Behavior of Cells in Excess Gravitational Field: Using Centrifuge
Haruka HINO, Hiromi SUGIMOTO, Yusuke TAKAHASHI, Shigehiro HASHIMOTO, Shoki MIURA
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACTThe effect of mechanical field on orientation and extension ofcells has been studied using centrifuge in vitro. Four kind ofcells were used in the test: C2C12 (mouse myoblast cell line
originated with cross-striated muscle of C3H mouse), L929
(fibroblast connective tissue of C3H mouse), HUVEC (Human
Umbilical Vein Endothelial Cells), Neuro-2a (a mouse neuralcrest-derived cell line). To apply the mechanical force fieldfor 24 hours to the cells adhered on the glass plate, the plate wasset in the tube in a conventional centrifugal machine, which wasplaced in an incubator. Variation was made at the angular
position of the plate in the tube to make variation at thedirection of the force on the surface of scaffold: normal andtangential. The experiment shows following results. The
elongation of neurites of Neuro2a is accelerated in the excess
gravitational force field. L929 tends to tilt to the direction of
the tangential force. C2C12 tends to make orientation at
diagonal direction of the tangential force. The cell tends toextend the area of adhesion at the excess gravitational force
field. C2C12 tends to change the shape at the excess
gravitational field.
Keywords: Biomedical Engineering, Cell Culture, Excess
Gravitational Field, C2C12, L929, Neuro-2a and HUVEC.

proliferation and differentiation of cells would be applied to theregenerative tissue technology.
In the present study, the effect of mechanical field onorientation and extension of cells has been studied using
centrifuge in vitro.

2. METHODSHyper Gravitational Force Field
The hyper-gravitational force was applied to cultured cells withthe centrifugal force.
A glass plate of 50 mm ×10 mm was used for the scaffold forcell culture. The glass plate is inserted in the tube, which iscontained in the rotor. The angle between the radial directionof the rotation of the rotor and the axial direction of the tube in
the rotor is 1 rad. The variation was made on the direction of
the glass plate in the tube. In the group X the glass plate wasset so that the culture surface is parallel to the radial direction of
the rotation. The centrifugal force is applied parallel to theculture surface in the group X. In the group Y, the glass plate
was set in the perpendicular position to the former group rotatedat the longitudinal axis of the tube. Both the tangential fore(Ft) and the normal force (Fn) at the culture surface are appliedby the centrifuge in the group Y (Fig. 1).

1. INTRODUCTION
The centrifugal force (Fc) is calculated by Eq. 1.
A biological cell is surrounded by mechanical force field in vivo.
The cell is sensitive to the mechanical stimulation, and shows
several responses: deformation, and migration. The response
includes passive one and active one. The cell has compliance,
and is deformed by force. The cell deforms, on the other hand,to minimize the intra force. The cell is moved by the force.
The cell moves in response to the force.

F c = m r ω2

(1)

In Eq. 1, m is mass, r is radius of the rotation, and ω is angularvelocity.

The object is exposed to the force at the surface with two kindsof directions: normal, and tangential. The object is pressed by
the normal force. The object is sheared on the surface with thetangential force.
The muscle tissue might decrease in the micro gravitational
field [1, 2]. The muscle tissue might increase, on the other
hand, in the hypergravity. The previous study shows that theexcess gravitational field thicken the myotubes in vitro [3].

Ft
Fn
Ft

Ft

The acceleration technique for orientation, proliferation anddifferentiation of cells has been studied to make tissue in vivo orin vitro [3-14].
Control methodology for orientation,
Fig. 1: Glass plate with mark: group X (right), group Y (left).

Cell Culture
Four kinds of cells were used in the experiment: C2C12 (mouse
myoblast cell line originated with cross-striated muscle of C3Hmouse), L929 (fibroblast connective tissue of C3H mouse),
HUVEC (human umbilical vein endothelial cells), andNeuro-2a (a mouse neural crest-derived cell line).
C2C12 of the passage between three and nine was cultured in
D-MEM (Dulbecco’s Modified Eagle’s Medium) containing
10% decomplemented FBS (fetal bovine serum) and 1%
penicillin/ streptomycin.
The medium was changed to
D-MEM (Dulbecco’s Modified Eagle’s Medium) containing 2%
decomplemented HS (horse serum) and 1% penicillin/
streptomycin for differentiation.

Fig. 2: Group X (left), and Y (right).

In the test with C2C12, the experiment of 150 G (r = 0.07 m by
ω = 140 rad/s) was added instead of 100 G.
L929 of the passage between three and nine was cultured in
D-MEM (Dulbecco’s Modified Eagle’s Medium) containing
10% decomplemented FBS (fetal bovine serum) and 1%
penicillin/ streptomycin.
HUVEC of the passage between three and eight was cultured in
EBM-2 (Endothelial Cell Basal Medium) containing 2%
decomplemented FBS (fetal bovine serum).
Neuro2a of the passage between two and nine was cultured in
D-MEM (Dulbecco’s Modified Eagle’s Medium) containing
10% decomplemented FBS (fetal bovine serum) and 1%
penicillin/ streptomycin. For differentiation, the medium waschanged to D-MEM (Dulbecco’s Modified Eagle’s Medium)
containing 2% decomplemented FBS (fetal bovine serum) andof Retinoic Acid for differentiation. The concentration ofRetinoic Acid in the medium is adjusted to 20 μM.
Fig. 3: Centrifuge in incubator.

In the gravitational field, gravitational force (Fg) is calculated
by Eq. 2, where g is gravitational acceleration.
Fg = m g(2)

In the present study, the centrifugal acceleration of 100 G (1 G
is equal to the gravitational acceleration) is generated at r =0.07 m by ω = 120 rad/s with the centrifugal machine (CN-1040,
Matsuuraseisakusyo. Ltd, Tokyo, Japan).
In the group X, the tangential force (Ft) of 100 G is applied tothe surface of the glass plate. In the group Y, both the normal
force (Fn) of 87 G and the tangential force (Ft) of 50 G are
simultaneously applied to the surface of the glass plate (Fig. 2).
Perpendicularly crossing lines of grooves, which make 25
squares (5 × 5) of 1 mm × 1 mm, are marked on the rear surfaceof the glass plate to trace the cell behavior (Fig. 1). One of thedirections of the lines corresponds to the direction of thetangential force by the centrifuge (Fig. 1). The grooves (0.016
mm width, 0.002 mm depth) were machined by the ultrashortpulse laser (IFRIT, Cyber Laser Inc., Tokyo, Japan).
During the stimulation test to cells, the centrifugal machine wasplaced in an incubator to keep the content of carbon dioxide of
5 % at 310 K (Fig. 3).

The cells were seeded on the glass plate at the density of 1000
cells/cm2. After the cells were cultured in the incubator for 24hours without stimulation, the cells were exposed to the excess
gravitational field by centrifuge for 24 hours. The glass plate,on which cells adhered, was set in the medium in the tube.
The angle of the glass plate in the tube was adjusted to that ofgroup X or Y (Fig. 2). Several tubes with the glass plate were
set in the rotor to cultivate cells of group X and Y
simultaneously. Each tube was capped with a film of gas
permeable paraffin [4]. After the centrifuge, the cells were
cultured successively in the incubator for another 24 hours.
To keep the content of carbon dioxide of 5 % at 310 K, the cells
were incubated in an incubator through the entire experimental
term including the term of exposure to the excess gravitational
field. Namely, the centrifugal machine was placed in the
incubator during the centrifugation (Fig. 3). In the control
group (normal force of 1 G), the cells were cultured without
centrifuge on the glass, which was placed in the polystyrene
dish (50 mm diameter) without collagen coating.
Morphological Study
The morphology of each cell was observed with an inverted
phase-contrast microscope (IX71, Olympus, Tokyo) every 24
hours during the test: after 24 hours of culture, in three minutes
after exposure to the excess gravitational field, after additional
24 hours. In the same area of the marked square, the behaviorsof the cells were traced. The outline of each single cell(except cells in the colony) is traced with “Image J” to calculate
parameters: the orientation, the projected area, and the shapeindex (Fig. 4).
To evaluate the orientation, the angle between the direction oftangential force by centrifuge and the longitudinal direction ofeach cell was measured. To evaluate the deformation, theprojected area was calculated as the area surrounded by theoutline of each cell. The shape index was calculated by theratio between the major axis and the minor axis of each cellprojected to the plate of the scaffold. The shape index
becomes unity at the circle.
To evaluate differentiation of Neuro-2a, the extension of neurite
was observed.

ControlGroup XGroup Y

Fig. 5a: Angle of C2C12. 90 deg : centrifuge.

3. RESULTS
Fig. 5 shows the angle between the direction of the tangential
force (on the surface of the scaffold by centrifuge) and thelongitudinal direction of each cell. The degree of 90 showsthat the longitudinal direction of the cell is parallel to thedirection of the tangential force. In fig. 5, zero degree means
the longitudinal direction of each cell is perpendicular to thedirection of tangential force. In Fig. 5, data are arranged in theascending order. When the angles are distributed at random,
the data align on the linear line. In the range of dense
population, the data align on the gentle inclination line. In therange of sparse population, on the other hand, the data align on
the steep inclination line.

Fig. 5b: Angle of L929.

Fig. 5 shows the following results. In the control study, theangles distribute at random. C2C12 shows tendency to alignon the direction to the tangential force of 50 G with normal
force of 87 G by centrifuge. C2C12 shows tendency to alignon the diagonal oblique direction to the tangential force of 150
G by centrifuge (to 70 and 130 degrees). L929 shows
tendency to align on the perpendicular direction to thetangential force of 50 G with normal force of 87 G bycentrifuge. L929 shows slight tendency to align on the
direction perpendicular (zero and 180 degrees) to the tangential
force of 100 G by centrifuge. HUVEC shows slight tendency
to align on the direction to the tangential force of 50 G withnormal force of 87 G by centrifuge.
Fig. 5c: Angle of HUVEC.

In Figs. 6-7, the column shows the mean value, and the barshows the range (±) of the standard deviation. The mean value
in Fig. 6 shows that the area of each single cell (C2C12, L929,
and HUVEC) tends to increase under the mechanicalstimulation.
Fig. 7 shows that there is not statistically significant differenceat the shape index of cell between control study and centrifuge
study. Only the shape index of C2C12 tends to increase. The
number of Neuro-2a with neurites increases after centrifugation.
Fig. 8 shows that the differentiation of Nero-2a [15] isaccelerated by the mechanical stimulation by centrifuge.

Fig. 4: Outline of each single cell is traced.
shows 1 mm × 1 mm.

Square markL929 changes the shape at the excess gravitational field (Fig.
9a). Some cells of HUVEC are elongated in the excess
gravitational force field (Fig. 9b). The direction of thecentrifugal force is vertical in Fig. 9.

Fig. 6a: Area of C2C12.

Mean: 1170, 1810, 1883 μm2.

Fig. 6b: Area of L929.

Mean: 876, 1371, 1696 μm2.

Fig. 6c: Area of HUVEC.

Fig. 7b: Shape index of L929. Control: n=638, Group X:
n=411, Group Y: n=344. Mean: 0.43, 0.44, 0.43.

Fig. 7c: Shape index of HUVEC. Control: n=2262, Group X:
n=658, Group Y: n=1269. Mean: 0.48, 0.45, 0.50.

Mean: 775, 1204, 1266 μm2.
Fig. 8: Ratio of Nero-2a with neurites.

Fig. 9a: L929 after stimulation: group X (left), group Y (right).
Square mark shows 1 mm × 1 mm.
Fig. 7a: Shape index of C2C12. Control: n=1810, Group X:
n=140, Group Y: n=149. Mean: 0.44, 0.49, 0.45.

moderate mechanical stimulation, on the other hand, might
accelerate differentiation of cells [3].
The mechanicalstimulation can decrease proliferation of cells [3]. 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 [3].

Cells passively follow the direction in the strong tangential
force field. Immediately after the centrifugation, cell mightstart to show active response to the mechanical stimulation.
Mild tangential force field induces the active reflection of cells
to tilt perpendicularly to decrease internal force of the cells.

The effect of mechanical field on orientation and extension ofcells has been studied using centrifuge in vitro.
Theelongation of neurites of Neuro2a is accelerated in the excess
gravitational force field, especially in the normal stress field.
The normal force field has effect similar to adhesion to thescaffold. The results of three kinds of cells (C2C12, L929,
HUVEC) show that the single cell tends to tilt to the directionof gravitational force. L929 extends the area of adhesion at
the tangential force field. L929 changes the shape at theexcess gravitational field.
Some cells of HUVEC are
elongated in the excess gravitational force field.

The cells might change the orientation at the differentiation.
C2C12 made perpendicular orientation of myotubes to the flow
direction in the previous study [16]. The orientation of C2C12
at diagonal direction of flow might be preparation to makeperpendicular orientation of myotubes in the successive
cultivation. C2C12 might differentiate earlier on polystyrene
dish than glass plate [3].

Exfoliation of cells tends to increase by the combination ofexcess normal force and shear force than pure shear force at thescaffold. The area of the cell projected to the scaffold tends toincrease under the excess gravitational field. The shape ofL929 and HUVEC tends to change under the excess
gravitational field. The response of the cell depends on the
direction of the gravitational field.

Fig. 9b: HUVEC after stimulation: group X (left), group Y(right). Square mark shows 1 mm × 1 mm.

4. DISCUSSION

The response of biological system to the microgravity field has
been studied using a space satellite. The cell cycle might
extend in the space [1, 2]. C2C12 differentiation might beaccelerated and the myotube might align to the direction
perpendicular to the centrifugal field [4].
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 [17-19].
To apply continuous
uniform mechanical stimulation to the cells, centrifugal force[3-5] is used in the present study.
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 [11, 21-23] andmagnetic stimulation [12].
Another study shows thatmechanical stimulation improves a tissue-engineered skeletal
muscle [24]. The results of the study will contribute toacceleration technique in regenerative medicine
The previous studies showed that a mechanical field governs
behavior of cells [3-10, 16-20, 24-29]. The shear flow governs
the orientation of endothelial cells [28, 29]. The shear stressaffects the orientation of the smooth muscle cells in the
biological tissue [17]. The direction of the mechanical field
affects fibroblasts [19]. The effect of shear flow on orientationof cells depends on the kinds of cells [16]. 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 [18].
Too strong mechanical stimulation damages cells.

The

5. CONCLUSION
The effect of mechanical field on orientation and extension ofcells has been studied using centrifuge in vitro. Four kind ofcells were used in the test: C2C12 (mouse myoblast), L929
(mouse fibroblast), HUVEC (Human Umbilical Vein
Endothelial Cells), Neuro-2a (a mouse neural crest-derived cell).
The experiment shows the elongation of neurites of Neuro2a,the tilt of L929, the diagonal tilt of C2C12, extension of the areaof adhesion of cells, and the change of the shape of C2C12 at
the excess gravitational field.

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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