Deformation of Cell Passing through Micro Slit
between Micro Ridges
Atsushi MIZOI, Yusuke TAKAHASHI, Haruka HINO, Shigehiro HASHIMOTO
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/andToshitaka YASUDA
Bio-systems Engineering, Department of Electronic Engineering,Tokyo National College of Technology, Tokyo, Japan
ABSTRACTThe micro slits have been designed between micro ridges, anddeformation of a biological cell passing through a micro slit has
been observed in vitro. The slit, of which width is 1.3 mm andheight is 0.0004 mm or 0.010 mm, has been designed between
the transparent polydimethylsiloxane disk and glass disk.
Each disk has the micro ridge. The slit is placed at the middle
part of a flow channel: 0.05 mm height, 3 mm width. The
suspension of swine red blood cells, or Hepa1-6 (mouse
hepatoma cell line of C57L mouse) was alternatively introduced
to the slits by drawing with a syringe pump at a constant flowrate of 2.8 ×10-11 m3/s (2.8 ×10-10 m3/s for Hepa1-6). The
deformation of cells passing through the micro slit wasobserved with an inverted phase-contrast microscope. The
experimental results show that cells deform to the flat disk andpass through the micro slit: red blood cell at 0.0004 mm height,
and Hepa1-6 at 0.010 mm height. The deformation isevaluated with the ratio of the projected area of the disk during
the passing through the slit. The designed slit between microridges has capability to evaluate the deformability of cells.
Keywords: Biomedical Engineering, Red Blood Cell, Hepa1-6,
Deformation, Photolithography, and Micro-slit.

The photolithography technique enables manufacturing a
micro-channel [15-21]. 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.
In the present study, micro slits have been designed, andfabricated using the photolithography technique to observe
deformation of biological cells passing through the slit in vitro.

2. METHODS
Micro Slit
The slit, of which width (W) is 1.30 mm and height (H) is
0.0004 mm (narrow) or 0.010 mm (wide), has been designed
between a transparent polydimethylsiloxane (PDMS) disk and aborosilicate glass (Tempax) disk (Figs. 1 & 2). Both of thedisks have micro ridges. The upper disk of PDMS has a ridgeof 0.06 mm height: 0.10 mm width and 2 mm length. The
lower disk of glass has two ridges of 0.3 mm width (2.3 mm
length) with the interval (W) of 1.3 mm. At the ridges of thelower part, variation is made on the height (H): 0.0004 mm(narrow, H1) or 0.010 mm (wide, H2). These ridges makecontact each other in the perpendicular position, and make slits
between the ridges.

1. INTRODUCTION
The behavior of biological cells depends on the flow [1]. The
deformability of the biological cell plays an important role invivo. An erythrocyte, for example, has high flexibility [2-7]and deforms in the shear flow [8, 9]. It also passes throughmicro-circulation, of which the dimension is smaller than the
diameter of the red blood cell. After circulation through theblood vessels for days, the red blood cell is trapped in the
micro-circulation systems.
Several systems sorts cells according to the deformability invivo. One of the systems, which trap red blood cells, is aspleen. The spleen has special morphology in the blood flow
path to sort injured red blood cells [10-14].

Upper Disk
A silicon wafer (Type N, Matsuzaki Seisakusyo, Co., Ltd.,Tokyo, Japan) is used for a surface mold for the upper disk (Fig.
3). The diameter and the thickness of the wafer are 50 mm
and 0.30 mm, respectively.

W
Fig. 1: Slit between ridges.

H8.8 cm3 for the upper disk.
After degassing, PDMS was baked at 353 K for one hour 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 (Fig. 9). Two holes of 5 mmdiameter were machined with a punching tool at the upper disk
to make the inlet and the outlet for the flow channel.
Fig. 2: Upper PDMS (A) and lower glass (B) disks, and silicone
sheet (C).
1. Silicone wafer

5. Dry etching

2. OFPR-800

6. Residual photo-resist
exfoliation

3. Exposure

7. Transcription

4. Developing

8. PDMS

Fig. 3: Photolithography process of upper disk.

To increase affinity between the wafer and the photo-resist
material, hexamethyldisilazane (HMDS, 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 positive photo-resist material of OFPR-800(Tokyo Ohka Kogyo Co., Ltd, Tokyo, Japan) was coated on thewafer at 3500 rpm for 5 s with the spin coater. The
photo-resist was baked in an oven.
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). To increase the adhesiveness ofthe coating, the wafer was baked in the oven. The photo-resist
was developed with tetra-methyl-ammonium hydroxide(NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki, Japan).
For the upper disk, the wafer was etched with the Deep RIE
System (MUC-21 ASE-SRE, Sumitomo Precision Products Co.,
Ltd., Amagasaki, Japan) to make the deeper micro grooves.
To exfoliate the residual photo-resist material from the surface,
the disk was exposed to the oxygen gas of 30 milliliter per
minute at power of 100 W for five minutes using a compact
etcher (FA-1, Samco Inc., Kyoto): the oxygen plasma ashing.
The dimension of the grooves on manufactured mold wasmeasured with the laser microscope (VK-X200, Keyence
Corporation, Osaka, Japan) (Fig. 8). After the mold of thewafer 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 volume of PDMS is

Photomask for Lower Disk
Three kinds of the photomask were designed with variation ofpatterns: a vacancy of 3 mm × 30 mm (A), two vacancies of 0.3
mm × 2.3 mm with the interval of 1.3 mm (B), and two patternsof 0.3 mm × 2.3 mm with the interval of 1.3 mm (C). The
borosilicate glass (Tempax) disk (50 mm diameter, 1.1 mm
thickness) was used for the base of the mask (Fig. 4).
Titanium was coated on the surface with 150 nm thickness inthe electron beam vapor deposition apparatus (JBS-Z0501EVC,
JEOL Ltd., Japan). The positive photoresist material of
OFPR-800LB (Tokyo Ohka Kogyo Co., Ltd, Tokyo, Japan) was
coated on the disk at 3500 rpm for 5 s with the spin coater.
The photoresist was baked in the oven at 368 K for threeminutes.
The pattern was drawn on the mask with a laser drawing system.
The pattern was baked on the heated plate at 368 K for threeminutes.
The photoresist was developed withtetra-methyl-ammonium hydroxide (NMD-3, Tokyo Ohka
Kogyo Co., Ltd., Kawasaki, Japan) for 8 minutes. The disk
was rinsed by the ultrapure water, and dried by the spin-dryer.
The titanium coating disk was etched with the plasma gas using
RIE-10NR (Samco International, Kyoto, Japan).
Lower Disk (Narrow H1)
The positive photo-resist material of OFPR-800 (Tokyo Ohka
Kogyo Co., Ltd, Tokyo, Japan) was coated on the Tempax glass(50 mm diameter) at 3500 rpm for 5 s with the spin coater (Fig.
5). The photo-resist was baked in the oven at 368 K for threeminutes.
Two kinds of masks (A and C) coated with titanium werecombined and adhered on the surface of OFPR-800, and thephotoresist was exposed to the UV light through the mask in the
single sided mask aligner (M-1S, Mikasa Co. Ltd., Japan) at 10
V for 30 s. The photoresist was baked in the oven at 368 K for
three minutes.

1. Tempax glass

2. Ti deposit

5. Developing

6. Dry etching

3. SU-8-10

4. Exposure
Fig. 4: Photomask for lower disk.

7. Residual
photo-resist
exfoliation

The photoresist was developed with tetra-methyl-ammoniumhydroxide (NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki,
Japan) for eight minutes. The glass was immersed in abuffered hydrofluoric acid solution on a mixture of hydrofluoric
acid and ammonium fluoride for 20 minutes for etching.
Lower Disk (Wide H2)
The negative photoresist material of high viscosity (SU8-10:
Micro Chem Corp., MA, USA) was coated on the Tempax glass(50 mm diameter) at 2500 rpm for 30 s with a spin coater. The
photo-resist was baked in the oven at 333 K for three minutes.
The mask (B) coated with titanium was adhered on the surfaceof SU8-10, and the photoresist was exposed to the UV lightthrough the mask in the single sided mask aligner. The
aluminum sheet of 3 mm × 50 mm was adhered on the surfaceof the photoresist, and the resist was exposed to the UV lightthrough the mask in the single sided mask aligner to form theflow channel.

Fig. 7: Flow channel with micro slit.

After the resist was baked in the oven at 333 K for threeminutes, the photoresist was developed with SU8 developer(Nippon Kayaku Co., Ltd, Tokyo, Japan).

The flow channel (Fig. 2) consists of two transparent upper andlower disks and a thin sheet of silicone rubber (thickness of 0.05mm, ARAM Corporation, Osaka).

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 channel, a syringe pump, tubes and a microscope (Fig. 6).

A rectangular open space of 5 mm × 30 mm is cut off in the
sheet, and sandwiched between the disks. The open space
forms a channel of 30 mm length × 5 mm width × 0.05 mm
depth.

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.

After hydrophilization by the oxygen plasma ashing by RIE(FA-1, Samco International, Kyoto, Japan), the three parts stick
together with their surface affinity. The diameter of the glass
disks is 50 mm. The thicknesses of the upper and the lower
disks are 4.5 mm and 1.1 mm, respectively.

1. Tempax glass

2. OFPR-800

3. Photo-Mask
6. Wet Etching

The lower PDMS disk has two micro ridges on the uppersurface. The upper PDMS disk has one micro ridge on thelower surface. The upper disk has two holes. The silicone
tubes are stuck at the holes for the inlet and the outlet. To seal
the circumferential micro gap between disks, extra PDMS was
painted to fill the gap from the outside, and baked at 373 K for
one hour in the oven (Fig. 7).

4. Exposure

5. Developing

7. Residual Photo-Resist
Exfoliation

Fig. 5: Photolithography process of lower disk.

Flow Test
Two kinds of cells were used in the flow test: swine red blood
cells, and Hepa1-6 (passage < 10, mouse hepatoma cell line of
C57L mouse)
The swine blood (Tokyoshibaurazouki Co., Ltd., Tokyo, Japan)
was diluted by the saline solution to make a suspension of red
blood cells at the volume ratio of 0.03 percent. The red blood
cells were used immediately, or used after preservation in arefrigerator at 277 K in four weeks.
In the case of Hepa1-6, experimental protocol is as follows.
The cells were exfoliated from the bottom of the culture dishwith trypsin, and suspended in the medium of D-MEM(Dulbecco’s Modified Eagle’s Medium).
After the saline solution was prefilled in the flow channel, thesuspension of the cells was introduced into the flow channel.

Fig. 6: Flow (from right to left) test system: reservoir (right),
flow channel on the microscope, syringe pump (left).

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 admitted by thesyringe pump at 298 K (at the flow rate of 2.8 ×10-11 m3/s forred blood cell, 2.8 ×10-10 m3/s for Hepa1-6). In the flow pathof height of 0.05 mm (width of 5 mm), the flow rate makes
mean velocity of 1.1 mm/s at the flow rate of 2.8 ×10-10 m3/s.
At the microscopic image, the outline of each cell was traced
with “Image J”, and the area (A) was calculated. The
deformation ratio (A2 / A1) was calculated from the projectedarea of each cell before slit (A1) and that in the slit (A2).
Fig. 10: Tracing across groove (H1) on lower disk.
3. RESULTS
Fig. 8 exemplifies the tracing across the groove on the mold forthe ridge of the upper disk measured by the laser microscope(VK-X200, Keyence Corporation, Osaka, Japan). The tracingshows that the depth of the groove is 0.061 mm. Fig. 9
exemplifies the tracing across the ridge on the upper disk of
PDMS measured by the laser microscope. The tracing showsthat the height of the ridge is 0.061 mm. Fig. 10 exemplifies
the tracing across the groove (for the narrow slit H1) etched on
the glass measured by the stylus profiler (Dektak XT-E, Bruker
Corporation). The tracing shows that the depth is 0.00038 mm.
Fig. 11 exemplifies the tracing across the ridge (for the wide slit
H2) on the lower disk measured by the laser microscope. The
tracing shows that the height of the ridge is 0.0105 mm.

Fig. 11: Tracing across ridge (H2) on lower disk.

Fig. 12 exemplifies red blood cell flowing through slit H1.
The red blood cell deforms and passes through the slit of 0.0004
mm. Fig. 13 exemplifies Hepa1-6 flowing through slit H2.
The cell deforms and passes through the slit of 0.010 mm.

Fig. 8: Tracing across groove on mold for upper disk.

Fig. 12: Red blood cell flows through slit H1 from right to left
(A→H). Dimension from left to right is 0.4 mm.

Fig. 9: Tracing across ridge on upper disk.

Fig. 14 shows the projected area of the red blood cell: before
the slit (left), and in the slit (right). Every cell deforms to theflat disk, while passing through the slit. The projected area ofthe cell increases under deformation, and the mean deformation
ratio (A2 / A1) is 1.31 (Fig.15). Fig. 16 shows the projectedarea of Hepa1-6: before the slit (left), and in the slit (right).
Every cell deforms to the flat disk, while passing through theslit. The projected area of the cell increases under deformation,and the mean deformation ratio (A2 / A1) is 1.10 (Fig. 17).

4. DISCUSSION
The biological cells are sorted according to the shape, anddeformability in vivo. Several cells pass through the micro slit.
Some cells or fragments, which pass through the slit, aredecomposed. Some cells, which cannot pass through thenarrow channel, are captured, on the other hand. Erythrocyte
deforms from the biconcave disk to the parachute like shape,
when it is passing through micro capillary. Erythrocyte rotatesin the shear field. The most of biological cells, on the other
hand, keep the spherical shape, when they are flowing in themedium.
When passing through a micro slit, erythrocyte deforms to thethin disk and struggles to go through. The mechanicalproperty of erythrocyte is maintained with energy consumption.
To keep the biconcave shape, energy is consumed between
structural proteins in the red blood cell.
Because thedimension of the slit is mainly governed by the height of thelower ridge in the present study, the slight over dimension of theheight of the upper ridge does not vary the dimension of the slit.
The ridge of the PDMS has elasticity, so that the micro ridge
may deform with the small ratio.

A1

A2

Fig. 14: Projected area of red blood cell [μm2]: before slit (A1;left), in slit (A2; right).
A2 / A1

Fig. 15: Deformation ratio (A2 / A1) (mean: 1.31) of 16 red
blood cells.

A1

A2

Fig. 16: Projected area of Hepa1-6 [μm2]: before slit (A1; left),in slit (A2; right)
A2 / A1

Fig. 17: Deformation ratio (A2 / A1) (mean: 1.10) of 16 cells of
Hepa1-6.

Fig. 13: Hepa1-6 flows through slit H2 from right to left
(A→H). Dimension from left to right is 0.8 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. Some red blood cells can pass throughthe position out of the slit. In the present study, the micro slit
has been designed with narrower dimension than the previous
study [16] to observe the deformation of cells or to trap some
red blood cells. The fine architecture of the red pulp of thespleen has been investigated in the previous studies [13]. The
continuity between capillaries and splenic sinuses has been
examined with the microscope. The special morphology
might relate to the function for sorting erythrocytes.

In the previous studies, the typical diameter of the microchannel, which simulates the capillary blood vessel, is around0.005 mm. 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 deformation is evaluated with theratio of the projected area of the disk during the passing throughthe slit in the present study. The mean ratio of red blood celland Hepa1-6 are 1.1 (1.02 - 1.25) and 1.3 (1.07-1.71),
respectively. The deformation in the perpendicular direction
can be observed with slit between micro cylindrical pillars [16].

5. CONCLUSION
Deformation of a biological cell passing through a micro slit has
been observed in vitro. The slit, of which width is 1.3 mm andheight is 0.0004 mm or 0.010 mm, has been designed between aridge at transparent polydimethylsiloxane disk and ridges atglass disk. The experimental results show that red blood cells
deform to the flat disk and pass through the micro slit of 0.0004
mm height, and that Hepa1-6 deform to the flat disk and passthrough the micro slit of 0.010 mm height. The deformation isevaluated with the ratio of the projected area of the disk during
the passing through the slit. The mean ratio of red blood celland Hepa1-6 are 1.1 and 1.3, respectively. The designed slit
between micro ridges has capability to evaluate thedeformability of cells.

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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