Sorting of Cells with Flow Channel: Movement of Flowing
Myoblast Cell at Oblique Micro Grooves
Shigehiro HASHIMOTO, Akira HAYASAKA, Yuji ENDO
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACT

2. METHODS

The movement of a flowing cell near the oblique micro grooveon the bottom surface in the micro flow channel has beenmeasured to sort biological cells in vitro. The micro groove of
the rectangular shape (4.5 μm depth, and 0.2 mm length) has
been fabricated on the polydimethylsiloxane (PDMS) disk by
the photolithography technique. The angle between the flow
direction and the longitudinal axis of the groove is 45 degree.
Variation has been made on the width (0.03 mm < w < 0.05mm) of the groove. A rectangular flow channel (0.05 mm
height × 1 mm width × 25 mm length) has been constructed
between two transparent PDMS disks.
C2C12 (mouse
myoblast cell line) was used in the test. A flow velocity (0.04mm/s < vx < 0.80 mm/s) of the suspension of cells wascontrolled by the pressure difference between the inlet and theoutlet. The shifted distance of each cell along the obliquegroove depends on the diameter of the cell. In the present
device, the malnourished cell with the different density can be
distinguished by the shifted distance according to the velocityof the cell.

Micro Grooves
For trapping cells, several micro grooves of the rectangularshapes (4.5 μm depth, and 0.2 mm length) have been fabricatedon the surface of the polydimethylsiloxane (PDMS) plate withthe photolithography technique. Several grooves are arrangedon the same wall for the bottom of the micro flow channel.
The angle between the flow direction and the longitudinal axisof the groove is 45 degree. At the groove arrangement fromupstream to downstream, variation has been made on the width(w) of the groove: 0.03 mm, 0.04 mm, and 0.05 mm. The
angle between the longitudinal direction of the groove and theflow direction is 45 degrees.

Keywords: Biomedical Engineering, C2C12, Micro Groove,
Sorting, and Polydimethylsiloxane.

Photomask for Lower Plate
The slide glass (Matsunami Glass Ind., Ltd., Japan) plate (38mm length, 26 mm width, and 1.0 mm thickness) was used forthe base of the photomask. Before the deposition of titanium,
the surface of the glass plate was cleaned 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 International,
Kyoto, Japan). Titanium was deposited on the surface of theglass plate with 200 nm thickness in the spattering system (backpressure < 9×10－4 Pa, RF100W, 0.5 Pa, 8 min, L-210S-FH
Canon Anelva Corporation, Kawasaki, Japan).

1. INTRODUCTION
Several methods were proposed for the sorting of biologicalcells in vitro [1-12]. The micro fluidic systems were used insome methods [4-12]. In these systems, variety of propertiesof cells were picked up for the sorting. In the present study,
physical properties have been picked up: diameter, and specificgravity.
The technology of sorting of cells can be applied to regenerative
medicine to select the target cells. It also can be applied todiagnostics to handle the target cell [13].
To capture the target cell, several kinds of morphology were
designed in the micro fluidic systems: electrodes [10], microslits [14, 15], micro holes [16, 17], or micro grooves [18, 19].
The photolithography technique enables manufacturing the
micro-morphology [20].
In the present study, the movement of the single cell flowing
over the micro groove, which is manufactured by thephotolithography technique, has been analyzed in vitro.

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 withthe spin coater (1H-DX2, Mikasa Co. Ltd., Tokyo). The
positive photoresist material of OFPR-800LB (Tokyo Ohka
Kogyo Co., Ltd, Tokyo, Japan) was coated on the titanium withthe spin coater (at 2500 rpm for 30 s). The photoresist was
baked in the oven (DX401, Yamato Scientific Co., Ltd) at 368
K for three minutes.
The pattern for the groove (Fig. 1) was drawn on the mask witha 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 3.26 V, the velocity of 0.137mm/s, the acceleration of 0.5 mm/s2, and the focus offset at+3.17.

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 plate. The volume ratio of PDMS tocuring agent is ten to one. After degassing, PDMS was baked
at 368 K for twenty minutes in the oven.
Upper Plate
The polyimide tape (0.055 mm thickness, 1 mm width, and 25
mm length) was pasted at the center of thepoly-methyl-methacrylate plate for the mold.

0.02 mmFig. 1: Micro patterns for the groove at drawing.

After the plate was set in the mold of poly-methyl-methacrylate(Fig. 2), PDMS was poured with the curing agent on the plate.
The volume ratio of PDMS to curing agent is ten to one. After
degassing, PDMS was baked at 368 K for twenty minutes in theoven (DX401). Two holes (diameter of 5 mm) with theinterval of 20 mm were made for the inlet and the outlet (Fig.
3).

The photoresist was baked in the oven (DX401) at 368 K forfive minutes.
The photoresist was developed withtetra-methyl-ammonium hydroxide (NMD-3, Tokyo Ohka
Kogyo Co., Ltd., Kawasaki, Japan) for five minutes, rinsed withthe 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 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 six minutes. The
residual OFPR-800LB was removed by acetone dipped for one
minute. The plate was dipped in 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 distilledwater, and 1100 rpm for 30 s with N2 gas.
Lower Plate
The slide glass (Matsunami) plate (38 mm length, 26 mm width,and 1.0 mm thickness) was used for the base of the mold. 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).

30 mm

Fig. 2: Mold for upper plate.
The epoxy based negative photo-resist material (SU8-5: Micro
Chem Corp., MA, USA) was coated on the glass at 2700 rpm
for 120 s with a spin coater. The photoresist was baked in theoven (DX401) at 338 K for three minutes, and at 368 K forseven minutes successively.
The photoresist was exposed to the UV light through thephotomask in the mask aligner (M-1S, Mikasa Co. Ltd., Japan)
at 15 mW/cm2 for 15 s. The photoresist was baked in the ovenat 363 K for seven minutes. The photo-resist was developedwith SU8 developer (Nippon Kayaku Co., Ltd, Tokyo, Japan)
for five minutes to make micro rectangular ridges, where thelaser beam was radiated.

10 mm

The glass surface with the micro pattern was rinsed with IPA(2-propanol, Wako Pure Chemical Industries, Ltd.) 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.
After the plate was enclosed with a peripheral wall of polyimide

Fig. 3: Upper plate.

Fig. 4: Dimension of width of each groove.

0.05 mm

Flow System
Both the upper and the lower plates were exposed to the oxygengas (0.1 Pa, 30 cm3/min) in the reactive ion etching system(FA-1) (oxygen plasma ashing, 50 W, for thirty seconds).
Immediately after ashing, the upper disk adheres (plasma
bonding) to the lower disk to make the flow path (0.055 mm
height × 1 mm width × 25 mm length) between them. The
dimension of the width of each groove was measured on the
microscopic image (Fig. 4). The flow channel is placed on thestage of the inverted phase-contrast microscope (IX71,
Olympus Co., Ltd., Tokyo) (Fig. 5).
Flow Test
C2C12 (passage < 10, mouse myoblast cell line originated withcross-striated muscle of C3H mouse) was used in the test.
Cells 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) in the incubator for one
week.
For comparison with normal cells (group A), malnourished cells(group B) were prepared. In the group B, cells were kept in
the cryopreservation solution (the serum type Cellbanker1(including dimethylsuloxide: Nippon Zenyaku Kogyo Co., Ltd,
Koriyama, Japan)) in the incubator (at 310 K with 5% of CO2)
for one week without medium change after cryopreservation.

Fig. 5: Experimental system.

Each cell rolling over the micro grooves 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 each cell was analyzed by“Kinovea” at the video images (Fig. 6a): 30 frames per second.
At the images, the contour of each cell was traced with “ImageJ”, and approximated to the circle to calculate the diameter (D).
The velocity of each cell (vx) immediately before the groove
was traced on the component parallel to the direction of themain flow (Fig. 6b). The shifted distance of each cell along
the groove was traced on the component (y) perpendicular to themain flow direction.

The inner surface of the flow channel was hydrophilized by theoxygen (30 cm3/min, 0.1 Pa) plasma ashing for one minute at
100 W by the reactive ion etching system (FA-1), 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).
A part of thesuspension of cells (4000 cells/cm3, 0.06 cm3) was poured at the
inlet of the flow channel. The flow occurs by the pressure
difference between the inlet and the outlet. The inlet hole (thedepth of 3 mm and the diameter of 5 mm) makes the pressurehead.
The residual part of the suspension of cells were continuously
cultured in the medium for 30 minutes to confirm the vitality ofcells by adhesion to the scaffold. The specific gravity of thecell was measured in comparison with the density of thephthalic acid ester solution between 1.05 g/cm3 and 1.08 g/cm3
by centrifuge.

Fig. 6a: Tracings (blue line) of movement of cell passing overgroove: flow from left to right.
0.1 mmw

GrooveDCellyto the velocity (vx) of each cell: vx < 0.1 mm/s, 0.1 mm/s < vx <0.2 mm/s, and 0.2 mm/s < vx. At the low velocity, data scatters(Fig. 9). At the middle velocity, the tendency of decrease of
the shifted distance (y) with the ratio of D/w is clearer around
0.4 of D/w (Fig. 10).

vx
Shifted distance (y) [μm]Fig. 6b: Parameter of movement of cell passing over groove.
Shifted distance (y) [μm]D/w
Fig. 8: Shifted distance (y) vs. D / w: normal cell (circle), andmalnourished cell (triangle).
Diameter of cell (D) [μm]Shifted distance (y) [μm]Fig. 7: Shifted distance (y) of C2C12 vs. diameter of cell (D):0.03 mm (circle), 0.04 mm (triangle), and 0.05 mm (square).

3. RESULTS
The dimension of the width of each groove was confirmed at
the microscopic image as the designed value (Fig. 4). The
density of the normal C2C12 (group A) was 1.065 g/cm3. The
density of the malnourished C2C12 (group B) was lower than
1.06 g/cm3. Unlike cells of group A, cells of group B did notadhere to the scaffold within 30 minutes in the incubator. The
diameter of each cell was in the range between 13.1 μm and21.7 μm. The velocity of each cell (vx) immediately before thegroove was in the range between 0.038 mm/s and 0.80 mm/s
according to the pressure difference between the inlet and theoutlet. The velocity decreases with the time, because thepressure head at the inlet decreases with time.

D/w
Fig. 9: Shifted distance (y) vs. D / w: vx < 0.1 mm/s.
Shifted distance (y) [μm]The shifted distance was in the range shorter than 11.1 μm.
Data of the shifted distance (y) of C2C12 are collected inrelation to the diameter of each cell (D) in Fig. 7. The shifted
distance (y) at the groove (0.03 mm < w < 0.05 mm) scatters
between 4 μm and 12 μm, when the diameter of each cell is in
the range between 12 μm to 20 μm. The value of y tends toincrease with the width of the groove.
Fig. 8 shows y in relation to D/w. The ratio of D/w is unity,
when the diameter of the cell (D) is equal to the width of thegroove (w). The shifted distance (y) tends to decrease with theincrease of the ratio of D/w. When the diameter of the cell (D)is big compared with the width of the groove (w), the travel
along the groove is shorten.
The results are divided into three graphs (Figs. 9-11), according

D/w
Fig. 10: Shifted distance (y) vs. D / w: 0.1 mm/s < vx < 0.2
mm/s.

Several fluid flow systems were used in the previous studies.
In the previous studies, cylindrical [16] and half cylindrical [17]holes were used for the trap of cells. The asymmetrical hole[17] might be suitable for trap than the symmetrical hole. The
depths of the micro patterns were between 0.002 mm and 0.01
mm in the previous studies [16-20]. In the present study, thedepth of the grooves is (4.5 μm, which is smaller than the
diameter of the cells. The deeper hole may have advantage totrap every cell. At the shallower trap, on the other hand, it is
not easy to tarp a cell. The trap of the appropriate dimension
may distinguish cells. The duration of the trapped time of thecell might relate to interaction between the micro hole and thecell: affinity between the cell and the surface of the micro
pattern, or deformability of the cell.

Shifted distance (y) [μm]D/w
Fig. 11: Shifted distance (y) vs. D / w: 0.2 mm/s < vx.
Shifted distance (y) [μm]The results of the previous study show that the movement ofcell travelling on the wall is modified by the oblique micro
groove on the wall under the cell velocity lower than 1 mm/s
[18]. The angle of 45 degrees between the longitudinal
direction of the groove and the flow direction is effective toshift the streamline of the cell. The shift movement along theoblique groove depends on the several parameters: the diameterof cells, the width of the groove, the velocity of the cell, and thekinds of cells [18]. As the diameter of the cell decreases, thetraveling length along the groove increases. The movement
might be related not only to the diameter but also to thedeformability of the cell.
The movement of flowing cell at the bottom surface of the flow
channel might be related to the specific gravity of the cell: thedensity difference between the cell and the medium. The
shifted distance of malnourished cells by the oblique groove issmaller than that of normal cells in the present study.

D/w
Fig. 12: Shifted distance (y) vs. D / w: 0.25 mm/s < vx < 0.40
mm/s: normal cell (circle), and malnourished cell (triangle).

Data of malnourished cells are added in Fig. 8. The shifted
distance (y) of the malnourished cell is shorter than that of thenormal cell. The tendency is confirmed, when data are
selected according to the range of the velocity (vx) of each cell(Fig. 12).

4. DISCUSSION
Fluorescence technique was used in the previous sorting
systems [1, 2]. Non-destructive cell sorting systems, on theother hand, were designed in the previous studies [3]. The
label-free methods were designed with microfluidic systems [6,12]. Some of them were designed to capture cancer cells [8,13].
The rectangular grooves have been successfully manufactured
on the wall of the micro fluid channel. The dimension of thegrooves was confirmed by the laser microscope [18]. Several
kinds of systems were designed for the cell sorting in vitro[1-19]. The micromachining technique has been applied tocell technology. The microfluidic system was applied to sortbiological cells, and to trap biological cells [4-12]. The sorting
technique might be applied to selection of cells for regenerative
medicine and diagnostics of disease.

The cell density in the suspension is very low in this study to
reduce the interaction between the cells. Cells can be sortedby the traveling length along the micro groove.

5. CONCLUSION
The movement of a flowing cell near the micro groove on thebottom surface in the micro flow channel has been measured tosort biological cells in vitro. The micro groove of therectangular shape (4.5 μm depth, and 0.2 mm length) has been
fabricated on the bottom surface of the flow channel ofpolydimethylsiloxane by the photolithography technique. The
angle between the flow direction and the longitudinal axis of thegroove is 45 degree. C2C12 (mouse myoblast cell line) wasused in the test. The shifted distance of each cell along theoblique groove depends on the diameter of the cell. In thepresent device, the malnourished cell can be distinguished by
the shifted distance according to the velocity of the cell around
0.3 mm/s.

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