Measurement of Deformability of Biological Cell Passing throughSingle Micro Slit between Walls
Yusuke TAKAHASHI, Shigehiro HASHIMOTO, Atsushi MIZOI, Haruka HINO, Takeshi YAMAGUCHI
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
ABSTRACTA single micro slit has been designed between walls to observe
the deformation of a cell passing through the slit in vitro. The
micro slit of 0.015 mm width, 0.1 mm length and 0.05 mm
height was fabricated between walls of weirs on the glass plateusing the photolithography technique. The single micro slit isset in the flow path between parallel plates, of which dimensionof the cross section has 2 mm width and 0.05 mm height.
Three types of biological cells were used in the test
alternatively: C2C12 (mouse myoblast cell line), Hepa1-6(mouse hepatoma cell line), and Neuro-2a (mouse neuralcrest-derived cell line).
The suspension of cells wasintroduced into the slits by the pressure difference between theinlet and the outlet, which was kept by the gravitational level ofthe medium. The cell passing through the slit was observed by
the microscope. The movement and the shape of the cell was
analyzed at the video images. The elastic modulus ofmyoblast passing through the slit estimated by the velocity (1mm/s) and by the deformation ratio (0.5) of the cell in the slit
was 10－2 Pa. The experimental results show that the designed
slit has capability to measure deformability of the cell.
Keywords: Biomedical Engineering, C2C12, Hepa1-6,
Neuro2-a, Photolithography and Micro Flow Channel.

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 [1-16]. The techniquemight be applied to handle cells in diagnostics in vitro [17-21].
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. Somecells can pass through the slit narrower than the capillary.
The photolithography technique enables manufacturing a
micro-pattern [24-27]. Several micro-fabrication processes
have been designed to simulate the morphology of the
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 [3-9].
In the present study, a single micro slit has been designed
between walls to observe the deformation of a cell passing
through the slit in vitro.

2. METHODS
Micro Slit
A single micro slit has been designed between walls of weirs(Fig. 1). The micro slit of 0.015 mm width, 0.1 mm length (L)and 0.05 mm height (H) was fabricated between walls of weirs
on the glass plate using the photolithography technique. The
single micro slit is set in the flow path between parallel plates,of which dimension of the cross section has 1.5 mm width (W),
30 mm length and 0.05 mm height (H) (Fig. 2). The flow pathis constructed between parallel plates: the upper plate ofpolydimethylsiloxane (PDMS), and the lower plate of glass(S1111, Matsunami Glass, Japan). The upper plate has twoholes of 5 mm diameter for the inlet and for the outlet of theflow of the cell suspension.
The micro pattern for the micro slit was controlled by two
photomasks, which is made by the following process.
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. 3). 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
100 nm thickness in the electron beam vapor deposition
apparatus (3.1×10－4 Pa, 0.5 nm/s, 200 s, JBS-Z0501EVC JEOL
Ltd., Tokyo, Japan). The oxygen (0.1 Pa, 30 cm3/min) ashing
was applied on the surface of the titanium in the reactive ion
etching system (100 W, for five minutes, FA-1).
To improve affinity between glass and photoresist material,
HMDS (hexamethyldisilazane: Tokyo Chemical Industry Co.,
Ltd., Tokyo) was coated on the glass plate at 3000 rpm for 30 swith a 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 5000 rpm for 30s). The photoresist was baked in the oven (DX401, Yamato
Scientific Co., Ltd) at 373 K for one minute.

Fig. 4a: Photomask.

0.1

Cell

Slit

0.015
Fig. 4b: Magnified photomask at slit: dimension from left toright is 0.7 mm.

Fig. 1: Micro slit: unit of number, mm.

Φ5
30

25

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, and driedby the spin-dryer (1100 rpm, 30 s, with N2 gas, SF-250, Japan
Create Co., Ltd., Tokorozawa, Japan).

[mm]2Fig. 2: Dimension of flow channel.
Titanium

Glass Development

OFPR-800

Etching

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 3.2 V, the velocity of 0.151
mm/s, the acceleration of 0.34 mm/s2, and the focus offset at+0.45.

Laser drawing

OFPR-800 removal

Fig. 3: Photolithography process for photomask.

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 five 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 (Fig. 4).
Slit on Lower Plate
The glass slide was used for the base of micro slit, after
cleaning with acetone (Fig. 5). 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 etchingsystem (FA-1). The negative photoresist material of highviscosity (SU8-10: Micro Chem Corp., MA, USA) was coatedon the glass slide at 3000 rpm for 30 s with a spin coater. The
photoresist was baked in the oven at 338 K for three hours.

The photoresist was exposed to the UV light through the maskin the mask aligner (M-1S, Mikasa Co. Ltd., Japan) at 15
mW/cm2 for 16 s. The photoresist was baked at the hotplate in
two processes: at 338 K for one minute, and at 368 K for ten
minutes. The photoresist was developed with SU-8 Developer(Micro Chem) for ten minutes. The glass surface with themicro pattern was rinsed with IPA (2-propanol, Wako Pure
Chemical Industries, Ltd.) for one minute, and pure water for
one minute. The pattern was baked at the hotplate at 393 K(Fig. 6).

UV light
Glass

Mask

SU-8

Development

Fig. 5: Photolithography process for lower plate.

At the end of the process, the dimension of the micro pattern
was measured with a Stylus Profiler (Dektak XT-E, Bruker
Corporation). The height along the cross sectional line of
micro pattern was traced (Fig. 7).
Upper Plate
After the slide glass plate was enclosed with a peripheral wallof 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. The volume ratio of
PDMS to curing agent is ten to one. After degassing, PDMS
was baked at 368 K for one hour in an oven (DX401, Yamato
Scientific Co., Ltd). The baked plate of PDMS (3 mmthickness) was exfoliated from the slide glass plate. The
PDMS plate was cut to make a rectangular upper plate for theflow channel. Two holes with the interval of 25 mm were
punched by a punching tool (trepan MK405, Kai Industries Co.,
Ltd., Gifu, Japan) to make the inlet and the outlet. The upper
plate was stuck on the lower plate with micro pattern adjusting
the position of holes to make the flow channel. A rectangularparallelepiped channel of 30 mm length × 2 mm width × 0.05
mm height is formed between upper and lower plates. The
two plates stick together with their surface affinity (Fig. 8).
A one-way flow system has been designed to introduce thesuspension of cells. The system consists of a flow channel,
tubes and a microscope. A silicone tube of 3 mm internal
diameter and of 5 mm external diameter is used for theconnector to the flow channel. The flow channel is placed on
the stage of the inverted phase contrast microscope (IX71,
Olympus Co., Ltd., Tokyo) (Fig. 9).

Fig. 6: Optical microscopic image of slit of SU8: dimension
from left to right is 0.7 mm.

W
HFig. 7: Tracings of height of surface of lower plate of flow
channel: H=0.05 mm, W=1.5 mm.

Flow Test
Cells of the passage between four and nine were used in the test.
Three types of biological cells were used in the test
alternatively: C2C12 (mouse myoblast cell line originated withcross-striated muscle of C3H mouse), Hepa1-6 (mouse
hepatoma cell line of C57L mouse), or Neuro-2a (a mouseneural crest-derived cell line).
The flow channel washydrophilized by the oxygen (30 cm3/min, 0.1 Pa) plasma
ashing for one minute at 100 W by RIE (FA-1), and prefilledwith the saline solution.
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 flow path was carefully examined to avoid mixing of air
bubbles, which might stir the medium in the flow channel.
The suspension of cells (50 mm3) was injected into the inlet ofthe flow channel, and introduced into the slits by the pressure
difference between the inlet and the outlet, which was kept by
the gravitational level of the medium (2.5 mm).

Fig. 8: Flow channel.

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. The
movement of the cell was analyzed by Kinovea (Ver. 8.23,
Commons Attribution) at the video images: 30 frames per
second.

The deformation of the cell in the slit was calculated by thefollowing equations (Fig. 10).
Q = v A = v (D1 H)
F=ρQv
S = π (D0 / 2)2σ=F/Sε = (D0－D1) / D0
E = σ /ε = ρ (D1 H) v2 / (π (D0 / 2)2 (D0－D1) / D0)

(1)
(2)
(3)
(4)
(5)
(6)

Fig. 13 exemplifies Hepa1-6 passing through the slit. Fig. 14
shows velocity of Hepa1-6 passing through the slit. In thiscase, E = 1 × 10－2 Pa, calculated by D0 = 2.1 × 10－5 m, D1 =1.4 × 10－5 m (ε = 0.5), v = 1.4 × 10－3 m/s.
Fig. 15 exemplifies Neuro-2a passing through the slit. Fig. 16
shows velocity of Neuro-2a passing through the slit. In thiscase, E = 2 × 10－4 Pa, calculated by D0 = 4.2 × 10－5 m, D1 =1.2 × 10－5 m (ε = 0.7), v = 0.6 × 10－3 m/s.

Where Q is the flow rate in the slit, v is the flow velocity in theslit, A is the cross sectional area of the slit, H is the height of theslit (5 × 10－5 m), F is force applied on the cell, ρ is the densityof the fluid (1.0 ×103 kg/m3), S is the cross section of the cell, σ
is the stress applied on the cell, D0 is the initial diameter of thecell, D1 is the width of the slit, ε is the strain of the cell, and E isthe elastic modulus.

3. RESULTS
Fig. 4 shows the photomask for the flow channel and the slit.
The designed pattern is successfully etched on the titanium filmon the glass. Fig. 6 shows the optical microscopic image ofthe slit. After adjustment of the micromachining condition,
the designed pattern is successfully made on the glass. Fig. 7
shows the tracings of lower plate along the cross section of the
micro-machined flow channel on the glass. The depth of thegroove on the lower plate is 0.054 mm, which can be adjusted
to the height of the micro slit of 0.05 mm (H) in the flow
channel assembly. Fig. 8 shows the assembly of the upper andlower plate to make the flow channel.

Fig. 11: C2C12 through slit: flow from left to right, bar shows
0.1 mm.
Velocity [μm/s]

Fig. 11 exemplifies C2C12 passing through the slit. Fig. 12shows velocity of C2C12 passing through the slit. The zero
velocity for four seconds corresponds to the movement of thecell attacking at the entrance of the slit. In this case, E = 3 ×
10－2 Pa, calculated by D0 = 1.6 × 10－5 m, D1 = 1.3 × 10－5 m (ε= 0.2), v = 1.4 × 10－3 m/s.

Fig. 12: Velocity of C2C12 passing through slit.

Fig. 9: Experimental system.

Fig. 13: Hepa1-6 through slit: flow from left to right, bar shows
0.1 mm.

Time [s]Velocity [μm/s]
D0

Cell

L

v

Slit

D1
Time [s]Fig. 14: Velocity of Hepa1-6 passing through slit.

Fig. 10: Deformation of cell passing through slit.

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. In the present study, the flow rate is confirmedby the flow speed of the cell during microscopic observation.

Fig. 15: Neuro-2a through slit: flow from left to right, barshows 0.1 mm.
Velocity [μm/s]

The optical micro scope observation is difficult at the tall slit
with the narrow width, because of the scattering of the light.
The height of the wall is limited to 0.05 mm in the present study.
The wall of the flow channel has the taper to introduce cells
smoothly to the single slit in the present study. Although thedimension of the width of the slits (D1) was scattered between
0.012 mm and 0.014 mm, the morphology of the micro pattern
has been successfully manufactured by the photolithography
process in the present study.
The high speed camera is necessary to trace the deformation ofa cell in the slit, because the cell slips fast after entering into theslit. In some cases, on the other hand, it takes time to deformfrom sphere to disk to enter into the slit.

Time [s]Fig. 16: Velocity of Neuro-2a 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 make frictional
resistance. 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]. In anotherstudy, the micro slit was manufactured between ridges [2].
Only one projection can be traced in the present experimental
system. The cell can deform in the perpendicular direction.
To observe the deformation in the perpendicular plane, the slit
between upper and lower ridges is effective [2].
There are several methods to sort cells [3-10]. 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.
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.
In the previous study, several slits were designed between microcolumns [1]. The multiple slits are convenient to sort cells as
the filter. The fluid selected, however, a slit of parallel
multiple slits at random in the previous system. While a cell is
passing through a slit, the main fluid flows through another slit.
The driving force of the passing cell changes with the clogging
at the slit.
The resistance of the flow channel with a single slit is very highin the present experimental system. The resistance increases,

According to the experimental results of in the present study,
the ascending order of compliance of the cell estimated by theelastic modulus is C2C12, Hepa1-6, Neuro-2a. Hepa1-6 isable to pass the narrower slit than C2C12.

5. CONCLUSION
A single micro slit has been designed between walls to observe
the deformation of a cell passing through the slit. The microslit of 0.015 mm width, 0.1 mm length and 0.05 mm height was
fabricated between walls of weirs on the glass plate using thephotolithography 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. Both the
movement and the shape of the cell were analyzed at the video
images. The elastic modulus of myoblast passing through theslit estimated by the velocity (1 mm/s) and by the deformation(0.5) of the cell in the slit was 10－2 Pa. 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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