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

Sorting of Cells Using Flow Channel with Oblique Micro
Grooves
Yusuke TAKAHASHI, Shigehiro HASHIMOTO, Yoshinori HORI, Takuya TAMURA
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
Cells roll over on the surface of the wall in the shear flow, andmake adhesion to the wall. The surface was modified tocapture flowing cells [11-13, 19]. The technique will also be
applied to handle cells in diagnostics in vitro.

ABSTRACTMicro grooves have been designed to sort biological cells,
which flows through a micro channel in vitro. The micro
groove of the rectangular shape (0.0045 mm depth, and 0.2 mm
length) has been fabricated on the surface of thepolydimethylsiloxane (PDMS) disk by the photolithography
technique. Variation has been made on the width (0.03 mm <w < 0.05 mm) and on the angle between the longitudinal
direction of the groove and the flow direction (45 degrees, 90
degrees) of the groove. A rectangular flow channel (0.05 mm
height × 2 mm width × 15 mm length) has been constructed
between two transparent PDMS disks. Four kinds of cells
were used in the test: C2C12 (mouse myoblast cell line),
MC3T3-E1 (mouse osteoblast precursor cell), Hepa1-6 (mouse
hepatoma cell), and Neuro-2a (mouse neural crest-derived cell).
A flow velocity of the suspension of cells was controlled by thepressure difference between the inlet and the outlet. Neuro-2a
often moves to the counter direction against the flow in thegroove.
The shift movement along the oblique groove
depends on the several parameters: the diameter of cells, thewidth of the groove, the velocity of the cell, and the kinds ofcells.

In the present study, the movement of the single cell flowing at
the micro groove, which is manufactured by thephotolithography technique, has been analyzed in vitro.

2. METHODS
Micro Grooves
For trapping cells, several micro grooves of the rectangularshape (0.0045 mm depth, and 0.2 mm length) have been
fabricated on the surface of the polydimethylsiloxane (PDMS)
plate with the photolithography technique. Several grooves
are arranged on the same wall. At the groove arrangementfrom upstream to downstream, variation has been made on thewidth of the groove: 0.03 mm, 0.04 mm, and 0.05 mm (Fig. 1).
Variation has also been made on the angle between thelongitudinal direction of the groove and the flow direction: 45
degrees, and 90 degrees (Fig. 2).

Keywords: Biomedical Engineering, MC3T3E1, Hepa1-6,
C2C12, Neuro-2a, Micro Groove and Polydimethylsiloxane.

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 cleaned 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
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).

1. INTRODUCTION
A flowing cell is captured to a wall of a flow path. Several
cells adhere to the internal wall of the blood vessel, when thepart of the wall has a defect. The morphology of the defectmight govern the capture of cells. The capture also might
depend on the property of the cell. The cancer cell transits
from the original place to another place, and proliferates tomake a tumor at another place. The transition occurs throughthe blood vessels and the lymph vessels. The cancer cells
adhere to the inner wall of the vessels.
The effect of the surface property of the scaffold on the cell has
been studied in the previous studies [1-8].
Severalmicro-fabrication processes have been designed to control
adhesion of biological cells in vitro, and to simulate themorphology of the microcirculation [9, 10].
The
photolithography technique enables manufacturing the
micro-morphology. The micro-fabrication technique has also
been applied to design microfluidic systems in vitro [11-19].

Fig. 1: Dimension of three types of oblique grooves.

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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 7000 rpm for 30 s). The photoresist was
baked in the oven (DX401, Yamato Scientific Co., Ltd) at 368
K for five minutes.
The pattern for the groove 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 3.26 V, the velocity of 0.137mm/s, the acceleration of 0.5 mm/s2, and the focus offset at+3.17.

Fig. 3: Photolithography process for photomask.

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.

Developing

Fig. 4: Photolithography process for lower plate.

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 (Fig.
4). The surface of the glass plate was hydrophilized by theoxygen (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 exposed to the UV light through thephotomask in the mask aligner (M-1S, Mikasa Co. Ltd., Japan)
at 15 mW/cm2 for 25 s. The photoresist was baked in the ovenat 393 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.

The epoxy based negative photo-resist material (SU8-5: Micro
Chem Corp., MA, USA) was coated on the glass at 2500 rpm
for 30 s with a spin coater. The photoresist was baked in theoven (DX401) at 368 K for five minutes.

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
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 slide glass (Matsunami) plate (38 mm length, 26 mm width,and 1.0 mm thickness) was used for the base of the mold (Fig.
5). The polyimide tape (0.055 mm thickness, 10 mm width)
was pasted at the center of the glass plate. The tape was cut(mm × mm) by the ultra-short pulse laser (IFRIT, Cyber Laser
Inc., Tokyo, Japan) to make the mold for the upper part of theflow channel.

Fig. 2: Two patterns of microgrooves on photomask.

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After the glass plate was enclosed with a peripheral wall ofpolyimide, PDMS was poured with the curing agent on the plate(Fig. 6). The volume ratio of PDMS to curing agent is ten toone. After degassing, PDMS was baked at 368 K for twenty
minutes in the oven (DX401). Two holes (diameter of 5 mm)with the interval of 5 mm were punched by a punching tool tomake the inlet and the outlet (Fig. 7a).
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 (15 mm length
× 2 mm width × 0.055 mm height) between them. The flow
channel is placed on the stage of the inverted phase-contrast
microscope (IX71, Olympus Co., Ltd., Tokyo) (Fig. 7b).
Flow Test
Four kinds of cells were used in the test: C2C12 (passage < 10,mouse myoblast cell line originated with cross-striated muscle
of C3H mouse), MC3T3-E1 (passage nine, osteoblast precursor
cell line derived from mouse calvaria), Hepa1-6 (passage six,mouse hepatoma cell line of C57L mouse), and Neuro-2a(passage seven, mouse neural crest-derived cell line)

Fig. 6: Upper plate.

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.
Fig. 7a: Flow channel.

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). The suspension ofcells (20000 cells/cm3, 0.06 cm3) was poured at the inlet of theflow channel. The flow occurs by the pressure difference
between the inlet and the outlet. The inlet hole (the depth of 3
mm and the diameter of 5 mm) makes the pressure head.

Fig. 7b: 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: 30 frames per second. At theimages, the contour of each cell was traced with “Image J”, andapproximated to the circle to calculate the diameter (D) (Fig. 8).
The velocity of each cell around the groove was traced on thecomponent parallel to the direction of the main flow of thevelocity of the cell: immediately before the groove (v0) and in
the groove (v1) (Fig. 9). The travel distance of each cell along
the groove was traced on the component (x) perpendicular to themain flow direction.
Fig. 5: Mold for upper plate.

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Fig. 8: Evaluation of dimension of cell (white circle) at optical
microscope image (right).

Fig. 12: Velocity of C2C12 in three width type of oblique (45degrees) groove vs. time: 0.03 mm (circle), 0.04 mm (square),and 0.05 mm (triangle).

3. RESULTS

Fig. 9: Evaluation of movement of cell at groove: velocity
before groove (v0), in groove (v1): vertical travel distance (x).

Fig. 10 exemplifies the cell approaching to the groove (left) anddeparting from the groove (right): MC3T3-E1 (a), and Neuro2a(b). The direction of the flow is from left to right in Fig. 10.
Most of cells travel along the groove to the downstream to show
positive x (Fig. 10a). Many cells of Neuro2a migrate in thegroove to the upstream to show negative x (Fig. 10b).
Fig. 11 shows the velocity of C2C12 around the groove of 90
degrees: before the groove (v0) and in the groove (v1). Eachpair of data shows v0 and v1 of the same cell. The velocity
decreases with the time, because the pressure head at the inlet
decreases with time. The velocity in the groove is slower thanthat before the groove, except at the velocity of 0.62 mm/s.

Fig. 10a: MC3T3-E1 (red circle) before groove (left), in groove(right); flow from left to right: optical microscope image.

Fig. 12 shows the velocity (v1) of C2C12 in the groove of 45
degrees. Each group of three data shows v1 of the same cell ateach width of the groove, respectively. The velocity decreases
with the time, because the pressure head at the inlet decreases
with time. The velocity in the groove tends to be slower in thewider groove at the higher velocity.
Figs. 13, 14 and 15 show the shifted distance (x) along thegroove, which is the component perpendicular to the main flow
direction, in relation to the diameter (D) of each cell: C2C12(Fig. 13), MC3T3-E1 (Fig. 14), and Hepa1-6 (Fig. 15),
respectively. Each marker shows the variation of the width of
the groove: 0.03 mm, 0.04 mm, and 0.05 mm, respectively.
Each dotted line shows the approximated linear relationship at
the corresponding data. The distance (x) tends to increase withdecrease of the diameter of the cell of C2C12 (Fig. 13), and of
MC3T3-E1 (Fig. 14). The tendency is remarkable at thegroove of the narrower width (0.03 mm). In Hepa1-6, on theother hand, the distance (x) varies with no significant relation to
the diameter (D) of each cell.

0.1 mmFig. 10b: Neuro2a (red circle) before groove (left), after groove(right); flow from left to right: optical microscope image.

Fig. 16 show the shifted distance (x) along the groove in
relation to the velocity immediately before the groove (v0) of
Hepa1-6. Each marker shows the variation of the width of thegroove: 0.03 mm, 0.04 mm, and 0.05 mm, respectively. Eachdotted line shows the approximated linear relationship at thecorresponding data. The distance (x) tends to increase withdecrease of the velocity immediately before the groove (v0).
Fig. 11: Velocity of C2C12 vs. time: before groove (circle), andin groove (triangle): 90 degrees groove.

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Fig. 16: Velocity (v0) of Hepa1-6 in relation to shifted distancealong groove (x): circle, 0.03 mm; triangle, 0.04 mm; square,
0.05 mm width: dotted line, approximated line.

Fig. 13: Diameter (D) of C2C12 in relation to shifted distancealong groove (x): circle, 0.03 mm; triangle, 0.04 mm; square,
0.05 mm width: dotted line, approximated line.

4. DISCUSSION
The rectangular grooves has been successfully manufactured on
the wall of the micro fluid channel. The dimension of thegrooves were confirmed by the laser microscope [13]. Several
kinds of systems were designed for the cell culture in flow
[11-21] and for the cell sorting in vitro [22-25]. The
micromachining technique has been applied to cell technology.
The microfluidic system was applied to sort biological cells,
and to trap biological cells [11-13]. The experimental results
might contribute to analyze adhesive mechanism of cancer cell
during metastasis. The micro trap might simulate adhesivemechanism of flowing cells. Neuro-2a, for example, might behighly adhesive.
Several fluid flow system were used in the previous studies.
In the previous studies, cylindrical [11] and half cylindrical [12]holes were used for the trap of cells. The asymmetrical holemight be suitable for trap than the symmetrical hole. The
depths of the micro holes were between 0.002 mm and 0.01 mm
in the previous studies [11-13]. In the present study, the depthof the grooves is 0.0045 mm, which is smaller than the diameterof the cells. The deeper hole may have advantage to trap cells.
At the shallower trap, on the other hand, it is not easy to tarp acell. The trap of the appropriate dimension may distinguishcells. The duration of the trapped time of the cell might relate
to interaction between the micro hole and the cell: affinity
between the cell and the surface of the micro pattern, or
deformability of the cell.

Fig. 14: Diameter (D) of MC3T3-E1 in relation to shifted
distance along groove (x): circle, 0.03 mm; triangle, 0.04 mm;
square, 0.05 mm width: dotted line, approximated line.

The results of the present study show that the movement of celltravelling on the wall is modified by the oblique micro grooveon the wall under the cell velocity lower than 1 mm/s. The
angle of 45 degrees between the longitudinal direction of thegroove and the flow direction is effective to shift the streamlineof the cell. The shift movement along the oblique groove
depends on the several parameters: the diameter of cells, thewidth of the groove, the velocity of the cell, and the kinds ofcells. As the diameter of the cell decreases, the travelinglength along the groove increases. The movement might berelated not only to the diameter but also to the deformability of
the cell. The cell density in the suspension is very low in thisstudy to reduce the interaction between the cells. Cells can besorted by the traveling length along the microgroove.

Fig. 15: Diameter (D) of Hepa1-6 in relation to shifted distancealong groove (x): circle, 0.03 mm; triangle, 0.04 mm; square,
0.05 mm width: dotted line, approximated line.

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5. CONCLUSION
Oblique micro grooves have been designed to sort biologicalcells, which flow through a micro channel in vitro. The micro
groove of a rectangular shape has been fabricated on the surfaceof the polydimethylsiloxane plate with the photolithography
technique. Four kinds of cells were used in the test: C2C12(mouse myoblast), MC3T3-E1 (mouse osteoblast), Hepa1-6(mouse hepatoma cell), and Neuro-2a (mouse neuralcrest-derived cell). The results show that the cell is trapped by
the micro grooves and that the traveling length along the groove
is related to the diameter of the cell, to the width of the groove,to the velocity of the cell, and to the kinds of cells.

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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