Behavior of Cell Passing through Micro Slit
Shigehiro HASHIMOTO, Atsushi MIZOI, Haruka HINO, Kenta NODA, Kenichi KITAGAWA
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
ABSTRACT

2. METHODS

Behavior of a biological cell passing through a micro slit has
been observed in vitro. The photolithography techniqueenables manufacturing the micro slit. A silicone disk is used
for a mold, and a dry etching process is applied for the
micro-fabrication. The slit, of which width is 0.85 mm andheight is 0.001 mm, has been designed between two parts oftransparent polydimethylsiloxane disks, which have microridges. The suspension of swine red blood cells or C2C12(mouse myoblast cell line originated with cross-striated muscle
of C3H mouse) has alternatively been introduced to the slits bydrawing with a syringe pump. The behavior of cells passing
through the micro slit has been observed with an inverted
phase-contrast microscope. The experimental results showthat several red blood cells can pass through the micro slit,
although C2C12 cannot pass through the micro slit.

Micro Slits
The slit, of which width is 0.85 mm and height is 0.001 mm,
has been designed between two parts of transparent
polydimethylsiloxane disks (Figs. 1 & 2). They have microridges. The lower part has three ridges of 0.001 mm height(0.1 mm width, 1 mm length) with the interval of 0.85 mm.
The upper part has a ridge of 0.057 mm height: 0.05 mm widthand 2 mm length (Fig. 3). These ridges make contact eachother in the perpendicular position, and make slits between theridges.

Keywords: Biomedical Engineering, Red Blood Cell, C2C12,Photolithography and Micro-slit.

1. INTRODUCTION

Mold Base
A 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.
The surface of the wafer is cleaned three times in an ultrasoniccleaner: with 2-propanol for five minutes, with hydrogen
peroxide solution for five minutes, and with ultrapure water for
five minutes. Then, the wafer was dried with an air gun.

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 red blood cell.
After circulation through the blood vessels for days, the red
blood cell is trapped in the micro-circulation systems.
One of the systems, which trap red blood cells, is a spleen.
The spleen has special morphology in the blood flow path tosort injured red blood cells [4-6].
The photolithography technique enables manufacturing a
micro-channel [7-9]. Several micro-fabrication processes havebeen designed to simulate morphology of microcirculation.
The technique also will be applied to handle cells in diagnostics
in vitro [10].
In the present study, micro slits have been designed to control
behavior of biological cells in vitro.

Fig. 1: Channel with slit.

of the grooves on manufactured mold without coating wasmeasured with the laser microscope (VK-X200, Keyence
Corporation, Osaka, Japan).
Silicone sheet

Fig. 2: Cross-section view of slit: A-A’ in Fig.1.

PDMS Disk
The surface of the wafer with micro pattern was coated with
0.001 mm thickness of parylene in the parylene coater(PDS-2010, Speciality Coating Systems, Indianapolis). Afterthe wafer was enclosed with a peripheral wall of polyimide,
PDMS (Sylgard 184 Silicone Elastomer Base, Dow Corning
Corporation) was poured together with the curing agent (Dow
Corning Corporation) on the wafer. The volume ratio of
curing agent is ten percent of PDMS. The volumes of PDMS
are 4.4 cm3 for the upper disk and 2.2 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. The dimension of theridges on manufactured PDMS was measured with the laser
microscope.

0.1 mm2 mm
1 mm
0.05 mm
2 mm
Fig. 3: Dimension of micro ridges: upper (left), lower (right).

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. The photo-resist
was baked on the heated plate at 383 K for 90 s.
The pattern of ridges (Fig. 3) was drawn on the wafer with alaser drawing system (DDB-201K-KH, Neoark Corporation,
Hachioji, Japan).
The width of the trace of laser is
proportional to the voltage, although the width is inversely
proportional to the velocity. To control the dimension of theridges of the mold with the laser drawing system, theparameters were selected as follows: the voltage of 3 V, thevelocity of 0.1 mm/s, the acceleration of 0.5 mm/s2.
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 with the air gun. To increase theadhesiveness of the coating, the wafer was baked at 383 K for 5
minutes.
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 grooves. On thelower disk, the gas of SF6 (50 cm3/min at 1013 hPa) with O2 (25cm3/min at 1013 hPa) and with Ar (25 cm3/min at 1013 hPa)
was applied at 50 W at 10 Pa for five minutes. On the upper
disk, the switching mode between C4F8 gas and SF6 gas wasapplied.
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 grooves wasdried with the air gun, and used for the concave mold to makemicro convex ridges in the following process. The dimension

Flow Test System
A one-way flow system was designed to observe the behavior ofcells through the micro slits in vitro (Fig. 4). The system
consists of a flow 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 for the connector to the flow chamber.
The flowchamberconsistsof two transparent
polydimethylsiloxane (PDMS) disks and a thin sheet of silicone
rubber (thickness of 0.05 mm, ARAM Corporation, Osaka) (Fig.
1).
A rectangular open space of 2 mm × 30 mm is cut off in the
sheet, and sandwiched between the PDMS plates. The open
space forms a channel of 30 mm length × 2 mm width × 0.05
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 4 mm and
2 mm, respectively.
The PDMS disk, which has three micro columns on the uppersurface, was placed in the bottom. The upper PDMS disk has
one micro column on the lower surface, and has two holes of 5
mm diameter machined with a punching tool. The silicone
tubes are stuck at the holes with an adhesive for the inlet andthe outlet.
The assembled disks are contained on thepolystyrene dish of 70 mm diameter and the circumferential
crevice between the parts is filled with adhesive from outside(Fig. 4).
Flow Test
Two kinds of cells were used in the flow test: C2C12 (mouse
myoblast cell line originated with cross-striated muscle of C3Hmouse), and swine red blood cells.
In the case of C2C12, cells were suspended in the medium of
D-MEM (Dulbecco’s Modified Eagle’s Medium) with thedensity of the cells 15000 cells/cm3, and pumped at flow rate of
0.005 cm3/hour.

Fig. 4: Flow system: channel and microscope.

Swine red blood cells in sodium acid citrate aqueous solution(ACD-A, Terumo Corporation, Japan) were used after
preservation in a refrigerator for two days.
After
centrifugation of the swine blood, the red blood cells wereseparated from plasma. The cells were suspended in thephosphate buffer solution to make a suspension of red blood
cells at the volume ratio of 0.05 percent.

Fig. 5: Micro slit. Cells flow from left to right under verticalridge (0.05 mm height). Horizontal ridge has 0.001 mm height(1 mm length, 0.1 mm width).

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 0.003 cm3 /hour at 298 K (Fig. 4). In the flow path ofdepth of 0.05 mm (width of 2 mm), the flow rate makes mean
velocity of 0.08 mm/s.

3. RESULTS
Fig. 5 shows the manufactured slit observed with the inverted
microscope. Cells flow from left to right over the verticalridge (0.05 mm height).
The horizontal ridge crossing
perpendicularly to the vertical ridge makes the slit between
vertical ridge and the surface of the lower disk.
Fig. 6 exemplifies the dimension of the groove on the
manufactured mold of the lower part measured by the laser
microscope. The width and the length are 0.1 mm and 1 mm,
respectively. The tracings of the depth along the three lines(Fig. 6(A)) and the cross section (Fig. 6(B)) of the groove show
slightly scattered values around the mean value of 0.00088 mm.
Fig. 7 exemplifies dimension of the groove of the upper parts.
The tracings of the depth along the three lines of the grooveshow slightly scattered values around the mean value of 0.053
mm.
Figs. 8 & 9 exemplify dimension of the ridges on the
manufactured lower and upper parts measured by the laser
microscope, respectively. The mean values of the height are
0.0011 mm and 0.055 mm, respectively. The mean value of
thickness of the sheet of silicone rubber measured by the laser
microscope is 0.057 mm.
Figs. 10-12 exemplify the red blood cell passing through the slit.
The cell flows from left to right. The velocity of the cellmoving through the slit is not accelerated compared with thedecrease of cross section of the flow path. Fig. 13 exemplifies
C2C12 approaching to the slit. C2C12 was not able to passthrough the slit.

Top view

Cross-section of three lines

Fig. 6(A): Dimension of groove on manufactured mold of lower
part measured by laser microscope: tracings of cross section
shows mean depth of 0.00088 mm.

4. DISCUSSION
In the present study, the micro slit has been designed withnarrower dimension than the previous study [11] to observe thedeformation of cells or to trap some red blood cells in thepresent study (Fig. 14). Deformation has not been observed
while the red blood cell is passing through the slit. The low
velocity of the red blood cell might show the friction between
the wall of the slit and the cell during movement.

Fig. 6(B): Dimension of groove on manufactured mold of lower
part measured by laser microscope: top view (upper), andtracings (lower) of cross section between 2 to 2’ (upper), whichshows mean depth of 0.00088 mm.

Top view

Fig. 7(B): Dimension of groove on manufactured mold of upper
part measured by laser microscope: top view (upper), crosssection (middle) shows mean depth of 0.053 mm, threedimensional view (lower).

Cross-section of three lines
Fig. 7(A): Dimension of groove on manufactured mold of upper
part measured by laser microscope: tracings of cross section
shows mean depth of 0.053 mm.

The dimension of the slit might be extended during theassembly process of two disks so that most of red blood cells
pass through the slit.
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.

Top view

Cross-section of three lines

Fig. 8(A): Dimension of ridge on manufactured lower partmeasured by laser microscope: mean height is 0.0011 mm.

(Tracings of cross-section)

Red blood cellFig. 10: Red blood cell approaches to slit. Flow from left toright. The bar shows 0.02 mm.

(Three-dimensional view)
Red blood cellFig. 8(B): Dimension of ridge on manufactured lower partmeasured by laser microscope: mean height is 0.0011 mm.

Fig. 11: Red blood cell passing through slit. Flow from left toright. The bar shows 0.02 mm.

Top view

Red blood cellFig. 12: Red blood cell passed through micro slit. Flow fromleft to right. The bar shows 0.02 mm.

Cross-section of three lines

Flow(Cross-section perpendicular to longitudinal direction)
Fig. 9: Dimension of ridge on manufactured upper partmeasured by laser microscope: mean height is 0.055 mm.

C2C12
100 µm

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.

Fig. 13: C2C12 approaches to slit. Flow from right to left.
The bar shows 0.1 mm.

Red blood cell passing through slit
Fig. 14: Deformed cell might pass through slit.

The effect of flow on cells has been investigated in the previous
studies [12, 13].
A micro channel could simulate themicrocirculation system. Fatigue of erythrocyte was evaluatedthrough the narrow path [14, 15].
To simulate themicrocirculation system with a fabricated channel, the three
dimensional curvature of the wall of the flow channel might beimportant. Cells are responsive to the micro morphology ofthe scaffold. The micro groove governs the behavior of cells.
The micro-slit is, on the other hand, useful for treatment of cellin diagnostics [10].
The micro-channel devices may
contribute to the development of biotechnology.

5. CONCLUSION
The behavior of a biological cell through a micro slit has been
observed in vitro. The slit, of which width is 0.85 mm andheight is 0.001 mm, has been designed between two parts oftransparent polydimethylsiloxane (PDMS) disks.
Thesuspension of swine red blood cell, or C2C12 has been
introduced to the slits, and the movement of cells has been
observed with a microscope. The experimental results showthat several cells can pass through the micro slit.

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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