Behavior of Cells through Micro Slit
Shigehiro HASHIMOTO, Tsutomu HORIE, 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

ABSTRACTThe behavior of a biological cell through a micro slit has been
observed in vitro. An erythrocyte deforms and passes throughthe micro-circulation, of which the dimension is smaller thanthe diameter of the erythrocyte. The spleen, for example, hasspecial morphology in the blood flow path to sort erythrocytes.
The photolithography technique enables manufacturing themicro slit. A silicone disk was used for a mold, and a dryetching process was applied for the micro-fabrication. The slit,of which width is 0.002 mm and height is 0.08 mm, has beenmadebetweenmicro
cylindricalcolumns
ofpolydimethylsiloxane. The suspension of swine red blood
cells or L929 (fibroblast-like mouse cells) has alternatively been
introduced to the slits, and the behavior of cells has been
observed with a microscope. The experimental results showthat several cells can pass through the micro slit of 0.002 mm.
Keywords: Biomedical Engineering, Red Blood Cell, L929,
Photolithography and Micro-slit.

1. INTRODUCTION
An erythrocyte has flexibility [1] and deforms in the shear flow
[2, 3]. It also passes through micro-circulation, of which thedimension is smaller than the diameter of the erythrocyte.
After circulation through the blood vessels for days, theerythrocyte is trapped in the micro-circulation systems.
One of the systems, which trap erythrocytes, is a spleen. Thespleen has special morphology in the blood flow path to sort
injured erythrocytes [4-6].

The photolithography technique enables manufacturing a
micro-channel [7, 8]. 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, micro slits have been designed to control
behavior of biological cells in vitro.

2. METHODS
Micro Slits
Tow patterns of micro slits between micro cylindrical columnsof 0.05 mm diameter have been designed on a disk oftransparent polydimethylsiloxane (PDMS): one with 0.056 mm
height columns (type 1) and the other with 0.08 mm heightcolumns (type 2). Variation is made on the interval of thecolumns, which makes variation of the width of the slits: 0.002
mm, 0.003 mm and 0.004 mm.
MoldA silicon wafer (Type P, Matsuzaki Seisakusyo, Co., Ltd.,Tokyo, Japan) is used for a surface mold for the disk. The
diameter and the thickness of the wafer are 50 mm and 0.30 mm,respectively (Fig. 1).
The surface of the wafer is cleaned two times: with 2-propanol
for five minutes in an ultrasonic cleaner, and with ultrapure
water for ten minutes. Then, the wafer was dried on the hotplate (AHP-300, Asahi-rika, Chiba, Japan) at 383 K for 10minutes.

The pattern of holes (Fig. 3) was drawn on the wafer with alaser drawing system (DDB-201K-KH, Neoark Corporation,Hachioji, Japan). The 19 cylindrical holes with a diameter of
0.05 mm were arranged on the line with a small interval
between 0.003 mm and 0.005 mm. The width of the trace oflaser is proportional to the voltage, although the width isinversely proportional to the velocity.
To control thedimension of the holes of the mold with the laser drawing
system, the parameters were selected as follows: the voltage of3 V, the velocity of 0.1 mm/s, the acceleration of 0.5 mm/s2.

Fig. 1: Silicon wafer.

The photo-resist was developed with tetra-methyl-ammoniumhydroxide (NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki,Japan) for several minutes. The wafer was rinsed with thedistilled water, and dried on the heated plate. To increase theadhesiveness of the coating, the wafer was baked at 383 K for 5minutes.
The wafer was etched with the plasma gas using Si Deep RIE
System (MUC-21 ASE-SRE, Sumitomo Precision Products Co.,
Ltd., Amagasaki, Japan) to make the micro cylindrical holes.
The switching mode between C4F8 gas and SF6 gas was appliedon the disk.

Fig. 2: Photo-resist material on wafer: before (left) and after(right) spinning.

The residual photo-resist was exfoliated in the separating
solution (Hakuri 105, Tokyo Ohka Kogyo Co., Ltd, Kawasaki,Japan). The wafer was dipped in 2-propanol, before rinsed
with the distilled water. Then, the wafer with the holes wasdried on the hot plate, and used for the concave mold to makemicro cylindrical columns in the following process.
PDMS Disk
The surface of the wafer with micro pattern was coated with0.001 mm thickness of parylene in the parylene coater(PDS-2010, Speciality Coating Systems, Indianapolis). Afterthe wafer was enclosed with a peripheral wall of polyimide (Fig.
4), PDMS (Sylgard 184 Silicone Elastomer Base, Dow CorningCorporation) was poured with the curing agent (Dow CorningCorporation) on the wafer (Fig. 5). The volume ratio between
PDMS and the curing agent is ten to one. The volumes of
PDMS are 2 cm3 for the upper disk and 10 cm3 for the lower
disk, respectively.
After degassing, PDMS was baked at 383 K for one hour in anoven (DX401, Yamato Scientific Co., Ltd). The baked disk of
PDMS is exfoliated from the mold (Fig. 6).
Measurement of Morphology
The dimension of the manufactured slit was measured withEDX WETSEM (SEM, Scanning Electron Microscope,JSM-6360LA), JEOL Ltd., Tokyo, Japan) without coating.

Fig. 3: Pattern of holes (type 1): dimension (mm), 0.005 (A),0.004 (B), 0.003 (C); depth, 0.056 mm.

Photolithography
The photo-resist material of OFPR-800 (Tokyo Ohka Kogyo
Co., Ltd, Tokyo, Japan) was coated on the wafer with 0.002 mm
thick at 5000 rpm for 30 s with a spin coater (Fig. 2). The
photo-resist was baked on the heated plate at 383 K for 90 s.

Flow Test System
A one-way flow system was designed to observe the behavior ofcells through the micro slits in vitro. The system consists of aflow chamber, a syringe pump, tubes and a microscope. The
micro-syringe-pump (Fusion200, CXF1020, ISIS Co., Ltd.,Osaka) was used for the syringe pump. A plastic tube of 3 mm
internal diameter and of 5 mm external diameter was used forthe connector to the flow chamber.
The flowchamberconsistsof two
transparent
polydimethylsiloxane (PDMS) disks and a thin sheet of silicone
rubber (Fig. 7). The thickness of the sheet is 0.1 mm.

Micro columns

Fig. 4: Mold enclosed with peripheral wall of polyimide.

Fig. 5: PDMS poured with the curing agent on the wafer.

Fig. 7: Flow chamber consists of two PDMS disks and a thinsheet of silicone rubber.

Fig. 8: Upper disk of PDMS with two holes for connection withtube.

The flowchamberconsistsof two transparent
polydimethylsiloxane (PDMS) disks and a thin sheet of silicone
rubber (Fig. 7). The thickness of the sheet is 0.1 mm.
A rectangular open space of 1 mm × 20 mm is cut off in thesheet, and sandwiched between the PDMS plates. The open
space forms a channel of 20 mm length × 1 mm width × 0.1 mm
depth.
The three parts stick together with their surface affinity without
an adhesive. The diameter of two PDMS plates is 50 mm.
The thicknesses of the upper and the lower disks are 10 mm and2 mm, respectively.

Fig. 6: Disk of PDMS.

The PDMS disk, which has micro columns on the upper surface,
was placed in the bottom. The upper PDMS disk has no micro
pattern, and has two holes of 5 mm diameter machined with apunching tool (Fig. 8). The silicone tubes are stuck at theholes without an adhesive for the inlet and the outlet (Fig. 9).

Fig. 9: Flow chamber with inlet and outlet tubes.

Fig. 11: Flow system.

Fig. 10: Swine blood.

Flow Test
Two kinds of cells were used in the flow test: L929
(fibroblast-like, mouse connective tissue, RCB1422, Riken Bio
Resource Center, Tsukuba), and swine red blood cells.
In the case of L929, cells were suspended in the phosphatebuffer solution.
Swine red blood cells were used after preservation in therefrigerator for two days. After centrifugation of the swine
blood, the red blood cells were separated from plasma (Fig. 10).
The cells were suspended in the phosphate buffer solution to
make a suspension of red blood cells at the volume ratio of 0.1
percent.
The movement of cells near the slits was observed with aninverted phase-contrast microscope (IX71, Olympus Co., Ltd.,Tokyo), while the suspension of cells was pumped at the flowrate of 1 cm3 /hour at 298 K (Fig. 11). In the flow path ofdepth of 0.1 mm (width of 1 mm), the flow rate makes mean
velocity of 2.8 mm/s.

Fig. 12: Micro cylindrical columns in the flow path of whichwidth is 1 mm. The bar shows 0.1 mm.

AB

Fig. 13: Micro slits between micro cylindrical columns. The barshows 0.05 mm.

CFig. 15: L929 (A) flows through micro slits of type 1. Cell
flows above column (B). Cell stuck in slit (C). The barshows 0.05 mm.

Fig. 14: Micro slits between micro cylindrical columns.
Fig. 16: L929 moves to wider slit of type 2 (from top tobottom). The bar shows 0.05 mm.
3. RESULTS
Fig. 12 shows the top view of the twelve micro cylindricalcolumns in the flow path.
The microscopic measurement shows that the minimum widthof the slits between columns is 0.002 mm (Figs. 13 & 14).
In Figs. 15-18, the medium flows from left to right. Figs.
15-17 show L929 flows near the micro columns. Some cells
are passing through the micro slit (Fig. 15(A)). Some cells are
flowing over the micro column in type 1 (Fig. (B)). Few sells
are flowing over the column in type 2. Some cells get stuck
between the micro columns (Fig. 15(C)). Some cells move towider slit (Fig. 16).
Fig. 18 shows red blood cells flowing near the micro columns.
Every cell is passing through the slit.

Fig. 17: L929 flows through micro slits (from left to right) oftype 2. The bar shows 0.05 mm.

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.

REFERENCES

Fig. 18: Red blood cells flow through micro slits of type 2.
The diameter of columns is 0.05 mm.

4. DISCUSSION
The micro-slit is useful for treatment of cell in diagnostics [9].
The fine architecture of the red pulp of the spleen has been
investigated in the previous studies [4-6]. The continuity
between capillaries and splenic sinuses has been examined withthe microscope. The special morphology might relate to thefunction for sorting erythrocytes.
In the previous studies, the typical diameter of the microchannel, which simulates the capillary blood vessel, is around0.005 mm [8]. The red blood cell, on the other hand, passes
through micro slit narrower than 0.001 mm in the spleen. The
small dimension of passage has been applied to biological cells
in the present study.
The effect of flow on cells has been investigated in the previous
studies [10-11].
A micro channel could simulate themicrocirculation system. To simulate the microcirculation
system with a fabricated channel, the three dimensional
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The micro-channel devices may contribute to the developmentof biotechnology.

5. CONCLUSION
The behavior of a biological cell through a micro slit has been
observed in vitro. The slit, of which width is 0.002 mm andheight is 0.08 mm, has been made between micro cylindricalcolumns. The suspension of swine red blood cells, or L929
has been introduced to the slits, and the movement of cells has
been observed with a microscope. The experimental results
show that several cells can pass through the micro slit of 0.002
mm.

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