Proceedings of The 24th World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2020)

Velocity of Flowing Myoblast Cell at Oblique Micro Grooves
Shigehiro HASHIMOTO, Taku MATSUMOTO, Yuji ENDO, Shono KUWABARA, Hiroki YONEZAWA
Biomedical Engineering, Department of Mechanical Engineering, Kogakuin University
Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
Kiyoshi YOSHINAKA
Health and Medical Research Institute, National Institute of Advanced Industrial Science & Technology
Tsukuba, 305-8564, Japan
ABSTRACTthe micro groove, which is manufactured byphotolithography technique, has been analyzed in vitro.

The velocity change of a flowing cell near the oblique micro
groove on the bottom surface in the micro flow channel has
been measured in vitro. The micro groove of the rectangularshape (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 thelongitudinal axis of the groove is 45 degree. Variation has
been made on the width (0.03 mm, 0.04 mm, and 0.05 mm) 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. In the test, malnourished C2C12(mouse myoblast cell line) was used in comparison with thenormal C2C12. A flow velocity (0.02 mm/s < vx < 0.23 mm/s)of the suspension of cells was controlled by the pressure
difference between the inlet and the outlet. The change of thevelocity (perpendicular to the main flow direction) of eachflowing normal cell at the oblique groove is higher at the slower
flowing velocity on the wider groove.

the

2. METHODS
Micro Grooves
For changing the velocity of each cell, three micro grooves of
the rectangular shapes (4.5 μm depth, and 0.2 mm length) havebeen fabricated on the surface of the polydimethylsiloxane(PDMS) plate with the photolithography technique [15]. The
grooves are arranged on 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.
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).

Keywords: Biomedical Engineering, C2C12, Malnourished
Cell, Micro Groove and Velocity Change.

1. INTRODUCTION
The technology of sorting of cells can be applied to regenerative
medicine to select the target cells [1]. It also can be applied todiagnostics to handle the target cell [2].

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.

Several methods were proposed for the sorting of biologicalcells in vitro. The microfluidic systems were used in somemethods. In these systems, variety of properties of cells were
picked up for the sorting: electric, magnetic, dimension, or
deformability [3, 4]. In the present study, physical properties
have been picked up: diameter, and specific gravity.

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.

To capture the target cell, several kinds of morphology were
designed in the microfluidic systems: electrodes [5-8], microslits [9, 10], micro holes [11, 12], or micro grooves [13-15].
The photolithography technique enables manufacturing the
micro-morphology [16, 17]. The velocity of each cell canchange at the groove on the bottom wall of the flow channel[15].

The inner surface of the flow channel was hydrophilized by theoxygen (30 cm3/min, 0.1 Pa) plasma ashing for one minute at

In the present study, the velocity of the single cell flowing over31

Proceedings of The 24th World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2020)

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 viability 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.

0.02 mmFig. 2: Cell approaches to groove: contour of cell is traced: flowfrom left to right.
w

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 on the eyepiece of the
microscope. Two-dimensional movement of each cell parallelto the bottom plane was analyzed by “Kinovea” at the video
images (Fig. 1): 30 frames per second. At the images, theprojected contour of each cell was traced with “Image J” (Fig.
2). The area (S) enclosed by the outline was calculated. The
equivalent diameter (D) at the approximated circle wascalculated by Eq. (1).
S = 0.25 π D2

Groove
vy
vx

Δy

Flow

(1)
D
Cell

GrooveFig. 3: Parameter of movement of cell passing over groove.

The velocity of each cell near the groove was traced on thecomponents parallel (vx) and perpendicular (vy) to the directionof the main flow (Fig. 3). The shifted distance of each cellalong the groove was traced on the component (Δy)
perpendicular to the main flow direction.
0.1 mm
3. RESULTS

Fig. 1: Tracings (yellow line) of movement of cell passing overgroove: left, w = 30 µm; middle, w = 40 µm; right, w = 50 µm:
flow from left to right.

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
not adhere to the scaffold within 30 minutes in the incubator.
The diameter of each cell was in the range between 13.1 μm
and 21.7 μm. The velocity of each cell (vx) immediately
before the groove was in the range between 0.02 mm/s and 0.23mm/s according to the pressure difference between the inlet andthe outlet. The velocity gradually decreases with the time,
because the pressure head at the inlet decreases with time. The
tracings of the velocity of each cell are displayed in Fig. 4.
Fig. 4a shows the component of the velocity parallel to the main
flow direction (vx). Each cell flows at constant velocity

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Proceedings of The 24th World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2020)

component on vx. Fig. 4b shows the component of the velocity
perpendicular to the main flow direction (vy). Each cell flows
with the peak velocity component of vy (three peaks: at x = –0.15 mm, 0 mm, 0.15 mm) at each groove. The component ofvy is very small at the other part of the channel.
The shifted distances (Δy) were in the range shorter than 10 μm
at each groove (w: 0.03 mm, 0.04 mm, and 0.05 mm). Data of
the shifted distances (Δy) are collected in relation to the area of
each cell (S) in Fig. 5: normal cells (Fig. 5a), and malnourishedcells (Fig. 5b). The shifted distance (Δy) at the groove (w =0.04 mm) scatters between 2 μm and 10 μm, when the area of
each cell is in the range between 50 μm2 to 350 μm2. The
value of Δy tends to decrease with the area of each cell at thegroove width of 0.04 mm, especially in the case of
malnourished cells (Fig. 5b).
Fig. 5a: Δy vs. cell area (S): w = 40 µm: normal cell: regression
line (Δy = – 0.0088 S + 7.3): correlation coefficient, r2 = 0.08.

Fig. 4a: Position (x) vs. velocity of cell (vx): the same color
shows the same cell.
Fig. 5b: Δy vs. cell area (S): w = 40 µm: malnourished cell:
regression line (Δy = – 0.020 S + 8.0): correlation coefficient, r2= 0.55.

Fig. 6 shows relationships between the shifted distance (Δy) andthe velocity (vx) of each cell: normal cells (Fig. 6a), andmalnourished cells (Fig. 6b). The value of Δy decreases withthe velocity (vx) of each cell at the groove width of 0.04 mm in
the case of normal cells (Fig. 6a).
Fig. 7 shows relationships between the velocity ratio (vy max /vx) and the length ratio (D / w): normal cells (Fig. 7a), andmalnourished cells (Fig. 7b). The velocity ratio is higher at
normal cells than malnourished cells. At the normal cell,
velocity component perpendicular to the main flow changes atthe oblique groove more than at the malnourished cell. The
velocity ratio is higher at the narrower width (w = 0.03 mm)
than at the wider width (w = 0.05 mm). At some normal cells,
the velocity ratio (vy max / vx) is higher at the small length ratio(D / w).

Fig. 4b: Position (x) vs. velocity of cell (vy): the same color
shows the same cell.

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Proceedings of The 24th World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2020)

Fig. 7b: Velocity ratio (vy max / vx) vs. D / w: circle, w = 30 µm;
square, w = 40 µm; triangle, w = 50 µm: malnourished cell.

Fig. 6a: Δy vs. velocity (vx): w = 40 µm: normal cell: regression
line (Δy = – 2.2 vx + 7.7): correlation coefficient, r2 = 0.56.

4. DISCUSSION
Filtration is one of the basic methods of sorting [18].
Fluorescence techniques [19-21] was used in the previous
sorting systems with flow cytometry technique [22].
Non-destructive cell sorting systems, on the other hand, were
designed in the previous studies [23]. The label-free methods
were designed with microfluidic systems [24, 25]. Some ofthem were designed to capture cancer cells [26].
The microfluidic system was applied to sort biological cells[27-31], and to trap biological cells [32, 33]. The sorting
technique might be applied to selection of cells for regenerative
medicine and diagnostics of disease.
Several kinds of systems were designed for the cell sorting invitro [9, 20, 21]. The micromachining technique has been
applied to cell technology [34].
Fig. 6b: Δy vs. velocity (vx): w = 40 µm: malnourished cell:
regression line (Δy = 2.4 vx + 2.6): correlation coefficient, r2 =0.06.

Several fluid flow systems 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 hole[12] might be suitable for trap than the symmetrical hole.
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 [13].
The depths of the micro patterns were between 2 µm and 10 µm
in the previous studies [19-22]. In the present study, the depthof the grooves is (4.5 μm, which is smaller than the diameter ofthe cells. The deeper hole may have advantage to trap every
cell. At the shallower trap, on the other hand, it is not easy totarp a cell. The trap of the appropriate dimension candistinguish cells. The duration of the trapped time of the cellmight 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.
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
[21]. The angle of 45 degrees between the longitudinal
direction of the groove and the flow direction is effective to

Fig. 7a: Velocity ratio (vy max / vx) vs. D / w: circle, w = 30 µm;
square, w = 40 µm; triangle, w = 50 µm: normal cell.

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Proceedings of The 24th World Multi-Conference on Systemics, Cybernetics and Informatics (WMSCI 2020)

shift 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 thecell types [13]. As the diameter of the cell decreases, thetraveling length along the groove increases. The movement
may be related not only to the diameter but also to
deformability of the cell. Deformability was measured on red
blood cells in previous studies [35].

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The movement of flowing cell at the bottom surface of the flow
channel may 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
was smaller than that of normal cells in the previous study [15].
In the present study, cells are sparsely suspended in the medium
flow to reduce the interaction between cells. Cells can besorted by the velocity change at the micro groove according to
the diameter and the width of the groove [36].

5. CONCLUSION
The velocity change of a flowing C2C12 near the oblique micro
groove on the bottom surface in the micro flow channel has
been measured in vitro. The oblique micro groove of therectangular shape (4.5 μm depth, and 0.2 mm length) has been
fabricated on the polydimethylsiloxane (PDMS) disk by thephotolithography technique. The change of the velocity(perpendicular to the main flow direction) of each flowing
normal cell at the oblique groove is higher at the slower flowing
velocity on the wider groove of 0.05 mm.

ACKNOWLEDGMENT
The authors thank to Mr. Akira Hayasaka for his assistance ofthe experiment.

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