Design of Micro-channel forControlling Behavior of Cells in Vitro
Fumihiko SATO, Shigehiro HASHIMOTO, Takahiro OOSHIMA
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/andKei OYA
Research Institute for Science and Technology,
Kogakuin University, Tokyo, JapanandHiromichi FUJIE
Human Mechatronic Systems, Faculty of System Design,
Tokyo Metropolitan University, Tokyo, Japan
andToshitaka YASUDA
Bio-systems Engineering, Department of Electronic Engineering,Tokyo National College of Technology, Tokyo, JapanABSTRACT

1. INTRODUCTION

A micro-channel has been designed to control behavior of
biological cells in vitro. An erythrocyte deforms and passes
through the micro-circulation, of which the dimension issmaller than its diameter. The spleen, for example, has special
morphology in the blood flow path to sort injured erythrocytes.
The micro-channel is useful for treatment of cell in diagnostics,too. The photolithography technique enables manufacturing
the micro-channel. A borosilicate glass disk was used for amold, and a wet etching process was applied for
micro-fabrication. The study shows that the curvature of themicro groove can be controlled with the isotropic etchingprocess in the photolithography machining and that thephotolithography technique is effective to manufacture themicro-channel for the manipulation of a biological cell.

An erythrocyte has flexibility [1] and deforms in the shear flow[2]. It also passes through micro-circulation, of which thedimension is smaller than its diameter. After circulation
through the blood vessels for days, the erythrocyte is trapped in
the micro-circulation systems.

Keywords: Biomedical Engineering, Cell, Micro-channel andEtching.

One of the systems, which trap erythrocytes, is a spleen. Thespleen has special morphology in the blood flow path to sort
injured erythrocytes [3-5].
The photolithography technique enables manufacturing a
micro-channel [6-13]. Several micro-fabrication processes
have been designed to simulate morphology of microcirculation.
The technique also will be applied to handle cells in diagnostics
in vitro [9].
In the present study, a micro-channel has been designed tocontrol behavior of biological cells in vitro. The fabrication
technique has been investigated with the photolithography
process.

Developing

CoatingLaserdrawing

Etching

Removing

Fig. 3: Disk coated with chromium.
Fig. 1: Photolithography and etching processes.

Fig. 4: Disk coated with photo-resist material.
Fig. 2: Borosilicate glass disk.

2. METHODS
Photolithography
The micro channel has been fabricatedphotolithography and etching processes (Fig. 1).

withtheA borosilicate glass disk (Tempax, Matsunami Glass Ind., Ltd.,Tokyo, Japan) of 50 mm diameter and 1.1 mm thick was used
for a mold (Fig. 2).
The disk was cleaned with 2-propanol (IPA, Taisei Kagaku,Tokyo, Japan) in an ultrasonic cleansing apparatus for fiveminutes. After cleaning, the disk was rinsed with distilledwater, and dried on a heated plate (PXW-4, Asahi-rika, Chiba,Japan).
The disk was coated with chromium in the sputtering system(L-210S-FH, Canon Anelva Corporation, Kawasaki, Japan) (Fig.
3). In the system, the thickness of coating increases inproportion to the time. The time for coating was adjusted to 3
min to control the thickness to 0.1 mm.

The photo-resist material of Ethyl-Cellosolve-Acetate(2-ethoxyethyl acetate) (OFPR-800, Tokyo Ohka Kogyo Co.,
Ltd, Kawasaki, Japan) was coated with a spin coater (Fig. 4).
The photo-resist was baked for ninety seconds on the plate
heated at 383 K.
The groove pattern (Fig. 5) was drawn on the disk with a laser
drawing system (DDB-201K-KH, Neoark Corporation,Hachioji, Japan). The pattern was baked for five minutes on
the plate heated at 393 K.
To control the width of the groove of the mold with the laser
drawing system, variations were made in the parameters: thevoltage, the velocity, the acceleration, and the focus. In thepresent study, the target minimum value of the width is one
micrometer for the channel of cells. Several bars were drawn
with the variations in the parameters, and the each width of thegrooves was measured (Fig. 6).

Fig. 5: Groove pattern.

Unit of dimension is mm.

Fig. 7: Developed pattern.

Fig. 6: Control the width of the groove.

The photo-resist was developed with tetra-methyl-ammoniumhydroxide (NMD-3, Tokyo Ohka Kogyo Co., Ltd, Kawasaki,Japan) for five minutes (Fig. 7). The disk was rinsed with thedistilled water, and dried on the heated plate.
The disk was dipped in TW (Nissin-Kasei Co., Ltd, Tokyo,Japan) to etch chrome (Fig. 8). The disk was rinsed with thedistilled water, and dried on the heated plate.
Wet Etching
The disk was etched with the hydrofluoric acid (LAM2680,Wako Pure Chemical Industries, Ltd., Osaka, Japan) (Fig. 9).
Variation was made on the time for etching. After etching, thedisk was rinsed with the distilled water to stop etching.
The photo-resist was exfoliated in separating solution (Hakuri
105, Tokyo Ohka Kogyo Co., Ltd, Kawasaki, Japan) heated at353 K., and dipped in IPA, before rinsed with the distilled water(Fig. 10).

Fig. 8: After chrome etching.

The disk, again, was dipped in TW (Nissin-Kasei Co., Ltd,Tokyo, Japan) to etch chrome. The disk was rinsed with thedistilled water, and dried on the heated plate.
Measurement of Morphology
The dimension of the groove was measured with a laser
microscope (VK-9510, Keyence Corporation, Osaka, Japan).
The width of the groove was measured, when the parameters of
the laser drawing system were varied. Both the width and thedepth of the groove were measured, when the time of etching
was varied (Fig. 11).

Wet etchingabFig. 9: After hydrofluoric acid etching.
Fig. 11: Extension of width (a) and depth (b) were measured,
after wet etching.

Fig. 12: Some patches along the narrow groove.
Fig. 10: After photo-resist removal.

3. RESULTS
The experimental results show following relations between thewidth of the groove and the parameters of the drawing system:
the width increases with the voltage, the width decreases withthe velocity, and the width decreases with focus. To decrease
the voltage of the single drawing, the each pattern was traced
three times. The experimental results show that the too highvelocity makes some patches along the narrow groove (Fig. 12).
The minimum width of 540 nm has been attained in laser
drawing of three times at the voltage of 3.15 V, the velocity of 2
mm s-1, acceleration of 0.5 mm s-2, and the focus offset of 0.4 V(Fig. 13). The width of 0.022 mm has been attained in laser
drawing of three times at the voltage of 5.0 V, the velocity of
0.5 mm s-1, acceleration of 0.5 mm s-2, and the focus offset of-0.9 V (Fig. 14).

Fig. 13: Channel of minimum width of 540 nm before wetetching.

The effect of flow on cells has been investigated in the previous
studies [14-16].
A micro channel could simulate themicrocirculation system. To simulate the microcirculation
system with a fabricated channel, the three dimensional
curvature of the wall of the flow channel might be important.
Cells are responsive to the micro morphology of the scaffold[17]. The microgroove governs the behavior of cells [18].
The Tempax has enough thermostability during thephotolithography process.
Several factors govern themorphology of the groove at etching on glass [19]. The
control of the temperature during the photolithography process
is important to control the uniform dimension of the groove.
The wet etching process has been investigated on glass andapplied to a monitoring system for a cell [19].
Fig. 14: Channel of width of 0.022 mm before wet etching.
Magnification ratio is the same as Fig. 13.

Length [mm]The uniformity of thickness of coating of the photo-resist
material governs the uniformity of the laser drawing through thefocusing position. To control the penetration depth in the wet
etching process, the timing of using the stop solution isimportant.
The narrow groove decreases the speed ofpenetration during the etching process.
When the width of the micro groove is narrow enough, the ratioof penetration of the hydrofluoric acid to the laminar direction
increases. The isotropic etching produces round sidewalls.
This process controls curvature of the micro groove on the mold.
It enables making semi-cylindrical grooves.

0.007
0.006
0.005
0.004
0.003

ab0.002

After etching process, the mold will be applied topolydimethylsiloxane [16] to make a micro-channel forbiological cells. The micro-channel devices may contribute tothe development of biotechnology [10].

5. CONCLUSION
0.001

0510Time [s]1520Fig. 15: Extension of width (a) and depth (b) related to the timeof etching. See Fig. 11.

Both the width and the depth after application of the wet
etching on the above mold (Figs. 13&14) have been measured(Fig. 12) and illustrated for the wider channel (Fig. 14) in Fig.
15.
The depth of the groove increases in proportion to the time ofetching. The width of the groove also increases with the timeof etching, but extension of width is above one micrometer evenat the 0.5 s of etching.

4. DISCUSSION
The fine architecture of the red pulp of the spleen has been
investigated in the previous studies [3-5]. The continuity
between capillaries and splenic sinuses has been examined withthe microscope. The special morphology might relate to thefunction for sorting erythrocytes.

A micro-channel has been designed to control behavior of cells
in vitro. The study shows that the curvature of the microgroove can be controlled with the isotropic wet etching process
after the photolithography machining and that thephotolithography technique is effective to manufacture themicro-channel for the manipulation of a biological cell.

6. ACKNOWLEDGMENT
This work was supported by a Grant-in-Aid for Strategic
Research Foundation at Private Universities (2008-2012) fromthe Japanese Ministry of Education, Culture, Sports andTechnology.

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