
Cells in Response to Hydrostatic Pressure: Comparison with
Response to Shear Stress”, JSME International Journal,

Series C, Vol. 46, No. 4, 2003, pp. 1248-1255.
[16] P. Uttayarat, M. Chen, M. Li, F.D. Allen, R.J. Composto
and P.I. Lelkes, “Microtopography and Flow Modulate the
Direction of Endothelial Cell Migration”, Am. J. Physiol.
Heart Circ. Physiol., Vol. 294, 2008, pp. H1027-H1035.
[17] N. Azuma, S. A. Duzgun, M. Ikeda, H. Kito, N. Akasaka, T.
Sasajima and B. E. Sumpio, “Endothelial Cell Response to
Different Mechanical Forces”, Journal of Vascular Surgery,Vol. 32, No. 4, 2000, pp. 789-794.
[18] D.M. Pedrotty, J. Koh, B.H. Davis, D.A. Taylor, P. Wolf
and L.E. Niklason, “Engineering Skeletal Myoblasts: Rolesof Three-dimensional Culture and Electrical Stimulation”,
American Journal of Physiology: Heart and Circulatory
Physiology, Vol. 288, No. 4, 2005, pp. H1620-H1626.
[19] M. Marotta, R. Bragós and A.M. Gómez-Foix, “Design and
Performance of an Electrical Stimulator for Long-term
Contraction of Cultured Muscle Cells”, Bio Techniques, Vol.
36, No. 1, 2004, pp. 68-73.
[20] S.H. Ku, S.H. Lee and C.B. Park, “Synergic Effects of
Nanofiber Alignment and Electroactivity on Myoblast
Differentiation”, Biomaterials, Vol. 33, No. 26, 2012, pp.
6098-6104.
[21] S. Curtze, M. Dembo, M. Miron and D.B. Jones, “Dynamic
Changes in Traction Forces with DC Electric Field in
Osteoblast-like Cells”, Journal of Cell Science, Vol. 117,
No. 13, 2004, pp. 2721-2729.
[22] S. Hashimoto, F. Sato, R. Uemura and A. Nakajima, “Effectof Pulsatile Electric Field on Cultured Muscle Cells in Vitro”,
Journal of Systemics Cybernetics and Informatics, Vol.
10, No. 1, 2012, pp. 1-6.
[23] S. Hashimoto and K. Tachibana, “Effect of Magnetic Field
on Adhesion of Muscle Cells to Culture Plate”, Journal of
Systemics, Cybernetics and Informatics, Vol. 11, No. 4,
2013, pp. 7-12.