Cell Deformation Passing through Slit between Micro-Machined
Surfaces in Vitro: Comparison among Cell Types
Shigehiro HASHIMOTO, Keisuke KAKISHIMA, Yusuke TAKAHASHI

Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACT
Deformation of cell passing through the slit between
micro-machined surfaces has been observed in vitro. At the
middle part of the flow channel, the slit (0.8 mm width, and0.01 mm height) has been made between the transparent
polydimethylsiloxane plate and glass plate, of which surfaces
are micro-machined by photolithography technique. Fourkinds of cells were used in the test: C2C12 (mouse myoblast
cells), Hepa1-6 (mouse hepatoma cells), L929 (fibroblast
connective tissue of mouse), and Neuro-2a (mouse neuralcrest-derived cell line). The suspension of each kind of cells
was injected to the slits. The deformation of each cell passing
through the micro slit was observed with an inverted
phase-contrast microscope. The experimental results showthat each cell deforms to the flat disk and passes through themicro slit of 0.01 mm height. Both the projected area and thepassing velocity of each cell in the slit were measured. The
passing velocity of each cell tends to decrease with the higher
deformation. At some cells of C2C12, the velocity in the slit
is higher than that before the slit even at the higher deformation
ratio. The designed slit between micro-machined surfaces hascapability to sort the deformability of cells.
Keywords: Biomedical Engineering, C2C12, Hepa1-6, L929,
Neuro-2a, Photolithography and Micro-slit.

1. INTRODUCTION
The deformability of the biological cell plays an important rolein vivo. A red blood cell, for example, has high flexibility. The
elasticity of the membrane [1], and fatigue [2] were evaluated in
the previous studies. The aging effect on deformability [3]and the sublethal damage [4] were also detected in the shear
flow in the previous studies.
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 sort cells according to the deformability in vivo.
One of the systems, which trap red blood cells, is a spleen.
The spleen has special morphology at the blood flow path tosort injured red blood cells [5]. A slit is one of the systems,
which sorts biological cells in vivo. The sorting at the slit
depends on the deformability of each cell. Some cells are able
to pass through a very narrow slit.

A photolithography technique enables manufacturing micro
grooves [6] or micro structures [7] in the flow-channel for cell
tests in vitro. Several micro-fabrication processes have been
designed to simulate morphology of the microcirculation [8].
The technique also has been applied to handle cells in
diagnostics in vitro [9]. The photolithography technique canbe applied to make a micro slit. The slit between micro
cylinders was made to sort cells in the previous study [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 the walls of the slit, another type of the slit isdesigned with the combination of micro ridges in the previous
study [11].
In the present study, a micro slit has been fabricated between
micro-machined surfaces by the photolithography technique,and deformation of a biological cell passing through the microslit has been compared among cell types experimentally invitro.

2. METHODS
Micro Slit
The slit has been designed between a transparent
polydimethylsiloxane (PDMS) plate and a borosilicate glass(Tempax) plate (Fig. 1). The upper plate of PDMS has arectangular ridge of 0.05 mm height, 0.10 mm width, and 2 mm
length. The lower plate of glass has a rectangular groove of
0.010 mm depth (H), 2 mm width, and 20 mm length, which
has a narrow part of 0.8 mm width. These plates make contact
to make slits (width (W) of 0.8 mm, length (L) of 0.1 mm andheight (H) of 0.010 mm) between them.
Photomask for Upper Plate
The glass plate of 0.2 mm thick was used for the base of themask (Fig. 2). Before the vapor deposition of titanium, thesurface of the glass plate was hydrophilized by the oxygen (30cm3/min, 0.1 Pa) plasma ashing for five minutes at 100 W by
the reactive ion etching system (FA-1, Samco Inc., Kyoto).
Titanium was coated on the surface with 150 nm thick in theelectron beam vapor deposition apparatus (JBS-Z0501EVC,
JEOL Ltd., Japan). The positive photoresist material of
OFPR-800LB was coated on the titanium at the plate at 7000
rpm for 60 s with a spin coater. The photoresist was baked on
the heated plate at 368 K for five minutes. The pattern was
drawn on the mask with a laser drawing system

(DDB-201K-KH, Neoark Corporation, Hachioji, Japan). The
pattern was baked on the heated plate at 368 K for five minutes.
The photoresist was developed with NMD-3 for 5 minutes. The
plate was rinsed by the ultrapure water, and dried by the
spin-dryer. The titanium coating plate was etched with theplasma gas (SF6, Ar) using RIE-10NR (Samco International,
Kyoto, Japan). The residual OFPR-800LB on the surface was
exfoliated by acetone.
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 the spincoater. 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
1000 rpm for 30 s with the spin coater again. After thephotoresist was prebaked on the heated plate at 338 K for fiveminutes, the plate was baked on the heated plate at 368 K for
one hour. The photomask was mounted on the surface ofSU8-10, and the photoresist was exposed to the UV lightthrough the mask in the mask aligner (M-1S, Mikasa Co. Ltd.,
Japan) at 15 mW/cm2 for 20 s. After the photoresist wasprebaked 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). The glass surface with themicro pattern was rinsed with IPA (2-propanol, Wako Pure
Chemical Industries, Ltd.) for five minutes, and with theultrapure water. The plate was dried by the spin-dryer. The
plate was baked on the heated plate at 368 K for three minutes.
Upper Plate
After the mold of the glass plate was enclosed with a peripheral
wall of polyimide tape, PDMS (Sylgard 184 Silicone Elastomer
Base, Dow Corning Corporation) was poured together with thecuring agent (Dow Corning Corporation) on the mold (Fig. 3).
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. Two holesof 8 mm diameter were machined with a punching tool at theupper disk to make the inlet and the outlet for the flow channel(Fig. 5).
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 thesurface with 150 nm thick in the electron beam vapor deposition
apparatus. The part of Titanium coating was covered with thepolyimide tape to make the flow channel of 2 mm width(narrow part: 0.8 mm width) × 20 mm length. The titanium
coating disk was etched with the plasma gas (SF6, Ar) using
RIE-10NR.
After the etching, the polyimide tape was
exfoliated.

Lower Plate
A glass plate (Fig. 4) was cleaned by an ultrasonic cleaner withalkaline solution, and rinsed by the ultrapure water. The
surface of the glass plate was hydrophilized by the oxygen (30cm3/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 (SU8-10) was coated on
the glass plate at 2000 rpm for 30 s with the spin coater. Afterthe photoresist was baked on the heated plate at 338 K for threeminutes, the plate was baked on the heated plate at 368 K forseven minutes. The photomask was mounted on the surface ofSU8-10, and the photoresist was exposed to the UV lightthrough the mask in the mask aligner (M-1S) at 15 mW/cm2 for
20 s. After the photoresist was prebaked 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 for three minutes. The glass surface wasrinsed with IPA for two minutes, and with the ultrapure water.
The plate was dried by the spin-dryer.

W
H
Fig. 1: Microslit.

Fig. 2: Photolithography process of mask.

Fig. 3: Upper plate of PDMS.

Fig. 4: Lower plate of glass.

Lphase-contrast microscope (IX71, Olympus Co., Ltd., Tokyo) at
298 K (Fig. 8).

Channel
10 mm

The microscopic movie images of thirty frames per second at
the shutter speed of 1/2000 s were recorded by the 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 (Fig. 9). The deformation
ratio (Ra) was calculated at each cell by Eq. 1.
Ra = (A2 － A1)/ A1(1)

Fig. 5: Flow channel device：diameter of each hole is 8 mm.
In Eq. 1, A1 is the area before the slit, and A2 is the area in the
slit.

Slit

W

The velocity of the cell passing through the slit (v) was
calculated at the movie by “Kinovea” (Fig. 10). Most of cells
pass through the slit in a few seconds, and Data of clogging cellin the slit are not included in the following figures.
The velocity ratio (Rv) was calculated at each cell by Eq. 2.
Rv = (v2 － v1)/ v1

0.5 mm(2)

In Eq. 2, v1 is the velocity before the slit, and v2 is the velocity
in the slit.

Fig. 6: Micro slit in flow channel.

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 (Aminopropyl-triethoxysilane) 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 to make the slit (Figs. 5& 6), and was baked on the heated plate at 338 K for fiveminutes.
Flow Test
Four kinds of cells (passage < 10) were used in the flow test:
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse), Hepa1-6 (mouse hepatoma cell line of
C57L mouse), L929 (fibroblast connective tissue of C3Hmouse), and Neuro-2a (a mouse neural crest-derived cell line).
D-MEM (Dulbecco’s Modified Eagle’s Medium) containing
10% FBS (decomplemented fetal bovine serum) and 1%
penicillin/ streptomycin (GIBCO, Life Technologies Japan Ltd.,Tokyo, Japan) was used for the medium.
The cells were exfoliated from the bottom of the culture dishwith trypsin, and suspended in the culture medium. The innersurface of the flow channel was hydrophilized by the oxygen(30 cm3/min, 0.1 Pa) plasma ashing for thirty seconds at 50 W
by the reactive ion etching system (FA-1). After the bovine
serum albumin solution was prefilled in the flow channel, thesuspension (250 μl) of the cells was poured at the inlet (8 mmdiameter) of the flow channel (Fig. 7). 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
behavior of cells near the slit was observed with an inverted

Fig. 7: Flow test.

Flow channel

Microscope

CameraFig. 8: Experimental system.

decrease with the increase of the deformation ratio. In Fig. 16,
each line shows the approximate straight line of data belong toeach kind of cells. Every line has negative slope. The line of
C2C12 has the steepest negative slope. The slope of each lineis –3.3 (C2C12), –2.1 (Hepa1-6), –1.7 (L929), and –0.6
(Neuro-2a), respectively.
Slit
Fig. 9: Tracings of projected area of each cell: area before slit
(A1), in slit (A2): flow from left to right.

4. DISCUSSION
For the reason of the limitation of the photolithography process,the edge of the ridge is not sharp, but it has the edge with thesmall width. The biological system might have the sharperedge, so that a cell passes easily through the slit with the shorter
travel distance in vivo [5]. The cell has to struggle to passthrough the slit in the present experimental device with thelonger travel distance.
A

0.1 mm

B

Slit
Slit

Slit

Fig. 10: Tracings of movement of each cell: velocity before slit(v1), in slit (v2): flow from left to right.
CD

3. RESULTS

The area of every cell increases in the slit, which shows
deformation to a flat disk in the slit (Figs. 12a, 13a, 14a, 15a).
None of the cell L929 has the projected area smaller than 300
μm2 before the slit (A1) (Fig. 14a). The velocity of L929 isslow in the slit (v2) (Fig. 14b).
In Fig. 15b, the velocity of Neuro-2a in the slit (v2: triangle)
tends to decrease with increase of the projected area before slit
(A1). The tendency shows the passing velocity decreases withthe own higher deformation ratio of each cell. Both at C2C12(Fig. 12b) and at Hepa1-6 (Fig. 13b), on the other hand, thevelocity in the slit (v2) scatters, which shows some cells passed
through the slit relatively fast with the high deformation ratio.
Especially at several cells of C2C12, the velocity in the slit (v2)
is higher than that before the slit (v1) (Fig. 12b).
The relationship between the velocity ratio (Rv) and thedeformation ratio (Ra) is summarized in Fig. 16. Some cellsof L929 have the deformation ratio above 1.0, which shows thelarge deformation of the cell in the slit. Several cells of
C2C12 have the velocity ratio above 1.0, which shows
accelerated velocity in the slit. The velocity ratio tends toSlit

0.1 mmFig. 11: Cell (traced by yellow line) approaches to slit (A),enters into slit (B), is passing through slit (C), and leaves fromslit (D): flow from left to right.

Projected area [µm2]Figs. 12–15 show the projected area (Fig. 12a, Fig. 13a, Fig.
14a, and Fig. 15a) and the velocity (Fig. 12b, Fig. 13b, Fig. 14b,
and Fig. 15b) of each cell: C2C12 (Fig. 12), Hepa1-6 (Fig. 13),
L929 (Fig. 14), and Neuro-2a (Fig. 15), respectively. In thesefigures, each mark of circle and of triangle shows datum before
the slit and in the slit, respectively. Data are arranged in theascending order of the projected area before the slit (A1) in Figs.
12a, 13a, 14a and 15a. The datum on each cell is arranged at
the corresponding cell number.

Slit

1000

750
500
250
0

0

10

20

30

Cell number
Fig. 12a: Area of C2C12: before slit (circle), in slit (triangle).

Velocity [µm / s]Fig. 11 exemplifies cell passing through the slit. A cellapproaches to the slit (A), enters the slit (B), is passing throughthe slit (C), and leaves from the slit (D).

3000
2000
1000
0

0

10

20

30

Cell number
Fig. 12b: Velocity of C2C12: before slit (circle), in slit
(triangle).

750
500
250
0

0

3

6

9

12

Projected area [µm2]Projected area [µm2]

1000

1000

750
500
250
0

0

6

Cell number

Fig. 15a: Area of Neuro-2a: before slit (circle), in slit (triangle).

Velocity [µm / s]

Velocity [µm / s]

1200
800

400

0

3

6

9

1800
1200
600
0

12

0

6

Cell number

800
400

10

20

Cell number

4.5
3
1.5
0

0

0.4

0.8

1.2

Deformation ratio [-]Fig. 14a: Area of L929: before slit (circle), in slit (triangle).

Velocity [µm / s]18

Fig. 15b: Velocity of Neuro-2a: before slit (circle), in slit
(triangle).

Velocity ratio [-]Projected area [µm2]

1200

0

12

Cell number

Fig. 13b: Velocity of Hepa1-6: before slit (circle), in slit
(triangle).

018

Cell number

Fig. 13a: Area of Hepa1-6: before slit (circle), in slit (triangle).

0

12

2400

Fig. 16: Relationship between velocity ratio (Rv) anddeformation ratio (Ra): C2C12 (circle), Hepa1-6 (triangle),
L929 (square), Neuro-2a (rhombus): approximate straight line is
added.

1800
1200
600
0

0

10

20

Cell number
Fig. 14b: Velocity of L929: before slit (circle), in slit (triangle).

The dimension of the slit was confirmed by the passing velocity
of porcine red blood cells in the present study. The ratio of thecross-sectional area between the flow channel (0.06 mm × 2mm) and the slit (0.01 mm × 0.8 mm) is 15, so that the mean
flow velocity of the media in the slit is 15 times faster than that
before the slit. Every datum of the velocity ratio in Fig. 16 issmaller than 5, which depends on the friction between the celland the wall of the slit.
The moving velocity of the cell suspended in the media follows
the media velocity. In the previous study [10], the flow rate
was controlled by the syringe pump, but the flow rate varied
because of several factors: the compliance of the wall of theflow path, and clogging of the flow path.

The flow rate is controlled by the pressure difference between
inlet and outlet of the flow channel in the present study, which
has advantage to keep the inner pressure of the flow channel forthe morphological stability of the flow channel. The moving
velocity also depends on the interaction between the cell and thesurface of the slit (friction). To keep the surface property of
the channel stable, bovine serum albumin is pre-coated on thesurface of the flow path by prefilling the bovine serum albumin
solution in the channel in the present study. The sphere withthe diameter of 18 μm makes the projected circle area of 250
μm2. Because the projected area is larger than 250 μm2 in theslit, every cell is forced to be deformed in the slit of 10 μm
height in the present study (Figs. 12, 14, 16, and 18). 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, on the other hand, captured at the channel.
A red blood cell has a high deformability. It deforms from thebiconcave disk shape to the parachute like shape, when it ispassing through the micro capillary. In the shear field, the red
blood cell rotates and deforms to the ellipsoid shape. The
most of biological cells, on the other hand, mostly keep thespherical shape, when they are flowing in the medium. The
deformability of a red blood cell changes with aging [3]. The
deformability of the biological single cell depends on several
factors. The deformability has been analyzed in several
studies: using microfluidics [9], measuring local viscoelasticity[12], using atomic force microscopy [13, 14], and using cellmechanics model [15]. The methodology can also be applied
to the soring technology on cells [16, 17]. The deformation isevaluated with the ratio of the projected area of the plan view ofthe disk-like shape during the passing through the slit in thepresent study. The deformation in the perpendicular direction
can be observed at another type of the slit between micro
cylindrical pillars [10].

5. CONCLUSION
Deformation of a cell through the slit (0.8 mm width, and 0.01
mm height) between micro-machined (photolithography
technique) surfaces has been observed in vitro. Both the
projected area and the passing velocity in the slit have been
evaluated at the microscopic images. The passing velocity ofeach cell tends to decrease with the higher deformation ratio.
The tendency depends on cell types. Some cells of C2C12(mouse myoblast cells) and of Hepa1-6 (mouse hepatoma cells)
pass through the slit relatively fast at the higher deformation
ratio. At some cells of C2C12, the velocity in the slit is higher
than that before the slit.
The designed slit between
micro-machined surfaces has capability to sort cells according
to their deformability.

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