Micro Hole for Trapping Flowing Cell
Shigehiro HASHIMOTO, Yusuke TAKAHASHI, Haruka HINO, Reona NOMOTO
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, Japan

ABSTRACTMicro holes have been designed to trap a biological cell, whichflows through a micro channel in vitro. Several micro traps ofa half cylindrical hole of 0.002 mm depth (0.02 mm diameter)
have been fabricated on the surface of the polydimethylsiloxane(PDMS) disk with the photolithography technique.
A
rectangular flow channel (0.1 mm depth × 1 mm width × 20
mm length) has been constructed with a silicone film of 0.1 mm
thick, which has been sandwiched by two transparent PDMS
disks. Two types of biological cells were used in the test
alternatively: C2C12 (mouse myoblast cell line originated withcross-striated muscle of C3H mouse), or MC3T3-E1 (mouse
osteogenic cell line). A constant flow (2.8 ×10-10 m3/s or 8.3
×10-10 m3/s) 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 results show that the cell is trapped with the micro hole
under the wall shear rate less than 100 s-1 for several seconds.

vessel is thin enough to take in things, the cancer cell easily
metastasizes through the membrane.
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 [1-4]. Several
micro-fabrication processes have been designed to control
adhesion of biological cells in vitro [2-8], and to simulatemorphology of microcirculation [9]. The micro-fabrication
technique has also been applied to design microfluidic systems
in vitro [10-16]. Cells are rolling on the surface of the wall inthe shear flow, and make adhesion to the wall [6]. The surface
was modified to capture flowing cells [8, 17, 18]. The
technique will also be applied to handle cells in diagnostics invitro [19]. In the present study, micro holes have been
designed to trap a biological cell, which flows through a microchannel in vitro.

2. METHODS
Keywords: Biomedical Engineering, C2C12, MC3T3-E1,
Micro Trap and Polydimethylsiloxane.

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 topatient’s death. The tumor permeates through the lymph
vessels. The cancer cell transits from the original place toanother place, and proliferates to make tumor in another place.
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

Micro Traps
Several micro traps of a half cylindrical hole of 0.002 mm depth(Fig. 1) have been fabricated on the surface of thepolydimethylsiloxane (PDMS) disk with the photolithography
technique (Fig. 2). The diameter of the half cylinder is 0.02
mm. Sixteen holes are arranged in two lines. The interval
between holes is 0.02 mm. Variation is made on the directionof the half cylindrical holes (Fig. 3).
A rectangular flow channel has been constructed with a silicone
film, which has been sandwiched by two transparent PDMS
disks (Fig. 4).
A silicon wafer (Type P, 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 plasma
ashing).
The photo-resist material of low viscosity (SU8-2: Micro
Chem Corp., MA, USA) was coated on the wafer with 0.002
mm thick at 2000 rpm for 30 s with a spin coater. The
photo-resist was baked on the heated plate with two processes:
at 338 K for 1 minute, before at 368 K for 3 minutes.
The pattern of holes (Fig. 3) to make half cylindrical columnson the mold was drawn on the wafer with a laser drawing
system (DDB-201K-KH, Neoark Corporation, Hachioji, Japan).
To control the dimension of the pattern on the mold with thelaser drawing system, the parameters were selected as follows:
the voltage of 4 V, the velocity of 0.01 mm/s, the acceleration of
0.5 mm/s2. The pattern was baked on the heated plate withtwo processes: at 338 K for 1 minute, before at 368 K for 3
minutes.
The photo-resist was developed with SU8 developer (Nippon
Kayaku 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 ofthe coating, the wafer was baked at 423 K for 5 minutes.

Traps
Fig. 3: Pattern of holes: dimension (mm).
Cell

Hole for connection to tube

Fig. 1: Several micro traps in flow channel.

Silicon wafer
Coating

Mold
SU8

Developing
Transcription
Micro traps

Laser drawing
PDMS

Fig. 2: Photolithography technique.

Assembling

Fig. 4: The flow channel consists of two transparent
polydimethylsiloxane (PDMS) disks and a thin silicone rubber
sheet.

After development, the dimension of the half cylindrical microcolumns on the mold was measured with a laser microscope(VK-X200, Keyence Corporation, Osaka, Japan). The convex
mold with micro pattern is used only for the lower disk of
PDMS to make the micro holes on the surface.

(NaHCO3, 75 g/L) and 1% of Antibiotic-Antimycotic (penicillin,
streptomycin and amphotericin B, Life Technologies). Before
the flow test, the cells were exfoliated from the plate of theculture dish with trypsin, and suspended in the alpha-MEM.

The surface of the wafer was coated with 0.001 mm thicknessof Parylene (Specialty Coating Systems, Inc., IN, USA).

The suspension was introduced to the chamber at the constantflow with the micro syringe pump. Variation was made on theflow rate: 2.8 ×10-10 m3/s and 8.3 ×10-10 m3/s.

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.

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 holes in the channel wasobserved with the microscope.

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. 4) are machined by a punching tool. The silicone tubes
are stuck at the holes without an adhesive.
Flow System
A one-way flow system is designed to control the wall shear
rate at the disk of PDMS (Fig. 5) [14]. The system consists ofa flow channel, a micro syringe pump, tubes and a microscope(Fig. 6). A plastic 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
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. 4). 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 channel was exposed tothe oxygen gas in a reactive ion etching system (FA-1, Samco
Inc., Kyoto) to be characterized as hydrophilic (oxygen plasma
ashing), before assembled.

Fig. 5: Flow test system: flow chamber and microscope(middle), syringe pump (right).
Syringe pump

Flow
Tube

Tube

Immediately after the characterization, the flow path of thechamber was rinsed with a saline solution, and the suspensionof cells was introduced, successively.

Flow channel

One of the tubes is connected to the syringe pump (Fig. 6).
The room temperature was maintained at 25 degrees Celsius.
The chamber is placed on the inverted phase-contrast
microscope (IX71, Olympus Co., Ltd., Tokyo).

Cell
Microscope
Fig. 6: Flow to syringe pump through flow chamber.

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 MC3T3-E1 (mouse osteogenic cellline, Riken Bio Resource Center, Tsukuba).
C2C12 was cultured with the D-MEM (Dulbecco’s Modified
Eagle Medium) containing 10% FBS 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.
MC3T3-E1 was cultured with the alpha-MEM (alpha modified
Eagle’s minimal essential medium) containing 10% FBS (fetal
bovine serum), in the incubator for one week. The medium
also ontains 2.4% of sodium bicarbonate aqueous solution

Plate

q
D
b

G
Plate

Fig. 7: Parabolic velocity profile between parallel plates.

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. 7).

In the case of MC3T3-E1, one of the cells is trapped in the holefor one second in the flow rate of 2.8 ×10-10 m3/s (Fig. 11).
Every cell rolls over the hole in the flow rate of 8.3 ×10-10 m3/s
(Fig. 12).

G = 6 q / (b D2)

The time of trapped in the hole is longer with C2C12 than with
MC3T3-E1.

(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. 7). The wall
shear rate G varies from 35 to 100 per second, when the flowrate q varies from 2.8 ×10-10 m3/s to 8.3 ×10-10 m3/s.

3. RESULTS
The laser measurement of surface morphology of the moldshows that the diameter of half cylindrical column is 0.02 mm,
as the designed dimension. The laser measurement also showsthat the mean height of micro columns is 0.0015 mm (Fig. 8).
The flow tests show the following results.
The moving cells over the holes are able to be observed with themicroscope in the flow test system. The diameter of thesuspended cell is approximately 0.01 mm. The trapped time of
the cell is measured compared with the movement of floatingcell.
In the case of C2C12, one of the cells is trapped in the hole for
5 seconds in the flow rate of 2.8 ×10-10 m3/s (Fig. 9). Anothercell is trapped in the hole for 8 seconds in the flow rate of 8.3
×10-10 m3/s (Fig. 10).

Fig. 9: C2C12 in red lower circle approaches to the hole (upper
left), enters (upper right), is trapped for 5 s (lower left), andexits (lower right). Flow (2.8 ×10-10 m3/s) from right to left.

Fig. 10: C2C12 enters (left), is trapped for 8 s (middle), andexits (right). Flow (8.3 ×10-10 m3/s) from right to left.
Fig. 8: Laser measurement of morphology of convex column at
surface of the mold. Cross section at line A-B (bottom).

study. The asymmetrical hole might be suitable for trap thansymmetrical hole.
The depth of the micro holes was 0.01 mm in the previous study[8]. In the present study, the depth of the micro holes of 0.002
mm is smaller than diameter of the cells. The deeper hole may
have advantage to trap cells. The shallow trap, on the other
hand, may distinguish cells. The duration of the trapped timeof the cell might relate to interaction between the micro holeand the cell: adhesiveness between the cell and the surface ofthe micro pattern, or deformability of the cell.
Behavior of biological cells might depend on several factors:
magnetic field [20], electric field [21], or mechanical field [2224].

5. CONCLUSION

Fig. 11: MC3T3-E1 in red small circle approaches to the hole
(upper left), enters (upper right), is trapped for 1 s (lower left),and exits (lower right). Flow (2.8 ×10-10 m3/s) from right toleft.

Micro holes have been designed to trap a biological cell, whichflows through a micro channel in vitro. Several micro traps ofa half cylindrical hole of 0.002 mm depth (0.02 mm diameter)
have been fabricated on the surface of the polydimethylsiloxane(PDMS) disk with the photolithography technique. Two typesof biological cells were used in the experiment alternatively:
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse), or MC3T3-E1 (mouse osteogenic cellline). The experiments show that the cell is trapped with themicro hole under the wall shear rate less than 100 s-1 for several
seconds.

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.

Fig. 12: MC3T3-E1 in red small circle approaches to the hole(left), is on the hole (middle), exits from hole (right). Flow
(8.3 ×10-10 m3/s) from right to left.

4. DISCUSSION
The photolithography technique has been applied to fabricate
the micro channel. The microfluidic system has been applied
to sort biological cells [13], and to trap biological cells [14, 17,18]. The system also used to study local environment aroundthe cultured cell [1, 4]. The micro pattern of the surface has
been applied to study the surface effect of adhesion of cells [2,3].
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
adhesive mechanism of cancer cell during metastasis. The
micro trap might simulate adhesive mechanism of flowing cells.
In the previous study, cylindrical holes were used for trap ofcells. The half cylindrical holes are designed in the present

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