Micro Groove for Trapping of Flowing Cell
Yusuke TAKAHASHI, Shigehiro HASHIMOTO, Haruka HINO, Atsushi MIZOI, Nariaki NOGUCHI
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/
ABSTRACTThe micro groove has been designed to trap a biological cell,which flows through a micro channel in vitro. The micro
groove of the rectangular shape (0.002 mm depth, 0.025 mm
width and 0.2 mm length) has been fabricated on the surface ofthepolydimethylsiloxane(PDMS)
disk
with
the
photolithography technique. Variation has been made on theangle between the longitudinal direction of the groove and theflow direction: zero, 45, and 90 degrees. A rectangular flow
channel (0.1 mm depth × 5 mm width × 30 mm length) has
been constructed with a silicone film of 0.1 mm thick, which
has been sandwiched by two transparent PDMS disks. Twotypes of biological cells were alternatively used in the test:
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse), or 3T3-L1 (mouse fat precursor cells).
A constant flow (2.8 ×10-11 m3/s) of the suspension of cells wasintroduced with a syringe pump. The behavior of cells moving
over the micro groove was observed with an inverted phase
contrast microscope. The results show that the cell is trappedwith the micro grooves under the wall shear rate of 3 s-1 for a
few seconds and that the trapped interval depends on the kind ofcells.
Keywords: Biomedical Engineering, C2C12, 3T3-L1, Micro
Flow Channel, Micro Groove and Polydimethylsiloxane.

morphology of the microcirculation. The micro-fabrication
technique has also been applied to design microfluidic systems
in vitro [14-20]. Cells roll over on the surface of the wall inthe shear flow, and make adhesion to the wall [10]. The
surface was modified to capture flowing cells [10, 11, 21, 22].
The technique will also be applied to handle cells in diagnostics
in vitro [23].
In the present study, a micro groove has been designed to trap abiological cell, which flows through a microchannel in vitro.

2. METHODS
Micro Traps
For trapping cells, several micro grooves of the rectangularshape (0.002 mm depth, 0.025 mm width, and 0.2 mm length)
have been fabricated on the surface of the polydimethylsiloxane(PDMS) disk with the photolithography technique.
Sixgrooves are arranged in two lines. The interval between
grooves is 0.2 mm. Variation is made on the angle between
the longitudinal direction of the groove and the flow direction:
zero, 45, and 90 degrees (Fig. 1).
A silicon wafer (Type N, Matsuzaki Seisakusyo, Co., Ltd.,Tokyo, Japan) was used for a surface mold for the disk. The
diameter and the thickness of the wafer are 50 mm and 0.30 mm,
respectively.

1. INTRODUCTION
A biological cell adheres to the scaffold, and reveals several
behaviors: migration, deformation, proliferation, anddifferentiation. The behavior of cells depends on the various
environmental factors [1-5]. A flowing cell is captured to awall of a flow path. Several cells adhere to the internal wall of
the blood vessel, when the part of the wall has a defect. The
morphology of the defect might govern the capture of cells.
The capture might depend on the property of the cell.
The cancer cell transits from the original place to another place,and proliferates to make a tumor at another place. The
transition occurs through the blood vessels and the lymph
vessels. The cancer cells adhere to the inner wall of thevessels.
The photolithography technique enables manufacturing a
micro-channel. The effect of the surface of the scaffold on cell
culture has been studied in the previous studies [6-9]. Several
micro-fabrication processes have been designed to control
adhesion of biological cells in vitro [6-13], and to simulate the

The photo-resist material of low viscosity (SU8-2: Micro Chem
Corp., MA, USA) was coated on the wafer with 0.002 mm thickat 2000 rpm for 30 s with a spin coater. The photo-resist
baked on the heated plate with two processes: at 338 K for 1
minute, before at 358 K for 3 minutes.
The pattern for grooves (Fig. 1) on the mold was drawn on thewafer with a laser drawing system (DDB-201K-KH, Neoark
Corporation, Hachioji, Japan). To control the dimension of thepattern on the mold with the laser drawing system, theparameters were selected as follows: the voltage of 4 V, thevelocity of 0.01 mm/s, the acceleration of 0.5 mm/s2. The
pattern was baked on the heated plate with two processes: at
338 K for 1 minute, before at 358 K for 3 minutes.
The photo-resist was developed with SU8 developer (Nippon
Kayaku Co., Ltd, Tokyo, Japan) for six minutes to make micro
rectangular ridges, where the laser beam was radiated. The
wafer was rinsed with the isopropyl alcohol and the ultrapure
water, before dried with the air gun.

50 mm5 mm

A1

B

Fig. 1: Six grooves. Flow direction is vertical.
30 mm
After development, the dimension of the micro rectangularridges on the mold was measured with a laser microscope(VK-X200, Keyence Corporation, Osaka, Japan).
Themorphology along the longitudinal and transverse lines of eachridge was traced. The concave mold with micro patterns isused only for the lower disk of PDMS to make the micro
grooves on the surface for trapping cells.
The surface of the wafer was coated with 0.001 mm thicknessof Parylene (Specialty Coating Systems, Inc., IN, USA).

5 mm

C

A2

Fig. 3: The flow channel consists of two transparent
polydimethylsiloxane (PDMS) disks (A1, A2) and a thin silicone
rubber sheet (B). C is magnified pattern on A2.

After the wafer was enclosed with a peripheral wall ofpolyimide, 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 wafer. The volume ratio of PDMS tocuring agent is ten to one. After degassing, PDMS was baked
at 383 K for one hour in an oven.
Flow System
A one-way flow system has been designed to control the wall
shear rate at the wall of PDMS [4]. The system consists of aflow channel, a micro syringe pump, tubes and a microscope(Fig. 2). A silicone tube of 3 mm internal diameter and of 5mm external diameter is used for the connector to the flow
channel.
The flow channel consists of two transparent PDMS disks and athin sheet of silicone rubber.
A thin sheet (0.1 mm thick) of silicone rubber, which has arectangular void space of 5 mm × 30 mm, is sandwiched
between two transparent PDMS plates (Fig. 3). The void
space forms a channel of 30 mm length × 5 mm width × 0.1 mm
depth. The three parts stick together with their surface affinity.

Fig. 4: The flow channel on the stage of microscope.

At the upper disk, two holes of 5 mm diameter (Fig. 4) aremachined by a punching tool. The silicone tube is inserted toeach hole of the upper PDMS disk. To seal the leak at the gap
between elements, the liquid of PDMS was pasted on thejunction of elements, and baked at 333 K for 90 minutes in anoven.
One of the tubes is connected to the syringe pump, and the othertube is connected to the reservoir of the suspension (Fig. 2).
The chamber is placed on the inverted phase-contrast
microscope (IX71, Olympus Co., Ltd., Tokyo).
Flow Test
Two types of biological cells were used in the test alternatively:
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse), or 3T3-L1 (mouse fat precursor cells).

Fig. 2: Flow test system: flow chamber and microscope(middle), syringe pump (left).

Cells were cultured with the D-MEM (Dulbecco’s Modified
Eagle’s Medium) containing 10% FBS and 1% of
Antibiotic-Antimycotic(penicillin,
streptomycin
andamphotericin B, Life Technologies) in the incubator for one
week. Before the flow test, the cells were exfoliated from theplate of the culture dish with trypsin, and suspended in the
D-MEM.

Plate

q
D
b

G
Plate

Fig. 5: Parabolic velocity profile between parallel plates.

The suspension of 4000 cells/cm3 was introduced to the channel
at the constant flow rate of 2.8 ×10-11 m3/s with the microsyringe pump.

Fig. 6: Manufactured six grooves.
horizontal.

Flow direction is

The flow path was carefully examined to avoid mixing of air
bubbles, which might stir the medium in the flow channel.
The behavior of cells moving over the grooves in the channel
was observed with the microscope.
Shear Rate on Wall
The shear rate (G, [s-1]) on the wall of the disk is calculated by
Eq. 1, in which a parabolic velocity profile between parallelwalls is hypothesized (Fig. 5).
G = 6 q / (b D2)(1)

In Eq. 1, q is the flow rate [m3 s-1], b is the width of the canal[m] and D is distance [m] between two parallel walls. In thepresent study, D is 0.1 mm, and b is 5 mm. The wall shear rate
G is 3 s-1, when the flow rate q is 2.8 ×10-11 m3/s. The mean
flow velocity (v) in the cross section of the channel is calculated
by Eq. 2. In the present study, v is 0.00006 m/s.
v = q / (b D)(2)

3. RESULTS

Fig. 7: Laser measurement of morphology of groove. Cross
section (bottom) at longitudinal line A-B of groove.

In the case of C2C12, one of the cells is trapped in the grooveof 45 degrees for four seconds in the flow rate of 2.8 ×10-11
m3/s (Fig. 8). Another cell is trapped in the perpendiculargroove for twenty-three seconds in the flow rate of 2.8 ×10-11
m3/s (Fig. 9).

The manufactured micro grooves observed by the inverted
phase-contrast microscope are shown in Fig. 6. The laser
measurement of surface morphology of the pattern (Fig. 7)
shows the following dimension: the depth of the groove scatters
between 0.00197 mm and 0.00206 mm, the width of the groove
scatters between 0.026 mm and 0.028 mm, the length scatters
between 0.196 mm and 0.198 mm.

In the case of 3T3-L1, one of the cells moves along the fringe ofthe groove of zero degree for eight seconds in the flow rate of
2.8 ×10-11 m3/s (Fig. 10). Another cell is trapped in the grooveof 45 degrees for two seconds in the flow rate of 2.8 ×10-11 m3/s
(Fig. 11).

The flow tests show the following results.

C2C12 tends to be trapped in the groove and hold still for
longer time than 3T3-L1.

The moving cells over the grooves are able to be observed withthe microscope in the flow test system. The diameter of thesuspended cell is approximately 0.015 mm. The trapped timeof the cell is measured compared with the movement of floatingcell in the main flow.

4. DISCUSSION
The photolithography technique has been applied to fabricate
the micro channel. The microfluidic system has been applied
to sort biological cells [17], and to trap biological cells [18, 21,22]. The system also used to study local environment aroundthe cultured cell [7]. The micro pattern of the surface has been
applied to study the surface effect of adhesion of cells [6].

(A)

(B)

(B)

(C)

(D)
(C)

(D)

(E)

(F)
Fig. 8: C2C12 in red circle approaches to groove (A), is trappedin groove for 4 s (B, C), and leaves groove (D). Another
C2C12 in black circle moves with main flow (2.8 ×10-11 m3/s)
from right to left.

(A)

Fig. 9: C2C12 in red circle approaches to groove (A), and istrapped in groove (B-F) for 23 s. Another C2C12 in black
circle moves with main flow (2.8 ×10-11 m3/s) from right to left.

(A)

(A)

(B)
(B)

(C)
(C)

(D)
(D)

(E)

Fig. 11: 3T3-L1 in red circle approaches to groove (A), istrapped in groove for 2 s (B, C), leaves groove (D). Another
3T3-L1 in black circle moves with main flow (2.8 ×10-11 m3/s)
from right to left.

Fig. 10: 3T3-L1 in red circle approaches to groove (A), traces
fringe of groove for 8 s (B-D), depart from groove (E).
Another 3T3-L1 in black circle moves with main flow (2.8
×10-11 m3/s) from right to left.

The morphology of micro channel has simulated the lymph
system in the circulatory system in vivo. In several studies,permeability has been tried to control in designing artificial
vessels.

The experimental results might contribute to analyze adhesivemechanism of cancer cell during metastasis. The micro trap
might simulate adhesive mechanism of flowing cells.
In the previous study, cylindrical [10] and half cylindrical [11]holes were used for trap of cells. The asymmetrical hole might
be suitable for trap than the symmetrical hole. The depth ofthe micro holes was 0.01 mm in the previous study [10]. In
the present study, the depth of the grooves of 0.002 mm issmaller than diameter of the cells. The deeper hole may haveadvantage to trap cells. The shallow trap, on the other hand,
may distinguish cells. The duration of the trapped time of thecell might relate to interaction between the micro hole and thecell: adhesiveness between the cell and the surface of the micro
pattern, or deformability of the cell.
The property of the surface might govern the affinity between
the surface and the cell. When the air trapped in the groove,
every cell moves along the boundary of the groove. Behaviorof biological cells might depend on several factors: magnetic
field [1], electric field [2], or mechanical field [3-5].

5. CONCLUSION
Micro groove has been designed to trap a biological cell, whichflows through a micro channel in vitro. The micro groove of arectangular shape has been fabricated on the surface of thepolydimethylsiloxane disk with the photolithography technique.
Two types of biological cells were used in the test alternatively:
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse), or 3T3-L1 (mouse fat precursor cells).
The results show that the cell is trapped with the micro grooves
and that the trapped interval depends on the kind of cells.

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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