Effect of Medium Flow on Cultured Cells
Haruka IWATA, Shigehiro HASHIMOTO, Saori OKUDA and Hiroshi NAKAOKA
Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, 535-8585, Japanhashimoto@bme.oit.ac.jp http://www.oit.ac.jp/bme/~hashimoto

2. METHODSABSTRACTAn effect of the flow on migration and on deformation ofcultured cells has been studied in vitro. A flow chamber was
designed to observe behavior of adhered cells on a plane in a
culture medium flow under a microscope. A thin sheet ofsilicone rubber was sandwiched by two plates of transparent
glass to form a rectangular flow channel of 2 mm width × 52
mm length × 0.1 mm depth. The chamber was tilted to make aslope of 0.17 rad perpendicular to the longitudinal flow
direction. Five kinds of cells were used in the experiment:
C2C12 (mouse myoblast), L6 (rat skeletal muscle cell), A7r5
(rat aortic smooth muscle cell), CS-2P2-C75 (porcine aortic
endothelial cell), and L929 (mouse fibroblast). After several
cells adhered to the glass plate, the medium steady flow
between 4 and 60 mL/hour was applied on the cells with thesyringe pump, and behavior of adhered cells in the flow wasobserved. The flow generates wall shear stress between 0.3
and 5 Pa. The experimental results show that cells tend to tilt,
deform and migrate to the direction of downstream anddownward before exfoliation. CS-2P2-C75 and L929 did notadhere to the wall of the glass chamber.
Keywords: Biomedical Engineering, Muscle Cells, Cell
Culture, Flow, Migration and Orientation

1. INTRODUCTION
Behavior of biological cells depends on various environmental
factors, such as electric [1-3], magnetic [4, 5] and mechanical[6-8] fields.

Culture Medium Flow System
A one-way flow system was designed to observe an effect offlow and slope on cells adhered to a plate. The system
consists of a flow chamber, a syringe pump, tubes and a
microscope (Fig. 1). TE-331S (Terumo Co., Ltd. Tokyo) wasused for the syringe pump. A plastic tube of 2 mm internal
diameter and of 3 mm external diameter was used for the flow
channel.
The flow chamber consists of two transparent glass plates and athin silicone rubber sheet (Fig. 2). The dimension of two glass
plates is 76 mm length, 26 mm width and 1.5 mm thick, each.
A rectangular open space of 2 mm × 52 mm is cut off in a thinsheet of silicone rubber of 0.1 mm thick, and sandwiched
between the supporting plastic plates (Fig. 3). The sheet issandwiched between two plates of glass to form a rectangularchannel of 2 mm width × 52 mm length × 0.1 mm depth. The
three plates stick together with their surface affinity withoutbond. At the upper glass plate, two holes of 2.5 mm diameteris machined by a grinder, and adhered to the plastic tube by abond of polyurethane resin (Fig. 3). On the outer surface ofthe lower glass plate, grooves of dotted line were machined tomake the marks of position in the chamber.
The tube is connected to the plastic syringe pump. The
chamber is placed on the tilted plate to make a slope of 0.17 radperpendicular to the longitudinal flow direction (Fig. 3). The
tilted plate is heated at 37 degrees Celsius to maintaintemperature of the medium. The chamber is placed on the
inverted phase-contrast microscope (IX71, Olympus Co., Ltd.,Tokyo).

Cell culture technique has been progressed and myoblasts havebeen clinically applied to ischaemic cardiomyopathy in the fieldof regenerative medicine.
Acceleration technique fororientation and proliferation of cells has been studied to makemuscle tissue in vivo and in vitro [2, 6]. Control methodology
for orientation and proliferation of cells would be applied toregenerative tissue technology.
Several methods have been designed to apply mechanical stressto cells [6-8]. The transmission point of stress to a specimen isimportant. When fixation between the cell and the scaffold is
not enough, stress is not transmitted to the cell. A flow can beused, on the other hand, to apply stress to a specimen [9-13].
The specimen receives shear stress in the shear flow.
In the present study, the effect of flow and slope on orientationof cultured muscle cells has been studied in vitro.
Fig. 1: Experimental system.

Upper Plate

Silicone Sheet

Flow Test
After the chamber was set on the microscope out of the
incubator, the constant flow of the medium was applied toadhered cells with the syringe pump (Fig. 1). The flow path
was carefully examined to avoid inclusion of air bubbles, whichmight stir the medium in the flow chamber and induce
exfoliation of cells. The behavior of cells on the plate of thechamber was observed with the microscope. The photo ofcells was taken during the flow test for one hour. Variation
was made in flow rate between 4 and 60 mL/hour. The flowrate started with 4 mL/hour and was escalated to 60 mL/hour: 4,
5, 8, 20, 30, 60 mL/hour.
Shear Rate on Wall
The shear rate (G, s-1) on the wall of the glass plate is calculated
by Eq. 1, which is assuming a parabolic velocity profile
between parallel plates.
G = 6 q / (b D2)

Mark

(1)

In Eq. 1, q is flow rate (m3 s-1), b is width of the canal (m) and
D is distance (m) between two parallel walls. In the present
study, b is 2 mm, and D is 0.1 mm.
Lower Plate
The shear stress (T, Pa) is product of viscosity (N, Pa s) of thefluid and the shear rate (G, s-1) of flow (Eq. 2).
T=NG

Fig. 2: Flow chamber of three plates; upper plates of glass,silicone sheet, lower plates of glass with mark.

(2)

The viscosity of the medium was measured with the cone andplate type of viscometer (TVE-22L, Toki-Sangyo Co., Ltd.
Tokyo).

3. RESULTS
The result of measurement with the viscometer shows that theviscosity of the medium is 0.0010 Pa s at 37 degrees Celsius at
the shear rate of 600 s-1. The calculated shear rate on the glass
wall of the flow chamber by Eq. 1 varies between 330 and 5000
s-1, when the flow rate varies between 4 and 60 mL/hour. The
calculated shear stress, thus, varies between 0.3 and 5 Pa forviscosity of 0.001 Pa s, when the shear rate varies between 330
and 5000 s-1.

Fig. 3: Chamber placed on the tilted heating plate.

Cell Culture
Five kinds of cells were used in the experiment: C2C12 (mouse
myoblast), L6 (rat skeletal muscle cell), A7r5 (rat aortic smoothmuscle cell), CS-2P2-C75 (primary normal porcine aortic
endothelial cell), and L929 (mouse fibroblast). The cells ofeach kind were cultured on a dish with the Dulbecco’s Modified
Eagle’s Medium (D-MEM) in an incubator for one week.
Then, Cells were exfoliated from the plate of the culture dishwith trypsin, and suspended in the D-MEM. The suspension
was introduced to the chamber and cultured in the incubator for
24 hours to make cells adhere to the glass plate of the chamber
before the flow test.

Fig. 4: C2C12 on the glass wall of the chamber before flow
test. The bar shows 0.1 mm.

AC
0.108 mm0.116 mm
Flow

B

Flow

D

0.115 mm

0.118 mmFlow

Fig. 4 exemplifies C2C12 adhered to the plate of the chamber in
24 hours without medium flow.
The slope goes down from upper to lower in Figs. 5-10. The
medium flows right to left in Figs. 5-10. C2C12 elongates tothe downstream along the streamline of the flow (Fig. 5). The
C2C12 marked with the line in Fig. 5 elongates from 0.108 mm
to 0.121 mm in thirty minutes under flow of 4 mL/hour, which
generate a wall shear stress of 0.3 Pa estimated by Eqs. 1 & 2.
Another C2C12 marked with the circle in Fig. 5 rolls downalong the slope. The cell marked with the circle in Fig. 6 tilts
for 30 minutes. Under steady flow of 30 mL/hour, most of
C2C12 cells rolled downstream. Fig. 7 shows cells before andafter flow stimulation for three minutes. Almost all of C2C12
cells are exfoliated in three minutes under steady flow of 60
mL/hour. The calculated wall shear rate and wall shear stressare 5000 s-1 and 5 Pa, respectively (Fig. 7).
Fig. 8 shows L6 migration for eleven minutes. L6 marked
with the circle in Fig. 8 migrates downstream under steady flowof 4 mL/hour. In Fig. 9, L6 marked with the circle elongatesdownstream in 20 min under steady flow of 5 mL/hour.
Fig. 10 shows A7r5 under steady flow of 4 mL/hour. The
most of cells are exfoliated in fifteen minutes. The rest ofcells elongates downstream in sixty minutes. CS-2P2-C75(Fig. 11) and L929 (Fig.12) does not adhere to the wall of glassin the chamber.

Flow

E

0.121 mmFlow

Fig. 5: C2C12 under steady flow (arrow: from right to left)of 4 mL/hour. Slope goes down from upper to lower.
Cells were observed every 15 minutes: A, B, C, D, E.

4. DISCUSSION
Several movements occur on adhered cells in the flow:
deformation, tilting to downstream, tilting to downward (Fig. 13(c)), elongation along the streamline (b), deformation to berounded (d), exfoliation (e), rolling to downstream (Fig. 13).
The deformation and migration of cells in the flow depend on
the adhering point of the cell to the wall. The free part of the

Flow
Flow

Flow
Flow

Fig. 6: C2C12 movement in 30 min (from above tobottom) under steady flow (from right to left) of 4
mL/hour. The bar shows 0.1 mm.
Fig. 8: L6 migrates downstream in 11 min under steadyflow (from right to left) of 4 mL/hour. The bar shows 0.1
mm.

Flow

Acceleration of proliferation and orientation of cells areimportant targets in the research field of regenerative medicine
on cultured biological tissue. The previous study shows thatelectric stimulation enhances differentiation of muscle cells [1].
Another study shows mechanical stimulation improves
tissue-engineered human skeletal muscle [6].
Anotherprevious study shows that muscle cells can adhere andproliferate under electric stimulation with periodical pulses, andthat adhesion of muscle cells can be controlled with theamplitude of pulse [3].
Bioreactors have been developed to control the environment
around the cultured cell [14].

FlowFig. 7: C2C12 exfoliated in 3 min under steady flow (fromright to left) of 60 mL/hour. The bar shows 0.1 mm.
cell might rotate around the adhering point. Collagen coating
might be necessary for the flow test of CS-2P2-C75 and L929.

The previous studies show that mechanical field, on the other
hand, affects on cells’ behavior. Erythrocytes are very flexible,
and are rolled and deformed in the shear flow [9]. The shear
flow also affects on the orientation of endothelial cells [10].
The shear stress affects on the orientation of smooth muscle
cells in the biological tissue [7]. The direction of mechanicalfield affects on fibroblast [8]. The previous study shows thatthe micro-grooves affects on the orientation of cells [15].
The effect of slope has been examined in the present experiment,
because a tilted rotating disk was used for another study of cell
culture environment [16]. To evaluate the effect of slope
independently of the effect of flow, the direction of slope has
been set perpendicularly to that of flow. The present study
shows that the shear flow and slope affects on behavior of cells
adhered to the plate. The combined effect of flow and slope

Flow
Flow

Flow

Flow

Fig. 9: L6 elongates downstream in 20 min under steadyflow (from right to left) of 5 mL/hour. The bar shows 0.1
mm.
on each cell might govern the orientation of cells in the vortex
flow on the tilted rotating disk.

5. CONCLUSION
The effect of flow on cultured muscle cells has been studied invitro. The experimental results show that cells tend toelongate along the streamline and tilt to the direction ofdownstream and downward.

Flow

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] 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.
[2] 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.

Fig. 10: A7r5 cells are exfoliated and elongateddownstream under steady flow (from left to right) of 4
mL/hour. Middle, 15 min; Bottom, 60 min. The barshows 0.1 mm.
9, No. 4, 2005, pp. 883-892.
[3] E. Yamada, S. Hashimoto, K. Tachibana, M. Okada, K.
Yamasaki, H. Kondo, K. Imoto, S. Mochizuki, T. Fujisato, M.
Ohsuga and H. Otani, “Effect of Electric Stimulation on
Adhesion and Proliferation of Cultured Muscle Cells”, Proc.
12th World Multi-Conference on Systemics Cybernetics
and Informatics, Vol. 2, 2008, pp. 124-129.

Tilt
Elongated
Flow

Down

Adhered cellFig. 11: CS-2P2-C75 in culture dish.
mm.

The bar shows 0.1

Adhered point

Adhered point

RoundedExfoliatedFig. 13: Cells deformation and migration under flow.

Fig. 12: L929 in culture dish.

The bar shows 0.1 mm.

[4] K. Yamasaki, S. Hashimoto, M. Okada, K. Ono, T. Fujisato,
S. Mochizuki, M. Yoshiura, H. Tsutsui, K. Akazawa,“Design of Environment for Arrangement of Cultured
Muscle Cells”, Proc. 12th World Multiconference on
Systemics Cybernetics and Informatics, Vol. 2, 2008,
pp.130-134.
[5] S. Hashimoto, S. Mochizuki, Y. Morita, H. Tsutsui, M.
Yoshiura, K. Akazawa, M. Ohsuga, S. Uto, H. Otani and T.
Fujisato, “Environmental Design for Muscle Cell Culture
with Magnetic Field”, Proc. 2007 Inaugural IEEE
International Conference on Digital Ecosystems andTechnologies (IEEE-DEST 2007), 2007, pp. 468-472.
[6] 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.
[7] 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.
[8] J.H.-C. Wang, G. Yang, Z. Li and W. Shen, ”Fibroblast
Responses to Cyclic Mechanical Stretching depend on Cell
Orientation to the Stretching Direction”, Journal of
Biomechanics, Vol. 37, 2004, pp. 573-576.

[9] S. Hashimoto, H. Oku, N. Komoto, Y. Murashige, S. Manabe,
K. Ikegami, C. Miyamoto, “Effect of Pulsatile Shear Flowon Migration of Endothelial Cells Cultured on Tube”, Proc.
6th World Multiconference on Systemics Cybernetics
and Informatics, Vol. 2, 2002, pp. 296-300.
[10] M. Toda, K. Yamamoto, N. Shimizu, S. Obi, S. Kumagaya,
T. Igarashi, A. Kamiya and J. Ando, “Differential Gene
Responses in Endothelial Cells Exposed to a Combination of
Shear Stress and Cyclic Stretch”, Journal of Biotechnology,
Vol. 133, No. 2, 2008, pp. 239-244.
[11] A. M. Malek and S. Izumo, “Mechanism of Endothelial
Cell Shape Change and Cytoskeletal Remodeling in
Response to Fluid Shear Stress”, Journal of Cell Science,Vol. 109, 1996, pp. 713-726.
[12] Y. Sugaya, N. Sakamoto, T. Ohashi and M. Sato,“Elongation and Random Orientation of Bovine Endothelial
Cells in Response to Hydrostatic Pressure: Comparison with
Response to Shear Stress”, JSME International Journal,
Series C, Vol. 46, No. 4, 2003, pp. 1248-1255.
[13] P. Uttayarat, M. Chen, M. Li, F. D. Allen, R. J. Composto
and P. I. Lelkes, “Microtopography and Flow Modulate the
Direction of Endothelial Cell Migration”, Am. J. Physiol.
Heart Circ. Physiol., Vol. 294, 2008, pp. H1027-H1035.
[14] E. Cimetta, M. Flaibani, M. Mella, E. Serena, L. Boldrin, P.
De Coppi and N. Elvassore, “Enhancement of Viability ofMuscle Precursor Cells on 3D Scaffold in a Perfusion
Bioreactor”, The International Journal of Artificial
Organs, Vol. 30(5), 2007, pp. 415-428.
[15] E.T. den Braber, J.E. de Ruijter, H.T.J. Smits, L.A. Ginsel,
A.F. von Recum and J.A. Jansen, “Quantitative Analysis of
Cell Proliferation and Orientation on Substrata with Uniform
Parallel Surface Micro-grooves”, Biomaterials, Vol. 17 No.
11, 1996, pp. 1093-1099.
[16] S. Hashimoto, M. Okada, S. Mochizuki, T. Fujisato, M.
Yoshiura and K. Nishimura, “Orientation of Cultured
Myotubes in Vortex Flow of Medium with Swinging Plate”,
Proc. 13th World Multi-Conference on Systemics
Cybernetics and Informatics, Vol. 2, 2009, pp. 196-201.