Effect of Wall Shear Stress in Flow on Myoblast
Haruka HINO, Masashi OCHIAI, Shigehiro HASHIMOTO, Kazutaka KIMURA, Yusuke TAKAHASHI
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
shashimoto@cc.kogakuin.ac.jp http://www.mech.kogakuin.ac.jp/labs/bio/andToshitaka YASUDA
Bio-systems Engineering, Department of Electronic Engineering,Tokyo National College of Technology, Tokyo, Japan
ABSTRACTAn effect of wall shear stress on adhered myoblast has beenstudied in vitro. A flow channel was designed to observe theresponse of cells to the fluid shear stress under a microscope.
A Polydimethylsiloxane disk with a concaved part is placed on
the inner bottom of a culture dish to form a parallelepiped flow
channel of 25 mm length × 5 mm width × 0.1 mm depth. The
flow channel is placed in a chamber, where temperature andCO2 content are kept 310 K and 5 percent, respectively. The
chamber is placed on the stage of an inverted phase contrast
microscope to observe the behavior of cells adhered on the disk
under a flow. The shear stress on the wall is calculated with anestimated parabolic velocity profile between the parallel disks.
After several cells adhered to the disk, the shear stress wasapplied on the cells in the medium flow with a syringe pump for
1 hour. The variation of the flow rate produces the wall shear
stress between 0.1 Pa and 3 Pa. C2C12 (mouse myoblast cellline) was exposed to the wall shear stress with the methodology.
The experimental results show that the myoblast deforms mostfrequently at the wall shear stress of 1 Pa.
Keywords: Biomedical Engineering, Cell Culture, Myoblast,
Shear stress, Migration and Flow.

1. INTRODUCTION

proliferation of cells would be applied to the regenerative tissue
technology.
A flow can be used to apply a stress field to a cell [6-15]. The
cell directly receives the shear stress in the shear flow. The
high shear flow might deform cell, peel cells off the scaffold,
and inhibit proliferation. The mild shear flow, on the other
hand, might accelerate the active deformation, migration, andproliferation.
In the present study, the effect of wall shear stress on adhered
myoblast has been studied in vitro.

2. METHODS
Flow Channel
A flow channel was designed to observe the response of cells to
the fluid shear stress under a microscope. The flow channel
consists of a polystyrene dish of 35 mm diameter, a disk ofpolydimethylsiloxane (PDMS), three glass capillaries, and three
polyvinylchloride tubes.
For PDMS disk, a die of carbon steel with a central rectangularconvex part was machined by a flat-surface grinding machine(Fig. 1). The dimension of the convex part is as follows: 25
mm length, 5 mm width, 0.1 mm height. The dimension of theconvex part was measured with a laser microscope (Fig. 2).

Cells are exposed to mechanical stimulation in vivo. The shear
stress is one of the mechanical stimulations. The endothelial
cells are exposed to the shear stress in the blood flow at the wallof the blood vessels. The other cells are also exposed to theshear stress among the deformation of the tissue.
Cells adhere, migrate, deform, proliferate, and differentiate.
The mechanical stress might affect the behavior of cells.
Cell culture technique has been developed and several
methodologies have been clinically applied to regenerative
medicine. The acceleration technique for orientation andproliferation of cells has been studied to make tissue in vivo orin vitro [1-5]. Control methodology for orientation and

Fig. 1: Dimension (mm) of die with a central rectangularconvex.

After the die was enclosed with a peripheral wall of polyimide,
PDMS (Dow Corning Corporation, MI, USA) was poured withthe curing agent on the die. PDMS was baked at 333 K for
one hour in an oven. The PDMS disk was cut at diameter of
30 mm with the concaved center part. Three holes were
punched at the ends of the concaved part of the PDMS disk.
Two of them are for the inlet, and the other is for the outlet.

The shear rate on the wall is calculated with an estimatedparabolic velocity profile between the parallel walls [8]. The
shear stress is the product of the viscosity of the medium andthe wall shear rate. The shear rate (G, [s-1]) on the wall of theplate is calculated by Eq. 1, in which a parabolic velocity
profile between parallel plates is hypothesized.
G = 6 q / (b D2)

The PDMS disk, which has a concaved part at the center, wasplaced on the inner bottom of a culture dish to form aparallelepiped flow channel of 25 mm length × 5 mm width ×
0.1 mm depth (Fig. 3). Three glass capillaries connected to the
polyvinylchloride tube were inserted to the hole of the PDMS
disk. The liquid of PDMS was pasted on the junction ofelements, and baked at 333 K for 90 minutes in an oven (Fig.
4).

(1)

In Eq. 1, q is the flow rate [m3 s-1], b is the width of the canal[m] and D is distance [m] between two parallel walls. In thepresent study, D is 0.1 mm, and b is 5 mm.
The shear stress T [Pa] is the product of viscosity N [Pa s] of thefluid and the shear rate G [s-1] of the flow (Eq. 2).
T=NG

(2)

The viscosity of the medium was measured with the cone andplate type of a viscometer (TVE-22L, Toki-Sangyo Co., Ltd.
Tokyo) at 310 K.
Cell
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) of the passage between fourth and ninth
was used in the tests.

Fig. 2: Die of steel with a central rectangular convex.

D-MEM (Dulbecco’s Modified Eagle Medium) containing 10%
FBS (Fetal Bovine Serum) and 1% penicillin/ streptomycin wasused for the medium. After the medium was drawn into theflow channel through the first inlet tube by the syringe pump(dotted arrow in Fig. 4, Fig. 5), the suspension of C2C12 in
D-MEM was introduced into the channel through the second
inlet tube by the syringe pump (solid arrow in Fig. 4, Fig. 6).

Pump

MediumFig. 3: Cross section of flow channel.

Fig. 5A: Channel is prefilled with medium.

Fig. 5B: Medium is introduced into channel with syringe pump.
Fig. 4: Flow channel. Arrow shows flow direction.

Pump

Pump

MediumCell suspension
Fig. 6: Suspension of cells is introduced through another inlet
into channel with syringe pump.

Fig. 9A: Flow stimulation on cells in channel.

Ultrapure Water

Fig. 7: Cells are incubated in channel.
Fig. 9B: Flow stimulation on cells in channel placed inincubator on microscope.

Fig. 8: Channel is placed in incubator on microscope.

After every tube was occluded (Fig. 7), the channel was placedin an incubator, where both the temperature of 310 K and thecarbon dioxide partial pressure of 5 percent are maintained.
The ultrapure water was poured over the dish to cover thechamber to avoid evaporation of medium.
After incubation without flow stimulation, the flow channel is
move from the incubator to the microscope system.
The microscope system consists of a chamber of incubation, amicroscope, and a camera (Fig. 8). The channel is placed in
the chamber, where temperature and CO2 content are kept 310
K and 5 percent, respectively. The chamber is placed on thestage of the inverted phase contrast microscope (IX71, Olympus,
Tokyo) to observe the behavior of cells adhered on the dishunder the flow.
After several cells adhered to the disk, the shear stress wasapplied on the cells in the medium flow pulled with the syringe
pump for 1 hour (Fig. 9). Variation was made on the flow rate.
The constant flow rate between 1.3 cm3 hour-1 and 39 cm3
hour-1 produces the constant wall shear stress between 0.1 Pa
and 3 Pa.

Fig. 10: Dimension of the convex part measured with a laser
microscope.

The observation area was selected in the downstream side of thechannel to apart from turbulent flow. To trace the same cell,the observation area was marked on the channel with the circle,
and time-lapse pictures were taken with the constant interval of
five minutes.

3. RESULTS
Fig. 10 shows the tracings of the surface morphology of theconvex part of the machined die. The height of the convex
part varies between 0.096 mm and 0.106 mm. The widthvaries between 4.96 mm and 5.00 mm. The length is 25.0 mm.
Mean roughness is 0.0018 mm.
The viscosity of the medium measured with the cone and plate
type of a viscometer was 0.002 Pa s at the shear rate of 40 s-1.
Fig. 11 shows cells for one hour without flow, after incubationfor 48 hours. A few cells migrate in short distance, anddeform slightly for one hour.

Fig. 11: Cells before (A), and after 20 minutes (B), 40 minutes(C), 60 minutes (D) of cultivation without flow. Dimension
from left to right is 2 mm.

Fig. 12: Cells before (A), and after 20 minutes (B), 40 minutes(C), 60 minutes (D) of cultivation with flow (wall shear stress:
0.2 Pa). Direction of flow is from right to left. Dimension
from left to right is 2 mm.

In the first flow test, cells were exposed to the wall shear stressof 0.1 Pa for one hour, after incubation for 48 hours. Most ofcells keep the shape, and stay in the same position. One cellexfoliates within one hour of exposure to the flow.
Fig. 12 shows cells exposed to the wall shear stress of 0.2 Pa forone hour, after incubation for 48 hours. One cell extends thepseudo to the direction of the flow. One cell deforms, andextends perpendicular to the direction of the flow.

Fig. 13: Cells before (A), and after 20 minutes (B), 40 minutes(C), 60 minutes (D) of cultivation with flow (wall shear stress:
0.3 Pa). Direction of flow is from right to left. Dimension
from left to right is 2 mm.

Fig. 14: Cells before (A), and after 20 minutes (B), 40 minutes(C), 60 minutes (D) of cultivation with flow (wall shear stress:
1 Pa). Direction of flow is from right to left. Dimension
from left to right is 2 mm.

Fig. 13 shows cells exposed to the wall shear stress of 0.3 Pa forone hour, after incubation for 48 hours. One cell migrates andre-adheres. Several cells deform, and extend to the random
direction regardless of that of flow.
Fig. 14 shows cells exposed to the wall shear stress of 1 Pa forone hour, after incubation for 24 hours. The number ofexfoliated cells is higher after incubation of 24 hours than afterthat of 48 hours. The cell, which extends the pseudo to thedirection of flow, shortens the pseudo, and deforms to thesphere in one hour (in the circle in Fig.14). One cell migrates
to the upstream direction of the flow.
Fig. 15 shows cells exposed to the wall shear stress of 3 Pa forone hour, after incubation for 24 hours. A lot of cells keepadhesion to the wall. The number of cells of deformation islower at 3 Pa than that at 1 Pa.
The myoblast (C2C12) deforms regardless of the direction of
the flow at the wall shear stress lower than 0.2 Pa. The
myoblast deforms to the direction of flow at the wall shear
stress higher than 0.3 Pa. The myoblast, which extends thepseudo in parallel to the direction of flow, shortens the pseudo,and deforms to the sphere at the wall shear stress of 1 Pa in one
hour. More than 90 percent of myoblasts keep their adhesion,and keep their shape at the wall shear stress of 3 Pa. Somecells proliferate at the wall shear stress of 3 Pa (in the circle inFig. 15). The cell does not exfoliate during proliferation.

4. DISCUSSION
The flow channel system of the parallel disks has been used in
the previous experiments to study the effect of the shear stresson the cells [8, 9]. Although the sandwiched elastic film
between the disks is convenient to avoid air leakage, the shapeof the film may make the geometric fluctuation. The die witha central rectangular convex part was machined for PDMS disk
in the present study. The geometric accuracy of the wall ofchannel improves the laminar flow between the parallel walls.

It is not easy to estimate the shear stress value on the wall, whenthe medium has the free surface [6, 7]. The parallelepipedchamber is convenient to observe the response of cells undercontrolled shear stress [8, 9].
To apply mechanical stimulation to the cells, centrifugal force[3] or shear flow [10] is useful.
Both acceleration of proliferation and orientation of cells areimportant targets in the research field of regenerative medicineon the cultured biological tissue. The behavior of biologicalcells depends on electric [1] and magnetic [2] fields.
The previous studies show that a mechanical field, on the other
hand, governs behavior of cells. The shear flow governs theorientation of endothelial cells [6, 13].
Too strong mechanical stimulation damages cells.
The
moderate mechanical stimulation, on the other hand, might
accelerate differentiation of cells [3]. The mechanical stressexfoliates several cells, which makes vacancy around theadhesive cell [14].
The effect of shear flow on orientation of cells depends on thekinds of cells [6]. Although HUBEC orients along the streamlines, C2C12 tilts from the stream lines to make myotubes.
The previous study shows orientation of cells perpendicular tothe stretch direction [5].
The cells might deform to minimize the internal stress [5].
Myoblast, of which the longitudinal axis is parallel to the flow
direction, deforms to be rounded in the present experiment.
The permeability of the wall of the flow path and thetemperature difference may form bubbles. The contaminationof bubbles disturbs the uniform shear flow.
The short incubation time might reduce the affinity between thecell and the scaffold so that exfoliation of cells increases under
the shear flow.
Although the adhesive force of a cell to a scaffold might
decrease during proliferation, the myoblast (C2C12) is able tokeep adhesion during proliferation at the wall shear stress of 3
Pa. The experimental results show that deformation of C2C12
occurs most frequently at the wall shear stress of 1 Pa. The
deformation of a myoblast depends not only on dimension of
the shear stress, but also on exposure time to the shear stress.
C2C12 may form orientation at the wall shear stress of 1 Pa forseveral hours.

5. CONCLUSION
The effect of the wall shear stress on the adhered myoblast has
been studied in vitro. A flow channel was designed to observe
the response of cells to the fluid shear stress under observation
with a microscope. The experimental results show that the
myoblast deforms most frequently at the wall shear stress of 1
Pa.
Fig. 15: Cells before (A), and after 20 minutes (B), 40 minutes(C), 60 minutes (D) of cultivation with flow (wall shear stress:
3 Pa). Direction of flow is from right to left. Dimension
from left to right is 2 mm.

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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