Responses of Cells to Fluid Shear Stress in Vitro
Fumihiko SATO, Shigehiro HASHIMOTO
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/andKei OYA
Research Institute for Science and Technology,
Kogakuin University, Tokyo, Japan
and
Hiromichi FUJIE
Human Mechatronic Systems, Faculty of System Design,
Tokyo Metropolitan University, Tokyo, Japan

ABSTRACT
Responses of cells to a fluid shear stress have been studied invitro. A rhombus flow chamber was designed to observe theresponse of cells to the fluid shear stress under a microscope.
A thin sheet of silicone rubber was sandwiched by twotransparent polydimethylsiloxane disks to form a parallelepiped
flow channel of 20 mm length × 0.1 mm depth. Variation wasmade on the width of the channel (from 1 mm to 3 mm) with arhombus shape to vary the wall shear stress. Behavior of cellson the disk was observed under a flow with an inverted phase
contrast microscope. The shear stress on the wall is calculatedwith an estimated parabolic velocity profile between the paralleldisks. After several cells adhered to the disk, the shear stress
was applied on the cells in the medium flow with a syringe
pump. The constant flow rate of 18 mL/hour produces thewall shear stress between 1 Pa and 3 Pa. L929 (fibroblast-like,mouse connective tissue) was examined with the methodology.
The experimental results show that the response of cells can beobserved under stimulation by the fluid at the controlled wall
shear stress in the flow chamber.
Keywords: Biomedical Engineering, L929, Cell Culture,
Polydimethylsiloxane and Shear Stress.

Cell culture technique has been developed and several
methodologies have been clinically applied to regenerative
medicine.
Acceleration technique for orientation andproliferation of cells has been studied to make muscle tissue invivo or in vitro [1-15]. Control methodology for orientationand proliferation of cells would be applied to regenerative tissue
technology.
The mechanical stress is one of the interested points in theenvironment of cells, because they receive mechanical forces invivo.
Several methods have been designed to apply
mechanical stress to cells [6-15].
A transmission point of the stress to a specimen is important.
In many studies, the stress is applied to a scaffold. Whenfixation between the cell and the scaffold is not enough, thestress is not transmitted to the cell. A flow can be used, on theother hand, to apply a stress field to a specimen [9-15]. The
specimen directly receives the shear stress in the shear flow.
A flow chamber with a laminar flow is effective to study the
responses of cells to a fluid shear stress quantitatively. In thepresent study, responses of cells to the fluid shear stress havebeen studied with a parallel piped rhombus chamber in vitro.

2. METHODS
1. INTRODUCTION
Biological cells respond to various environmental factors, suchas electric [1-3], magnetic [4, 5] and mechanical [6-15] fields.

Rhombus Chamber
A one-way flow system was designed to observe responses ofcells to a fluid shear stress in vitro. The system consists of aflow chamber, a syringe pump, tubes and a microscope (Fig. 1

& 2). The micro-syringe-pump (Fusion200, CXF1020, ISIS
Co., Ltd., Osaka) was used for the syringe pump. A plastic
tube of 3 mm internal diameter and of 5 mm external diameter
was used for the connector to the flow chamber.
Theflow chamberconsistsof twotransparent
polydimethylsiloxane (PDMS) disks and a thin sheet of silicone
rubber (Fig. 3).

PDMS disk

A silicon wafer was used for a surface mold for the disk (Fig. 4).
The diameter and the thickness of the wafer are 50 mm and 0.30mm, respectively. The surface of the wafer was cleaned with
the isopropyl alcohol, and coated with 0.001 mm thickness ofparylene in the parylene coater (PDS-2010, Speciality Coating
Systems, Indianapolis). After the wafer was enclosed with aperipheral wall of polyimide, PDMS (Sylgard 184 Silicone
Elastomer Base, Dow Corning Corporation) was pored with thecuring agent (Dow Corning Corporation) on the wafer. After
degassing (Fig. 5), PDMS was baked at 383 K for one hour inan oven (DX401, Yamato Scientific Co., Ltd) (Fig. 6).

Silicone sheet
PDMS disk

Fig. 3: The flow chamber consists of two transparent
polydimethylsiloxane (PDMS) disks and a thin silicone rubber
sheet.

The diameter of two PDMS plates is 50 mm. The thicknessesof the upper and the lower disks are 10 mm and 2 mm,
respectively.

Fig. 4: Silicon wafer (diameter: 50 mm).

Fig. 1: Flow test system: flow chamber and microscope(middle), syringe pump (right).
Syringe pump

Flow
Tube

Tube

Flow chamber
Cell
Microscope
Fig. 5: Degassing of PDMS.

Fig. 2: Flow to syringe pump through flow chamber.

Plate

q
D
b

G
Plate

Fig. 8: Parabolic velocity profile between parallel plates.

Fig. 6: PDMS baked at 383 K for one hour.
A rhombus open space of 3 mm × 20 mm is cut off in a thinsheet of silicone rubber being 0.1 mm thick, and sandwiched
between the PDMS plates (Fig. 3). The open space forms achannel of 20 mm length × 0.1 mm depth, where the widthvaries from 1 mm to 3 mm. The three plates stick together
with their surface affinity without an adhesive. At the upper
plate, two holes of 5 mm diameter (Fig. 3) are machined by apunching tool. The silicone tube is stuck at the holes without
an adhesive (Fig. 7). The inner surface of the chamber was
exposed to the oxygen gas in a reactive ion etching system(RIE-10NR, Samco Inc., Kyoto) to be characterized ashydrophilic (oxygen plasma ashing), before assembled.
Immediately after the characterization, the flow path of thechamber was rinsed with a saline solution, and the suspensionof cells was introduced, successively.
One of the tubes is connected to the plastic syringe pump (Fig.
1). The room temperature was maintained at 25 degrees
Celsius. The chamber is placed on the inverted phase-contrast
microscope (IX71, Olympus Co., Ltd., Tokyo).

Cell Culture
L929 (fibroblast-like, mouse connective tissue, RCB1422,
Riken Bio Resource Center, Tsukuba) was used in theexperiment. The cells were cultured on a dish with the
E-MEM (Eagle’s minimal essential medium) in an incubator for
one week. Then, Cells were exfoliated from the plate of theculture dish with trypsin, and suspended in the E-MEM. The
suspension was introduced to the chamber and cultured in the
incubator for 3 hours to make cells adhere to the PDMS plate of
the chamber before the flow test.
Flow Test
After the chamber was set on the microscope out of the
incubator, the constant flow at 18 mL/hour of the medium wasapplied to the adhered cells with the syringe pump (Fig. 1).
The flow path was carefully examined to avoid mixing of air
bubbles, which might stir the medium in the flow chamber andinduce exfoliation of cells. The behavior of cells on the plateof the chamber was observed with the microscope. The photosof cells were taken during the flow test for one hour.
Shear Stress on Wall
The shear rate [G, s-1] on the wall of the plate is calculated by
Eq. 1, in which a parabolic velocity profile between parallel
plates is hypothesized (Fig. 8).
G = 6 q / (b D2)(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 ranges from 1 mm to 3 mm
(Fig. 7). The rhombus chamber designed in the present study
has a variation on the width of the canal. The width at the
inlet of the chamber is 1 mm, and increases from 1 mm to 3 mm
in proportion to the distance from the inlet. At the middle
point, the width is 3 mm, and then decreases from 3 mm to 1
mm in proportion to the distance from the inlet. The width is
1 mm at the outlet. The variation of the width makes variationon the wall shear stress in the chamber, so that cells at variety ofwall shear stresses can be simultaneously observed in theconstant flow rate.
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

Fig. 7: The flow chamber consists of two transparent
polydimethylsiloxane (PDMS) disks and the thin silicone
rubber sheet.

(2)

The viscosity of the medium was measured with the cone andplate type of a 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 25 degrees Celsius at
the shear rate of 600 s-1. At the flow rate of 18 mL/hour, thecalculated shear rate on the PDMS wall of the flow chamber by
Eq. 1 varies from 3000 s-1 to 1000 s-1, when the width of theflow path varies from 1 mm to 3 mm. The calculated shear
stress, thus, varies from 3 Pa to 1 Pa for viscosity of 0.001 Pa s,
when the shear rate varies from 3000 s-1 to 1000 s-1.
Fig. 9 shows cells under the flow of 18 mL/hour, which
generates a wall shear stress between 2.9 Pa and 3 Pa estimatedby Eqs. 1 & 2. The medium flows from left to right in Figs.
9-15. The distance from left to right is 0.5 mm in Figs. 9-15.
The inlet of the chamber is located near left end of Figs. 9-15,
and the width of the flow path linearly increases from 1.0 mm
to 1.1 mm in the figures. As the increase of the width of theflow path, the wall shear stress decreases. The estimated shear
stress varies from 3.0 Pa (at left-end) to 2.9 Pa (at right-end).
Some cells elongate to the downstream along the streamline of
the flow in 40 min (left upper A in Fig. 13). Some cells(center B in Fig. 14) exfoliate, and flow to the downstream in
43 min.

0.1 mmFig. 10: Cells after flow stimulation of 18 mL/hour for 20 min.
Dimension from left to right is 0.5 mm.

4. DISCUSSION
To estimate the wall shear rate in the chamber, a parabolic
velocity profile is hypothesized as a laminar flow in the present
experiment. Reynolds number (Re) is useful index for
estimation of laminar flow.
Re = d D v / N(3)

0.1 mmFig. 11: Cells after flow stimulation of 18 mL/hour for 25 min.
Dimension from left to right is 0.5 mm.

0.1 mmFig. 9: Cells after flow stimulation of 18 mL/hour for ten
minutes. Dimension from left to right is 0.5 mm.

In Eq. 3, d is density of the fluid, D is distance [m] between twoparallel walls, N is viscosity [Pa s] of the fluid, and v is themean velocity of the flow [m s-1]. The mean velocity is
calculated by Eq. 4.
v = q / (b D)(4)

In Eq. 4, q is the flow rate [m3 s-1], and b is the width [m] of thecanal.

B

0.1 mmFig. 12: Cells after flow stimulation of 18 mL/hour for 30 min.
Dimension from left to right is 0.5 mm.

0.1 mmFig. 14: Cells after flow stimulation of 18 mL/hour for 43 min.
Dimension from left to right is 0.5 mm.

A

0.1 mmFig. 13: Cells after flow stimulation of 18 mL/hour for 40 min.
Dimension from left to right is 0.5 mm.
Re is in the range between 1.7 and 5, calculated with d (103 kgm-3), N (10-3 Pa s), D (10-4 m), q (5 × 10-9 m3 s-1), and b (ranges
from 1 mm to 3 mm) in the present experiment. The number
is small enough small to estimate the laminar flow.
The parallel piped rhombus chamber designed in the present
study realizes wall shear stress field, where the shear stressvaries linearly along the stream line.
The chamber isconvenient to observe the response of cells simultaneously withthe variation of the wall shear stress, while the wall shear stressis constant in the parallel piped rectangular chamber of theprevious study [15].

0.1 mmFig. 15: Cells after flow stimulation of 18 mL/hour for 60 min.
Dimension from left to right is 0.5 mm.

Both acceleration of proliferation and orientation of cells areimportant targets in the research field of regenerative medicineon the cultured biological tissue. The previous study showsthat electric stimulation enhances differentiation of muscle cells[1]. Another study shows mechanical stimulation improves atissue-engineered human skeletal muscle [7].
The previous studies show that a mechanical field, on the other
hand, governs behavior of cells. The shear flow governs theorientation of endothelial cells [9-13]. The shear stress affects
on the orientation of the smooth muscle cells in the biologicaltissue [8]. The direction of the mechanical field affects
fibroblasts [6].

The present experiment shows that the critical value of the shear
stress for exfoliation of L929 from PDMS is higher than that ofcells from glass, which has been studied in the previous
experiment. Affinity between L929 and PDMS may be higher
than that between cells and glass [15].
Too strong mechanical stimulation damages cells.
The
moderate mechanical stimulation, on the other hand, might
accelerate differentiation of cells [9].
The mechanicalstimulation decreases proliferation of cells [14].
The
mechanical stress also exfoliates several cells, which makes
vacancy around the adhesive cell. The differentiation might be
optimization of cells to the changing environment.

5. CONCLUSION
Responses of cells to a fluid shear stress have been studied invitro.
The rhombus flow chamber has been designed toobserve the response of cells to the fluid shear stress under amicroscope.
The experimental results show that bothdeformation and exfoliation of cells can be observed understimulation with the fluid at the controlled wall shear stress inthe flow chamber.

6. ACKNOWLEDGMENT
This work was supported by a Grant-in-Aid for Strategic
Research Foundation at Private Universities (2008-2012) fromthe Japanese Ministry of Education, Culture, Sports andTechnology.

REFERENCES[1] S. Hashimoto F. Sato, R. Uemura and A. Nakajima, “Effectof Pulsatile Electric Field on Cultured Muscle Cells in
Vitro”, Journal of Systemics Cybernetics andInformatics, Vol. 10, No. 1, 2012, pp. 1-6.
[2] 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.
[3] 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.
[4] Y. Sakatani, S. Hashimoto and J. Yoriki, “Effect of Static
Magnetic Field on Muscle Cells in Vitro”, Proc. 14th World

Multi-Conference on Systemics Cybernetics andInformatics, Vol. 2, 2010, pp. 280-284.
[5] J. Yoriki, S. Hashimoto, K. Tachibana, et al., “Effect ofmagnetic field on adhesion of muscle cells to culture plate”,
Proc. 13th World Multi-Conference on Systemics
Cybernetics and Informatics, Vol. 2, 2009, pp. 223-228.
[6] 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.
[7] 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.
[8] K. Nagayama and 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] S. Hashimoto and M. Okada, “Orientation of Cells Cultured
in Vortex Flow with Swinging Plate in Vitro”, Journal of
Systemics Cybernetics and Informatics, Vol. 9, No. 3,
2011, pp. 1-7.
[10] 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.
[11] 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.
[12] 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.
[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] S. Hashimoto, T. Ooshima, F. Sato, Y. Sakatani and Aki
Nakajima, “Effect of Vortex Flow on Cultured Cells in
Vitro”, Proc. 15th World Multi-Conference on Systemics
Cybernetics and Informatics, Vol. 2, 2011, pp. 112-117.
[15] A. Nakajima, H. Iwata, S. Hashimoto, T. Susa and F. Sato,“Effect of Shear Flow on Cultured Cartilage Cells”, Proc.
15th World Multi-Conference on Systemics Cybernetics
and Informatics, Vol. 2, 2011, pp. 124-128.