Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

Effect of Hysteresis of Stimulation of Tangential Force Field on
Alignment of 3T3-L1 Cultured on Micro Striped Pattern
Yuji ENDO, Shigehiro HASHIMOTO, Takuya TAMURA, Ryota MATSUZAWA
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACT

These behaviors might depend on the history of each cell.
While the strong stimulation above the threshold damages thecell, the stimulation below the threshold remains in the cell as amemory for the response in the next step. To trace thehysteresis effect of the mechanical stimulation on the single cell,the time-lapse images are effective. In the previous study, thelongitudinal axis of C2C12 (mouse myoblast cell line) tends toalign to the direction of the excess gravity. The axis tilts to theperpendicular direction, on the other hand, after stopping of theexcess gravity [9]. A single cell migrates at random on thescaffold. The cell tends to align to the longitudinal directionof the micro ridge line [10].

The effect of hysteresis of the tangential force field at thesurface of the scaffold on the single cell has been studied invitro. The striped pattern (0.7 μm height, 3 μm width, and 3
μm interval) were made on the surface of the scaffold plate tocontrol the orientation of each cell. Variation was made on theangle between the longitudinal direction of the ridge and the
direction of the tangential force: 0 degree, 45 degrees, and 90
degrees. 3T3-L1 (mouse fat precursor cells) was used in theexperiment. To apply the tangential force field (100 G) to thecells, the scaffold plate was set in the tube in a conventional
centrifugal machine placed in an incubator.
After thecentrifugation for 5 hours, the behavior of each cell at the timelapse microscope images was traced for 24 hours to analyze theangle between the longitudinal axis of the cell and the directionof the centrifugation. The experimental results show that thetendency of cells to align the longitudinal direction of the
striped pattern is strengthened by the hysteresis of the exposure
to the tangential force field. The tendency continues before
the proliferation of each cell.
Keywords: Biomedical Engineering, Cell Culture, Excess
Gravity and 3T3-L1.

In the present study, centrifugal force has been used to apply thetangential force field on the surface of the scaffold. The
hysteresis effect of the force field on the single cell attached on
the scaffold has been investigated. To examine the effect ofthe direction of the force field on the cell at the specificalignment, the orientation of the cell was controlled by the
striped pattern on the surface of the scaffold. The effect of themechanical field on orientation and deformation of a single cellon the micro striped pattern has been studied by centrifuge invitro, and the behavior of each cell has been traced after
stopping of the stimulation of the excess gravity.

1. INTRODUCTION

2. METHODS

The recent cell culture technique enables observation of the
behavior of each cell in vitro. A biological cell adheres on thescaffold and shows several active behaviors: migration,
deformation, and proliferation. The cell is deformed by theforce, because of its compliance. The cell deforms, on theother hand, to minimize the intra force. The cell is moved by
the force. The cell, on the other hand, moves by itself.
3T3-L1 is cell line of mouse fat precursor cells. In theprevious study, both the migration and the deformation of
3T3-L1 were restricted by the wall shear stress higher than 1 Pa
in flow in vitro [1]. Deformability of 3T3-L1 was observed by
the slit in the previous study in vitro [2, 3]. 3T3-L1 extended
along the lines of micro ridges on the scaffold surface [4].
Vibration decelerated adhesion of 3T3-L1 on the micro ridges[5]. 3T3-L1 migrated regardless of the direction of thevibrating micro ridges. The orientation of 3T3-L1 was tried tobe measured by the electric impedance in vitro [6]. Bothdifferentiation and growth of 3T3-L1 were delayed with electricpulses in the previous study in vitro [7]. The floating cells of
3T3-L1 were tried to be sorted by the micro groove on the wallof the flow channel [8].

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 centrifuge: 0 degree (parallel), 45
degree, and 90 degree (perpendicular). Three partial area wasmade on the same surface of the scaffold plate in parallel
position, so that the behavior of cells on each area can becompared simultaneously (Fig. 2). The pattern of each area
was also used as a marker to trace each cell.
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, the7Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

oxygen (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).

In the present study, the centrifugal acceleration of 100 G (1 G
is equal to the gravitational acceleration) ((Fc / Fg) = 100) is
generated with the centrifugal machine (CN-1040,
Matsuuraseisakusyo. Ltd, Tokyo, Japan).

To improve affinity to photoresist material, HMDS
(hexamethyldisilazane: Tokyo Chemical Industry Co., Ltd.,Tokyo) was coated on the glass plate at 3000 rpm for 30 s witha spin coater. The positive photoresist material of OFPR-800(Tokyo Ohka Kogyo Co., Ltd, Tokyo, Japan) was coated on theglass with the spin coater (at 3000 rpm for 20 s). The
photoresist was baked in the oven (DX401, Yamato ScientificCo., Ltd) at 373 K for ninety seconds.

Cell Culture
3T3-L1 (mouse fat precursor cells, a cell line derived from cellsof mouse 3T3) of the passage between fourth and tenth wasused 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 the glass plate at the density of 3000
cells/cm2. After the cells were cultured for 12 hours in thestatic state (1 G: normal), excess gravitational stimulation wasapplied for five hours. The gravity of 100 G (r = 0.09 m) wereapplied at ω = 104 rad/s.

The micro pattern 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 with tetra-methyl-ammoniumhydroxide (NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki,
Japan) for two minute, rinsed with the ultrapure water for threeminutes twice, and dried by the spin-dryer (SF-250, Japan
Create Co., Ltd., Tokorozawa, Japan). The glass plate with thephotoresist material was etched with the 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 apply the excess gravity, the plate, on which cells adhered,was set in the medium in the tube. Several tubes with theglass plate were set in the rotor of the centrifugation to cultivate
cells, simultaneously. To keep the content of carbon dioxideof 5 % at 310 K, the cells were cultured in an incubator throughthe entire experimental term including the term of exposure to
the hyper-gravitational field. Namely, the centrifugal machine
was placed in the incubator during the centrifugation.

To remove the residual OFPR-800LB on the surface of the glass,the oxygen (0.1 Pa, 30 cm3/min) plasma ashing was applied tothe surface of the glass at 100 W for five minutes by a reactive
ion etching system (FA-1).

In the control group, the cells were cultured without centrifuge
on the plate, which was placed in the polystyrene dish. In thecontrol group, no tangential force field was applied, and only
the gravitational force (1 G) normal to the scaffold surface plate
was applied.

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 the cell
culture.

Image Analysis
After stimulation of the excess gravity, cells on the glass plate
were moved from the centrifugal tube to the culture 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 images captured every
five 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.
1). On the ellipsoid, the length of the major axis (a), and theminor axis (b) were measured. The ratio of axes is calculated
as the shape index (P) by Eq. (3).
P=1 － b/a

Tangential Force Field
The tangential force field was applied to the culture surface
with the centrifugal force. The culture plate is inserted into thetube, which is contained in the rotor. The angle between theradial 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 so that the culture surface is parallel to the radial
direction of the rotation. The centrifugal force is applied
parallel to the culture surface. The centrifugal force (Fc) is
calculated by Eq. (1).
Fc = m r ω2

(1)

10 μm

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(3)

Fig. 1: On microscopic image, outline of each cell was traced(left), and contour of each cell was approximated to ellipsoid(right).

(2)

8

Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

At the circle, P = 0.
approaches to 1.

As the ellipsoid is elongated, P
0The angle (0 degree < θ < 90 degrees) between the direction of
the centrifuge and the direction of the major axis of each cell
was measured at the microscopic image. When the major axis
is parallel to the direction of centrifuge, θ = 0. When the
major axis is perpendicular to the direction of centrifuge, θ = 90
degree.

45

90
3. RESULTS
Centrifugal force

By the time-lapse images, continuous activity of each cell was
confirmed: migration, deformation, and proliferation (Figs. 2a& 2b). Each cell repetitively extends pseudopods. The
centrifuge direction was easily found by the stripe patterns of 0
degree. The behavior of each cell was easily traced with thehelp of the strip patterns as markers.

(a)0

In Figs. 3 & 5, each datum point shows the value of each cell at
12 hours, 17 hours, 23 hours, 29 hours, 35 hours, and 41 hours
after seeding. The timing of every 6 hours coincides to thecentrifugal force stimulation group.

45

In Figs. 4 & 6, each datum point shows the value of each cell
before centrifuge (before), immediately after stopping
centrifuge (after), and every 6 hours after stopping centrifuge,
respectively: before centrifugal force stimulation, immediately
after centrifugal force stimulation for 5 hours, 6 hours after
centrifugal force stimulation, 12 hours after centrifugal force
stimulation, 18 hours after centrifugal force stimulation, andafter 24 hours after centrifugal force stimulation.

90

Centrifugal force(b)

0.5 mmFig. 2: Cells immediately after stopping centrifuge (a) at 24hours after stopping centrifuge (b): 0 degree (upper), 45 degree(middle), and 90 degree (lower) in each picture.

Each cell elongates at random orientation on the flat surface
with no striped pattern without centrifugation: the angles θscatters in Fig. 3a. The tendency is the same on the flat
surface after centrifugal force stimulation of 100 G (Fig. 4a).

Angle [degree]

90

Without centrifugation, each cell not only elongates along thelongitudinal direction of the striped pattern, but also tilts torandom orientation on the striped pattern: 0 degree in Fig. 3b,
45 degree in Fig. 3c, and 90 degree in Fig. 3d.

45Each cell tends to align along the striped pattern by thecentrifugal force stimulation of 100 G: 0 degree in Fig. 4b, 45
degree in Fig. 4c, and 90 degree in Fig. 4d. The tendency isrelieved in 6 hours after stopping the stimulation, and cells tend
to apart from the centrifugal force direction. No cell is in thearea of 45 degree at 12 hours, after stopping of the centrifugal
force stimulation (Fig. 4c).

0
012

17
1

23
2

29
3

35
4

415

Time
6 [hour]Fig. 3a: Angle of 3T3-L1: control, no pattern.
Angle [degree]

On the flat surface with no striped pattern, each cell randomly
repeats deformation between elongation and shrinkage without
centrifugation: the shape index (P) scatters (Figs. 5a & 6a).
On the striped pattern, on the other hand, cells tend to elongate
more frequently than on the flat surface in 24 hours. The
shape index (P) tends to increase on the striped pattern: 0
degree in Fig. 5b, 45 degree in Fig. 5c, and 90 degree in Fig. 5d.
Some cells deform to the round shapes and proliferate after 6hours from stopping the centrifugal force stimulation on the
striped pattern (after 24 hours from seeding): 0 degree in Fig. 6b,
45 degree in Fig. 6c, and 90 degree in Fig. 6d.

12

17

23

29

35

41

Time [hour]Fig. 3b: Angle of 3T3-L1: control, 0 degrees.

9Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

Angle [degree]

12

17

23

Angle [degree]

29

35

41

Time [hour]
BeforeFig. 3c: Angle of 3T3-L1: control, 45 degrees.

17

23

29

35

41

Time [hour]

1224Time [hour]

17

23

29

35

41

Time [hour]

Shape index (P)

After

6

12

18

24

12

Time [hour]

17

23

29

35

41

Time [hour]Fig. 5b: Shape Index (P) of 3T3-L1: control, 0 degree.

Fig. 4a: Angle of 3T3-L1: 100 G, no pattern.

Shape index (P)Angle [degree]

12
After

18

Fig. 5a: Shape Index (P) of 3T3-L1: control, no pattern.

Angle [degree]

Before12

Shape index (P)Fig. 3d: Angle of 3T3-L1: control, 90 degrees.

Before6

Fig. 4d: Angle of 3T3-L1: 100 G, 90 degrees.

Angle [degree]

12

After

6

12

18

24

Time [hour]

17

23

29

35

41

Time [hour]Fig. 5c: Shape Index (P) of 3T3-L1: control, 45 degrees.

Fig. 4b: Angle of 3T3-L1: 100 G, 0 degree.

Shape index (P)Angle [degree]

Before

After

6

12

18

24

12

Time [hour]Fig. 4c: Angle of 3T3-L1: 100 G, 45 degrees.

17

23

29

35

41

Time [hour]Fig. 5d: Shape Index (P) of 3T3-L1: control, 90 degrees.

10Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

The average diameter of the floating cell in the medium is 20
μm. The low ridge and the narrow width were selected so as
not to disturb cell migration. Both deformation and migrationof 3T3-L1 are at random over the striped pattern in the present
study. Immediately after the tangential force stimulation of
100 G, 3T3-L1 frequently tilts to the striped pattern in thepresent study (Figs. 4b–d). C2C12, on the other hand, tended
to elongate and align along the striped pattern even in the casewithout the tangential force stimulation in the previous study[9]. The tilting tendency of C2C12 was enhanced by thetangential force of 100 G. C2C12 tends to elongate during thetangential force stimulation. The mild tendency was observedon MC3T3-E1 in the previous study [11]. The behavior ofeach cell can be easily traced with the aid of the micro striped
pattern on the scaffold as the marker. The tracking ability bothto the tangential force and to surface morphology was higher inC2C12 than in MC3T3-E1 in the previous study. The tracking
ability of 3T3-L1 is much lower than that of MC3T3-E1. On
the striped pattern parallel to the tangential force (0 degree),every type of cell tracked along the striped pattern. The
tracking ability on another direction of striped pattern depends
on the cell types.

Shape index (P)Before

After

6

12

18

24

Time [hour]Fig. 6a: Shape Index (P) of 3T3-L1: 100 G, no pattern.
Shape index (P)Before

After

6

12

18

24

Time [hour]

Most of cells do not peel off, but keep adhesion on the scaffold
and randomly migrate on the scaffold. 3T3-L1 tends tomigrate from the area of 45 degree, and to refrain from
migration to the area of 45 degree in 12 hours after hysteresis of
the tangential force stimulation of 100 G (Figs. 4c & 6c).
C2C12, on the other hand, tended to stay on the micro striped
pattern regardless of the angle between the longitudinal
direction of the concave-convex striped pattern and thetangential force. In the previous study, C2C12 was mostfrequently elongated on the striped pattern of 45 degree
between the tangential force direction and the longitudinal
direction of the stripe [9]. The elongated direction of thepseudopods of the cell aligns to the longitudinal direction of thepattern on the scaffold. The cell elongates more frequently on
the striped pattern than on the flat surface.

Fig. 6b: Shape Index (P) of 3T3-L1: 100 G, 0 degree.
Shape index (P)Before

After

6

12

18

24

Time [hour]Fig. 6c: Shape Index (P) of 3T3-L1: 100 G, 45 degrees.
The gravity in the fluid is reduced by the buoyancy.
Measurement of the density of cells by Phthalate ester method
shows that the mean density of each cell is 1.07×103 kg/m3.
When the cells floating in the medium of the density of
1.00×103 kg/m3, the effective centrifugal force ratio calculated
from the difference of two density is 7 G at centrifuge of 100 G.

Shape index (P)Before

After

6

12

18

24

The previous studies showed that the mechanical [12] and theelectrical [13–15] fields governs behavior of cells. The effectof shear flow on orientation of cells depends on the kinds ofcells [16, 17]. The response of biological system to themicrogravity field has been studied using a space satellite.
C2C12 differentiation might be accelerated and the myotube
might align to the direction perpendicular to the centrifugal
field [18, 19].
The previous study shows that thehyper-gravitational field thickens the myotubes in vitro [20].
The hypergravity promotes proliferation and differentiation ofcells [21, 22]. The acceleration technique for orientation,
proliferation and differentiation of cells has been studied tomake tissue in vivo or in vitro. Control methodology fororientation, proliferation and differentiation of cells would beapplied to the regenerative tissue technology.

Time [hour]Fig. 6d: Shape Index (P) of 3T3-L1: 100 G, 90 degrees.

4. DISCUSSION
The hysteresis of centrifugation might make each cell toincrease the contact area with the surface of the scaffold. Eachcell might adapt to the tangential force field by maximization of
the contact area between the cell and the scaffold.
To control the alignment 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 isslightly higher than threshold to make orientation of cells [10].

The present experimental results show following results.
3T3-L1 shows migration, deformation, and proliferation, evenafter the tangential centrifugal force stimulation of 100 G.

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Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

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Without the tangential centrifugal force stimulation, each cell
not only elongates along the longitudinal direction of the striped
pattern, but also tilts to random orientation on the striped
pattern. The tendency of cells to align the longitudinal
direction of the striped pattern is strengthened by the hysteresisof the exposure to the tangential force field. The tendency ofeach cell continues before its proliferation.

5. CONCLUSION
The effect of hysteresis of the tangential force field at thesurface of the scaffold on the single cell has been studied invitro. The striped pattern (0.7 μm height, 3 μm width, and 3
μm interval) were made on the surface of the scaffold plate tocontrol the orientation of each cell. Variation was made on theangle between the longitudinal direction of the ridge and the
direction of the tangential force: 0 degree, 45 degrees, and 90
degrees. The experimental results show that the tendency of
3T3-L1 (mouse fat precursor cells) to align to the longitudinal
direction of the striped pattern is strengthened by the hysteresisof the exposure for 5 hours to the tangential centrifugal force
field (100 G). The tendency continues before the proliferationof each cell. The average cell cycle of proliferation is 24
hours after seeding.

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