Effect of Culture Medium Flow on Orientation of Muscle Cells
Saori OKUDA, Shigehiro HASHIMOTO, Kohei ONO, Masahide OKADA,Shuichi MOCHIZUKI, Toshia FUJISATO, Hiroshi NAKAOKA, Masahiko YOSHIURA
Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, 535-8585, Japanhashimoto@bme.oit.ac.jp http://www.oit.ac.jp/bme/~hashimoto

ABSTRACTThe effect of slope and flow on orientation of cultured muscle
cells has been studied in vitro. A flow chamber was designedto observe behavior of adhered cells on a tilted plane in a culture
medium flow under a microscope. A thin sheet of silicone
rubber of 0.1 mm thick, which has a rectangular open space,
was sandwiched by two plates of transparent glass to form arectangular channel of 2 mm width × 52 mm length × 0.1 mm
depth. The chamber was tilted to make a slope of 0.17 radperpendicular to longitudinal flow direction, and placed on an
inverted microscope. C2C12 (Mouse myoblast cell line) cells
are suspended in the Dulbecco’s Modified Eagle’s Medium, andintroduced to the chamber via a tube of 2 mm internal diameterwith a syringe pump. After several cells adhered to the glassplate, the medium steady flow between 3 and 10 mL/hour wasapplied on the cells with the syringe pump, and behavior ofadhered cells in the flow is observed. The flow generates wall
shear stress between 0.5 and 1.7 Pa. The experimental results
show that cells tend to tilt to the direction of downstream anddownward.

Fig. 1: Experimental system.

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

1. INTRODUCTION
Behavior of biological cells depends on various environmental
factors, such as electric, magnetic and mechanical fields [1, 2].
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 [3-5]. Control methodology
for adhesion and proliferation of cells would be applied toregenerative tissue technology.
In the present study, the effect of slope and flow on orientationof cultured muscle cells has been studied in vitro.

2. METHODS
Culture Medium Flow System
A one-way flow system was designed to observe an effect ofslope and flow on cells adhered to a plate. The system consistsof a flow chamber, a syringe pump, tubes and a microscope (Fig.
1). TE-331S (Terumo Co., Ltd. Tokyo) was used for thesyringe pump. The plastic tube of 2 mm internal diameter andof 3 mm external diameter was used for the flow channel.

Fig. 2: Silicone sheet.

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 supporting plastic plates (Fig. 3).
After being
exfoliated from the supporting plastic plates, 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 without bond because of their surface
affinity. At the upper glass plate, two holes of 2.5 mm
diameter is machined by a grinder, and adhered to the plastic
tube by a bond of polyurethane resin (Fig. 4).
The tube is connected to the plastic syringe pump. Thechamber is placed on the tilted plate to make slope of 0.17 radperpendicular to longitudinal flow direction (Fig. 5). The tilted

Upper Plate

Silicone Sheet
Fig. 5: Chamber placed on the tilted heating plate.

Lower Plate

Fig. 3: Flow chamber of three plates; upper plates of glass,silicone sheet, lower plates of glass.

Fig. 6: Flow test with syringe pump and with microscope.
with the syringe pump (Fig. 6). The behavior of cells on theplate of the chamber was observed with the microscope. The
photo of cells was taken every 15 minutes during the flow testfor 12 hours. Variation was made in flow rate between 3 and10 mL/hour.
Shear Rate at Wall
The shear rate (G) at the glass plate is calculated by Eq. 1,
assuming a parabolic velocity profile between parallel plates.
Fig. 4: Flow chamber.
plate is heated at 37 degrees Celsius to maintain temperature ofthe medium. The chamber is placed on the inverted
phase-contrast microscope (IX71, Olympus Co., Ltd., Tokyo).
Cell Culture
C2C12 (Mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) were cultured on a dish with theDulbecco’s Modified Eagle’s Medium (D-MEM) in an
incubator for one week. Then, Cells were exfoliated from theplate of the culture dish with trypsin, and suspended in theD-MEM. The suspension was introduced to the chamber andcultured for 18 hours to make cells adhere to the glass plate ofthe chamber.
Flow Test
The constant flow of the medium was applied to adhered cells

G = 6 q / b D2

(1)

In Eq. 1, q is flow rate, b is width of the cannel (2 mm) and D isdistance (0.1 mm) between two parallel walls.
The shear stress is product of viscosity (N) of the fluid and theshear rate (G) of flow by Eq. 2.
T=NG

(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 shows that the viscosity of themedium is 0.002 Pa s at 37 degrees Celsius. The calculated

Fig. 8 exemplifies cells under flow of 4 mL/hour. The marked
cell in Fig. 8 elongates from 0.22 mm to 0.25 mm in thirty
minutes under flow of 4 mL/hour, which generate a wall shear
stress of 0.7 Pa estimated by Eqs. 1 & 2. Several movements
occur on adhered cells in the flow: deformation, tilting todownstream, tilting to downward, elongation along thestreamline, deformation to be rounded, exfoliation, rolling todownstream.

4. DISCUSSION
Acceleration of proliferation and orientation of cells areimportant tasks in the research field of regenerative medicine toculture biological tissue. The previous study shows thatelectric stimulation enhances differentiation of muscle cells [3].
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 [7].

Fig. 7: Adhered cells after 18 hours.

Bioreactors have been developed to control the environment
around the cultured cell [8-10].
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 [11]. The shear
flow also affects on the orientation of endothelial cells [12].
The shear stress affects on the orientation of smooth muscle
cells in the biological tissue [13]. The direction of mechanicalfield affects on fibroblast [14].

0.22 mm

0.1 mmFig. 8(a): Cells under flow of 4 mL/hour.

The previous study shows that the micro-grooves affects on theorientation of cell [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 [1]. The present study shows that theshear flow and slope affects on behavior of cells adhered to theplate.

5. CONCLUSION

0.25 mm

The effect of slope and flow on orientation of cultured muscle
cells has been studied in vitro. The experimental results showthat cells tend to tilt to the direction of downstream anddownward.

6. ACKNOWLEDGMENT

Fig. 8(b): Cells under flow of 4 mL/hour at 30 minutes
after Fig. 8(a).
shear rate varies between 250 and 830 sec-1, when the flow ratevaries between 3 and 10 mL/hour. The calculated shear stress,
thus, varies between 0.5 and 1.7 Pa for viscosity of 0.002 Pa s,
when the shear rate varies between 250 and 830 sec-1.
Cells adhered to the plate of the chamber in 18 hours without
medium flow (Fig. 7). Under steady flow of 10 mL/hour, cells
were entirely exfoliated in five minutes. The calculated wall
shear rate and wall shear stress are 830 sec-1 and 1.7 Pa,
respectively.

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

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