Micro Trap for Flowing Cell
Shigehiro HASHIMOTO, Reona NOMOTO, Shuhei SHIMEGI, Fumihiko SATO
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/andToshitaka YASUDA
Bio-systems Engineering, Department of Electronic Engineering,Tokyo National College of Technology, Tokyo, JapanandHiromichi FUJIE
Human Mechatronic Systems, Faculty of System Design,
Tokyo Metropolitan University, Tokyo, Japan

ABSTRACT
A micro channel has been designed to trap flowing biologicalcells in vitro. Several micro traps of a cylindrical hole of 0.01mm depth (diameter between 0.01 mm and 0.04 mm) have been
fabricated on the surface of the polydimethylsiloxane disk withthe photolithography technique. A rectangular flow channel(0.1 mm depth × 1 mm width × 20 mm length) has been madeof the disk with a silicone film of 0.1 mm thick. Two types of
biological cells were used in the test alternatively: L929
(fibroblast-like, mouse connective tissue, RCB1422, Riken Bio
Resource Center, Tsukuba) or C2C12 (mouse myoblast cell line
originated with cross-striated muscle of C3H mouse). A
constant flow of a suspension of cells was introduced with asyringe pump. The behavior of cells moving over the microholes was observed with an inverted phase contrast microscope.
The experimental results show that the cell is trapped with themicro hole under the wall shear rate less than 80 s-1.
Keywords: Biomedical Engineering, C2C12, L929, Micro Trap
and Polydimethylsiloxane.

patient’s death. The tumor permeates through the lymph
vessels. The cancer cell transits from the original place toanother place, and proliferates to make another tumor. The
transition occurs through the blood vessels and the lymph
vessels. The cancer cells adhere to the endothelial cells, which
cover the inner wall of the vessels. The lymph nodes areclinically ablated to avoid metastasis. Wastes and bacteria aretransported through the lymph vessel and dissolved at thelymph node. Because the basement membrane of the lymph
vessel is thin enough to take in things, the cancer cell easily
metastasizes through the membrane.
The photolithography technique enables manufacturing a
micro-channel [1, 2]. The effect of the surface of the scaffold
on cell culture has been studied in the previous studies [3-6].
Several micro-fabrication processes have been designed tocontrol adhesion of biological cells in vitro [4-6], and tosimulate morphology of microcirculation [7].
The
micro-fabrication technique has also been applied to design
microfluidic systems in vitro [8-13]. The technique will also
be applied to handle cells in diagnostics in vitro. In the present
study, a micro channel has been designed to trap flowing
biological cells in vitro.

1. INTRODUCTION
Recently, one per three persons dies according to cancer inJapan. A biological cell alters to a cancer cell by an internal oran external factor. The alteration is called “canceration”.
The cancer cell is out of the control system of the whole body.
The cancer cell has several characters: morphological change,
infinite proliferation, tumorigenic transformation, and metabasis.
Metabasis often causes the cancer recurrence, which leads to

2. METHODS
Micro Traps
Several micro traps (Fig. 1) of a cylindrical hole of 0.01 mm
depth (diameter between 0.01 mm and 0.04 mm) have beenfabricated on a polydimethylsiloxane (PDMS) disk with thephotolithography technique (Fig. 2).

the coating, the wafer was baked at 423 K for 5 minutes.
After development, the dimension of the micro columns on themold was measured with a laser microscope (VK-9510,Keyence Corporation, Osaka, Japan). The convex mold withmicro pattern is used only for the lower disk of PDMS to makethe micro holes.
Traps
The surface of the wafer was coated with 0.001 mm thicknessof Parylene (Specialty Coating Systems, Inc., IN, USA).
Cell

After the wafer is enclosed with a peripheral wall of polyimide,PDMS (Dow Corning Corp., MI, USA) is poured with thecuring agent on the wafer. After degassing, PDMS is baked at
353 K for two hours in an oven (Fig. 5).

Fig. 1: Several micro traps in flow chamber.

Silicon wafer
Coating

MoldSU8Developing
Transcription

The diameter of two PDMS disks is 50 mm. The thicknessesof the upper and the lower disks are 10 mm and 2 mm,respectively. At the upper disk, two holes of 5 mm diameter(Fig. 6) are machined by a punching tool. The silicone tubes
are stuck at the holes without an adhesive.

Laser drawing
PDMS

Assembling

Fig. 2: Photolithography technique.

A silicon wafer (Type N, Matsuzaki Seisakusyo, Co., Ltd.,Tokyo, Japan) is used for a surface mold for the disk (Fig. 3).
The diameter and the thickness of the wafer are 50 mm and 0.30mm, respectively.
The surface of the wafer is cleaned three times: with theisopropyl alcohol for ten minutes, with hydrogen peroxide
solution for ten minutes, and ultrapure water for ten minutes.
Then, the wafer was dried on the hot plate (PXW-4, Asahi-rika,Chiba, Japan) at 383 K for 10 minutes, and exposed to theoxygen gas in a reactive ion etching system (FA-1, Samco Inc.,Kyoto) to be characterized as hydrophilic (oxygen plasmaashing).

Fig. 3: Silicon wafer (diameter: 50 mm).

The photo-resist material of SU8 10 (Micro Chem Corp., MA,USA) was coated on the wafer with 0.015 mm thick at 1000rpm with a spin coater. The photo-resist was baked on theheated plate with two processes: at 338 K for 3 minutes, before
at 368 K for 7 minutes.
The pattern of holes (Fig. 4) to make columns on the mold wasdrawn on the wafer with a laser drawing system(DDB-201K-KH, Neoark Corporation, Hachioji, Japan). Tocontrol the dimension of the pattern on the mold with the laser
drawing system, the parameters were selected as follows: thevoltage of 4 V, the velocity of 0.1 mm/s, the acceleration of 0.5mm/s2. The pattern was baked on the heated plate with two
processes: at 338 K for 1 minute, before at 368 K for 3 minutes.
The photo-resist was developed with SU8 developer (NipponKayaku Co., Ltd, Tokyo, Japan) for five minutes to make microcolumns, where the laser beam was radiated. The wafer wasrinsed with the distilled water, and dried on the heated plate. To
decrease remaining stress and to increase the adhesiveness ofFig. 4: Pattern of holes: dimension (mm).

Immediately after the characterization, the flow path of thechamber was rinsed with a saline solution, and the suspensionof cells was introduced, successively.
One of the tubes is connected to the plastic syringe pump (Fig.
8). The room temperature was maintained at 25 degrees
Celsius. 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:
L929 (fibroblast-like, mouse connective tissue, RCB1422,Riken Bio Resource Center, Tsukuba) or C2C12 (Mouse
myoblast cell line originated with cross-striated muscle of C3Hmouse).

Fig. 5: PDMS baked on the mold.
Hole for connection to tube

L929 was cultured on a dish with the E-MEM (Eagle’s minimal
essential medium) containing 10% FBS (fetal bovine serum) inthe incubator for one week. Before the flow test, the cells
were exfoliated from the plate of the culture dish with trypsin,and suspended in the E-MEM.
C2C12 was cultured on a dish with the D-MEM (Dulbecco’s
Modified Eagle Medium) containing 10% FBS in the incubatorfor one week. Before the flow test, the cells were exfoliatedfrom the plate of the culture dish with trypsin, and suspended in
the D-MEM.

Micro traps

Fig. 6: The flow chamber consists of two transparent
polydimethylsiloxane (PDMS) disks and a thin silicone rubber
sheet.

Flow System
A one-way flow system is designed to control the wall shear
rate at the disk of PDMS (Fig. 7) [14]. The system consists ofa flow chamber, a micro syringe pump (International ScientificInstruments Supply Co., Ltd.), tubes and a microscope (Fig. 8).
A plastic tube of 3 mm internal diameter and of 5 mm external
diameter is used for the connector to the flow chamber. The
flow chamber consists of two transparent polydimethylsiloxane(PDMS) disks and a thin sheet of silicone rubber.
A thin sheet (0.1 mm thick) of silicone rubber, which has arectangular void space of 1 mm × 20 mm, is sandwiched
between the PDMS plates (Fig. 6). The void space forms achannel of 20 mm length × 1 mm width × 0.1 mm depth. The
three plates stick together with their surface affinity withoutadhesives. The inner surface of the chamber was exposed tothe oxygen gas in a reactive ion etching system (FA-1, Samco
Inc., Kyoto) to be characterized as hydrophilic (oxygen plasmaashing), before assembled.

Fig. 7: Flow test system: flow chamber and microscope(middle), syringe pump (right).

Syringe pump

Flow
Tube

Tube

Flow chamber
CellMicroscope
Fig. 8: Flow to syringe pump through flow chamber.

the suspended cell is approximately 0.01 mm. When cells
flow at the flow rate of 0.3 ×10-10 m3/s and 0.7 ×10-10 m3/s, cells
roll on the surface of the disk and are trapped in the holes.
When cells flow at the flow rate of 1.4 ×10-10 m3/s, some ofcells are trapped in the holes, while others flow pass over theholes. When cells flow at the flow rate of 2.8 ×10-10 m3/s,
every cell passes over the holes on the surface of the disk.
Several cells, which are trapped in a hole, flow out from thehole with the flow (Fig. 14).

PlateqDbG
Plate

Fig. 9: Parabolic velocity profile between parallel plates.

The suspension was introduced to the chamber at the constantflow. Variation was made on the flow rate: 0.3 ×10-10 m3/s,
0.7 ×10-10 m3/s, 1.4 ×10-10 m3/s and 2.8 ×10-10 m3/s. Variation
was made on the direction of the flow: from large holes to small
holes, and vice versa.
The flow path was carefully examined to avoid mixing of air
bubbles, which might stir the medium in the flow chamber andinduce exfoliation of cells. The behavior of cells on the plateof the chamber 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 parallel
plates is hypothesized (Fig. 9).
G = 6 q / (b D2)

Fig. 10: Laser measurement of morphology of convex column
at surface of the mold.

(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 1 mm (Fig. 9). The wall
shear rate G varies from 16 to 160 per second, when the flowrate q varies from 0.3 ×10-10 m3/s to 2.8 ×10-10 m3/s.
0.1 mm

3. RESULTS
The laser measurement of surface morphology of the moldshows that the diameter ranges from 0.01 mm to 0.04 mm, as
the designed dimension. The laser measurement also showsthat the mean height of micro columns is 0.01 mm (Fig. 10).
The dimension of the height scatters around the mean value.
Although the top surface of the column has asperity, the moldcan be used to make micro holes on the surface of the disk ofPDMS.

Fig. 11: L929 flows (1.4 ×10-10 m3/s).

Figs. 11-14 exemplify L929 in the flow in the chronologicalorder. In the figures, the circle shows interested cells, whichflow to a hole. After the cells are trapped in the hole, one of
the cells get out from the hole.
Figs. 15-17 exemplify C2C12 in the flow at 0.3 ×10-10 m3/s, at
0.7 ×10-10 m3/s and at 1.4 ×10-10 m3/s, respectively. In thefigure, the circle shows interested cells, which are trapped in thehole.
The observed behavior of cells is as follows. The diameter of0.1 mm

Fig. 12: L929 is going to be trapped (1.4 ×10-10 m3/s).

0.1 mm0.05 mmFig. 13: L929 cells are trapped in the hole (1.4 ×10-10 m3/s).

Fig. 16: C2C12 is trapped in the hole (0.7 ×10-10 m3/s). Flowfrom right to left.

0.1 mmFig. 14: L929 gets out from the hole (1.4 ×10-10 m3/s).

0.05 mmFig. 17: C2C12 is trapped in the hole (1.4 ×10-10 m3/s). Flowfrom right to left.

The experimental results show that a cell can be trapped with amicro hole under the wall shear rate less than 80 s-1. A cell istrapped in a hole with diameter of every size between 0.01 mm
and 0.04 mm, regardless of the order of holes in the flow.

4. DISCUSSION
0.05 mmFig. 15: C2C12 is trapped in the hole (0.3 ×10-10 m3/s). Flowfrom right to left.

The photolithography technique has been applied to fabricate
the micro channel [1, 2]. The microfluidic system has been
applied to sort biological cells [11], and to trap biological cells[12]. The system also used to study local environment aroundthe cultured cell [3, 6]. The micro pattern of the surface has
been applied to study the surface effect of adhesion of cells [4,5].
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 artificialvessels. The experimental results will contribute to analyze
adhesive mechanism of cancer cell during metastasis. Themicro trap might simulate adhesive mechanism of flowing cells.

5. CONCLUSION
A micro channel has been designed to trap flowing biologicalcells in vitro. Several micro traps of a cylindrical hole of 0.01
mm depth (diameter between 0.01 mm and 0.04 mm) have been
fabricated on the polydimethylsiloxane disk with thephotolithography technique. The experimental results showthat a cell is trapped in the micro hole with the diameter ofevery size between 0.01 mm and 0.04 mm under the wall shear
rate less than 80 per second.

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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