Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)

Slit between Micro Machined Plates for Observation of Passing
Cell: Deformation and Velocity
Shigehiro HASHIMOTO, Yusuke TAKAHASHI, Keisuke KAKISHIMA, Yuki TAKIGUCHI
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/the walls of the slit, the slit is designed with the combination of
micro ridges in the present study.

ABSTRACT
A micro slit between micro ridges has been used for observationof a biological cell passing through the micro slit in vitro. At
the middle part of the flow channel, the slit (0.8 mm < width <2 mm, 0.01 mm height) has been made between the microridges on the transparent polydimethylsiloxane plate and theglass plate by photolithography technique. Four kinds of cells
were used in the test: C2C12 (mouse myoblast cells), Hepa1-6(mouse hepatoma cells), MC3T3-E1 (mouse osteoblast
precursor cell line), and Neuro-2a (mouse neural crest-derived
cell line). The suspension of each kind of cells was injected tothe slits. The deformation of cells passing through the microslit was observed with an inverted phase-contrast microscope.
For the reference, the passing speed of the porcine red blood
cells through the slit was observed. The experimental results
show that cells deform to the flat circular disk and pass throughthe micro slit. The passing velocity through the slit depends
on the deformation ratio of the cell. The designed slit between
micro ridges has capability for observation of the deformationand the passing velocity of the single cell.

In the present study, a micro slits have been fabricated between
micro ridges by the photolithography technique, anddeformation of a biological cell passing through the micro slit
has been observed in vitro.

2. METHODS
Micro Slit
The slit, of which width (w) is 0.8 mm, length (l) is 0.1 mm andheight (h) is 0.010 mm, has been designed between atransparent polydimethylsiloxane (PDMS) plate and aborosilicate glass (Tempax) plate [5] (Fig. 1). The upper plateof PDMS has a rectangular ridge of 0.05 mm height, 0.10 mm
width, and 2 mm length. The lower plate of glass has arectangular groove of 0.010 mm depth (h), 2 mm width, and 20
mm length, which has a narrow width (W) part (0.8 mm < W <2 mm). These plates keep contact to make slits between them.
The slit is located at the middle part of the flow channel, which
has the following dimension: height of 0.064 mm, width of 2mm, and length of 30 mm.

Keywords: Biomedical Engineering, C2C12, Hepa1-6,
Neuro-2a, MC3T3-E1, Photolithography and Micro-slit.

Photomask for Upper Plate
The glass plate of 0.2 mm thick was used for the base of themask. Before the vapor deposition of titanium, the surface ofthe glass plate was hydrophilized by the oxygen (30 cm3/min,
0.1 Pa) plasma ashing for five minutes at 100 W by the reactive
ion etching system (FA-1, Samco Inc., Kyoto). Titanium wascoated on the surface with 150 nm thick in the electron beamvapor deposition apparatus (JBS-Z0501EVC, JEOL Ltd., Japan)
at the deposition rate of 0.1 nm/s. The positive photoresist
material of OFPR-800LB was coated on the titanium at the disk
at 7000 rpm for 60 s with the spin coater (1H-DX2, Mikasa Co.
Ltd., Tokyo, Japan). The photoresist was baked on the heatedplate at 368 K for five minutes.

1. INTRODUCTION
The deformability of the biological cell plays an important rolein vivo. An erythrocyte, for example, has high flexibility, anddeforms in the shear flow [1-3]. 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. A slit is one of the systems, whichsorts biological cells in vivo [4]. The sorting at the slit
depends on the deformability of the cell. Several cells are able
to pass very narrow slits.
The photolithography technique enables manufacturing the
micro-morphology in the flow channel [5-14]. The behaviorof cells in the flow channel will be applied to the cell sorting[9-16]. The photolithography technique can be applied tomake a micro slit [5-10]. The slit between micro cylinders
was made to sort cells in the previous study [9, 10]. The
deformation of the depth direction between cylinders, however,cannot be observed by the conventional optical microscope.
To observe the deformed cell at the direction perpendicular to

Fig. 1: Microslit in flow channel.

102Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)

The pattern was drawn on the mask with a laser drawing system(DDB-201K-KH, Neoark Corporation, Hachioji, Japan). To
control the dimension of the pattern on the mask with the laser
drawing system, the parameters were selected as follows: thevoltage of 3.3 V, the velocity of 0.1 mm/s, the acceleration of
0.5 mm/s2, and the focus offset at +0.1. The pattern was bakedon the heated plate at 368 K for five minutes. The photoresist
was developed with NMD-3 for 5 minutes. The disk wasrinsed by the ultrapure water, and dried by the spin-dryer. The
titanium coating disk was etched with the plasma gas (SF6, Ar)
using RIE-10NR (Samco International, Kyoto, Japan). For
etching, the gas of SF6 (50 cm3/min at 1013 hPa) with Ar (50cm3/min at 1013 hPa) was applied at 100 W at 4 Pa for ten
minutes.
The residual OFPR-800LB was exfoliated byacetone,surface with 150 nm thick in the electron beam vapor deposition
apparatus at the deposition rate of 0.1 nm/s. A part ofTitanium coating was covered with the polyimide tape to makethe flow channel of 2 mm width (narrow part: 0.8 mm width) ×
20 mm length. The titanium coating disk was etched with theplasma gas (SF6, Ar) using RIE-10NR. For etching, the gas ofSF6 (50 cm3/min at 1013 hPa) with Ar (50 cm3/min at 1013hPa) was applied at 100 W at 4 Pa for ten minutes. Afrter theetching, the polyimide tape was exfoliated.
Lower Plate
A glass plate was cleaned by an ultrasonic cleaner with alkaline
solution, and rinsed by the ultrapure water. The surface of theglass plate was hydrophilized by the oxygen (30 cm3/min, 0.1
Pa) plasma ashing for five minutes at 100 W by the reactive ion
etching system (FA-1). The negative photoresist material ofhigh viscosity (SU8-10) was coated on the glass plate at 2000
rpm for 30 s with a spin coater. After the photoresist was
baked on the heated plate at 338 K for three minutes, the plate
was baked on the heated plate at 368 K for seven minutes.
The photomask was mounted on the surface of SU8-10, and thephotoresist was exposed to the UV light through the mask in themask aligner (M-1S) at 15 mW/cm2 for 20 s. After thephotoresist was baked on the heated plate at 338 K for one
minute, the plate was baked on the heated plate at 368 K forfive minutes. The photoresist was developed with SU-8
developer for three minutes. The glass surface was rinsed with
IPA for two minutes, and with the ultrapure water. The plate
was dried by the spin-dryer. The height of the lower ridge wasmeasured by the stylus profiler (Dektak XT-E, Bruker
Corporation).

Mold for Upper Plate
A glass plate (38 mm × 26 mm × 1 mm: Matsunami Glass Ind.,
Ltd., Osaka, Japan) is used for a surface mold for the upper disk.
The plate was cleaned by an ultrasonic cleaner with alkaline
solution, and rinsed by the ultrapure water. The surface of theglass plate was hydrophilized by the oxygen (30 cm3/min, 0.1
Pa) plasma ashing for five minutes at 100 W by the reactive ion
etching system (FA-1). The negative photoresist material ofhigh viscosity (SU8-10: Micro Chem Corp., MA, USA) was
coated on the glass plate at 1000 rpm for 30 s with a spin coater.
After the photoresist was baked on the heated plate at 338 K for
two minutes, the plate was baked on the heated plate at 368 K
for seven minutes. SU8-10 was coated on the plate at 1000rpm for 30 s with a spin coater again. After the photoresist
was baked on the heated plate at 338 K for one minutes, theplate was baked on the heated plate at 368 K for five minutes.
The photomask was mounted on the surface of SU8-10, and thephotoresist was exposed to the UV light through the mask in themask aligner (M-1S, Mikasa Co. Ltd., Japan) at 15 mW/cm2 for
20 s. After the photoresist was baked on the heated plate at
338 K for one minute, the plate was baked on the heated plate at
368 K for five minutes. The photoresist was developed with
SU-8 developer (Nippon Kayaku Co., Ltd, Tokyo, Japan) forten minutes. The glass surface with the micro pattern wasrinsed with IPA (2-propanol, Wako Pure Chemical Industries,Ltd.) for five minutes, and with the ultrapure water. The plate
was dried by the spin-dryer.

Flow Channel
The upper plate of PDMS was rinsed with IPA, and with theultrapure water. The plate was dried by the spin-dryer. The
surface of the PDMS plate was hydrophilized by the oxygen (30cm3/min, 0.1 Pa) plasma ashing for thirty seconds at 50 W by
the reactive ion etching system (FA-1). The plate was rinsed
with APTES (Aminopropyltriethoxysilane) for five minutes,
and with the ultrapure water. The plate was dried in the ovenat 338 K for three minutes. The upper plate of PDMS wasadhered on the lower plate of SU8-10, and was baked on theheated plate at 338 K for five minutes.

Upper Plate
After the mold of the glass plate was enclosed with a peripheral
wall of polyimide, PDMS (Sylgard 184 Silicone Elastomer
Base, Dow Corning Corporation) was poured together with thecuring agent (Dow Corning Corporation) on the mold. The
volume ratio of curing agent is ten percent of PDMS. After
degassing, PDMS was baked at 373 K for one hour in an oven
(DX401, Yamato Scientific Co., Ltd, Tokyo, Japan). The
baked plate of PDMS is exfoliated from the mold. The
dimension of the ridges on manufactured PDMS was measured
with the laser microscope (VK-X200, Keyence Corporation,
Osaka, Japan). Two holes of 8 mm diameter were machinedwith a punching tool at the upper disk to make the inlet and theoutlet for the flow channel.

Flow Test
Four kinds of cells (passage < 10) were used in the flow test:
Hepa1-6 (mouse hepatoma cell line of C57L mouse), C2C12(mouse myoblast cell line originated with cross-striated muscle
of C3H mouse), MC3T3-E1 (an osteoblast precursor cell line
derived from Mus musculus (mouse) calvaria), and Neuro-2a (amouse neural crest-derived cell line). D-MEM (Dulbecco’s
Modified Eagle Medium) was used for the medium, except
αMEM for MC3T3-E1. Each medium contains 10% FBS(decomplemented fetal bovine serum) and 1% penicillin/
streptomycin (GIBCO, Life Technologies Japan Ltd., Tokyo,
Japan).
The cells were exfoliated from the bottom of the culture dishwith trypsin, and suspended in the culture medium (1000cells/cm3). In the case of porcine red blood cells, the bloodcollected through the carotid artery was diluted by thousand
times with the saline solution. The bovine serum albumin
solution was prefilled in the flow channel, and incubated for ten
minutes at 310 K in the incubator.

Photomask for Lower Plate
The glass plate of 0.2 mm thick was used for the base of themask. Before the vapor deposition of titanium, the surface ofthe glass plate was hydrophilized by the oxygen (30 cm3/min,
0.1 Pa) plasma ashing for five minutes at 100 W by the reactive
ion etching system (FA-1). Titanium was coated on the103Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)

The suspension of the cells was poured at the inlet of the flow
channel. The suspension flows by the pressure head of 5 mm,
which makes the pressure difference of 49 Pa between the inlet
and the outlet. The flow rate decreases gradually with thedecrease of the pressure head. The behavior of cells near theslit was observed with an inverted phase-contrast microscope(IX71, Olympus Co., Ltd., Tokyo) (Fig. 2).

0.1 mmFig. 3: Tracing across ridge on upper disk: unit, μm.

The microscopic images of thirty frames per second at the
shutter speed of 1/2000 s were captured by a camera(DSC-RX100M4, Sony Corporation, Tokyo, Japan). At theimages, the outline of each cell was traced with “Image J”, andthe projected area (A) was calculated. The deformation ratio(Ra) was calculated at each cell by Eq. 1.
Ra = A2 / A1

0.05 mmFlow

0.1 mm(1)

In Eq. 1, A1 is the area before the slit, and A2 is the area in theslit. Ra is unity, when the area in the slit (A2) is equal to that
before the slit (A1).

Fig. 4: Cells passing through slit: dimension from left to right is
0.8 mm; flow direction is from left to right.

The velocity of the cell passing through the slit (v) was
calculated at the movie by “Kinovea”. Most of cells passthrough the slit in a few seconds, and Data of clogging cell inthe slit are not included in the following figures.

Flow

The velocity ratio (Rv) was calculated at each cell by Eq. 2.
0.2 mmRv = (v2 － v1)/ v1(2)

In Eq. 2, v1 is the velocity before the slit, and v2 is the velocity
in the slit. Rv is zero, when the velocity in the slit (v2) is equal
to that before the slit (v1).

3. RESULTS
The tracing across the ridge on the lower disk measured by thestylus profiler shows that the height of the ridge is 0.010 mm.
Fig. 3 exemplifies the tracing across the ridge on the upper disk.
The tracing shows that the height of the ridge is 0.06 mm. Fig.
4 exemplifies the microscopic image near the slit. Some cells
are stopping at the inlet of the slit. Others are passing throughthe slit. Fig. 5 exemplifies C2C12 (in red circle) passing
through slit. The cell is approaching to the slit (a), enters to
the slit (b), is passing through the slit (c), and is leaving fromthe slit (d).

Fig. 5: C2C12 (in red circle) passing through slit: dimension
from left to right is 2 mm: approaching to slit (a), passing
through slit (c), departing from slit (d).
Fig. 2: Experimental system: channel, microscope, camera.

104

Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)10a). At the most of cells of Hepa1-6, the velocity of the cellin the slit is much lower than that before the slit (Fig. 9b). At
C2C12, the velocity of the cell in the slit is lower than that
before the slit especially in the case of the higher velocity
before the slit (v1 > 1 mm/s) (Fig. 8b). At MC3T3-E1, on theother hand, the velocity of the cell in the slit is sometimes
higher than that before the slit (Fig. 9b), even at the higher
velocity before the slit (v1 > 0.8 mm/s).

Deformation ratio (Ra)Data of the projected area of each cell are collected in Fig.11
accroding to the kind of cells. The black column shows themean area before the slit (A1), and the white column shows themean area in the slit (A2). Each bar shows the standard
deviation of each mean value. The area is relatively smaller at
MC3T3-E1, which corresponds to the smaller size of the cell.

Projected area in slit (A2) [μm2]Fig. 6: Relationship between deformation ratio (Ra) and area inslit (A2) of each cell: Neuro-2a.
v2 [mm/s]

v1 [mm/s]
Fig. 8a: Area of C2C12: before slit (circle), in slit (rhombus).

Fig. 7: Velocity of red blood cell before slit (v1) vs. in slit (v2):
at middle of slit (circle), at fringe of slit (triangle).

Fig. 6 shows the relationship between the deformation ratio(Ra) and the projected area in the slit (A2) of each cell. Theline shows the approximate linear relationship by the leastsquares method. Data scatters, but the bigger cell of the larger
area (A1) makes the larger area in the slit (A2) with the higher
deformation ratio (Ra). The approximate line shows that themean area of the cell before slit (A1) is 300 μm2.
Fig. 7 shows the relationship between the velocity of eachporcine red blood cell before slit (v1) and that in slit (v2). The
data of circles show the velocity at middle of slit, and those oftriangles show the velocity at fringe of slit. The line shows thelinear relationship between v1 and v2, which is inversely
proportional to the cross sectional area of the flow pass. The
ratio of the cross sectional area between the flow channel (0.064
mm height × 2 mm width) and the slit (0.01 mm height × 0.8
mm width) is 16. The velocities of some cells in the fringearea of the slit (v2) are slightly slower than the linear
relationship between v1 and v2, which corresponds to the
reduced flow velocity in the fringe area in the slit.

Fig. 8b: Velocity of C2C12: before slit (circle), in slit
(rhombus).

Figs. 8-10 show the projected area (Fig. 8a, Fig. 9a, and Fig.
10a) and the velocity (Fig. 8b, Fig. 9b, and Fig. 10b) of eachcell: C2C12 (Fig. 8), Hepa1-6 (Fig. 9), and MC3T3-E1 (Fig.
10), respectively. In these figure, each mark of circle and ofrhombus shows datum before the slit and in the slit, respectively.
Data are arranged in the ascending order of the data before theslit (A1, v1). The datum on the each cell is arranged at thecorresponding cell number. The projected area (A2) scatters,
but tends to increase with the increase of A1, which
proportionally relates to the size of the cell (Figs. 8a, 9a, andFig. 9a: Area of Hepa1-6: before slit (circle), in slit (rhombus).

105

Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)

Fig. 9b: Velocity of Hepa1-6: before slit (circle), in slit
(rhombus).

Fig. 12: Relationship between velocity ratio (Rv) anddeformation ratio (Ra): C2C12 (circle), Hepa1-6 (triangle),
MC3T3-E1 (rhombus).

800
Before Slit

Fig. 12 shows the relationship between the velocity ratio (Rv)
and the deformation ratio (Ra).
Data are categorizedaccording to the kind of cells: C2C12 (circle), Hepa1-6(triangle), and MC3T3-E1 (rhombus). The line shows theapproximate linear relationship by the least squares method ateach kind of cells. As the deformation ratio increases, thevelocity ratio decreases. The tendency is remarkable at
MC3T3-E1. Every velocity ratio is lower than 3 in Fig. 12.
Every velocity ratio of red blood cell on the other hand, ishigher than five (Fig. 7).

600
In SlitArea [µm2 ]

400
200
0

0

5

10

15

20

25

30

Cell number
Fig. 10a: Area of MC3T3-E1: before slit (circle), in slit
(rhombus).

4. DISCUSSION
When the diameter of the sphere is same as the height of the slit
(d = 10 μm), the projected area (A) is 79 μm2. In Figs. 8-10,
the projected area of each cell before the slit (A1) exceeds 200
μm2, so that the height of the slit is small enough to makedeformation of each cell, which is passing through the slit.
The dimension of the width of the slit has been selected wider
than 0.8 mm, which is enough wide for a cell to pass through.
The wider width of the slit has possibility to make deformationof the wall, which cannot keep precise dimension of the slit [5].
The ridge of the PDMS has elasticity, so that the micro ridge
may deform with the small ratio. The reduced velocity of the
porcine red blood cell at the fringe area of the slit might
correspond to the reduced cross sectional area or the side wall
effect (Fig. 7). The velocity ratio in Fig. 12 is smaller than 5,
which depends on the friction between the cell and the wall of
the slit. The cell of the larger diameter is forced to be
deformed at the higher deformation ratio in the slit (Fig. 6).
The higher deformation ratio makes the larger projected area inthe slit. The higher deformation ratio might increases thefrictional resistance at the slit (Fig. 13).

Fig. 10b: Velocity of MC3T3-E1: before slit (circle), in slit
(rhombus).
Projected area (A1, A2) [μm2]

The fine architecture of the red pulp of the spleen has been
investigated in the previous study [4]. In the previous studies,
the typical micro channel has cylindrical shape with 0.005 mm
diameter, which simulates the blood vessel capillary. The red
blood cell, on the other hand, passes through micro slit narrower
than 0.001 mm in the spleen. The deformability of erythrocytechanges with aging [1]. The deformation is evaluated with theratio of the projected area of the disk during the passing throughthe slit in the present study.
The deformation in theperpendicular direction can be observed with slit between micro
cylindrical pillars [9, 10].

Fig. 11: Projected area of cell: before slit (black), in slit (white):mean (column), standard deviation (bar).

106Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)[3] A.W.L. Jay, “Viscoelastic Properties of the Human Red
Blood Cell Membrane: I. Deformation, Volume Loss, andRupture of Red Cells in Micropipettes”, BiophysicalJournal, Vol. 13, No. 11, 1973, pp. 1166-1182.
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Proc. 21st World Multi-Conference on Systemics
Cybernetics and Informatics, Vol. 2, 2017, pp. 239-244.
[6] A. Mizoi, Y. Takahashi, H. Hino, S. Hashimoto and T.
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Cell
Slit

Fig. 13: Cell deformation in slit between plates of PDMS andof SU-8.

Reynolds number (Re) is calculated by Eq. 3.
Re = ρ v w/ η

(3)

In Eq. 3, ρ is density of the fluid, and q is the flow rate. Re is
1, when ρ, v, w, and η are 103 kg m－3, 1×10－3 m/s, 2×10－3 m,
and 2×10－3 Pa s, respectively. The turbulent flow may notoccur in the flow of small value of Reynolds number.

5. CONCLUSION
A micro slit between micro ridges has been used for observationof a biological cell passing through the micro slit in vitro. At
the middle part of the flow channel, the slit (0.8 mm < width <2 mm, 0.01 mm height) has been made between the microridges on the transparent polydimethylsiloxane plate and theglass plate by photolithography technique. Four kinds of cells
were used in the test: C2C12 (mouse myoblast cells), Hepa1-6(mouse hepatoma cells), MC3T3-E1 (mouse osteoblast
precursor cell line), and Neuro-2a (mouse neural crest-derived
cell line). For the reference, the passing speed of the porcine
red blood cells through the slit was observed.
Theexperimental results show that the passing velocity through theslit depends on the deformation ratio of the cell. The designed
slit between micro ridges has capability for observation of thedeformation and the passing velocity of the single cell.

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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Deformation of Erythrocyte in Shear Flow”, Journal of
Systemics Cybernetics and Informatics, Vol. 12, No. 3,
2014, pp. 41-46.

107