Measurement of Deformability of Cell by Slits between Micro
Cylindrical Pillars
Yusuke TAKAHASHI, Shigehiro HASHIMOTO, Haruka HINO, Hiromi SUGIMOTO
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACT

2. METHODS

Deformation of a cell has been measured using slits between
micro cylindrical pillars in vitro. Micro cylindrical pillars(0.02 mm diameter, 0.06 mm height) were fabricated on theglass plate using the photolithography technique. Variation
was made on the gap between pillars: 0.025 mm, 0.020 mm,
0.015 mm, and 0.010 mm. The micro pillars were set in theflow path between parallel plates, of which dimension of thecross section has 1 mm width and 0.06 mm height. Two typesof biological cells were used in the test alternatively: C2C12(mouse myoblast cell line), Hepa1-6 (mouse hepatoma cell line).
The suspension of cells was introduced into the slits, and thedeformation of the cell at the slit was observed by the
microscope. The experimental results show that several cells
clogged in the slit of 0.01 mm, and the passing time of C2C12
is shorter than that of Hepa1-6 at the smaller deformation ratio.
The designed slit has capability for evaluation of deformabilityof a cell.

Micro Slits
The micro slits have been designed between micro cylindricalpillars. The dimension of each micro cylindrical pillar is asfollows: 0.025 mm diameter, and 0.06 mm height (Fig. 1)

Keywords: Biomedical Engineering, Deformability, C2C12,
Hepa1-6, 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 vivo. The mechanism might
be applied to handle cells in diagnostics in vitro.
The deformability of cell was measured by several preparations
in viro: through micro capillaries, and between counter rotating
disks in the previous studies [1, 2]. The biological cell has thevisco-elastic property.
The photolithography technique enables manufacturing a
micro-channel. Several micro-fabrication processes have been
designed to simulate the morphology of the microcirculation.
In the previous studies, the micro slits were designed between
micro cylindrical pillars [3, 4], or between micro ridges [5].
The micro-fabrication technique has also been applied to design
microfluidic systems in vitro [6-12].
In the present study, deformation of a cell has been measured byslits between micro cylindrical pillars in vitro.

Cylindrical pillars are arranged along the four lines by thephotolithography technique. Each line is perpendicular to theflow direction of the channel. The pitch of lines is 0.5 mm.
Each line has its own uniform gaps (w: the width of slits)
between pillars. The gaps are 0.025 mm at the first line. The
gap is 0.020 mm at the second line. The gaps are 0.015 mm at
the third line. The gaps are 0.010 mm at the fourth line.
The micro slits are located at the middle part of the flow path
between parallel plates, of which dimension of the cross section
has 1 mm width (W), 30 mm length and 0.06 mm height (H)(Fig. 1). The flow path is constructed between parallel plates:
the upper plate of polydimethylsiloxane (PDMS), and the lower
plate of glass (S1111, Matsunami Glass Ind., Ltd., Japan). The
upper plate has two holes of 5 mm diameter for the inlet and forthe outlet of the flow of the cell suspension.
Photomask
The slide glass (S1111) plate (38 mm length, 26 mm width, and
1.0 mm thickness) was used for the base of the photomask (Fig.
2). The disk was dipped in the pure water and cleaned by theultrasonic washer for five minutes.

y
x

z
x
Fig. 1: Micro pillars in flow channel.

Before the vapor deposition of titanium, the surface of the glass
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, Samco Inc., 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 wasapplied on the surface of the titanium in the reactive ion etchingsystem (100 W, for one minute, 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 it with the spin coater (at 5000 rpm for 30 s). The
photoresist was baked in the oven (DX401, Yamato ScientificCo., Ltd) at 368 K for three 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 2.3 V, the velocity of 0.08
mm/s, the acceleration of 0.5 mm/s2, and the focus offset at+0.4. The photoresist was baked in the oven (DX401, Yamato
Scientific Co., Ltd) at 368 K for three minutes. The
photoresist was baked in the oven (DX401) at 393 K for fiveminutes.
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). After confirmation offree of residual resists by the microscope, the titanium coatedplate was etched with the plasma gas using RIE-10NR (Samco
International, Kyoto, Japan). For etching, the gas of SF 6 (50cm3/min at 1013 hPa) with Ar (50 cm3/min at 1013 hPa) wasapplied at 100 W at 4 Pa for five minutes. The residual
OFPR-800LB was removed by acetone. The plate was dippedin the distilled water in one minute, after it is dipped in 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).

Micro Cylindrical Pillars on Lower Plate
The slide glass (26 mm × 38 mm × 1 mm) was used for the baseof micro cylindrical pillars, after cleaning with acetone (Fig. 3).
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 negative
photoresist material of high viscosity (SU8-100: Micro Chem
Corp., MA, USA) was coated on the slide glass at 3000 rpm for
30 s with a spin coater. The photoresist was baked at the
hotplate at 338 K for three hours. The photoresist was
exposed to the UV light through the mask in the mask aligner(M-1S, Mikasa Co. Ltd., Japan) at 15 mW/cm2 for 14 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 Corp., USA)for ten minutes. The glass surface with the micro pattern wasrinsed 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 for five minutes.
At the end of the process, the micro morphology of the surface
with the micro pillars was observed by a scanning electron
microscope (JSM6360, JEOL Ltd., Japan) (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 (Fig. 5D). The volume
ratio of PDMS to curing agent is ten to one. After degassing,
PDMS was baked at 333 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 patternadjusting the position of holes to make the flow channel. A
rectangular parallelepiped channel is formed between upper andlower plates (Fig. 4). The two plates stick together with theirsurface affinity.

Glass

Glass

Glass

Glass

1. Coating 2. Exposure

3. BakingGlass

4. Development

Fig. 3: Process for fabrication of pillars.
Glass

1. Ti Coating

Glass

2. OFPR-800

Glass

3. Exposure

Cell suspension
Saline solution

Glass

4. Development

Glass

5. Etching

PDMS

Glass

6. Exfoliation
Slits

Fig. 2: Process for fabrication of photomask.
Fig. 4: Flow channel.

The flow channel is placed on the stage of the inverted phase
contrast microscope (IX71, Olympus Co., Ltd., Tokyo).
Flow Test
Two kinds 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). Cells before the passage tenth were used in
the flow test.

CelldCells (C2C12 and Hepa1-6) were cultured with the D-MEM(Dulbecco’s Modified Eagle’s Medium) containing 10% FBS
and 1% of Antibiotic-Antimycotic (penicillin, streptomycin andamphotericin B, Life Technologies Japan Ltd., Tokyo, Japan) in
the incubator. The medium was changed every two days.
Before the cells were proliferated to sub-confluent state, thecells were exfoliated from the plate of the culture dish withtrypsin, and suspended in the medium. The cell suspension
was diluted by the saline solution to be the suspension of theconcentration of 10000 cells/cm3 for the flow test.

w

Slit

PillarFig. 5: Cell caught by slit between pillars.

Before the cell flow test, the 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). The channel was filled with the 4 % bovine
albumin aqueous solution, and placed in the incubator for one
hour to coat stable proteins on the inner surface of the channel.

The passing time (t) of each cell through the slit was measured.
The relation between the passing time (t) and the deformation
ratio (R) was plotted in Fig. 9. The relation approximated to
the following linear equation.

The channel was washed out with the saline solution, and filledwith the saline solution of 60 mm3. The flow path was
carefully examined to avoid mixing of air bubbles, which mightstir the medium in the flow channel.

3. RESULTS

t=aR+b(2)

The 60 mm3 of suspension of cells was added into the inlet portof the flow channel. The suspension of cells was introduced tothe slits at the first line (w = 0.025 mm) by the pressure
difference between the inlet and the outlet, which was kept by
the gravitational level of the medium.

Fig. 6 shows the floating cell of Hepa1-6. The diameters ofcells suspended in the saline solution scatter around 0.02 mm.
Fig. 7 shows the scanning electron microscope image of the
micro pillars. Although several neighboring top surfaces ofthe micro cylindrical pillars make connections over the slit of
0.01 mm, the morphology of the gap between pillars keeps
rectangular slit. The micro pillars stand perpendicular to thesurface of the glass slide.

The capacity of the flow channel is 1.8 mm3 (30 mm × 1 mm ×0.06 mm). The additional volume of 60 mm3 makes the fluid
level difference of 3 mm between inlet and outlet. The
decrease ratio of the level difference between inlet and outlet issmaller than 10 % in ten seconds. The difference of 3 mm ofthe fluid (103 kg/m3 density) produces the pressure difference of0.03 kPa.

Fig. 8 exemplifies C2C12 passing through the slit (w = 0.01mm) between micro pillars. The cell approaches to the slit
between micro pillars (a), is captured between micro pillars (b),deforms to pass through the slit (c), and gets out from the slit(d). Several cells are clogged in the slits of 0.01 mm. Mostof cells, on the other hand, pass through the slits wider than
0.015 mm.

The cell passing through the slit was observed by the
microscope, and recorded by the video camera (DSC-RX100M4,
Sony Corporation, Japan), which is set at the eyepiece. Boththe movement and the shape of the cell were analyzed by“ImageJ”.
From the microscopic video image, the deformation ratio (R) of
each cell in the slit was calculated by the following equation(Fig. 5).
R=d/w

(1)

In Eq. 1, d is the diameter of the cell before deformation in theslit, and w is the width of the slit. R is unity, when the
diameter of the cell equals to the width of the slit.

Fig. 6: Hepa1-6 suspended in saline solution: bar shows 0.01mm.

Fig. 8b: C2C12 is captured between micro cylindrical pillars (w= 0.01 mm): diameter of cylinder is 0.025 mm: flow from left toright.

Fig. 7: Scanning electron microscope image of slit between
micro pillars: w = 0.010 mm (upper), and w = 0.015 mm(lower).

Fig. 8a: C2C12 (circled) approaches to micro cylindrical pillars(w = 0.01 mm): diameter of cylinder is 0.025 mm: flow fromleft to right.

Fig. 8c: Deformed C2C12 goes through slit between micro
cylindrical pillars (w = 0.01 mm): diameter of cylinder is 0.025
mm: flow from left to right.

Fig. 8d: C2C12 (circled) gets out from slit between micro
cylindrical pillars (w = 0.01 mm): diameter of cylinder is 0.025
mm: flow from left to right.

It is not easy to fabricate micro slit, keeping the rectangularshape smaller than 0.01 mm. The resistance of the flow
channel is very high in the present experimental system. The
resistance increases, when the cells clogged the slits in the flow
channel. Internal pressure of the flow channel should be
carefully controlled under the constant flow rate, otherwise theleakage or cavitation would occur. The flow path was
carefully examined to avoid mixing of air bubbles, which mightstir the medium in the flow channel. The small pressure
difference between the inlet and the outlet of the flow channel ismaintained around 0.03 kPa by the difference of the level of thefluid between the inlet and the outlet in the present study.

Time (t) [s]Deformation ratio (R)Fig. 9a: Time (t) [s] vs. deformation ratio (R): Hepa1-6: t =1.1×102 R－8.1×10.
Time (t) [s]Several parameters have been applied to evaluate thedeformability of biological cells: the maximum deformation
ratio, and shear stress responsiveness [2]. The higher a showsthat C2C12 takes longer time to be deformed to the higher ratio
in the present study. The shorter passing time of C2C12 at thesmaller deformation ratio shows that the frictional resistancemight be smaller at C2C12 than Hepa1-6.
The capture of the cell at the slit depends on several factors: thedimension of the cell and the slit, deformation of the cell, thewall sear stress (shear rate) at the slit, and the affinity (charge)of the cell to the wall of the slit. The channel was filled withthe 4 % bovine albumen aqueous solution, and placed in the
incubator for one hour to coat stable proteins on the innersurface of the channel in the present study.

Deformation ratio (R)Fig. 9b: Time (t) [s] vs. deformation ratio (R): C2C12: t =1.4×102 R－1.7×102.

Fig. 9 shows the relation between the passing time (t) and thedeformation ratio (R). In the range of R smaller than 1.5, thepassing time (t) of C2C12 (Fig. 9b) is shorter than that of
Hepa1-6 (Fig. 9a). In the range of R bigger than 1.5, data of
the passing time scatters wider at C2C12 than at Hepa1-6. The
coefficient (a) in Eq. 2 is higher at C2C12 (1.4×102 s) than
Hepa1-6 (1.1×102 s). Among the passed cells, the maximum
deformation ratios are 1.7 at Hepa1-6 and 1.9 at C2C12.

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 [4]. In anotherstudy, the micro slit was manufactured between ridges [5].
The slit in biological tissue has very short length (0.001 mm)
and very narrow width (0.001 mm) with sub millimeter heightin vivo. With the conventional machining technique, however,the slit with such kind of dimensions is not able to be made forin vitro test. The slit is fabricated between micro cylindricalpillars by photolithography technique in the present study.
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 [5].

The adhesion of the cell in the flow depends on the morphologyof the wall and the wall shear stress. The morphology of thedefect on the wall of flow path might govern the capture of cells.
The capture might depend on the property of the cell. The
mechanism of adhesion might be applied to sort cells [13].
The experimental results might contribute to analyze adhesivemechanism of cancer cell during metastasis. The micro trap
might simulate adhesive mechanism of flowing cells.
A red blood cell has flexibility and deforms in the shear flow [2].
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 cellis trapped in the micro-circulation systems. Some cells canpass through the slit narrower than the capillary.
There are several methods to sort cells [13-22]. Non-invasive
way is preferable to sort cells with minimum damage. Flowcytometry 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.

5. CONCLUSION
Deformation of a cell has been measured using slits between
micro cylindrical pillars in vitro. Slits of the width (0.01 mm< w < 0.025 mm) were fabricated between micro cylindricalpillars on the glass plate using the photolithography technique.
The deformation of C2C12 (mouse myoblast cell line) andHepa1-6 (mouse hepatoma cell line) at the slit was observed by
the microscope. The experimental results show that several
cells clogged in the slit of 0.01 mm, and the passing time of
C2C12 is shorter than that of Hepa1-6 at the smallerdeformation ratio.
The designed slit has capability forevaluation of deformability of a 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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