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

Observation of Cell Passing through Single Micro Slit between
Weir-walls
Shigehiro HASHIMOTO, Yusuke TAKAHASHI, Yusuke NAKANO,
Daisuke HASEGAWA, 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/andToshitaka YASUDA
Bio-systems Engineering, Department of Electronic Engineering,Tokyo National College of Technology, Tokyo, Japan
microcirculation. In the previous study, the micro slits havedesigned between micro cylindrical pillars [1], or between
micro ridges [2]. The micro-fabrication technique has also
been applied to design microfluidic systems in vitro [3-16].

ABSTRACTThe single micro slits of two kinds of width (10 μm, or 15 μm)
has been designed between weir-walls to investigate thedeformation of a cell passing through the slit related to the flowrate in vitro. The micro slit (0.1 mm length, and 55 μm height)
was fabricated between weir-walls of polydimethylsiloxane
using the micromachining technique. The single micro slit isset in the flow path (2 mm width, 55 μm height) between
parallel plates. Two types of biological cells were used in thetest alternatively: C2C12 (mouse myoblast cell), and Hepa1-6(mouse hepatoma cell).
The suspension of cells wasintroduced into the slits by the pressure difference between theinlet and the outlet, which was kept by the gravitational level
difference of the medium (< 4 mm). The each flowing cell
was observed by an inverted phase contrast microscope. The
movement and the shape of the cell was analyzed at the video
images by the camera at the eyepiece of the microscope. The
experimental results shows that some myoblasts pass the slit at
the higher deformation ratio with the higher flow rate. The
experimental results show that the designed slit has capability tomeasure deformability of the cell.
Keywords: Biomedical Engineering, C2C12,
Deformation, Micromachining and Micro Slit.

To analyze mechanical property of deformation of the single
cell, the single slit has an advantage than parallel arrangementof slits. With the parallel slits, the flow rate through each slit
cannot be maintained at constant value.
In the present study, the single micro slits of two kinds of width(0.010 mm, or 0.015 mm) has been designed between
weir-walls to investigate the deformation of a cell passing
through the slit related to the flow rate in vitro.

2. METHODS
Micro Slit
A single micro slit has been fabricated between weir-walls on
the glass plate using the photolithography technique: 0.1 mm
length (L) and 0.055 mm height (H). Variation was made on
the width of the slit: 0.010 mm, and 0.015 mm. The single
micro slit was set in the flow path between parallel
polydimethylsiloxane (PDMS) plates, of which dimension of
the cross section has 2 mm width (W), 30 mm length and 0.055
mm height (H). The upper plate has two holes of 5 mmdiameter for the inlet and for the outlet of the flow of the cellsuspension. The upper plate with the micro slit was made by
two kinds of molds, which were made by the following process.

Hepa1-6,

1. INTRODUCTION
Biological cells can pass through narrow gaps: micro capillaries,
or micro slits. The biological system sorts cells according to
the size, deformability, and adsorptivity of the cell. Cells are
sorted according to deformability through the gap or
adsorptivity on the membrane in vitro [1-17]. The techniquemight be applied to handle cells in diagnostics in vitro [13-17].

Photomask
The slide glass (Matsunami) plate (38 mm length, 26 mm width,and 1.0 mm thickness) was used for the base of the photomask(Fig. 1). Before the deposition of titanium, 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, Samco International, Kyoto, Japan).
Titanium was deposited on the surface of the glass plate with

The photolithography technique enables manufacturing a
micro-pattern [18-21]. Several micro-fabrication processes
have been designed to simulate the morphology of the120Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)

200 nm thickness in the spattering system (back pressure <9×10－4 Pa, RF100W, 0.5 Pa, 8 min, L-210S-FH Canon Anelva
Corporation, Kawasaki, Japan). The oxygen (0.1 Pa, 30
cm3/min) ashing was applied on the surface of the titanium inthe reactive ion etching system (RF 100 W, for five minutes,FA-1).

The titanium coated plate was etched with the plasma gas using
RIE-10NR (Samco International, Kyoto, Japan). For etching,
the gas of SF6 (50 cm3/min at 1013 hPa) with Ar (50 cm3/min at
1013 hPa) was applied at 100 W at 4 Pa for four minutes. The
residual OFPR-800LB was removed by acetone. The plate
was dipped in the distilled water in one minute, after it is dippedin ethanol for one minute. The plate was dried by the
spin-dryer: 300 rpm for 30 s with the distilled water, and 1100
rpm for 30 s with N2 gas.

To improve affinity to photoresist material, HMDS
(hexamethyldisilazane: Tokyo Chemical Industry Co., Ltd.,Tokyo) was coated on the glass plate at 3000 rpm for 30 s witha spin coater.
The positive photoresist material of
OFPR-800LB (Tokyo Ohka Kogyo Co., Ltd, Tokyo, Japan) was
coated on the titanium with the spin coater (at 7000 rpm for 30s). The photoresist was baked in the oven (DX401, Yamato
Scientific Co., Ltd) at 373 K for one minute.

The polyimide tape (0.055 mm thickness, 10 mm width), which
was cut to make the channel by the ultra-short pulse laser(IFRIT, Cyber Laser Inc., Tokyo, Japan), was pasted over theslit position at the center of the glass plate (Fig. 2). The
titanium coated plate was etched again with the plasma gas
using RIE-10NR. For etching, the gas of SF6 (50 cm3/min at
1013 hPa) with Ar (50 cm3/min at 1013 hPa) was applied at 100
W at 4 Pa for four minutes.

The pattern for the slit 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 mold with the laser drawing system, the parameters wereselected as follows: the voltage of 3.05 V, the velocity of 0.137mm/s, the acceleration of 0.5 mm/s2, and the focus offset at+2.4.

Mold AThe slide glass (Matsunami) plate (38 mm length, 26 mm width,and 1.0 mm thickness) was used for the base of the mold, after
cleaning for three minutes with an ultrasonic cleaning machine(Fig. 3). The plate was dried by the spin-dryer: 300 rpm for 30
s with the distilled water, and 1100 rpm for 30 s with N2 gas.
The surface of the 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).

The photoresist was developed with tetra-methyl-ammoniumhydroxide (NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki,
Japan) for one minute, rinsed with the distilled water (300 rpm,
30 s), and dried by the spin-dryer (1100 rpm, 30 s, with N2 gas,
SF-250, Japan Create Co., Ltd., Tokorozawa, Japan).

The epoxy based negative photoresist material (SU-8 100) was
coated on the glass with the spin coater (at 3000 rpm for 30 s).
The photoresist was baked in the oven (DX401) at 338 K for ten
minutes and at 368 K for one hour.
The photoresist was exposed to the UV light through thephotomask in the mask aligner (M-1S, Mikasa Co. Ltd., Japan)
at 16.2 mW/cm2 for 30 s. The photoresist was baked in theoven at 338 K for one minute, and at 368 K for five minutes.
The photoresist was developed with SU-8 developer (Micro
Chem Corp., MA, USA) for ten minutes. The glass surface
with the micro pattern was rinsed with IPA (2-propanol, Wako
Pure Chemical Industries, Ltd.) for one minute, and pure waterfor one minute. The pattern was baked for five minutes in theoven at 393 K.

Developing
Fig. 1: Photolithography process for photomask.

Mold BThe slide glass (Matsunami) plate (38 mm length, 26 mm width,and 1.0 mm thickness) was used for the base of the mold (Fig.
4). The polyimide tape (0.055 mm thickness, 10 mm width)
was pasted at the center of the glass plate. The tape was cut by
the ultra-short pulse laser (IFRIT) to make the mold for theupper part of the flow channel with the single slit. The
dimension of the slit on the manufactured mold was measured
with the laser microscope (VK-X200, Keyence Corporation,
Osaka, Japan). The height along the cross sectional line of theslit was traced.

Fig. 2: Polyimide tape was pasted over slit position.

Upper Plate
After the mold of the slide glass plate was enclosed with aperipheral wall of polyimide tape, PDMS (Sylgard 184 Silicone
Elastomer Base, Dow Corning Corp., MI, USA) was poured
with the curing agent (Sylgard 184 Silicone Elastomer Curing
Agent, Dow Corning Corp., MI, USA) on the mold (Fig. 5).
The volume ratio of PDMS to curing agent is ten to one.

Developing
Fig. 3: Mold A.

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

Two types of biological cells were used in the test alternatively:
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse), or Hepa1-6 (mouse hepatoma cell lineof C57L mouse). The inner surface of the flow channel washydrophilized by the oxygen (30 cm3/min, 0.1 Pa) plasma
ashing for one minute at 100 W by the reactive ion etchingsystem (FA-1, Samco International, Kyoto, Japan), and prefilledwith the bovine serum albumin solution for thirty minutes at
310 K.
Before the flow test, the cells were exfoliated from the plate of
the culture dish with trypsin, and suspended in the D-MEM(Dulbecco’s Modified Eagle’s Medium). The suspension ofcells (80 mm3) was poured at the inlet of the flow channel.
The flow occurs by the pressure difference between the inlet
and the outlet, which was kept by the gravitational level of themedium (< 3 mm).

Fig. 4: Mold B.

The cell passing through the slit was observed by the
microscope, and recorded by the camera (DSC-RX100M4,
Sony Corporation, Japan), which is set at the eyepiece of the
microscope. The movement of the cell was analyzed by“Kinovea (Ver. 8.23, Commons Attribution)” at the video
images: 30 frames per second. At the images, the outline ofeach cell was traced with “Image J (Ver. 1.48, National
Institutes of Health)”, and the diameter (D0) was calculated.

1. Mold

The deformation of the cell in the slit was evaluated by thefollowing equations (Fig. 7).
2. PDMS on moldFig. 5: Upper plate with slit.

Q = v A = v (b H)
ε = (D0－D1) / D0

(1)
(2)

After degassing, PDMS was baked at 368 K for 15 min in anoven (DX401, Yamato Scientific Co., Ltd). The baked plate of
PDMS (4 mm thickness) was exfoliated from the mold of theslide glass plate. The PDMS plate was cut to make arectangular upper plate for the flow channel. Two holes(diameter of 5 mm) with the interval of 25 mm were punchedby a punching tool (trepan MK405, Kai Industries Co., Ltd.,
Gifu, Japan) to make the inlet and the outlet. The morphologyof the slit was observed by a scanning electron microscope(SEM, JSM6360LA, JEOL Ltd., Tokyo, Japan).
Lower Plate
The slide glass plate (38 mm length, 26 mm width, and 1.0 mm
thickness) was used for the base of the lower plate. PDMS
was coated on the glass plate at 5000 rpm for one minute withthe spin coater, and baked in the oven at 368 K for ten minutes.
The surface of the lower plate was hydrophilized by the oxygen(30 cm3/min, 0.1 Pa) plasma ashing for one minute at 100 W by
the reactive ion etching system (FA-1).

Fig. 6: Flow channel is placed on stage of inverted phase
contrast microscope.

Flow Channel
By the surface affinity, the upper plate was stuck on the lower
plate to make the flow channel. A rectangular parallelepipedchannel of 30 mm length × 2 mm width × 0.05 mm height isformed between upper and lower plates. The two plates stick
together with their surface affinity.
The flow channel is placed on the stage of the inverted phase
contrast microscope (IX71, Olympus Co., Ltd., Tokyo) (Fig. 6).
Fig. 7: Schematic image of cross section of slit.

Flow Test
Cells of the passage between four and nine were used in the test.

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

Where Q is the flow rate in the channel, v is the flow velocity
before the slit, A is the cross sectional area of the channel, H isthe height of the slit (5 × 10－5 m), b is the width of the flow
channel (2 mm), D0 is the initial diameter of the cell, D1 is thewidth of the slit, and ε is the strain of the cell.

3. RESULTS
Fig. 8 shows the tracings of the surface of the tape on the glass
for the mold of slit by the laser microscope. The dimension(0.010 mm width) of the mold was confirmed at the tracings.
The designed pattern is successfully machined at the polyimide
tape pasted on the glass. Fig. 9 shows the scanning electron
microscope image of the slit between weir-walls. The image
shows the approximately rectangular groove. The top opening
side of the groove is covered by the counter plate to make theslit. Fig. 10 exemplifies the microscopic image of C2C12
passing through the slit. The cell approaches to the slit (Fig.
10a), enters in the slit (Fig. 10b), moves through the slit (Fig.
10c), and moves away from the slit (Fig. 10d).

0.01 mmFig. 9: SEM image of slit. Dimension from left to right is 0.1
mm.

Fig. 11 exemplifies the velocity of C2C12 passing through theslit. The zero velocity corresponds to the movement of the cellclogging in the slit. The term of 1.3 s between two peaks
correspond to the term of the cell passing through the slit. Fig.
12 shows the relationship between the flow rate and the strainof each cell, which passes through the slit. In Fig. 12, datumof the circle corresponds to that of C2C12, and datum of thetriangle corresponds to that of Hepa1-6. The data group of thelower strain correspond to data at the wider slit of 0.015 mm
width, and the data group of the higher strain correspond to dataat the narrower slit of 0.010 mm. Hepa1-6 does not passthrough the slit of 0.010 mm width. Some myoblasts pass theslit of 0.010 mm width at the higher deformation ratio with thehigher flow rate. There is positive correlation between flowrate and deformation ratio at C2C12. At the wider slit of 0.015
mm width, on the other hand, even the cell of the bigger
diameter passes through the slit at the lower flow rate.

0.1 mmFig. 10a: C2C12 (marked by the circle) approaching to slit:
flow from left to right, dimension from left to right is 0.5 mm.

Fig. 10b: C2C12 passing through slit.

0.1 mmFig. 10c: C2C12 passing through slit.

0.1 mmFig. 8: Tracings (lower) along the blue line (upper) of surfaceof tape on glass for mold of slit by laser microscope: unit, μm.

123Proceedings of The 22nd World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2018)higher strain) to pass through the slit (Fig. 12).
The
deformation of the harder part of the cell might be necessary topass through the narrower slit.
There are several methods to sort cells [1-12]. Non-invasive
way is preferable to sort cells with minimum damage. The
flow cytometry is one of the technologies, which is used for cellsorting. Cells suspended in the fluid are analyzed by the laser.
The fluorescently labelled components in the cell are analyzed
by the light emission.
The experimental results might
contribute to analyze adhesive mechanism of cancer cell duringmetastasis.
The micro trap might simulate adhesivemechanism of flowing cells.

Fig. 10d: C2C12 moves away from the slit.

A red blood cell has flexibility and deforms in the shear flow
[22, 23]. 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. Cells
has deformability [24], and some cells can pass through the slit
narrower than the capillary.
In the previous study, several slits were designed between microcolumns [1]. The multiple slits are convenient to sort cells as
the filter. The fluid selects, however, a slit of parallel multiple
slits at random. While a cell is passing through a slit, the main
fluid flows through another slit. The driving force of the cellpassing changes with the clogging at the slit.

Fig. 11: Velocity of C2C12 passing through slit.

Flow rate [×10－１２ m3/s]

25

The resistance of the flow channel with a single slit is very highin the present experimental system. The resistance increases,
when the cells clogged the slits in the flow channel. Internalpressure of the flow channel should be carefully controlled
under the constant flow rate, otherwise the leakage or cavitation
would occur. The flow path was carefully examined to avoid
mixing of air bubbles, which might stir the medium in the flow
channel.

20
15
10
5

0

0

0.2

0.4

0.6

In the present study, the flow is controlled by the pressure
difference between the inlet and the outlet, which was kept by
the gravitational level of the medium. The diameter (5 mm) of
the inlet port is much larger than cross sectional area of the slit
to keep the level of the inlet port. In the present study, theflow rate is confirmed by the flow speed of the cell during
microscopic observation.

0.8

StrainFig. 12: Flow rate vs. strain of C2C12 (circle) and Hepa1-6(triangle) passing through slit.

The capture of the cell at the slit depends on several factors: thedimension of the cell and the slit, deformability of the cell, thewall shear stress (shear rate) at the slit, and the affinity (charge,
friction) of the cell to the wall of the slit. The surface is notcompletely flat in the slit, which might increase frictional
resistance. The wall of the slit has roughness (Fig. 9), which
affects the movement of each cell passing through the slit. In
the present study, Neuro-2a (a mouse neural crest-derived cellline) tends to adhere to the wall of the flow channel, and isdifficult to be observed the movement of passing through theslit. Protein coating might make property of the surface stable.

4. DISCUSSION
Preparation of the slit of sub-micrometer is not easy. In theprevious study, the slit of micrometer between pillars was
manufactured by photolithography technique [1]. Only one
projection can be traced in the present experimental system.
The cell deforms in the three dimensional space. To observe
the deformation in the perpendicular plane, the slit between
upper and lower ridges is effective [2].
In the present study, the slit of the narrow width (0.010 mm, and0.015 mm) has been machined by micromachining technique:the ultra-short pulse laser, or photolithography. The results ofthe dimension of the machining was acceptable for the slit,
where the movement of each cell was able to be observed.
Two kinds of dimension of the width of the slit enable the wider
range of the strain in the experiment. At the narrower slit, thehigher flow rate is necessary for the bigger cell (bigger diameter,

5. CONCLUSION
The single micro slits of two kinds of width (0.010 mm, or
0.015 mm) has been designed between weir-walls to investigate
the deformation of a cell passing through the slit related to theflow rate in vitro. The micro slit of 0.1 mm length and 0.05

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

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mm height was fabricated between weir-walls of thepolydimethylsiloxane plates using the photolithography
technique. The suspension of cells (C2C12 or Hepa1-6) was
introduced into the slits, and the cell passing through the slit
was observed by the microscope. The experimental resultsshows that some myoblasts pass through the slit at the higher
deformation ratio with the higher flow rate.
Both themovement and the shape of the cell were analyzed at the video
images. The experimental results show that the designed slit
has capability to measure deformability of the 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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