Cell Behavior After Stimulation of Excess Gravity
Takuya TAMURA, Haruka HINO, Shigehiro HASHIMOTO, Hiromi SUGIMOTO, Yusuke TAKAHASHI
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACTThe effects of the excess gravitational field on orientation anddeformation of cells have been studied using centrifuge in vitro.
Two kinds of cells were used in the test: C2C12 (mouse
myoblast cell line), and Neuro-2a (mouse neural crest-derived
cell line). To apply the mechanical force field (<100 G) to thecells adhered on the glass plate, the plate was set in the tube in aconventional centrifugal machine placed in an incubator (310 K,
5% CO2). The behavior of cells was traced at the time lapse
images for 24 hours, after the excess gravitational stimulationfor 1 or 5 hours. The contour of each cell was traced andapproximated to ellipse to analyze the angle between thelongitudinal axis of the cell and the direction of the excess
gravity.
The experiment shows following results. The
longitudinal axis of C2C12 tends to align to the direction of theexcess gravity, and turns to the perpendicular direction after
stopping of the excess gravity. C2C12 aligns to the direction
perpendicular against the excess gravity at division. The
neurite of Neuro-2a extends along the direction of the excess
gravity, and tends to extend subsequently apart from the excess
gravity. The present study shows that the excess gravitational
stimulation affects the behavior of cells after stimulation.
Keywords: Biomedical Engineering, Cell Culture, Excess
Gravitational Field, C2C12 and Neuro-2a.

1. INTRODUCTION
A biological cell shows passive and active behaviors in anenvironment. While the shear stress deforms the cell, the cell
deforms by itself to adopt the shear field. While the strong
stimulation above the threshold damages the cell, thestimulation below the threshold remains in the cell as a memory
for the response in the next step. A biological cell issurrounded by mechanical force field in vivo. The cell issensitive to the mechanical stimulation, and shows several
responses: deformation, and migration. The response includes
passive one and active one. The cell has compliance, and isdeformed by force. The cell deforms, on the other hand, tominimize the intra force. The cell is moved by the force.
The cell moves in response to the force. The muscle tissue
might decrease in the micro gravitational field [1-3]. The
muscle tissue might increase, on the other hand, in thehypergravity [4-13]. The previous study shows that the excess
gravitational field thickens the myotubes in vitro [5]. The
acceleration technique for orientation, proliferation anddifferentiation of cells has been studied to make tissue in vivo orin vitro [5-13].
Control methodology for orientation,

proliferation and differentiation of cells would be applied to theregenerative tissue technology. In the present study, the effectof mechanical field on orientation and deformation of cells has
been studied by centrifuge in vitro, and the behavior of cells has
been traced after stopping of the stimulation of excess gravity.

2. METHODSHyper Gravitational Force Field
The hyper-gravitational force was applied to cultured cells withthe centrifugal force. A glass plate of 38 mm ×13 mm ×1 mm
was used for the scaffold for cell culture. The glass plate isinserted in the tube, which is contained in the rotor. The angle
between the radial direction of the rotation of the rotor and theaxial 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 thegroup X the glass plate was set so that the culture surface isparallel to the radial direction of the rotation. The centrifugal
force is applied parallel to the culture surface in the group X.
In the group Y, the glass plate was set in the perpendicular
position to the former group rotated at the longitudinal axis of
the tube. Both the tangential fore (Ft) and the normal force(Fn) at the culture surface are applied by the centrifuge in thegroup Y (Fig. 1) [6]. In the group X, the variation is made on
the position of the glass plate in the tube: at the middle of the
tube (X1), or at the bottom of the tube (X2). To stabilize theposition of the glass plate in the tube, Polydimethylsiloxane(PDMS) is filled in the tube to fill the vacancy below the glass
plate in the group X1 (Fig. 3a) and Y. The centrifugal force(Fc) is calculated by Eq. 1.
Fc = m r ω2

(1)

In Eq. 1, m is mass, r is radius of the rotation, and ω is angularvelocity. In the gravitational field, gravitational force (Fg) is
calculated by Eq. 2, where g is gravitational acceleration.
Fg = m g(2)

Fig. 1: Tube preparation of group X2, X1, X, and Y from left toright: direction of excess gravity from right to left.

streptomycin for 24 hours in the first step. For differentiation,
the medium was changed to D-MEM (Dulbecco’s Modified
Eagle’s Medium) containing 2% decomplemented FBS (fetal
bovine serum), 1% penicillin/ streptomycin, and of Retinoic
Acid for subsequent 144 hours in the second step. The
concentration of Retinoic Acid in the medium is adjusted to 20
μM. After differentiation, the cells were exposed to the excess
gravitational field by centrifuge for one hour. In the test with
Neuro-2a, the gravity of 5 G (r = 0.085 m) were applied at ω =24 rad/s.

Fig. 2: Position of cell culture observation on glass plate: 50 G
(left), and 100 G (right): Square marks, 1 mm × 1 mm.

Fig. 3a: Tube for centrifugation: 50 G (left), and 100 G (right).
Fig. 3b: Centrifuge in incubator (right).

In the present study, the centrifugal acceleration of < 100 G (1G is equal to the gravitational acceleration) is generated withthe centrifugal machine (CN-1040, Matsuuraseisakusyo. Ltd,Tokyo, Japan). Perpendicularly crossing lines of grooves,which make 25 squares (5 × 5) of 1 mm × 1 mm, are marked on
the rear surface of the glass plate to trace the cell behavior (Fig.
2). One of the directions of the lines corresponds to thedirection of the tangential force by the centrifuge (Fig. 2). The
grooves (0.016 mm width, 0.002 mm depth) were machined by
the ultrashort pulse laser (IFRIT, Cyber Laser Inc., Tokyo,
Japan). The glass plate was sterilized in an autoclave before
cell seeding.
During the stimulation test to cells, thecentrifugal machine was placed in an incubator to keep thecontent of carbon dioxide of 5 % at 310 K (Fig. 3b).
Cell Culture
Two kinds of cells were used in the experiment: C2C12 (mouse
myoblast cell line originated with cross-striated muscle of C3Hmouse), and Neuro-2a (a mouse neural crest-derived cell line).
The cells were seeded on the glass plate at the density of 1000
cells/cm2.
C2C12 of the passage eight was cultured in D-MEM(Dulbecco’s Modified Eagle’s Medium) containing 10%
decomplemented FBS (fetal bovine serum) and 1% penicillin/
streptomycin. After the cells were cultured for 12 hours in theresting state, excess gravitational stimulation was applied forfive hours. In the test with C2C12, the gravity of 50 G (r =0.045 m, group X1), and the gravity of 100 G (r = 0.09 m, groupX2) were applied at ω = 104 rad/s.
Neuro-2a of the passage four was cultured in D-MEM(Dulbecco’s Modified Eagle’s Medium) containing 10%
decomplemented FBS (fetal bovine serum) and 1% penicillin/

To apply the excess gravity, the glass plate, on which cells
adhered, was set in the medium in the tube in the position.
The angle of the glass plate in the tube was adjusted to that ofgroup X or Y (Fig. 1). Several tubes with the glass plate were
set in the rotor to cultivate cells of group X and Y
simultaneously. To keep the content of carbon dioxide of 5 %at 310 K, the cells were incubated in an incubator through theentire experimental term including the term of exposure to theexcess gravitational field. Namely, the centrifugal machine
was placed in the incubator during the centrifugation (Fig. 3b).
In the control group (normal force of 1 G), the cells were
cultured without centrifuge on the glass without collagencoating, which was placed in the polystyrene dish (60 mm
diameter) with collagen coating.
Image Analysis
After stimulation of the excess gravity, the glass plate was
moved from the centrifugal tube to the dish. Cells were
subsequently cultured in the dish placed in the incubator
combined with an inverted phase-contrast microscope(LCV110-SK, Olympus Co., Ltd., Tokyo). The behavior ofeach cell was analyzed on the time lapse image captured every
ten minutes for 24 hours after stimulation of the excess gravity.
“Image J” was applied to analyze the behavior of each cell.
On the microscopic image, the outline of each cell was traced,
and the contour of each cell was approximated to ellipsoid (Fig.
4a). As to the ellipsoid, the length of the major axis (a), andthe minor axis (b) were measured. The ratio of axes iscalculated as the shape index (P) by Eq. 3.
P=a/b

(3)

At the circle, P =1. As the ellipsoid becomes flat, P
approaches to zero. The angle (0 degree < θ < 90 degree)
between the direction of the centrifuge and the direction of the
major axis of each cell was measured at the microscopic image(Fig. 4b). When the major axis is parallel to the direction ofcentrifuge, θ = 0. When the major axis is perpendicular to thedirection of centrifuge, θ = 90 degree. At Neuro-2a, the lengthof neurite was measured, and the elongation was calculated by
Eqs. 4 & 5.
ΔL = L1 － L0
R =ΔL / L0

(4)
(5)

In Eq. 4, ΔL is elongation, R is elongation ratio, L0 is length at
the stopping of stimulation, L1 is length at 24 hours after
stopping of stimulation. The angle between the direction ofneurite and the direction of centrifuge was also measured. To
evaluate differentiation of Neuro-2a, the extension of neurite
was observed.

Fig. 4a: Outline of each single cell is traced, and approximatedto ellipsoid.

θFig. 4b: Angles θ between direction of centrifuge (horizontal)
and direction of major axis of cell.

3. RESULTS
Fig. 5 shows the angle between the direction of the tangential
force (on the surface of the scaffold by centrifuge) and the
major axis of each cell. In Figs. 5 and 7, data are arranged in
the ascending 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. 5 shows the following results.
In the control study, the angles distribute at random. C2C12
shows tendency to align on the direction to the tangential forceof 100 G by centrifuge just after centrifugation. In 18 hours,
the distribution shifts from 0 to 90 degrees, which shows theshift to the perpendicular direction against the tangential force.
The shift returns to random distribution in 24 hours. At 50 G,
C2C12 shows tendency to align on the diagonal (60 degrees)
direction to the tangential force by centrifuge in 24 hours (Fig.
5e).
Several cells are divided in 24 hours after stopping of thestimulation of centrifuge for 5 hours. Fig. 6 exemplifies cell
division of C2C12 after centrifuge of 100 G. The directions of
the major axes of both cells are perpendicular to the direction of
the tangential force by the centrifuge (Fig. 6). Fig. 7 shows
the distribution of the angle between the direction of thetangential force (by centrifuge) and the major axis of each cellat one hour after cell division. Most of cells distribute in therange of the angle lager than 45 degree after centrifuge of 100 G.
In the control group, on the other hand, the angle distribute at
random.
Fig. 8a exemplifies the shape index of each cell of C2C12 ofeach group immediately after stimulation of the centrifuge forfive hours. Fig. 8b shows the distribution of the shape index
traced every six hours. In Fig. 8b, the column shows the meanvalue, and the bar shows the range (±) of the standard deviationof 38 samples. Fig. 8 shows that data scatter and do not havestatistically significant differences among groups. In thegroup of 50 G, the value of shape index (P) tends to become
smaller than the other group after stimulation of the centrifuge.
The value of P becomes smaller, when the cell is elongated.

Fig. 9 exemplifies the elongation of the neurite of Neuro-2a
after stopping of the stimulation of 5 G for one hour. The
direction of the tangential force of the centrifuge is from right toleft in Fig. 9. Each triangle shows each end of neurite. The
neurite extend with time: 11 hours, 11 hours and 45 minutes, 12hours and 50 minutes, and 15 hours and 40 minutes after
centrifuge. The direction of the extension is parallel to that ofcentrifuge at first, and curves to perpendicular direction against
centrifuge with time. Fig. 10 shows the elongation ratio of theneurite of Neuro-2a. Each bar shows mean value of fivesample data from each group. The neurite extends in thegroup of X longer than in the control group. Fig. 11
exemplifies the tracing of orientation of C2C12 at each groupby the time lapse image of every ten minutes: control (normal
direction of 1G), X1 (50 G), and X2 (100 G). Data shows theactive random movement of each cell. In the group of 50 G,
the fluctuation decrease in the movement of each cell. Fig. 12
exemplifies the tracing of the shape index of C2C12 at eachgroup: control (normal direction of 1G), X1 (50 G), and X2 (100G). Data shows the active random movement of each cell.
The movement tends to decrease in the group of X 1.

θ [deg]Cell number

Fig. 5a: Angle of C2C12: immediately after centrifuge:rhombus, control; square, 50 G; triangle, 100 G.
θ [deg]Cell number

Fig. 5b: Angle of C2C12: 6 hours after centrifuge: rhombus,
control; square, 50 G; triangle, 100 G.
θ [deg]Cell number

Fig. 5c: Angle of C2C12: 12 hours after centrifuge: rhombus,
control; square, 50 G; triangle, 100 G.

θ [deg]

P

Cell number

Fig. 5d: Angle of C2C12: 18 hours after centrifuge: rhombus,
control; square, 50 G; triangle, 100 G.
θ [deg]Time [hour]Fig. 8b: Shape index of C2C12 every six hours: from left,
control, 50 G, 100 G: mean (column) ± S.D. (bar): n=38.

Cell number

Fig. 5e: Angle of C2C12: 24 hours after centrifuge: rhombus,
control; square, 50 G; triangle, 100 G.

Fig. 9: Elongation of neurite of Neuro-2a: 11 h after centrifuge(upper left), 11 h 45 m (upper right), 12 h 50 m (lower left), and
15 h 40 m (lower right): centrifuge from right to left; dimension
from left to right of each figure is 1 mm. Each triangle showseach end of neurite.
Fig. 6: Cell division (in circle): at 760 min (left) and at 820 min(right) after centrifuge of 100 G: arrow shows centrifuge, andbar shows 0.2 mm.

Elongation ratio (R)θ [deg]Cell number

Fig. 7: Angle of C2C12: one hour after division of cell,stimulated by centrifuge: rhombus, control; square, 50 G;
triangle, 100 G.

XYControl

Fig. 10: Elongation ratio of neurite of Neuro-2a: mean (bar): n= 5.
θ [deg]Shape index (P)Control

50 G

100 G

Fig. 8a: Shape index of C2C12 immediately after stimulation ofcentrifuge: horizontal bar shows mean value.

Time [min]Fig. 11a: Orientation tracing of C2C12 at control.

4. DISCUSSIONθ [deg]Time [min]Fig. 11b: Orientation tracing of C2C12 at 50 G.
θ [deg]Time [min]Fig. 11c: Orientation tracing of C2C12 at 100 G.
P

Time [min]Fig. 12a: Shape index tracing of C2C12 at control.
PTime [min]Fig. 12b: Shape index tracing of C2C12 at 50 G.
PTime [min]Fig. 12c: Shape index tracing of C2C12 at 100 G.

The previous studies showed that a mechanical field governs
behavior of cells [3-21]. The direction of the mechanical field
affects fibroblasts [15]. The effect of shear flow on orientationof cells depends on the kinds of cells [20]. Although HUVEC(human umbilical vein endothelial cells) orients along thestream lines, C2C12 tilts from the stream lines to makemyotubes. When mechanical stimulation is applied to thescaffold, the whole stimulation cannot always be transmitted tothe cells. When the tension applied to a scaffold, thedeformation of the scaffold generates compression and shear in
the different direction simultaneously [15].
To apply
continuous uniform mechanical stimulation to the cells,
centrifugal force is used in the present study. The effect of
mechanical field on orientation and deformation of several
kinds of cells was studied using centrifuge in vitro in theprevious studies [5-8]. The differentiation of cells was
accelerated by the mechanical stimulation by centrifuge in theprevious study [13]. The response of biological system to themicrogravity field has been studied using a space satellite.
The cell cycle might extend in the space [1, 2]. C2C12
differentiation might be accelerated and the myotube mightalign to the direction perpendicular to the centrifugal field [7].
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, on the other hand, sheared on
the surface with the tangential force. The variation was madeon the position (radius) of cell culture in the same rotor of thecentrifuge, to test in the different gravitational environment
simultaneously. Cells passively follow the direction in thestrong tangential force field.
Immediately after thecentrifugation, cell might start to show active response to themechanical stimulation. Mild tangential force field induces
the active reflection of cells to tilt perpendicularly to decreaseinternal force of the cells. The cell shows adaptation against
stimulation. The stimulation leaves hysteresis in the cell.
The gravitational stimulation governs the behavior of the cell
after stimulation. The cells might change the orientation at thedivision and at differentiation. C2C12 made perpendicularorientation of myotubes to the flow direction in the previous
study [20]. The orientation of C2C12 at diagonal direction offlow might be preparation to make perpendicular orientation of
myotubes in the successive cultivation.
C2C12 might
differentiate earlier on polystyrene dish than glass plate [5].
Migration of a cell on the scaffold plate was observed inprevious studies. The time lapse image of every ten minutes iseffective to trace the movement of each cell. The response of
the cell depends on the direction of the gravitational field.
Each cell changes its own direction during migration. The
migration depends on the morphology of the scaffold surface[17]. The movement of the cell is observed on the flat surfaceof plate in the present study. When a cell cannot keepadhesion under stimulation, the shape index approaches to “1”to be a sphere. Cells are exposed to 5 G for one hour in thepresent test of Neuro-2a, because Neuro-2a was not able tosurvive under centrifugation for one hour at 100 G. During
centrifugation, the direction of the force field tilts fromhorizontal direction because of the gravity (1 G) of the earth.
In the case of centrifuge of 5 G, the shift of the angle is 11
degree. The gravity in the fluid is reduced by the buoyancy.
The density of the cell of C2C12 is 1.065×103 kg m–3 measured
by the centrifugal method: each separator adjusted to various

density of the fluid of phthalate-ester is compared with thedensity of the cell in the glass capillary by centrifuge [22].
The density of medium of DMEM, on the other hand, is1.0055×103 kg m–3. The difference between two densities is0.0595×103 kg m–3. The effect of the centrifugal force on thecell in the medium is 6 G at centrifuge of 100 G (group X 2 inthe present study).

5. CONCLUSION
The effects of excess gravitational field on orientation andextension of cells have been studied using centrifuge in vitro.
Two kinds of cells were used in the test: C2C12 (mouse
myoblast cell line), and Neuro-2a (mouse neural crest-derived
cell line). The behavior of cells was traced at the time lapse
images for 24 hours, after excess gravitational stimulation(<100 G) for 1 or 5 hours. The experiment shows following
results. The longitudinal axis of C2C12 tends to turn to thedirection perpendicular direction.
C2C12 aligns to thedirection perpendicular against the excess gravity at division.
The neurite of Neuro-2a tends to extend apart from the excess
gravity. The present study shows that the excess gravitational
stimulation affects the behavior of cells after stimulation.

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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