Deformation of Cell Passing through Micro Slit
Atsushi MIZOI, Yusuke TAKAHASHI, Haruka HINO, Shigehiro HASHIMOTO
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
Deformation of a biological cell passing through a micro slit has
been observed in vitro. The photolithography techniqueenables manufacturing the micro slit. A silicon wafer is used
for a mold, and a dry etching process is applied for the
micro-fabrication of the surface. The slit, of which width is
0.05 mm and height is 0.0006 mm or 0.003 mm, has been
designed between two parts of transparent polydimethylsiloxane
disks, which have micro ridges. The slit is placed at the
middle part of a flow channel: 0.05 mm height, 3 mm width,and 30 mm length. The suspension of swine red blood cells,
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse), or 3T3-L1 (mouse fat precursor cells)
was alternatively introduced to the slits by drawing with asyringe pump at a constant flow rate of 2.8 ×10-10 m3/s. The
deformation of cells passing through the micro slit wasobserved with an inverted phase-contrast microscope. The
experimental results show that several red blood cells deformand pass through the micro slit of 0.0006 mm height, and thatsome cells of C2C12 and of 3T3-L1 deform and pass throughthe micro slit of 0.003 mm height.

in vitro [17].
In the present study, micro slits have been designed to observe
deformation of biological cells passing through the slit in vitro.

2. METHODS
Micro Slits
The slits, of which width (W) is 0.05 mm and height (H) is
0.0006 mm or 0.003 mm, have been designed between two
parts of transparent polydimethylsiloxane (PDMS) disks.
Both of the disks have micro ridges (Fig. 1). The lower parthas ten ridges of 0.2 mm width (1 mm length) with the interval(W) of 0.05 mm. Variation is made of the height (H) of thelower ridges: 0.0006 mm or 0.003 mm. The upper part has aridge of 0.05 mm height: 0.05 mm width and 2 mm length.
These ridges make contact each other in the perpendicular
position, and make slits between the ridges.

Keywords: Biomedical Engineering, Red Blood Cell, C2C12,
3T3-L1, Photolithography and Micro-slit.

Mold Base
A silicon wafer (Type N, 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.

1. INTRODUCTION

The surface of the wafer is cleaned by a spin dryer (Japan
Create Co., Ltd., Tokorozawa, Japan).

An erythrocyte has flexibility [1, 2] and deforms in the shear
flow [3-5]. It also passes through micro-circulation, of which
the dimension 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.

AS

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 [6-8].

BH

The photolithography technique enables manufacturing a
micro-channel [9-16]. Several micro-fabrication processes
have been designed to simulate morphology of microcirculation.
The technique also will be applied to handle cells in diagnostics

W

Fig. 1A: Upper (A) and lower (B) micro ridges to make microslits (S) for cell passing.

C

A
B
Fig. 1B: Upper (A) and lower (B) micro ridges to make microslits. Thin sheet of silicone rubber (C) with a rectangular holeis sandwiched between A and B.

Fig. 2: Lines for measurement of surface micro morphology oflower disk.

Photolithography
To increase affinity between the wafer and the photo-resist
material, hexamethyldisilazane (Tokyo Chemical Industry Co.,
Ltd., Tokyo, Japan) was coated on the wafer at 3000 rpm for 30
s with a spin coater (Mikasa Co., Ltd., Tokyo, Japan).
The photo-resist material of OFPR-800 (Tokyo Ohka Kogyo
Co., Ltd, Tokyo, Japan) was coated on the wafer at 3500 rpm
for 5 s with the spin coater. The photo-resist was baked on theheated plate at 373 K for 90 s.
The pattern of ridges was drawn on the wafer by a laser (wavelength of 405 nm) 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 0.02 mm/s, the acceleration of 0.3 mm/s2. To
increase the adhesiveness of the coating, the wafer was baked at
393 K for 300 s.
The photo-resist was developed with tetra-methyl-ammoniumhydroxide (NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki,
Japan) for one minute. The wafer was rinsed with theultrapure water and with the distilled water, and dried with thespin dryer.
For the upper disk, 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 thedeeper micro grooves. On the upper disk, the switching mode
between C4F8 gas and SF6 gas was applied.
For the lower disk, RIE-10NR (Samco International, Kyoto,
Japan) was used. For etching of 0.0006 mm depth, the gas ofSF6 (20 cm3/min at 1013 hPa) with O2 (10 cm3/min at 1013hPa) and with Ar (10 cm3/min at 1013 hPa) was applied at 50 W
at 20 Pa for three minutes. For etching of 0.003 mm depth, thegas of SF6 (20 cm3/min at 1013 hPa) with O2 (10 cm3/min at
1013 hPa) and with Ar (10 cm3/min at 1013 hPa) was applied at
200 W at 20 Pa for three minutes.
The residual photo-resist was exfoliated in the compact etcher
FA-1 (Samco International, Kyoto, Japan). The gas of O2 (30cm3/min) was applied at 100 W for five minutes.

Fig. 3: Lines for measurement of surface micro morphology ofupper 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).
The wafer with the grooves was used as the concave mold to
make micro convex ridges in the following process. The
dimension of the grooves on manufactured mold without
parylene coating was measured with the laser microscope(VK-X200, Keyence Corporation, Osaka, Japan) (Figs. 2 & 3).
PDMS Disk
After the wafer was enclosed with a peripheral wall ofpolyimide, 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 8.8 cm3 for the upper disk and 4.4 cm3 for the lower disk,
respectively.
After degassing, PDMS was baked at 433 K for two hours in anoven (DX401, Yamato Scientific Co., Ltd, Tokyo, Japan). The
baked disk of PDMS is exfoliated from the mold. The
dimension of the ridges on manufactured PDMS was measured
with the laser microscope.
Two holes of 5 mm diameter were machined with a punchingtool at the upper disk to make the inlet and the outlet for theflow channel.
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 channel, a syringe pump, tubes and a
microscope.

painted to fill the gap from the outside, and baked at 373 K for
one hour in the oven.
Flow Test
Three kinds of cells were used in the flow test: the passage
fourth of C2C12 (mouse myoblast cell line originated withcross-striated muscle of C3H mouse), the passage seventh of
3T3-L1 (mouse fat precursor cells), and swine red blood cells.
In the case of C2C12 and of 3T3-L1, experimental protocol is
as follows. The cells were exfoliated from the bottom of theculture dish with trypsin. Cells were suspended in the mediumof D-MEM (Dulbecco’s Modified Eagle’s Medium) with thedensity of 5000 cells/cm3. To adjust the density of cells, thenumber of cells is counted with Burker-Turk hemocytometer
under a phase contrast microscope (IX71, Olympus, Tokyo).

Fig. 4: Flow test system: pump (left), channel (middle), andmicroscope.

The swine blood (Tokyoshibaurazouki Co., Ltd., Tokyo, Japan)
was diluted by the phosphate buffer solution without calcium tomake a suspension of red blood cells at the volume ratio of 0.02
percent. The red blood cells were used immediately, or used
after preservation in a refrigerator at 277 K in four weeks.
The behavior 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. 5). In the flow path of depthof 0.05 mm (width of 3 mm), the flow rate makes mean velocityof 2 mm/s.

3. RESULTS
The dimension of the 0.0006 mm grooves on the manufactured
mold of the lower part measured by the laser microscope is asfollows. Although the data of depth at tracings along threelines (Fig. 2) across ten grooves show slightly scattered values
between 0.00051 mm and 0.00085 mm, the mean depth is
0.00063 mm.
Fig. 5: Flow channel on microscope.

The micro-syringe-pump (Fusion200, CXF1020, ISIS Co., Ltd.,
Osaka) was used for the syringe pump. The silicone tube of 3
mm internal diameter and of 5 mm external diameter was used
for the connector to the flow channel.
The flow channel consists of two transparent PDMS disks and athin sheet of silicone rubber (thickness of 0.05 mm, ARAM
Corporation, Osaka) (Fig. 1B, Fig. 5).
A rectangular open space of 3 mm × 30 mm is cut off in the
sheet, and sandwiched between the PDMS disks. The open
space forms a channel of 30 mm length × 3 mm width × 0.05
mm depth.
The three parts stick together with their surface affinity. The
diameter of two PDMS disks is 50 mm. The thicknesses of theupper and the lower disks are 4 mm and 2 mm, respectively.
The lower PDMS disk has ten micro ridges on the upper surface.
The upper PDMS disk has one micro ridge on the lower surface.
The upper disk has two holes. The silicone tubes are stuck at
the holes for the inlet and the outlet.
To seal thecircumferential micro gap between disks, extra PDMS was

The dimension of the 0.003 mm grooves on the manufactured
mold of the lower part measured by the laser microscope is asfollows. Although the data of depth at tracings along threelines (Fig. 2) show slightly scattered values between 0.0026 mm
and 0.0030 mm, the mean depth is 0.0028 mm.
The data of the depth at the trace along four lines (Fig. 3) acrossthe groove on the manufactured mold of the upper parts scatter
between 0.044 mm and 0.055 mm, and the mean depth is
0.0049 mm.
Fig. 6 exemplifies the laser microscopic image of the 0.0006
mm ridges on the manufactured PDMS disks of the lower part.
Although the heights at the tracings along three lines (Fig. 2)
across ten ridges show scattered values between 0.00053 mm
and 0.00077 mm, the mean height is 0.00066 mm.
Fig. 7 exemplifies the tracing of the 0.003 mm ridges on the
manufactured PDMS disks of the lower part measured by thelaser microscope. Although the heights at the tracings along
three lines (Fig. 2) across ten ridges show slightly scatteredvalues between 0.0028 mm and 0.0031 mm, the mean height is0.0029 mm.

The data of the height at the trace along four lines (Fig. 3)
across the ridge of the upper parts slightly scatter between 0.054
mm and 0.055 mm, and the mean height is 0.055 mm.
Fig. 8 shows the microscopic view of the slits assembled
between the ridges of upper and lower disks.
Figs. 9-11 exemplify the red blood cell passing through the slit.
The cell flows from right to left. The preserved red blood cells
pass through slits of 0.0006 mm more easily than the fresh red
blood cell. The cell marked with the circle in Fig. 10 cannot
pass through the slit.
Fig. 12 exemplifies C2C12 passes through slit of 0.003 mm.
One cell (marked with the circle) deforms to a flat disk (B-D),
is moving through the slit (E, F), and has passed (G) throughthe slit.
Fig. 13 exemplifies 3T3-L1 passes through slit of 0.003 mm.
One cell (marked with the circle) deforms to a flat disk (A), ismoving through the slit (B, C), and has passed (D) through theslit.

Fig. 6: Laser microscope image of micro ridges on lower disk
to make micro slit for red blood cells (upper), and tracing alongtransverse line (Fig. 2) of ridges (lower). Dimension from left
to right is 1.5 mm.

Fig. 7: Tracing along transverse line of micro ridges on lower
disk (Fig. 3) to make micro slit for cells (Fig. 1). Dimension
from left to right is 1.5 mm.
Fig. 9: Fresh red blood cells (photos of every two seconds: A, B,
C, D). Flow from right to left. The bar shows 0.2 mm.

Fig. 8: Micro slits. Each arrow shows the path for cell.
Dimension from left to right is 1.5 mm.

Fig. 10: Red blood cells (preserved for two weeks) passing
through slit (photos of every two seconds: A, B, C, D). Flowfrom right to left. The bar shows 0.2 mm.

Fig. 11: Red blood cell (preserved for three weeks) passing
through slit (photos of every two seconds: A, B, C, D). Flowfrom right to left. The bar shows 0.2 mm.

Fig. 13: 3T3-L1 passes through slit. Flow from right to left.
The bar shows 0.2 mm. Successive photos: A 0 s, B 3 s, C 6 s,
D 6.2 s.

4. DISCUSSION
3T3-L1 is a cell line derived from cells of 3T3 mouse. 3T3-L1
cells have a fibroblast-like morphology. The cells differentiate
into an adipocyte-like phenotype under appropriate conditions.
Because the dimension of the slit is mainly governed by theheight of the lower ridge, the slight over dimension of theheight of the upper ridge might not vary the dimension of theslit. The ridge of the PDMS has elasticity, so that the microridge may deform with the small ratio.
In the present study, the micro slit has been designed withnarrower dimension than the previous study [18] to observe thedeformation of cells or to trap some red blood cells.
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 [6-8]. 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 [10]. 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. 12: C2C12 passes through slit. Flow from right to left.
The bar shows 0.5 mm. Successive photos: A 0 s, B 6.3 s, C
6.7 s, D 6.9 s, E 9.97 s, F 10.3 s, G 10.3 s.

The effect of flow on cells has been investigated in the previous
studies [19, 20].
A micro channel could simulate themicrocirculation system. Fatigue of erythrocyte was evaluatedthrough the narrow path [21, 22].
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 [17].
The micro-channel devices may
contribute to the development of biotechnology.

5. CONCLUSION
Deformation of a biological cell passing through a micro slit has
been observed in vitro. The slit, of which width is 0.05 mm
and height is 0.0006 mm or 0.003 mm, has been designed
between two parts of transparent polydimethylsiloxane disks.
The experimental results show that several red blood cells
deform and pass through the micro slit of 0.0006 mm height,
and that some cells of C2C12 and of 3T3-L1 deform and passthrough the micro slit of 0.003 mm height.

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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