Design of Cross Type of Flow Channel
to Control Orientation of Cell
Haruka HINO, Shigehiro HASHIMOTO, Yusuke TAKAHASHI, Shusuke NAKANO
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACTA cross type of flow channel has been designed to control
orientation of cell by direction of flow in vitro. The flow path
(4 mm width, 0.2 mm height) is made between paralleltransparent plates. Flow rate of 48 cm3/hour is controlled by asyringe pump. The wall shear rate of 500 s-1 is calculated by
the parabolic velocity profile between the parallel plates. The
direction of the flow can be changed by alternative use of theinlet and outlet port of the cross type of the channel. The flow
channel is placed in a chamber, where temperature and CO2
content are kept 310 K and 5 percent, respectively. The
chamber is placed on the stage of an inverted phase contrast
microscope to observe the behavior of cells adhered on the wallunder a flow. After several cells adhered to the disk, the wall
shear rate was applied on the cells in the medium flow for 1
hour at each direction. C2C12 (mouse myoblast cell line) wasexposed to the wall shear rate with the methodology. The
experimental results show that the change of the direction ofmyoblasts can be observed by the cross type of flow channel.

A flow can be used to apply a stress field to a cell [16-22].
The cell directly receives the shear stress in the shear flow.
The high shear flow might deform cell, peel cells off thescaffold, and inhibit proliferation. The mild shear flow, on theother hand, might accelerate the active deformation, migration,and proliferation.
In the present study, a cross type of flow channel has been
designed to control orientation of cell by direction of flow invitro.

2. METHODS
Cross Type of Flow Channel
A cross type of flow channel has been designed to change
perpendicularly the direction of the flow during cell culture(Figs. 1-4).

Keywords: Biomedical Engineering, Flow Channel, Cell
Culture, Myoblast, Shear rate, and Flow.

1. INTRODUCTION
A biological cell deforms not only passively but also actively.
The flow deforms the cell along the stream line [1]. The cell
deforms, on the other hand, to minimize the intra force.
Cells are exposed to mechanical stimulation in vivo [2-4]. The
shear stress is one of the mechanical stimulations. Theendothelial cells are exposed to the shear stress in the bloodflow at the wall of the blood vessels [5-9]. The other cells arealso exposed to the shear stress among the deformation of thetissue.

Fig. 1: Change direction of flow at cross type of channel.

Cells adhere, migrate, deform, proliferate, and differentiate.
The mechanical stress might affect the behavior of cells [10].
Cell culture technique has been developed and several
methodologies have been clinically applied to regenerative
medicine. The acceleration technique for orientation andproliferation of cells has been studied to make tissue in vivo orin vitro [11-15]. Control methodology for orientation andproliferation of cells would be applied to the regenerative tissue
technology.

Fig. 2: Design of cross type of flow channel.

Fig. 3: Cross section of flow channel.
Fig. 6: Flow channel and tubes.

The square mark of groove (1 mm × 1 mm) was machined by
the ultrashort pulse laser (IFRIT, Cyber Laser Inc., Tokyo,Japan) at the center on the rear surface of the scaffold of thelower disk, to trace the same cell (Fig. 4).
The mold for the upper disk is made of aluminum (Fig. 5A).
The mold has a convex cross pattern with the following
dimension: the width of 4 mm, the height of 0.2 mm, and thelength of 30mm. At the crossing point, each corner has a filletof curvature of r = 2.5 mm (Fig. 4). Each end of the cross hasa hole with the female screw of M4. The dimension of theconvex part was measured with a laser microscope (VK-X200,Keyence Corporation, Osaka, Japan).

Fig. 4: Dimension (mm) of flow channel.

In each hole with the female screw, the bolt with the male screw
was inserted. Each bolt was covered with the silicone tube
(internal diameter 2 mm, external diameter 4 mm) (Fig. 5B).
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 (Fig. 5D). The volumeratio of PDMS to curing agent is ten to one. After degassing,
PDMS was baked at 373 K for one hour in an oven (DX401,Yamato Scientific Co., Ltd). The baked disk of PDMS (3.1mm thickness) with tubes was exfoliated from the mold, andsterilized in an autoclave.
The upper and lower disks were exposed to the oxygen gas of30 milliliter per minute at power of 50 W for one minute in areactive ion etching system (FA-1, Samco Inc., Kyoto) justbefore the flow test to be characterized as hydrophilic (oxygenplasma ashing).

Fig. 5: Upper disk on mold: mold (A), mold with tubes (B),wall with mold with tubes (C), PDMS with tubes on mold (D),PDMS with tubes (E).

The flow channel consists of two disks of 50 mm diameter.
The borosilicate glass (Tempax) disk (1.1 mm thickness) is used
for the lower disk for the scaffold of the cell culture. The
upper disk is made of polydimethylsiloxane (PDMS), which has
concave pattern for the flow path (Fig. 3).

The upper disk adheres to lower disk to make the flow path
between them. To strengthen the contact between two disks,
they were sandwiched between two transparent disks ofpolymethylmethacrylate (diameter of 65 mm, thickness of 2mm), and fixed with six screws (Fig. 6).
In the present experiment, the shear rate on the wall of thescaffold is approximated with an estimated parabolic velocity
profile between the parallel walls [19].
The shear rate (γ, [s-1]) on the wall of the plate is calculated by
Eq. 1, in which a parabolic velocity profile between parallelplates is hypothesized.
γ = 6 q / (b D2)(1)

In Eq. 1, q is the flow rate [m3 s-1], b is the width of the channel[m] and D is distance [m] between two parallel walls. In thepresent study, D is 0.2 mm, and b is 4 mm. When q = 48
cm3/hour, γ= 500 s-1.
The shear stress τ [Pa] is the product of viscosity η [Pa s] of thefluid and the shear rate γ [s-1] of the flow (Eq. 2).
τ=ηγ

(2)

On the microscopic image, the outline of each cell was traced,
and the contour of each cell was approximated to ellipsoid.
The length of the major axis (a) and the minor axis (b) were
measured, and the ratio of axes is calculated as the shape index(I) by Eq. 3.
I=a/b
At the circle, I =1.
to zero.

(3)
As the ellipsoid becomes flat, I approaches

The angles between the direction of the flow and the directionof the major axis of each cell are also measured at themicroscopic image.

When η = 0.002 Pa s [17], τ = 1 Pa.
The silicone tube connects the flow channel with the syringe
pump, with the reservoir of the suspension of the cells, or withthe reservoir of the medium (Fig. 7).
The direction of the flow can be changed by alternative use of
the inlet and outlet port of the cross type of the channel. Thechannel is placed on the stage of an inverted phase contrast
microscope (IX71, Olympus, Tokyo) to observe the behavior ofcells adhered on the disk under the flow (Figs. 8&9). The CO2
gas is introduced into the reservoir of the medium to maintainthe carbon dioxide partial pressure at five percent in themedium through the channel (Fig. 10). The reservoir is placedin the thermostatic bath to maintain the temperature at 310 K.
Flow Test
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) of the passage ninth was used in thetests.

Fig. 8: Flow channel on stage of microscope.

D-MEM (Dulbecco’s Modified Eagle Medium) containing 10%
FBS (Fetal Bovine Serum) and 1% penicillin/ streptomycin wasused for the medium. After the medium was drawn into theflow channel through the one inlet tube and through three outlet
tubes by the syringe pump to remove the air bubble, thesuspension (100000 cells/cm3) of C2C12 in D-MEM wasintroduced into the channel through the one inlet tube by thesyringe pump (Fig. 7).
After several cells adhered to the disk for 1.5 hour or for 3 hour,
the wall shear stress was applied on the cells by the flow
through one inlet tube and one outlet tube pulled with thesyringe pump for 1 hour. To trace the same cell, the time-lapse
pictures were taken with the constant interval of thirty seconds.

Fig. 9: Experimental system.

Fig. 7: Filling of medium before test.
Fig. 10: CO2 supply to medium.

3. RESULTS
The measurement by the laser microscope of the mold showsthat the height is 0.2 mm (Fig. 11). By the movement of theflowing cells, the control of the alternating flow direction along
the rim of the square of the mark is confirmed (Fig. 12).
Under the flow, the shape indexes (I) of adhered cells are 0.6(mean) ± 0.2 (standard deviation).
Fig. 13 shows exfoliation of cell under the shear flow. Fig. 14
shows cell rounded after the change of the flow direction.
Fig. 15 shows cells under shear flow. The cells are exposed to
the flow of respective direction for one hour each, after
cultivation for three hours for adhesion to the bottom of thechannel.

Fig. 14: Cell immediately after exposure to flow in the first
direction (A), for 30 s (B), for 30 min (C), for 1 hour (D),immediately after exposure to flow in the second direction (E),for 30 s (F).

Fig. 11: Dimension of mold for upper disk of flow channel.

Fig. 12: Alternating flow direction (arrow).

Fig. 13: Cell exposure to flow for 30 s (A), for 33 min (B), for38 min (C), exfoliation of cell (C). Arrow shows flow
direction. Square mark is 1 mm × 1 mm.

Fig. 15a: Cell in the first flow direction. Immediately after
exposure to flow (A), for 30 s (B), for 30 min (C), for 1 hour
(D). Square mark is 1 mm × 1 mm.

Fig. 16 shows the angle between the major axis of the cell andthe flow direction. Data of angle (360 degree is 6.26 rad, zero
degree is horizontal in the figure) are shown in ascending orderin Fig. 16. Data would rise along the straight line at random
distribution of the angles. When the data make curved line,
the cell tends to tilt to the angle in the range of low slope. Fig.
16a and Fig. 16b show different distribution of the angles,
which shows the change of orientation of cells after the changeof the flow direction. Fig. 16a shows more cells tend to tilt tothe angle around 90 degrees, which is flow direction in Fig. 16a,in 1 hour. Fig. 16b shows more cells tend to tilt to 45 and 135
degrees in 1 hour: the angle of zero is flow direction in Fig.
16b.

processing to decrease the leakage.
In the present study, the inner surface of the flow channel wastreated to be hydrophilic to avoid contamination with bubbles.
The hydrophilic property of the wall might affect the migrationof the cell. To avoid generation of bubbles, the internalpressure of the flow path was controlled by the opening of inlet
tubes in the present study.
The extended cultivation after exposure to the shear flow mightshow the effect of the shear flow on proliferation anddifferentiation of cells. In the dense state of cell, theorientation of cells might depend on the interaction between
cells.
The response of cell to the shear flow depends on point ofadhesion to the scaffold. Enough points of adhesion arenecessary for the active response to the shear field.

Fig. 15b: Cell in the second flow direction. Immediately after
exposure to flow (A), for 30 s (B), for 30 min (C), for 1 hour
(D). Square mark is 1 mm × 1 mm.

Several elongated cells recover to the rounded shape after
stopping exposure to the shear flow. The immediate recovery
shows that the deformation of cell might be passive.
The cells might change the orientation at the differentiation.
C2C12 made perpendicular orientation of myotubes to the flow
direction in the previous study [1]. The orientation of C2C12
at diagonal direction of flow might be preparation to makeperpendicular orientation of myotubes in the successivecultivation.
The wall shear stress of 1 Pa is selected in the present study as
the typical value, which affected cells in the previous study[17].

Fig. 16a: Cell orientation in the first flow direction.

The flow channel system of the parallel disks has been used in
the previous experiments to study the effect of the shear stresson the cells. Although the sandwiched elastic film between
the disks is convenient to avoid air leakage, the shape of thefilm may make the geometric fluctuation. The geometricaccuracy of the wall of channel improves the laminar flow
between the parallel walls.
It is not easy to estimate the shear stress value on the wall, whenthe medium has the free surface [1, 16]. The parallelepipedchamber is convenient to observe the response of cells undercontrolled shear stress [19].

Fig. 16b: Cell orientation in the second flow direction, after 3hours exposure to the first direction.

4. DISCUSSION
To decrease the turbulence of the flow around the crossing, theside wall has the curvature at the crossing (Fig. 17). The tubes
for inlet and outlet are embedded in the PDMS disk during theFig. 17: Calculated stream line at the cross type of flowchannel.

5. CONCLUSION
A cross type of flow channel has been designed to control
orientation of cell by direction of flow in vitro. The flow pathis made between parallel transparent plates. The direction of
the flow can be changed by alternative use of the inlet andoutlet port of the cross type of the channel. The flow channel
is placed in a chamber, where temperature and CO2 content are
kept 310 K and 5 percent, respectively. The chamber is placedon the stage of an inverted phase contrast microscope to observe
the behavior of cells adhered on the wall under a flow. The
experimental results show that the change of the direction ofmyoblasts can be observed by the cross type of flow channel.

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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