Orientation of Cultured Myotubes in Vortex Flow of Medium
with Swinging Plate
Shigehiro HASHIMOTO, Masahide OKADA, Shuichi MOCHIZUKI,
Toshia FUJISATO, Masahiko YOSHIURA, Kazutomo NISHIMURA
Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, 535-8585, Japanhashimoto@bme.oit.ac.jp http://www.oit.ac.jp/bme/~hashimotoandHajime OTANI
Cardiovascular Center, Kansai Medical University, Moriguchi, Japan

ABSTRACT

2. METHODS

A novel culture methodology with the vortex flow of themedium has been developed to make myotubes array in vitro.
A silicone plate of 3 mm thick was placed in the center ofculture dish of 52 mm internal diameter. The culture dish wasplaced on a tilted plate, which rotates to make a vortex flow
around the silicone plate with the swing motion. Variations
were made on the diameter (20 mm, 30 mm, and 40 mm) of
silicone plate and rotational speed (2.1 rad/sec, 5.2 rad/sec) of
swinging plate, which tilts six degree (0.1 rad). C2C12(Mouse myoblast cell line) cells are cultured in the vortex flowof Dulbecco’s Modified Eagle’s Medium (1.0 × 107 cells permL) for twelve days. The volume of the medium is 2 mL for
40 mm diameter, and 3 mL for 30 mm and 20 mm diameter.
The width of oriented area was measured under observation
with a phase-contrast microscope. The experimental results
show that cells tilt perpendicular to the flow direction in every
condition and that rotational speed affects on the growth oforiented area with muscle cells.

Vortex Flow System
A muscle cell culture system has been designed with vortex
flow to orient myotubes in vitro. A polystyrene culture dishwithout collagen coating (Iwaki, 3010-060, Asahi Glass Co.,
Ltd, Tokyo) was used. A silicone rubber disk of 3 mm thick(K-125, Togawa Rubber Co., Ltd., Osaka) was attached on thebottom at the center of the culture dish of 52 mm internal
diameter to restrict the space for the flow of the medium (Fig. 1).
Variation was made in the disk’s diameter between 20 mm and
40 mm. The silicone rubber disk is stuck on the bottom of thedish with affinity between their surfaces without bond.

Keywords: Biomedical Engineering, Muscle Cells, Cell Culture,
Flow, Adhesion and Orientation

1. INTRODUCTION
Cell culture technique has been progressed and myoblasts havebeen clinically applied to ischaemic cardiomyopathy in the fieldof regenerative medicine.
Acceleration technique fororientation of cells has been studied to make muscle tissue invivo and in vitro [1-4]. Control methodology for orientation ofcells would be applied to regenerative tissue technology.

The culture dish is placed on the plate, which tilted by 0.1 rad tothe horizontal plane (Fig. 2). The plate rotates to generate aswing motion (WAVE-SI, Taitec, Co., Ltd., Koshigaya).
Variatiopn was made in the rotating speed of the plate between
20 and 50 revolutions per minute (rpm). The motion produces
a one-way clockwise vortex flow in the medium around thesilicone rubber disk in the dish.
Fifteen dishes are
simultaneously acceptable to be placed on the plate, which
generates the same vortex flow in the medium in each dish.
The continuously swinging plate was placed in an incubator,
where temperature of 37 degrees Celsius and carbon dioxidepressure of 5 percent are maintained.
Cell Culture
C2C12 (Mouse myoblast cell line originated with cross-striatedSilicone disk
Flow direction
Culture area

Behavior of biological cells depends on various environmental
factors, such as electric, magnetic and mechanical fields.
Erythrocytes, for example, are oriented under blood flow [5].
Several systems for medium circulation have been designed toapply flow stimulation on cell culture [6]. Most of them use
pumps and tubes, which might cause contamination or materials
problem.
In the present study, a novel simple culture methodology withthe vortex flow of the medium has been developed to makeoriented myotubes in vitro.

52 mm dish
3 mm
40 mm

30 mm

Fig. 1: Culture dish with silicone disk. Torus area isdivided into twelve sectors.

Fig. 2: Culture dish on swinging plate in incubator.

Fig. 3: Spindle-shaped C2C12 on the first day of culture.
Bar indicates 0.05 mm.

muscle of C3H mouse) cells were suspended in the Dulbecco’s
Modified Eagle’s Medium (D-MEM) with density of 1.0 × 107
cells per mL. Fetal bovine serum (FBS) was added to themedium with the volume rate in 10 percent of FBS and 90
percent of D-MEM. The suspension was poured into the dishand cultured in an incubator for several days, while the plate
was continuously rotating to make a steady vortex flow aroundthe silicone rubber disk in the medium.
The volume of the suspension is 2 mL for silicone disk of 40
mm diameter, and 3 mL for that of 30 mm diameter and 20 mm
diameter. The volume of the medium was adjusted to coverwhole surface of the bottom of the culture dish around thesilicone disk, and not to flow over the superior surface of thesilicone disk during the swing motion of the plate. C2C12
cells were cultured in the vortex flow of the medium, while theplate was continuously rotating at 37 degrees Celsius in an
incubator. The medium was exchanged every two days.
After four days of cultivation, ten volume percent of FBS in themedium was replaced with seven volume percent of horse
serum to differentiate cells into myotubes.
For comparison, cells were cultured in the dish without asilicone rubber sheet at the counter position on the desk.
Measurement of Orientation
The directions of orientation of myotubes were observed withan inverted phase-contrast microscope.
On the externalsurface of the bottom plate of the culture dish, lines wereattached to mark the radial direction. The culture area of
torus-shape between the outer fringe of the silicone disk and theinner fringe of the dish was evenly divided into 12 sectors tomeasure area of myotubes array (Fig. 1). Each sector has acentral angle of 30 degrees (0.52 rad). The area is counted,
where the longitudinal direction of each myotube is within 15
degrees (0.26 rad) declination compared to the radial direction.
The area of myotubes array was calculated in each sector along
the radial direction of the dish. The distance between the topof myotube array, which is oriented to radial direction, and thefringe of silicone disk was measured in every radial direction.

3. RESULTS
The medium flow around the silicone disk synchronously withthe movement of the swinging plate was observed with the
movement of a tracer particle.

Fig. 4: Myotubes on the seventh day of culture contracted
by electric stimulation. Bar indicates 0.05 mm.

Muscle cells were not oriented with a vortex flow, after
cultivation for three days without a vortex flow. The adheredcells did not move and fuse to extend array. The experimental
results show that cells array is extended to the area, where cells
have not adhered yet.
When the diameter of the silicone disk was changed to 10 mmin the culture dish of 52 mm internal diameter, myotubes were
not arrayed in the vortex flow of medium in a week ofcultivation. When the internal diameter of the culture dish waschanged to 85 mm with the silicone disk of 40 mm, myotubes
were not arrayed in the vortex flow of medium in a week ofcultivation, either. The cells were arrayed on the swinging
plate, on the other hand, even when the tilting angle varies from
0.1 rad to 0.07 rad.
The muscle cells adhered to the bottom of the culture dish in
two days (Fig. 3) and were arranged along the circumferential
streamline around the silicone rubber disk, while the cells
proliferated and fused to become myotubes in the vortex flow of
the medium (Fig. 4). The experimental results show that

SiliconeFig. 5: C2C12 on the first day of culture in vortex flow
without silicone disk. Bar indicates 0.05 mm.

Fig. 8: C2C12 on the fifth day of culture in vortex flow
with silicone disk of 30 mm. Bar indicates 0.05 mm.

SiliconeFig. 6: C2C12 on the fifth day of culture in vortex flow
without silicone disk. Bar indicates 0.05 mm.

SiliconeFig. 7: C2C12 on the first day of culture in vortex flow
with silicone disk of 30 mm. Bar indicates 0.05 mm.
regular arrays of myotubes are grown up in the vortex flow
around the silicone disk, while the directions of myotubes are
random without the silicone disk (Figs. 5&6). The array of
myotubes grew around the silicone disk day by day, and thealignment curved to the radial direction near the disk (Figs.
7-10).
Figs. 11 and 12 show the orientation of mytubes compared to
the radial direction around the silicone disk, where the radial
direction is indicated with a straight line. The myotube arraygrows near the silicone disk (Fig. 11), while the orientation
decreases in the area away form the silicone disk (Fig. 12).
Fig. 13 exemplifies the array area ratio in each sector on theseventh day of culture in the vortex flow around the silicone

Fig. 9: C2C12 on the first day of culture in vortex flow
with silicone disk of 40 mm. Bar indicates 0.05 mm.

SiliconeFig. 10: C2C12 on the fifth day of culture in vortex flow
with silicone disk of 40 mm. Bar indicates 0.05 mm.
disk. The experimental results show that myotube arrays growto occupy one percent of culture area in every sector in a weekof cultivation (Fig. 13). Figs. 14-19 show the myotube arraydistance from the fringe of the silicone disk at each sector. The
results on two or three dishes are simultaneously displayed withdifferentiated marks in Figs. 14-19. The same legend symbols
of data indicate data in the same dish in Figs. 14-21. The
myotube arrays grow up to 1 mm distance from the fringe of thesilicone disk at every sector at 20 rpm (Figs. 14-16), while thegrowth decreases around the disk of 20 mm diameter at 50 rpm(Fig. 17). Around the silicone disk of 40 mm diameter,
myotubes array is maximized in the vortex flow at the speed of
50 rpm (Fig. 19).

1.6
1.4
Distance [mm]

1.2
1
0.8
0.6
0.4
0.2
0
1

2

3

4

5

Radial Line

6

7

8

9 10 11 12

SectorFig. 14: Myotube array length in each sector aroundsilicone disk of 20 mm at 20 rpm.

Fig. 11: C2C12 near silicone disk of 30 mm on the 12th
day of culture in vortex flow. Line shows radial
direction. Bar indicates 0.05 mm.

1.6

Distance [mm]

1.4
1.2
1
0.8
0.6
0.4
0.2
0

Radial Line

1

2

3

4

5

6

7

8

9

10 11 12

SectorFig. 15: Myotube array length in each sector aroundsilicone disk of 30 mm at 20 rpm.

Fig. 12: C2C12 away from silicone disk of 30 mm on the
12th day of culture in vortex flow. Line shows radial
direction. Bar indicates 0.05 mm.

1.8
1.6

0.14

1.4
Distance [mm]

0.12

Ratio

0.1
0.08
0.06

1.2
1
0.8
0.6
0.4

0.04

0.2
0

0.02
0

1

1 2

3 4 5

6 7

8 9 10 11 12

2

3

4

5

6

7

8

9

10 11 12

Sector

SectorFig. 13: Array area ratio in each sector on the seventh dayof culture.

Fig. 16: Myotube array length in each sector aroundsilicone disk of 40 mm at 20 rpm.
circumferential direction adjacent to the disk (Figs 10 & 11).

Figs. 20 and 21 exemplify the array area ratio along the radial
direction between the outer fringe of the silicone disk and theinner fringe of the dish. The ratio of unity indicates that every
myotube is arrayed to radial direction in Figs. 20 and 21. The
results show that myotubes array to the radial direction grows in
the inner side of the torus-shape flow path between the disk andthe fringe of the dish. The myotubes tend to array to

The myotubes (Fig. 4) contracted synchronously with electricpulses, which was applied to the surrounding medium at aperiod of 1 second through an electrode of a platinum wire.
After repetitive contraction for thirty minutes with electricpulses (pulse width of 1 ms, period of 1 s, amplitude of 70 V),
the myotube peeled off from the bottom of the dish and shrank.

1

0.7
0.6

0.8

Distance [mm]0.5
Ratio of area

0.4
0.3
0.2

0.6
0.4

0.1
0.2

0
1

2

3

4

5

6

7

8

9 10 11 120

Sector15

20

25

Radius [mm]Fig. 17: Myotube array length in each sector aroundsilicone disk of 20 mm at 50 rpm.

Fig. 20: Relation between array area ratio and radial
position on the twelfth day of culture (30 mm disk).

1
2.5

0.8
Ratio of area

Distance [mm]

2
1.5
1

0.6
0.4
0.2

Silicone

0.5

0
15

0
1

2

3

4

5

6

7

8

9 10 11 12

20

25

Radius [mm]SectorDistance [mm]Fig. 18: Myotube array length in each sector aroundsilicone disk of 30 mm at 50 rpm.

Fig. 21: Relation between array area ratio and radial
position on the twelfth day of culture (40 mm disk).

3.5

4. DISCUSSION

3

Muscle cells were not oriented with a vortex flow, when thecells adhere to the bottom of the dish after normal cultivationfor 3 days. Following the experimental results, cells were
exposed to the vortex flow before adherence to the bottom of
the dish.

2.5
2
1.5

The previous study shows that electric stimulation enhances
differentiation of muscle cells [7]. Another study shows
mechanical stimulation improves tissue-engineered humanskeletal muscle [3].

1
0.5
0
1

2

3

4

5

6

7

8

9 10 11 12

SectorFig. 19: Myotube array length in each sector aroundsilicone disk of 40 mm at 50 rpm.

A polystyrene culture dish without collagen coating was used in
the present study, to decelerate adhesion of cells to the bottom
the dish.
The experimental results show that cells tilt perpendicular to theflow direction in every condition and that rotational speed
affects on the growth of oriented area with muscle cells. The
cells might be stabilized in the direction, where the stress oncells is minimized [8]. The cells, on the other hand, might
accept stress being stretched to the flow direction. The slope,which is generated by the swing motion of the tilted plate, also
affects to the radial direction of the myotube array.
The silicone disk placed in the center of the dish might fix thecircumferential steady flow, which enhances orientation of
myotubes. The vortex flow in the dish without the center disk
is unsteady and turbulent. Turbulent flow might disturb toorient myotubes in a flow of higher velocity at higher rotational
speed, and in a wider space between the silicone disk and thefringe of the dish.
Orientation of myotubes might affect the effect of contraction ofeach myotube.

5. CONCLUSION

A novel culture methodology with the vortex flow of themedium has been developed to make myotubes array in vitro.
The experimental results show that cells tilt perpendicular to theflow direction in every condition and that rotational speed
affects on the growth of oriented area with muscle cells.

6. ACKNOWLEDGMENT

This work was supported by a Grant-in-Aid for Academic
Frontier from the Japanese Ministry of Education, Culture,
Sports and Technology.

REFERENCES[1] J. Stern-Straeter, A.D. Bach, L. Stangenberg, V.T. Foerster,R.E. Horch, et al., “Impact of Electrical Stimulation on
Three-dimensional Myoblast Cultures- A Real-time RT-PCR
Study”, Journal of Cellular and Molecular Medicine, Vol.
9, No. 4, 2005, pp. 883-892.
[2] F. H. Andrade, M. B. Reid, D. G. Allen and H. Westerblad,“Effect of Hydrogen Peroxide and Dithiothreitol onContractile Function of Single Skeletal Muscle Fibers from
the Mouse”, Journal of Physiology, Vol. 509.2, 1998, pp.
565-575.
[3] C. A. Powell, B. L. Smiley, J. Mills and H. H. Vandenburgh,“Mechanical Stimulation Improves Tissue-Engineered
Human Skeletal Muscle”, American Journal of
Physiology: Cell Physiology, Vol. 283, 2001, pp.
C1557-C1565.
[4] K. Yamasaki, S. Hashimoto, M. Okada, K. Ono, T. Fujisato,
S. Mochizuki, M. Yoshiura, H. Tsutsui, K. Akazawa, “Designof Environment for Arrangement of Cultured Muscle Cells”,
Proc. 12th World Multi-Conference on Systemics
Cybernetics and Informatics, Vol. 2, 2008, pp. 130-134.
[5] K. Yamamoto, T. Takahashi, T. Asahara, N. Ohura, T. Sokabe,
A. Kamiya and J. Ando, “Proliferation, Differentiation, andTube Formation by Endothelial Progenitor Cells in Response
to Shear Stress”, Journal of Applied Physiology, Vol. 95,
2003, pp. 2081-2088.
[6] S. Hashimoto, H. Oku, N. Komoto, Y. Murashige, S. Manabe,
K. Ikegami, C. Miyamoto, “Effect of Pulsatile Shear Flow on
Migration of Endothelial Cells Cultured on Tube”, Proc. 6th
World Multiconference on Systemics Cybernetics andInformatics, Vol. 2, 2002, pp. 296-300.
[7] Y. Kawahara, K. Yamaoka, M. Iwata, M. Fujimura, T.
Kajiume, T. Magaki, M. Takeda, T. Ide, K. Kataoka, M.
Asashima and L. Yuge, “Novel Electrical Stimulation Sets
the Cultured Myoblast Contractile Function to ‘on’”,Pathobiology, Vol.73, 2006, pp. 288-294.
[8] K. Nagayama, T. Matsumoto, “Mechanical Anisotropy of Rat
Aortic Smooth Muscle Cells Decreases with Their
Contraction (Possible Effect of Actin Filament Orientation)”,
JSME International Journal, Series C, Vol. 47, No. 4, 2004,
pp. 985-991.
[9] W. Gu, X. Zhu, N. Futai, B. S. Cho, S.Takayama,“Computerized Microfluidic Cell Culture Using Elastomeric
Channels and Braille Displays”, PNAS, 2004, Vol. 101, No.
45, pp. 15861-15866.
[10] A.Takamura, S. Hayasi, K. Nagayama T. Matumoto,“Mechanical Characterization of Brain Tissue in High-Rate
Extention”, J Biomech Sci Eng, Vol. 3, 2008, pp. 263-274.