Alignment and Deformation of MC3T3-E1 Cultured on Micro
Striped Pattern after Stimulation of Tangential Force Field
Yuji ENDO, Shigehiro HASHIMOTO, Takuya TAMURA
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACTThe effect of hysteresis of the tangential force field on the
single cell cultured at the surface of the scaffold with the micro
striped pattern has been studied in vitro. The lines of parallel
micro quadrangular ridges (0.7 μm height, 3 μm width, and 3
μm interval) were made on the surface of the scaffold plate by
the photolithography technique to control the orientation ofeach cell. Variation was made about the angle between thelongitudinal direction of the ridge and the direction of thecentrifugal force field: 0 degree, 45 degrees, and 90 degrees.
To apply the tangential force field (50 G, 100 G) on the surfaceof the scaffold, the plate was set in the tube in a conventional
centrifugal machine placed in an incubator.
After thecentrifugation for 5 hours, the contour of each cell at the time
lapse microscope images was traced for 24 hours in the
incubator, and the angle between the longitudinal axis of thecell and the direction of the centrifugation was analyzed. The
experimental results show that the MC3T3-E1 (mouse
osteoblast precursor cell) elongates to the longitudinal directionof the striped pattern by the hysteresis of the exposure to thetangential force field more frequently at 100 G than at 50 G.
Keywords: Biomedical Engineering, Cell Culture, Excess
Gravity and MC3T3-E1.

1. INTRODUCTION
The microgravity environment might weaken the mechanicalsupporting system of the biological body. MC3T3-E1 is thecell line of mouse osteoblast precursor cell. The bone
remodeling is controlled by the balance between osteoblast andosteoclast. In the previous study, myotube thickness increasedin the hyper gravity environment in vitro [1]. The shear flow
affected migration of osteoblasts in the previous experiment invitro [2]. MC3T3-E1 made orientation along the stream line
after the stimulation of the wall shear stress [3]. The floating
cells of MC3T3-E1 were tried to be sorted by the micro hole [4]or by the oblique micro groove [5]. The recent cell culture
technique enables observation of the behavior of each cell in
vitro. A biological cell adheres on the scaffold and shows
several active behaviors: migration, deformation, andproliferation. These behaviors might depend on the history ofeach cell. While the strong stimulation above the thresholddamages the cell, the stimulation below the threshold remains in
the cell as a memory for the response in the next step. To trace
the hysteresis effect of the mechanical stimulation on the single

cell, the time laps images are effective. In the previous study,
the longitudinal axis of C2C12 (mouse myoblast cell line) tends
to align to the direction of the excess gravity, and to turn to theperpendicular direction after stopping of the excess gravity [6,7]. A single cell migrates at random on the scaffold. The cell
tends to align to the longitudinal direction of the micro ridge
line [8].
In the present study, the centrifugal force has been used to apply
the tangential force field on the surface of the scaffold. The
hysteresis effect of the force field on the single osteoblast cellattached on the scaffold has been investigated. To examine theeffect of the direction of the force field on the cell at the
specific alignment, the orientation of the cell has been
controlled by the striped pattern on the surface of the scaffold.

2. METHODS
Micro-pattern on Scaffold Plate
The micro striped pattern has been made in three partial
rectangular areas of 0.4 mm × 1.6 mm on the PDMS
(polydimethylsiloxane)
plate
of
the
scaffoldbyphotolithography technique. The height (H), the width (W),and the interval (I) of the quadrangular ridges are 0.7 μm, 3 μm,
and 3 μm, respectively. Each area has its own specificdirection of the striped pattern. Namely, variation has been
made on the angle (θ) between the longitudinal direction of theridge and the direction of centrifugal force in the twodimensional scaffold surface: 0 degree (parallel), 45 degree, and90 degree (perpendicular). Three partial area was made on thesame surface of the scaffold plate in parallel position, so that the
behavior of cells on each area can be compared simultaneously.
The pattern of each area was also used as a marker to trace eachcell.
Mold for Micro Pattern
The borosilicate glass (Tempax) disk was used for the base of
the mold through micromachining process. The diameter andthe thickness of the disk are 50 mm and 1.1 mm, respectively.
To remove micro particles on the surface of the glass, theoxygen (0.1 Pa, 30 cm3/min) plasma ashing was applied to thesurface of the glass at 100 W for five minutes by a reactive ion
etching system (FA-1, Samco Inc., Kyoto, Japan). To improveaffinity to photoresist material, HMDS (hexamethyldisilazane:Tokyo Chemical Industry Co., Ltd., Tokyo) was coated on theglass plate at 3000 rpm for 30 s with a spin coater. The
positive photoresist material of OFPR-800 (Tokyo Ohka Kogyo

Co., Ltd, Tokyo, Japan) was coated on the glass with the spincoater (at 3000 rpm for 20 s). The photoresist was baked in
the oven (DX401, Yamato Scientific Co., Ltd) at 373 K for
ninety seconds.
The pattern for the slit was drawn on the mold with a laser
drawing system (DDB-201K-KH, Neoark Corporation,
Hachioji, Japan). The pattern was baked in the oven at 373 K
for five minutes.
The photoresist was developed withtetra-methyl-ammonium hydroxide (NMD-3, Tokyo Ohka
Kogyo Co., Ltd., Kawasaki, Japan) for two minutes, rinsed withthe ultrapure water for three minutes, and dried by the
spin-dryer (SF-250, Japan Create Co., Ltd., Tokorozawa,
Japan).
The glass plate with the photoresist material was etched withthe plasma gas using RIE-10NR (Samco International, Kyoto,
Japan). For etching, the gas of CF4 (30 cm3/min at 1013 hPa)
was applied at 100 W at 2 Pa. To remove the residual
OFPR-800LB on the surface of the glass, the oxygen (0.1 Pa,
30 cm3/min) plasma ashing was applied to the surface of theglass at 100 W for five minutes by a reactive ion etching system(FA-1).
After the mold of the glass disk was enclosed with a peripheral
wall of polyimide tape, PDMS (Sylgard 184 Silicone Elastomer
Base, Dow Corning Corp., MI, USA) was poured with thecuring agent (Sylgard 184 Silicone Elastomer Curing Agent,
Dow Corning Corp., MI, USA) on the mold. The volume ratioof PDMS to curing agent is ten to one. After degassing,
PDMS was baked at 338 K for one hour in an oven (DX401).
The baked plate of PDMS (1 mm thickness) was exfoliatedfrom the mold of the slide glass plate. The PDMS plate wascut to make a plate of 15 mm × 10 mm × 1 mm, and stacked on
the glass plate of 50 mm × 13 mm × 1 mm. The surface of the
PDMS plate was exposed to the oxygen gas (0.1 Pa, 30
cm3/min) in the reactive ion etching system (FA-1: oxygen
plasma ashing, 50 W) for thirty seconds just before cell culture.
Tangential Force Field
The tangential force field was applied to culture surface withthe centrifugal force. The culture plate is inserted in the tube,
which is contained in the rotor. The angle between the radial
direction of the rotation of the rotor and the axial direction of
the tube in the rotor is 60 degree. The glass plate was set sothat the culture surface is parallel to the radial direction of therotation. The centrifugal force is applied parallel to the culture
surface. 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, the gravitational force (Fg)is calculated by Eq. 2, where g is gravitational acceleration.
Fg = m g

Cell Culture
MC3T3-E1 (passage nine, osteoblast precursor cell line derived
from mouse calvaria) was used in the tests. Cells were
cultured in D-MEM (Dulbecco’s Modified Eagle’s Medium)
containing 10% decomplemented FBS (fetal bovine serum) and
1% penicillin/ streptomycin. The cells were seeded on theglass plate at the density of 3000 cells/cm2. After the cells
were cultured for 12 hours in the static state (1 G: normal), thecentrifugal force field stimulation was applied for five hours.
The centrifugal force field of 50 G (r = 0.045 m) or 100 G (r =0.09 m) were applied at ω of 104 rad/s.
To apply the centrifugal force field, the plate, on which cells
adhered, was set in the medium in the tube. Several tubes (50G, 100 G) with the glass plate were set in the rotor to cultivate
cells, simultaneously [6]. To keep the content of carbon
dioxide of 5 % at 310 K, the cells were cultured in an incubator
through the entire experimental term including the term of
exposure to the hyper-gravitational field.
Namely, thecentrifugal machine was placed in the incubator during thecentrifugation. In the control group, the cells were cultured
without centrifuge on the plate, which was placed in thepolystyrene dish. In the control group, no tangential force
field was applied (0 G). Namely, only the gravitational force(1 G) normal to the scaffold surface plate was applied.
After the stimulation of the centrifugation, the glass plate was
moved from the centrifugal tube to the culture dish. After the
stimulation, no tangential force field was applied (0 G). Only
the gravitational force (1 G) normal to the scaffold surface plate
was applied. Cells were subsequently cultured in the dishplaced in the incubator combined with an inverted
phase-contrast microscope (LCV110-SK, Olympus Co., Ltd.,Tokyo). The behavior of each cell was analyzed on the time
lapse image captured every five minutes for 24 hours after
stimulation of the excess gravity.
Image Analysis“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.
On the ellipsoid, the length of the major axis (a), and the minor
axis (b) were measured. The ratio of axes is calculated as theshape index (P) by Eq. 4.
P=1 － b/a

(4)

At the circle, P = 0. As the ellipsoid becomes flat (b < a), P
approaches to unity. The angle (0 degree < θ < 90 degrees)
between the direction of the centrifugal force and the directionof the major axis of each cell was measured at the microscopic
image. When the major axis is parallel to the direction ofcentrifugal force, θ = 0. When the major axis is perpendicular
to the direction of centrifugal force, θ = 90 degree.

(2)
3. RESULTS

In the present study, the centrifugal force is generated with thecentrifugal machine (CN-1040, Matsuuraseisakusyo. Ltd,Tokyo, Japan). Variation has been made at the centrifugal
force ratio (Fr) by adjusting the radius at the position of theculture plate: 50 G, and 100 G. The value of 1 G means thatthe centrifugal force is equal to the gravitational force.
Fr = Fc / Fg

(3)

By the time laps images, continuous activity of each cell was
easily traced: migration, deformation, and proliferation. Eachcell repetitively extends pseudopods (Fig. 1). In Figs. 2 & 5,each datum point shows the value of each cell at 12 hours, 17hours, 23 hours, 29 hours, 35 hours, and 41 hours after seeding.

0.5 mmFig. 1: Cells on striped pattern after 50 G (left) and 100 G(right): 0 degree (upper), 45 degree (middle), and 90 degree(lower): centrifugal force from left to right.

The timing of every 6 hours coincides to the centrifugal force
stimulation group. In Figs. 3, 4, 6, & 7, each datum pointshows the value of each cell before centrifuge (before),
immediately after stopping centrifuge (after), and every 6 hours
after stopping centrifuge, respectively: before centrifuge
stimulation, immediately after centrifuge stimulation for 5 hours,
6 hours after centrifuge stimulation, 12 hours after centrifuge
stimulation, 18 hours after centrifuge stimulation, and after 24hours after centrifuge stimulation. Fig. 2 shows the directionof the major axis of each cell without centrifuge (control study).
Each cell elongates at random orientation on the flat surface
without the striped pattern: the angles θ scatters.

centrifugal force direction (0 degree). Cells tend to alignalong the longitudinal direction of the striped pattern, which isparallel to the centrifugal force direction. The tendency isrelieved in 12 hours after the centrifugal force stimulation. Fig.
4c shows data on the striped pattern of 45 degree. The
tendency of cells tilting to the direction of striped pattern of 45
degrees at 100 G (Fig. 4c) is higher than that at 50 G (Fig. 3c).
Fig. 4d shows data on the striped pattern perpendicular to thecentrifugal force direction (90 degree). Cells tend to alignalong the longitudinal direction of the striped pattern, which isperpendicular to the centrifugal force direction. The tendency
continues for 24 hours after the centrifugal force stimulation.
The number of cells decreases after the centrifugal force
stimulation hysteresis: no cell is on the area of the striped
pattern of 90 degree at 6 hours, and at 18 hours after thecentrifugal force stimulation hysteresis.
Angle [degree]

12 17 23

29

35

41

Time [hour]Fig. 2: Angle of major axis of each cell on flat surface at 0 G.
Figs 3 & 4 show the angle between the direction of the major
axis of each cell and the tangential force (on the surface of thescaffold by centrifuge) of 50 G, and at 100 G, respectively. Fig.
3 shows the angle between the direction of centrifugal force of
50 G and the direction of the major axis of each cell on eachmicro pattern: 0 degree (Fig. 3b), 45 degrees (Fig. 3c), and 90
degrees (Fig. 3d), respectively. Fig. 3a shows data on the flat
surface.
The angle tends to increase after stimulation
hysteresis. Cells tend to tilt to the perpendicular direction to
the centrifugal force after proliferation: after 18 hours. The
number of cells decreases around the centrifuge direction. Fig.
3b shows data on the striped pattern parallel to the centrifugal
force direction (0 degree). Cells tend to align along thelongitudinal direction of the striped pattern, which is parallel tothe centrifugal force direction. The tendency is relieved in 12hours after the centrifugal force stimulation. Fig. 3c shows
data on the striped pattern of 45 degree. The direction of eachcell scatters around 45 degrees.
At 6 hours after thecentrifugal force stimulation, each cell tends to tilt to thedirection of the centrifugal force. In 12 hours after thecentrifugal force stimulation, the direction of each cell scattersagain. Fig. 3d shows data on the striped pattern perpendicular
to the centrifugal force direction (90 degree). Cells tend toalign along the longitudinal direction of the striped pattern,which is perpendicular to the centrifugal force direction. The
tendency continues for 24 hours after the centrifugal force
stimulation.
Fig. 4 shows the angle between the direction of centrifuge of
100 G and the direction of the major axis of each cell on eachmicro pattern: 0 degree (Fig. 4b), 45 degrees (Fig. 4c), and 90
degrees (Fig. 4d), respectively. Fig. 4a shows data on the flat
surface.
The angle tends to increase after stimulation
hysteresis. Cells tend to tilt to the perpendicular direction to
the centrifugal force after proliferation: after 18 hours. The
number of cells decreases around the centrifugal force direction.
Fig. 4b shows data on the striped pattern parallel to theAngle [degree]

BeforeAfter

6 12 18

24

Time [hour]Fig. 3a: Angle between major axis of each cell and tangential
force of 50 G on flat surface: before, after, 6h, 12 h, 18 h, 24 h.
Angle [degree]

Before After

6 12 18

24

Time [hour]Fig. 3b: Angle between major axis of each cell and tangential
force of 50 G on striped pattern of 0 degree.
Angle [degree]

BeforeAfter

6 12 18

24

Time [hour]Fig. 3c: Angle between major axis of each cell and tangential
force of 50 G on striped pattern of 45 degree.

Figs. 5, 6 and 7 show the shape index (P). Fig. 5 shows theshape index of each cell without centrifugal force stimulation(control study) on the flat surface. Cells are elongated within
12 hours after adhesion to the scaffold. Fig. 6 shows the shapeindex of each cell at 50 G on each micro pattern: 0 degree (Fig.
6b), 45 degrees (Fig. 6c), and 90 degrees (Fig. 6d), respectively.
Fig. 6a shows data on the flat surface. Cells tend to berounded after centrifugal force stimulation hysteresis.

Angle [degree]

BeforeAfter

6 12 18

24

Time [hour]Fig. 3d: Angle between major axis of each cell and tangential
force of 50 G on striped pattern of 90 degree.
Angle [degree]

BeforeAfter

6 12 18

24

Time [hour]Fig. 4a: Angle between major axis of each cell and tangential
force of 100 G on flat surface.

Fig. 7 shows the shape index of each cell at 100 G on eachmicro pattern: 0 degree (Fig. 7b), 45 degrees (Fig. 7c), and 90
degrees (Fig. 7d), respectively. The number of rounded cells(small value of P) on the ridges decreases immediately after
centrifugation (Fig. 7b & 7c). Cells tend to be elongated to thedirection of centrifugal force. The tendency is relieved in 12hours after stopping of the centrifugal force stimulation. The
elongation tendency is apparent, when the longitudinal directionof the micro ridge is parallel to the direction of centrifuge (Fig.
7b). The number of cells decreases after the centrifugal force
stimulation hysteresis (Fig. 7d).
Each cell extends along the striped pattern more frequently in 6
hours after centrifugal force stimulation.
The tendency
decreases in 12 hours after centrifugal force stimulation. At
proliferation, each cell tends to elongate to the direction
perpendicular to the hysteretic tangential force direction.

Angle [degree]
Shape index (P)BeforeAfter

6 12 18

24

Time [hour]Fig. 4b: Angle between major axis of each cell and tangential
force of 100 G on striped pattern of 0 degree.
Angle [degree]

BeforeAfter

6 12 18

12 17 23

29

35

41

Time [hour]Fig. 5: Shape index (P) of each cell on flat surface at 0 G.
Shape index (P)24Time [hour]Fig. 4c: Angle between major axis of each cell and tangential
force of 100 G on striped pattern of 45 degree.
Angle [degree]

Before After

6 12 18

24

Time [hour]Fig. 6a: Shape index (P) of each cell on flat surface after 50 G.
Shape index (P)BeforeAfter

6 12 18

24

Time [hour]Fig. 4d: Angle between major axis of each cell and tangential
force of 100 G on striped pattern of 90 degree.

Before After

6 12 18

24

Time [hour]Fig. 6b: Shape Index (P) of each cell on 0 degree after 50 G.

Shape index (P)BeforeAfter

6 12 18

Shape index (P)24Time [hour]Fig. 6c: Shape Index (P) of each cell on 45 degree after 50 G.

BeforeAfter

6 12 18

24

Time [hour]Fig. 7d: Shape Index (P) of each cell on 90 degree after 100 G.

Shape index (P)4. DISCUSSION

BeforeAfter

6 12 18

24

Time [hour]Fig. 6d: Shape Index (P) of each cell on 90 degree after 50 G.
Shape index (P)BeforeAfter

6 12 18

24

Time [hour]Fig. 7a: Shape index (P) of each cell on flat surface after 100 G.
Shape index (P)BeforeAfter

6 12 18

24

Time [hour]Fig. 7b: Shape Index (P) of each cell on 0 degree after 100 G.
Shape index (P)BeforeAfter

6 12 18

24

Time [hour]Fig. 7c: Shape Index (P) of each cell on 45 degree after 100 G.

The gravity in the fluid is reduced by the buoyancy.
Measurement of the density of cells by Phthalate ester method
shows that the density of MC3T3-E1 is between 1.06×103
kg/m3 and 1.07×103 kg/m3. When the cells floating in themedium of the density of 1.00×103 kg/m3, the effective
centrifugal force ratio calculated from the difference of two
density is 6.5 G at centrifuge of 100 G. During centrifugation,
the direction of the force field slightly tilts from horizontal
direction (vertical at the scaffold in the present study) becauseof the gravity (1 G) of the earth. In the case of centrifuge of 5G, the shift of the angle is 11 degree. The time lapse image ofevery ten minutes is effective to trace the movement of eachcell. The response of the cell depends on the direction of thehyper-gravitational field. Each cell changes its own direction
during migration. The migration depends on the morphologyof the scaffold surface [8]. When a cell cannot keep adhesion
under stimulation, the shape index (P) approaches to “0” to be asphere. Each cell deforms and migrates randomly. When thecell rounds, the contact between the cell and the scaffold
decreases. The cell easily exfoliates from the scaffold at thesmall contact area. After the cell rounds at proliferation,alignment of cells along the longitudinal direction of the striped
pattern is relieved. The average cell cycle of proliferation is
24 hours. The term for 41 hours from seeding is selected in
the present experiment. The elongated cell, on the other hand,tends to align along the longitudinal direction of the striped
pattern of ridges on the scaffold surface. The cell on the
striped pattern not parallel to the centrifugal force stimulation(at 45 degree, and at 90 degree) hardly makes contact to thescaffold surface at 100 G. The hysteresis of centrifugation
might make each cell to increase the contact area with thesurface of the scaffold. Each cell tends to align along thepattern on the scaffold surface at the higher tangential force
field (100 G) than at the lower tangential force field (50 G).
Data scatter in Fig. 3c than in Fig 4c. The variation was madeon the position (radius) of cell culture in the same rotor of thecentrifuge, to test in the different gravitational environment
simultaneously [6]. The main reason why number of cells
decrease in Fig. 4d and in Fig. 7d is not exfoliation, but cells
tend to migrate to the direction to keep away from the area ofthe stripes of 90 degree (perpendicular to the centrifugal force
direction). Cells passively follow the direction in the strong
tangential force field. Immediately after the centrifugation,
cell might start to show active response to the mechanicalstimulation. The mild tangential force field induces the active
reflection of myoblasts to tilt perpendicularly to decreaseinternal force of the cells. The cell shows adaptation against
stimulation. The stimulation leaves hysteresis in the cell.
The hyper-gravitational stimulation governs the behavior of thecell after stimulation. The number of cells, which do not alignto the longitudinal direction of ridges, tends to decrease by thehyper-gravitational stimulation. In the previous study, thetendency of the C2C12 to align along the longitudinal directionof ridges is enhanced on the ridge perpendicular to the directionof the excess gravity, after centrifuge [7]. To align the
direction of the single cell on the scaffold before stimulation,
the micro stripe pattern has been used in the present study.
The height of the ridge is designed as 0.7 μm, which is slightly
higher than threshold to make orientation of cells [8]. The
average diameter of the floating cell in the medium is 20 μm.
Both the low height and the small width of the ridge may not
disturb the migration of cells over the ridge. The behavior ofeach cell can be easily traced with the aid of the micro striped
pattern on the scaffold as the marker. To apply continuous
uniform mechanical stimulation to the cells, centrifugal force isused in the present study. The effect of mechanical field onorientation and deformation of several kinds of cells wasstudied using centrifuge in vitro in the previous studies [1, 6, 7,
9–12]. The response of biological system to the microgravity
field has been studied using a space satellite [13–15]. C2C12
differentiation might be accelerated and the myotube mightalign to the direction perpendicular to the centrifugal field [6].
The muscle tissue might decrease in the micro gravitational
field [15]. The muscle tissue might increase, on the other hand,in the hypergravity [1, 16]. The previous study shows that thehyper-gravitational field thickens the myotubes in vitro [1].
The acceleration technique for orientation, proliferation anddifferentiation of cells has been studied to make tissue in vivo orin vitro. Control methodology for orientation, proliferationand differentiation of cells would be applied to the regenerative
tissue technology.

5. CONCLUSION
The effect of hysteresis of the tangential force field on
MC3T3-E1 (mouse osteoblast precursor cell) cultured at thesurface of the scaffold with the micro striped pattern has beenstudied in vitro. The lines of parallel micro quadrangularridges (0.7 μm height, 3 μm width, and 3 μm interval) weremade on the surface of the scaffold plate to control theorientation of each cell. Variation was made about the angle
between the longitudinal direction of the ridge and the directionof the tangential force field: 0 degree, 45 degrees, and 90
degrees. To apply the tangential force field (50 G, 100 G) on
the surface of the scaffold, the plate was set in the tube in aconventional centrifugal machine placed in an incubator. The
experimental results show that the cell elongates to thelongitudinal direction of the striped pattern by the hysteresis of
the exposure to the tangential force field more frequently at 100
G than at 50 G. Some cells are rounded at proliferation, andrelieved to align along the longitudinal direction of the striped
pattern. Cells tend to elongate at the striped pattern parallel tothe tangential force, immediately after the centrifugal
stimulation of 100 G.

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