Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)

Effect of Flow on 3T3-L1 Oriented by Stripe Pattern of Micro
Ridges
Shigehiro HASHIMOTO, Haruka HINO, Shingo OKUZUMI, Yuji ENDO
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/[12]. Several types of flow systems have been applied to study
the response of cells to the shear stress in the flow in vitro [5-9,14].
In the present study, a flow channel with amicro-patterned scaffold has been designed to study
quantitatively the effect of flow on the oriented cell in vitro.

ABSTRACTThe effect of shear flow on an oriented cell has been studied by
the flow channel with the micro striped pattern of ridge lines on
the surface of the scaffold in vitro. The lines of parallel micro
quadrangular prism ridges (0.001 mm height, 0.003 mm width,and 0.003 mm interval) are made by the photolithography
technique on the lower surface of the channel as the scaffold to
make orientation of each cell. Variations are made about theangle between the longitudinal direction of the ridge and the
direction of the flow: 0, 45, 90 degrees. The flow rate of < 24cm3/hour is controlled by a syringe pump to make variation of
the wall shear stress of < 2 Pa. The movement of each cell of
3T3-L1 (mouse fat precursor cells) adhered on the micro pattern
was analyzed at the time lapse images for 10 hours: 2 hours(before the flow stimulation), 4 hours (during flow stimulation),and 4 hours (after stopping the flow stimulation). The
experimental results show that both the migration and thedeformation of each cell along the micro ridges are restricted by
the wall shear stress higher than 1 Pa.
Keywords: Biomedical Engineering,
Micro-pattern, 3T3-L1 and Shear stress.

Flow

2. METHODS
Flow Channel
A flow channel has been designed to observe behavior of cells
adhered on the micro-pattern in the flow during the cell culture
in vitro. The flow channel consists of two disks of 50 mm
diameter: the upper disk and the lower disk. The borosilicate
glass (Tempax) disk is used for the lower disk for the scaffoldof the cell culture. The upper surface of the lower disk has the
micro-pattern.
The
upper
disk
is
made
of
polydimethylsiloxane (PDMS), of which the lower surface has
concave part for the flow path.
Lower Disk with Micro-pattern
The micro-pattern is fabricated by the photolithography
technique at the central part of the upper surface of the lower
disk for the scaffold of the cell culture. Several parallel lines
of micro grooves have been made at the lower disk (Fig. 1).
The depth (D), the width (W), and the interval (I) of therectangular groove are 1 μm, 3 μm, and 3 μm, respectively.
Variation has been made on the angle (θ) between thelongitudinal direction of the groove and the flow direction: 0(parallel), 45, 90 (perpendicular) degrees. Each pattern isdrawn in the rectangle area of 1.6 mm × 0.4 mm, which is in theparallel position each other with the interval of 0.05 mm.

Channel,

1. INTRODUCTION
Biological cells are exposed to mechanical stimulation in vivo.
The shear stress is one of the mechanical stimulations. The
endothelial cells, for example, are exposed to the shear stress inthe blood flow at the wall of the blood vessels [1-8]. Someother cells are exposed to the shear stress among thedeformation of the tissue. A cell adheres on the scaffold,migrates, deforms, proliferates, and differentiates.
Themechanical stress might affect the action of the cell. The
biological cell takes action not only passively but also actively.
The cell is moved to downstream by the flow. The cell
migrates, on the other hand, to the upstream against the flow.
The flow deforms the cell along the stream line. The cellchanges the shape, on the other hand, to minimize the intra
force. Control methodology for orientation of cells would beapplied to the regenerative tissue technology. The action ofcell depends on the micro morphology of the scaffold [4-12].
The cell might be sensitive to the morphology of the similardimension to itself at the scaffold. The photolithography
technique [13] is available to make the micro patterns on thesurface. The previous study showed that the orientation ofmyoblast follows the longitudinal directions of the micro ridges

Fig. 1: Micro-pattern on lower disk of flow channel.

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Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)

Resist removal

the female screw, one end of the bolt with the male screw wasinserted. The other end of each bolt was inserted into thesilicone tube (internal diameter 4 mm, external diameter 6 mm).
After the mold was enclosed with a peripheral wall ofpolyimide tape, PDMS (Sylgard 184 Silicone Elastomer Base,
Dow Corning Corp., MI, USA) was poured with the curing
agent (Sylgard 184 Silicone Elastomer Curing Agent, Dow
Corning Corp., MI, USA) on the mold. The volume ratio of
PDMS to curing agent is ten to one. After degassing, PDMS
was baked at 373 K for one hour in an oven. The baked diskof PDMS (5 mm thickness) with tubes was exfoliated from themold to be used as the upper disk of the channel.

Developing, etching

Fig. 2: Photolithography process for micro-pattern.
Assembly of Disks
Both the upper and the lower disks were exposed to the oxygengas (0.1 Pa, 30 cm3/min) in the reactive ion etching system(FA-1) (oxygen plasma ashing, 50 W, for thirty seconds).
Immediately after ashing, the upper disk adheres (plasma
bonding) to the lower disk to make the flow path between them.
The assembly of disks was baked in the oven at 348 K for fiveminutes. Before cell culture, the flow channel was sterilized in
an autoclave at 394 K for forty minutes. After sterilization,
the channel was dried in the oven. In the clean bench, thechannel was sandwiched between the transparent disks(diameter of 65 mm, thickness of 2 mm) of
poly-methyl-methacrylate with six bolt/nuts as the supporter toadjust the fixation of the assembly of the flow channel.

The borosilicate glass disk was used for the base of the lower
disk in a photolithography process (Fig. 2). The diameter andthe thickness of the disk are 50 mm and 1.1 mm, respectively.
The surface of the glass disk was cleaned by the oxygen (0.1 Pa,
30 cm3/min) plasma ashing (100 W, for five minutes) in thereactive ion etching system (FA-1, Samco Inc., Kyoto, Japan).
To improve affinity between glass and photoresist material,
hexamethyldisilazane (HMDS: Tokyo Chemical Industry Co.,
Ltd., Tokyo) was coated on the glass plate at 3000 rpm for thirty
seconds with a spin coater (IH-DX2, Mikasa Co., Ltd., Tokyo,
Japan). The photoresist material of OFPR-800LB (Tokyo
Ohka Kogyo Co., Ltd., Tokyo, Japan) was coated on HMDS at
3000 rpm for twenty seconds with the spin coater. The
photoresist was baked in the oven (DX401, Yamato ScientificCo., Ltd) at 373 K for ninety seconds.

One of the silicone tubes connects the flow channel with thesyringe pump (Fig. 3). The other silicone tube connects theflow channel with the reservoir of the suspension of the cells, orwith the reservoir of the medium. The channel is placed on
the stage of an inverted phase contrast microscope (IX71,
Olympus, Tokyo) for the observation of cells adhered on the
micro pattern on the disk under the flow (Fig. 4). The CO2 gas
is blown into the reservoir of the medium to maintain the
carbon dioxide partial pressure at five percent in the medium.
The reservoir of the medium is placed in the thermostatic bathto maintain the temperature at 310 K (Fig. 4).
Thetemperature of the surface of the flow chamber was measured
by the infrared thermography (Fig. 6).

The pattern for the micro grooves was drawn on the photoresist
material with a laser drawing system (DDB-201K-KH, Neoark
Corporation, Hachioji, Japan). To control the dimension of thepattern with the laser drawing system, the parameters wereselected as follows: the voltage of 3.2 V, the velocity of 0.1
mm/s, the acceleration of 0.350 mm/s2. The pattern was bakedat the hotplate at 393 K for five minutes. The photo-resist wasdeveloped with tetra-methyl-ammonium hydroxide (NMD-3,
Tokyo Ohka Kogyo Co., Ltd., Kawasaki, Japan) for two
minutes. The disk was rinsed two times with the ultrapure
water, and dried by the spin-dryer (SF-250, Japan Create Co.,
Ltd., Tokorozawa, Japan).

Wall Shear Stress
In the present experiment, the shear rate on the wall of thescaffold is estimated with a parabolic velocity profile between
the parallel walls. The shear rate (γ, [s－1]) on the wall of theplate is calculated by Eq. 1.

The glass was etched with the plasma gas using a reactive ion
etching system (RIE-10NR, Samco Inc., Kyoto, Japan) to makelines of the micro grooves of 0.001 mm depth. For etching,
the gas of CF4 (30 cm3/min at 1013 hPa) with Ar (30 cm3/min at
1013 hPa) was applied at 100 W at 2 Pa for thirty minutes.
The residual OFPR-800LB was removed by the plasma ashing(Oxygen, 0.1 Pa, 30 cm3/min, 100 W, for five minutes) in thereactive ion etching system (FA-1).

γ = 6 q / (b d 2)

(1)

In Eq. 1, q is the flow rate [m3 s－1], b is the width of thechannel [m] and d is distance [m] between two parallel walls.
In the present study, d is 0.1 mm, and b is 4 mm. When q = 12
cm3/hour, γ= 500 s－1. The shear stress τ [Pa] is the product ofthe viscosity η [Pa s] of the fluid and the shear rate γ [s-1] of theflow (Eq. 2).

The morphology of the micro grooves was observed with thelaser microscope (VK-X200, Keyence Corporation, Osaka,
Japan). The height along the cross sectional line of the groove
was traced.

τ=ηγUpper Disk
The mold for the upper disk (diameter; 50 mm) is made ofaluminum. The mold has a convex rectangular pattern withthe following dimension: the width of 4 mm, the height of 0.1
mm, and the length of 30 mm. Each end of the rectangularpattern has a hole with the female screw of M4. The mold wascleaned in the ultrasonic cleaning machine. In each hole with(2)

When γ = 500 s－1 and η = 0.002 Pa s (at 310 K) [11], τ = 1 Pa.
Variation was made on the wall shear stress τ lower than 2 Pa byadjusting the flow rate q.
The flow adjacent to the micro grooves on the scaffold surfaceof the culture plate was traced by micro spheres (diameter 0.010

127Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)

mm, density 2.4 g/cm3: Thermo fisher scientific) rolling on thesurface at the microscopic video image in the extra flow test.
Cell Culture with Flow
3T3-L1 (mouse fat precursor cells, a cell line derived from cellsof mouse 3T3; passage between third and ninth) of the passage
before tenth was used in the tests. D-MEM (Dulbecco’s
Modified Eagle Medium) containing 10% FBS (Fetal Bovine
Serum) and 1% penicillin/ streptomycin was used for the
medium. After the medium was prefilled in the flow channel
through the inlet tube by the syringe pump, the suspension (5×104 cells/cm3) of 3T3-L1 in D-MEM was injected into thechannel through the inlet tube by the syringe pump (Fig. 3).
After several cells adhered on the micro-pattern in the flow
channel in two hours, the wall shear stress was applied on thecells by the flow pulled with the syringe pump for four hours.
After the flow stimulation for four hours, the microscopic
observation was continued for another successive four hours
without the flow stimulation. To trace the same cell, thetime-lapse pictures were taken with the constant interval of fiveminutes during the microscopic observation (Fig. 4).

Fig. 4: Experimental system: syringe pump (left), flow channel
on stage of microscope (middle), and reservoir in thermostaticbath (right).

Ro = No / Nt

In Eq. 4, No is the number of the oriented cells, and Nt is thetotal number of cells.
At the end of the test, the cell on the micro-pattern wasobserved by a scanning electron microscope (SEM,
JSM6360LA, JEOL Ltd., Tokyo, Japan) (Fig. 9).

As the control test, cells seeded on the micro pattern without
flow stimulation were also observed by time-lapse microscopic
images. The lower disk with micro-pattern was contained in
the culture dish, and placed in the incubator on the stage of theinverted phase-contrast optical microscope.

3. RESULTS

At the microscopic image, the outline of each cell was traced by“Image J”, and the contour of each cell was approximated toellipsoid. As to the ellipsoid, the coordinates of the centroid,
the length of the major axis (a), and the minor axis (b) were
measured. The centroid of each ellipsoid was used to trace themigration of the cell at the microscopic time lapse images. To
trace the migration of each cell by the coordinates, the x axis iscorrected to the direction of the flow. The ratio of axes iscalculated as the shape index (SI) by Eq. 3.
SI = 1 – b / a(4)

Fig. 5 shows the tracings of the lower disk by the laser
microscope. The dimensions of the depth (D), the width (W),and the interval (I) of the rectangular groove are confirmed tobe 0.001 mm, 0.003 mm, and 0.003 mm, respectively. Fig. 6
shows the thermographic image of the flow chamber. The
thermography shows the distribution of temperature around theflow chamber is maintained at 310 K. In Fig. 7, the red lines
shows the tracings of micro spheres rolling on the surface withthe micro grooves on the culture plate for 0.2 s in the extra flow
test. The micro spheres rolling over the micro grooves (theangle between the longitudinal direction of groove and the flow
is 45 degrees) shows the parallel steady stream lines. Fig. 8
shows the inverted-phase-contrast microscopic image of cellson the micro-pattern. Several cells adhere in two hours. Fig.
9 shows the scanning electron microscope images of cells on
the ridge between grooves at the end of the test. Some cells
adhere along the ridge line, while another cell makes a bridge
between ridges.

(3)

At the circle, SI = 0. As the ellipsoid becomes flat, SIapproaches to unity. The cell, of which SI is higher than 0.5,
was selected, and the angle (θ) between the major axis of thecell and the longitudinal direction of the groove was measured.
The number of the oriented cells (No) was counted as the cell,of which the angle (θ) is between –20 degree and 20 degree.
In the area of each micro-pattern, the orientation ratio (Ro) was
calculated by Eq. 4.

Fig. 10 shows the number of cells adhered on the micro pattern.
The number of cells increases without exposure to the shear
stress (Fig. 10A). Although the initial number of cells is
scattered, the number of cells increases after stopping (6 h) the
exposure to the shear stress. Most of cells are exfoliated by
the exposure to the shear stress of 1.5 Pa for four hours (Fig.
10D). Most of cells were exfoliated immediately after
exposure to the shear stress of 2.0 Pa. Fig. 11 shows theoriented ratio (%) of cells. Most of cells keep orientation
along the ridge between grooves. The orientation ratio at 90
degrees tends to decrease by the exposure to the shear stress.
Fig. 12 exemplifies the tracings (every hour) of the migration ofeach cell. The migration along the striped micro ridge
between grooves (including migration to the upstream)
decreases under exposure to the shear stress of 1 Pa (Fig. 12c).
Fig. 13 exemplifies the tracings (every hour) of the shape index
of each cell. Cells are exposed to the wall shear stress of 0.5
Pa for four hours (from 2 h to 6 h). The cell tends to be

Flow

Fig. 3: Cell culture with flow: reservoir (right), flow channel(middle), and syringe pump (left).

128Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)

rounded (decrease of SI), when the cell is exposed to the wall
shear stress (from 2 h to 6 h). After stopping exposure to theshear stress, the cell tends to be elongated except on the micro
pattern of 90 degrees (Fig. 13c).

4. DISCUSSION
In the present experimental device, the depth of the groove is 1
μm, which is minimum value for making orientation of cells[12].
The effect of the shear flow on myoblast wasinvestigated in the previous study [9]. The effect of the shear
flow on fibroblast [15] has been investigated in the present
study.

Fig. 8: Cells on micro-pattern at seeding (left), and at 2 hours
1 mmwithout flow after seeding (right).

Line for tracing(lower)

5 μm

2 μm

Fig. 9: SEM image of cell on micro ridge between grooves.
A

B

C

D

Fig. 5: Tracings (lower) of surface of mold along the line atlaser image (upper) for upper plate; unit, μm.

10 mmFig. 10: Number of cells adhered on each micro-pattern vs.
time: control (A), 0.5 Pa (B), 1 Pa (C), 1.5 Pa (D).

100
80
60
40
20
0

Fig. 6: Thermographic image of flow chamber.
Flow

0°

45°

2

45 degrees

B
100
80
60
40
20
0

6
10
Time[h]
100
80
60
40
20
0

200 μmFig. 7: Tracings of micro spheres rolling on the surface with themicro grooves (45 degrees) on the culture plate in the extra flow
test: red line shows tracing for 0.2 s.

0°

45°

90°

2

6
10
Time[h]

0°

45°

2

6
10
Time[h]D

Oriented rate…

C

90°

Oriented rate …

Oriented rate …

A

90°

Fig. 11: Oriented ratio (%) of cells vs. time on eachmicro-pattern: control (A), 0.5 Pa (B), 1 Pa (C), 1.5 Pa (D).

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A

200

y, μm

200

B

C

100

100
0

-200
0
-100

200
x, μm

-200

x, μm

-200
Fig. 12c: Migration of cell on micro pattern at 1 Pa: 0 degree(A), 45 degrees (B), and 90 degrees (C): unit μm.

200 y, μm

1
0.8

0
x, μm
-200-100 0 100 200
-100

SI

0.6
0.4

-200

0.2

Fig. 12a: Migration of cell on micro pattern without flow: 0
degree (A), 45 degrees (B), and 90 degrees (C): unit μm.
B 200

y, μm

200

0

y, μm

0

5
Time[h]10

Fig. 13a: shape index (SI) of each cell (0 degree): wall shear
stress of 0.5 Pa from 2 h to 6 h.

100

100
0
-200-100 0 100 200
-100
x, μm

0
-200-100 0 100 200
x, μm
-100

-200

-200

200

C

Flow

0
-200-100 0 100 200
-100

100

A

y, μm

100

0
-200-100 0 100 200
-100
x, μm

-200
C

200

y, μm

y, μm
Flow

100
Fig. 13b: shape index (SI) of each cell (45 degrees): wall shear
stress of 0.5 Pa from 2 h to 6 h.

0
-200
0
-100

200 x, μm-200Fig. 12b: Migration of cell on micro pattern at 0.5 Pa: 0 degree(A), 45 degree (B), and 90 degree (C): unit μm.
A

y, μm

B

200

y, μm

100

x, μm

Fig. 13c: shape index (SI) of each cell (90 degrees): wall shear
stress of 0.5 Pa from 2 h to 6 h.

0-200-100 0 100 200
-100
x, μm

The flow is one of the stimuli on cells in the experimental
systems: the donut type canal [3], the cylindrical pipe, thechannel between parallel walls [9], and Couette type flow using
rotating disk. For quantitative study, Couette type flow orPoiseuille type flow has advantage.

-200

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Takahashi, “Effect of Flow on Cultured Cell at Micro-patternof Ridge Lines”, Journal of Systemics, Cybernetics andInformatics, Vol. 15, No. 5, 2017, pp. 1–7.
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On the scaffold, the direction of each cell randomly scatters.
To control the direction of adhesion of the cell, the micro
pattern of the surface of the scaffold is available. Both the
migration and the deformation of the cell depend on the micro
morphology of the surface of the scaffold.
Thephotolithography technique can be applied to make the micro
morphology on the surface [13]. In the present study, the lines
of micro ridges of minimum height [12] are designed on thescaffold to make orientation of cell. The dimension of thewidth of the micro groove is smaller than the diameter of thecell, so that each cell does not fall into the groove. The wall
shear stress around 1 Pa is selected in the present study as the
typical value, which affected cells in the previous study [9].
Three types of direction (parallel, diagonal, and perpendicular)
has been tested for the orientation of cells in the flow. The
micro pattern on the scaffold can also be used as markers totrace the cell. Cells keep the active action (deformation,
migration, and proliferation) in the flow stimulation at the lower
shear stress (< 3 Pa). The cell also migrates to the counter
direction of the flow. The higher wall shear stress (> 3 Pa)
reduces the migration of cells. A cell deforms to a roundshape at the high wall shear stress. The wall shear stress of 1
Pa enhanced the migration of C2C12 along the ridge in theprevious study [9]. Cells restart migration after stopping thewall shear stress of 3 Pa within four hours, which shows that thecells are not damaged. The reaction might depend on thekinds of cell. C2C12 made perpendicular orientation of
myotubes to the flow direction in the previous study [3].

5. CONCLUSION
The effect of shear flow on an oriented cell has been studied by
the flow channel with the micro striped pattern of ridge lines on
the wall of the scaffold in vitro. The lines of parallel micro
quadrangular prism ridges (0.001 mm height, 0.003 mm width,and 0.003 mm interval) are made by the photolithography
technique on the lower surface of the channel as the scaffold to
make orientation of each cell. Variation is made about theangle between the longitudinal direction of the ridge and the
direction of the flow: 0, 45, 90 degrees. The experimental
results show that both the migration and the deformation ofeach cell of 3T3-L1 (mouse fat precursor cells) along the microridge are restricted by the wall shear stress higher than 1 Pa.
The flow channel system with the micro pattern of ridge lines invitro is effective to evaluate the effect of flow on oriented cells
quantitatively.

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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