Effect of Centrifugal Force on Cell Culture
Haruka HINO, Shigehiro HASHIMOTO
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 a mechanical force field on cell culture has beenstudied in vitro. C2C12 (mouse myoblast cell line originatedwith cross-striated muscle of C3H mouse) was cultured on apolystyrene plate coated with collagen for 13 days in themechanical force field. To apply the mechanical force field tothe cells on the plate, the plate was inserted into a centrifugal
tube. The tube was placed in a conventional centrifugal
machine, to set the surface of the culture plate in the parallel
position to the centrifugal field. The shape and the orientationof the cells were observed with a phase contrast microscopeduring the cell culture. The experiments show that C2C12
extends pseudo, proliferates to the confluent manner anddifferentiates to myotube even under continuous stimulation of
270 times of the gravitational force.
Keywords: Biomedical Engineering, C2C12, Cell Culture andCentrifugal Force Field.

1. INTRODUCTION
Cell culture technique has been developed and several
methodologies have been clinically applied to regenerative
medicine.
The acceleration technique for orientation,
proliferation and differentiation of cells has been studied tomake tissue in vivo or in vitro [1-9, 12]. Control methodology
for orientation, proliferation and differentiation of cells would
be applied to the regenerative 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 [2-10, 16].
A transmission point of the stress to a specimen is important.
In many studies, the stress is applied to a scaffold [2-5]. Whenfixation between the cell and the scaffold is not enough, thestress is not transmitted to the cell. A mechanical field, on theother hand, can be used to apply a continuous stress to a
specimen [6-10, 13]. The specimen fixed at a position
receives the shear stress in the mechanical field.
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 [11].
In the present study, the effect of mechanical stimulation on
cultured cells has been studied in centrifugal force field in vitro.

2. METHODS
Excess Gravitational Force Field
The excess gravitational force was applied to cultured cells withthe centrifugal force (Fig. 1). Cells were cultured on apolystyrene plate (30 mm × 8.5 mm × 2 mm) for two weeks inthe centrifugal force field. The plate has a line on the backside of the culture surface to mark the observation point. To
apply the mechanical force field to the cells on the plate, theplate was inserted into a centrifugal tube. The tube (Fig. 3)
was set on an angled rotor in the centrifugal machine (CN-1040,
Matsuuraseisakusyo. Ltd, Tokyo, Japan) (Fig. 4). The rotor(turning radius of 8 cm) was rotated with the constant speed
slower than 1750 revolutions per minute, which makes theexcess gravitational force lower than 270 G at the surface of theculture plate. The angle between the directions of thecentrifugal force and the surface of the cell culture plate is 1 rad.
The force is divided into two ingredients, which are thetangential force and the normal force at the culture plate. The
centrifugal force of 270 G makes 140 G of the tangential force
and 230 G of the normal force. To keep the balance of therotator in the centrifugal machine, two tubes were placed in the
counter position each other. The centrifugal machine wasplaced in an incubator to keep the partial pressure ofbicarbonate of 5 % at 310 K. The tube is shield with Parafilm(Pechiney Plastic Packaging Company, IL), through which gas
passes. For comparison, some tubes were shield with theplastic screw cap.

Cell Culture
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) of the passage between the third and thesixth was used in the test. C2C12 was cultured with the
D-MEM (Dulbecco’s Modified Eagle Medium) containing 10%
FBS. The medium containing penicillin and streptomycin wasrefreshed every two days.

Fig. 4: Electrodes for stimulation.
Para film

PlateFig. 1: Centrifugal force (arrow) applied on cell culture in tube.

The polystyrene plate cut from the bottom of a culture dish isdipped for 24 hours in ethanol for disinfection. After rinsed
with a phosphate buffer solution, the plate was placed in theculture dish of six wells, and the cells were seeded at thedensity of 3000 cells/cm2. After the cells were cultured for 24hours without centrifugal stimulation, the plate was moved fromthe dish to the tube and exposed to mechanical stimulation of
the centrifugal force for two weeks. The medium was changed
every two days.
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 observation, the rotation of
the centrifugal machine was stopped, and the plate with cells
was moved from the tube to the dish filled with the medium.
Electric Stimulation
At the end of tests, myotubes were stimulated with electricpulses to confirm differentiation of C2C12. The culture plate
was moved from the tube to the dish. The electric pulse(interval shorter than 0.5 s, amplitude lower than 25 V, duration
0.002 s) was generated with an electric stimulator (SEN5201,
Nihon Kohden Corporation, Japan). The pulses were appliedwith a couple of electrodes of platinum wire of 0.1 mm
diameter, which was dipped into the medium of the culture dish(Fig. 4). The movement of the myotubes was observed with
the inverted phase-contrast microscope.

Fig. 2: Cell culture in tube. Mark to trace the same area ofculture.

3. RESULTS
The experiments show the following results.
Cells deform from the round shape to the extended shape,
which shows adhesion on the culture plate in 24 hours (Fig.
5(A)). The cells are sparse on the plate, because most of cells
adhere to the bottom of the dish.

Fig. 3: Centrifugal machine in incubator.

C2C12 begins to fuse to make myotube after centrifugation
sealed with the cap for one day at 5.6 G (Fig. 5(B)).
Centrifugal force is downward direction in the following figures.
Myotubes are oriented not only to the parallel direction to thecentrifugal force field, but also perpendicular direction to thecentrifugal force field. Most of cells are exfoliated after
centrifugation sealed with the cap for four days (Figs.
5(C)&(D)). The result at 89 G (Fig. 6) is similar to that at 270
G (Figs. 7(C), 8(C), 9(C)).

Fig. 5(A): C2C12 before centrifugation of 5.6 G (250 rpm) withcap for 24 hours. Dimension from left to right is 2 mm.

Fig. 6(A): C2C12 before centrifugation of 89 G with cap for 24hours. Dimension from left to right is 2 mm.

Centrifugal force

MarkerFig. 5(B): C2C12 after centrifugation at 5.6 G for one day.
Centrifugal force is downward direction in the following
figures.

Fig. 5(C): C2C12 after centrifugation of 5.6 G with cap for twodays. Dimension from left to right is 2 mm.

Fig. 5(D): C2C12 after centrifugation of 5.6 G with cap for fourdays. Dimension from left to right is 2 mm.

Fig. 6(B): C2C12 after centrifugation of 89 G with cap for one
day. Dimension from left to right is 2 mm.

Fig. 6(C): C2C12 after centrifugation of 89 G with cap for twodays. Dimension from left to right is 2 mm.

Fig. 6(D): C2C12 after centrifugation of 89 G with cap for fourdays. Dimension from left to right is 2 mm.

Fig. 7(A): C2C12 before centrifugation of 270 G with film for
24 hours. Dimension from left to right is 2 mm.

Fig. 8(A): C2C12 after centrifugation of 270 G with film for 4
days. Dimension from left to right is 2 mm.

Fig. 7(B): C2C12 with film for 24 hours.
to right is 2 mm.

Fig. 8(B): C2C12 with film for 4 days.
to right is 2 mm.

Dimension from left

Dimension from left

Fig. 7(C): C2C12 before centrifugation of 270 G with cap for
24 hours. Dimension from left to right is 2 mm.

Fig. 8(C): C2C12 after centrifugation of 270 G with cap for 4
days. Dimension from left to right is 2 mm.

Fig. 7(D): C2C12 with cap for 24 hours.
to right is 2 mm.

Fig. 8(D): C2C12 with cap for 4 days.
right is 2 mm.

Dimension from left

Dimension from left toFig. 9(A): C2C12 after centrifugation of 270 G with film for 12
days. Dimension from left to right is 1 mm.
Fig. 10: Medium after two days of cell culture: centrifugation
with film (A), rest with film (B), centrifugation with cap (C),rest with cap (D).

When the tube sealed with the film, C2C12 proliferate to theconfluent manner and differentiates to myotube even undercontinuous stimulation of 270 times of the gravitational force
for 12 days (Figs. 7(A), 8(A), 9(A)).
Without excess gravitational force, C2C12 proliferate to theconfluent manner and differentiates to myotube even sealedwith cap (Figs. 7(D), 8(D), 9(D)).
Fig. 9(B): C2C12 with film for 12 days.
to right is 2 mm.

Dimension from left

C2C12 differentiates to myotube.
The repetitive cyclic
constriction of the myotube is observed in 13 days of culture,when the electric pulses are applied to the medium (Fig. 9(A)).
In two days of cell culture, the color of the medium in the tube
sealed with the cap does not change much (Fig. 10(C&D)),
although that in the tube sealed with the film changes (Fig.
10(B)).

4. DISCUSSION
The culture plate of polystyrene is cut from the bottom of theculture dish, because the material is standard in the cell culture.
Even after dipped in ethanol, C2C12 is proliferated anddifferentiated on the plate.
Fig. 9(C): C2C12 centrifugation of 270 G with cap for 12 days.
Dimension from left to right is 2 mm.

The response of biological system to the microgravity field has
been studied using a space satellite. The cell cycle might
extend in the space.
C2C12 differentiation might beaccelerated and the myotube might align to the direction
perpendicular to the centrifugal field in the atmospheric partialpressure [7, 8].
Compared to the controlled partial pressure in the incubator, theatmospheric partial pressure might decelerate proliferation of
C2C12 (Fig. 8(B) & (D)), although that might accelerate
differentiation of C2C12 (Fig. 8(A)).

Fig. 9(D): C2C12 with cap for 12 days.
to right is 2 mm.

Dimension from left

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 ofscaffold generates compression and shear in the different
direction simultaneously [14]. The parallelepiped chamber isconvenient to observe the response of cells under controlledshear stress [8]. To apply continuous uniform mechanicalstimulation to the cells, centrifugal force is used in the present
study [6].
Both acceleration of proliferation and orientation of cells areimportant targets in the research field of regenerative medicineon the cultured biological tissue. The previous study showsthat the behavior of cells depends on the electric [1] andmagnetic stimulation [12].
Another study shows thatmechanical stimulation improves a tissue-engineered humanskeletal muscle [2]. The results of the study will contribute toacceleration technique in regenerative medicine
The previous studies show that a mechanical field governs
behavior of cells [13-16]. The shear flow governs theorientation of endothelial cells [7, 9]. The shear stress affects
the orientation of the smooth muscle cells in the biologicaltissue [3]. The direction of the mechanical field affects
fibroblasts [5]. The effect of shear flow on orientation of cells
depends on the kinds of cells [7]. Although HUBEC orientsalong the stream lines, C2C12 tilts from the stream lines tomake myotubes. The previous study shows orientation of cells
perpendicular to the stretch direction [4].
Too strong mechanical stimulation damages cells.
The
moderate mechanical stimulation, on the other hand, might
accelerate differentiation of cells [6].
The mechanicalstimulation can decrease proliferation of cells [6]. 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 [6].

5. CONCLUSION
The effect of gravitational force on orientation of cell has beenstudied in vitro. To apply excess gravitational force on cells,
the cell culture tube was set in a conventional centrifugal
machine. The experimental results show that C2C12 extends
pseudo, proliferate to the confluent manner and differentiates tomyotube even under continuous stimulation of 270 times of thegravitational force.

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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