Effect of Pulsatile Shear Flowon Migration of Endothelial Cells Cultured on Tube
Shigehiro Hashimoto, Hiroshi Oku, Naoko Komoto, Yoshinori Murashige,Shirou Manabe, Kazuhiro Ikegami, Chiaki Miyamoto
Biomedical Systems, Dept. of Electronics, Information, and Communication Engineering,Osaka Institute of Technology, Osaka, 535-8585, Japan
hasimoto@elc.oit.ac.jp
andReiji Hattori, Hajime Otani, Hiroji Imamura
Dept. of Thoracic and Cardiovascular Surgery, Kansai Medical University
Moriguchi, JapanABSTRACT
The effect of pulsatile shear flow on migration ofendothelial cells was investigated in vitro. The innersurface of the silicone tube was sputtered in a vacuum tochange the surface character from hydrophobic tohydrophilic, and coated with collagen gel.
Theendothelial cells of rat's descending aorta were cultured
on the inner surface of the test tube. Two types of shear
flow were applied to the endothelial cells: a continuous
flow powered by a peristaltic roller pump, and a pulsatile
flow powered by a diaphragm pump. The behavior ofcells was observed through the tube wall by a phase
contrast microscope. In pulsatile flow, the cells did notpeel off at the mean shear rate of 15 1/s in 60 minutes.
The change in orientation of cell occurs gradually withrepeating peel off at the corner of the cell. The cultured
endothelial cells on the collagen-coated wall of silicone
rubber tube with sputtering did not peel off in pulsatile
shear flow at 37 degrees C for 24 hours. The
experimental results show that the longitudinal axes of
the cells tilt toward the flow direction and that pulsatile
flow may have an advantage for migration of cells.
Keywords: Endothelial Cells, Pulsatile Flow, Shear Rate,
Silicone Tube, Hydrophilic, Collagen Coating, SputterEtching
1. INTRODUCTION
Arterial endothelial-cells, which cover the inner surfaceof the blood vessel, are exposed to the pulsatile flow invivo. The cyclic shear stress is induced in cells with thepulsatile flow. The effects of shear stress to theendothelial cells have been studied and several chemical
responses were pointed out in the previous studies [1-5].
The longitudinal axes of endothelial cells are arranged
along the streamline. The orientation may occur bymigration of cells. In the present study, the effect ofpulsatile shear flow on migration of endothelial cells wasinvestigated in vitro.
2. METHODS
Endothelial cells
The descending aorta of an anesthetized rat was exposedand exfoliated from surrounding tissue underthoracotomy (Fig. 1). The descending aorta as long as
10 mm was excised from the rat. The excised aorta
segment was sunk in a medium including an antibiotic
substance (Fig. 2). The endothelial cells were gently
peeled off from the inner layer of descending aorta by
the flow of the culture medium with a syringe, andcollected at the downstream into another syringe (Fig. 3).
The surface of the inner wall were gently rubbed with asurgical knife to peel off endothelial cells (Fig. 4).
After cultivation for one week on a piece of glass plate ina dish preserved in an incubator to be cloned (Figs. 5 &6), the endothelial cells were cultured on the innersurface of the test tube.

Fig. 4: The surface of the inner wall were gently rubbed
with a surgical knife to peel off endothelial cells.
Fig. 1: The descending aorta of an anesthetized
rat was exposed and exfoliated from surrounding
tissue under thoracotomy.

Fig. 5: Endothelial cells were cultured on a piece ofglass plate in a dish preserved in an incubator to becloned.

Fig. 2: The excised aorta segment was sunk in a medium
including an antibiotic substance.

Fig. 6: Endothelial cells after cultivation for one week
on a piece of glass plate in a dish preserved in anincubator.

Fig. 3: The endothelial cells were gently peeled off fromthe inner layer of descending aorta by the flow of theculture medium with a syringe.

Test tubesTwo kinds of test tubes were prepared; one is made ofglass and the other of silicone-rubber. The internal andexternal diameters of the glass tube are 0.56 mm and 1.6mm, respectively. The internal and external diametersof the silicone-rubber tube are 1.5 mm and 2.0 mm,respectively. A 20mm-length segment of the silicone
rubber tube was turned inside out. The inner surface ofthe silicone tube was sputtered in a vacuum to change
the surface character from hydrophobic to hydrophilic(Fig. 7). After the sputter etching, the tube wasimmediately sunk in a water solution including collagenmolecules. Through the procedure, the inner surface ofthe silicone-tube wall was coated with collagen. Thecells contained in the suspension were poured into thetube to be cultured on the inner surface of the tube.

Fig. 7: The inner surface of the silicone tube wassputtered in a vacuum to change the surface characterfrom hydrophobic to hydrophilic.

Mock circulation circuit
A mock circulation circuit was constructed with thesilicone tube and the pump. Before cells were cultured
to be confluent on the surface, the tube with cells wascontained in a chamber made of the transparent styrene
plate (Fig. 8), and connected to the pump via silicone
tubes. The culture medium was filled in the chamber,where concentration of carbon dioxide was kept at 5 %in the incubator. The chamber also plays a role of thebuffer for the stroke volume in pulsatile flows. Somelines were marked on the bottom plate of the chamber toobserve cells at the same point of tube intermittently
during the test (Fig. 8). A liquid culture medium (0.002Pa s) was circulated in the circuit at 25 degrees C. In along-term test, the whole circulation system was set in
the incubator, and cells were exposed to the flow for 24hours at 37 degrees C (Fig. 9).

Fig. 8: Chamber made of the transparent styrene plate.

Fig. 9: Whole circulation system was set in theincubator.

Pumps
Two types of shear flow were applied to the endothelial
cells: a continuous flow and a pulsatile flow. The
continuous flow was powered with a peristaltic roller
pump (Fig. 10). To apply the pulsatile flow, a micro
diaphragm pump system was manufactured (Fig. 11).
The system consists of a battery, a DC motor, a crank, adiaphragm chamber, and leaflet one-way valves. In thepulsatile flow, the flow condition was controlled tosimulate that with the natural heart of rat: the beating
rate was 300 per minute and the stroke volume was 0.06
ml.

3. RESULTS
The most of cells were peeled off by continuous shear
flow with shear rate of 15 1/s in 10 minutes. By
pulsatile flow, however, the cells were not peeled off at
the mean shear rate of 15 1/s in 60 minutes. The cells
were repeatedly peeled off only at their corner, whilekeeping adhesion at another corner. The change inorientation of cell occurs gradually with repeating peel
off at the corner of the cell. The longitudinal axes ofendothelial cells tilted toward the direction of thestreamline (Figs. 12 & 13).

Fig. 10: The continuous flow was powered with aperistaltic roller pump.

The affinity between collagen-gel and silicone rubberincreased with sputtering, which made strong
adhesiveness between wall and cells. The cultured
endothelial cells on the collagen-coated wall of silicone
rubber tube with sputtering did not peel off in pulsatile
shear flow at 37 centigrade for 24 hours, while the cellson that without sputtering peeled off in 5 minutes.

Fig. 11: The pulsatile flow was powered with a micro
diaphragm pump.

Shear rate
The shear rate at the inner wall surface of the tube is
estimated by Eq. (1).
G = 4 Q / (P R3)

Fig. 12: The endothelial cells on the surface of the tube
before exposure to flow.

(1)

Where. G is shear rate at the wall (1/s), Q is flow rate(cm3/s), P is the ratio of the circumference of a circle toits diameter, and R is radius of the tube (cm). The
calculated value is an approximation under Poiseuilleflow. The flow rate (Q) was measured with anelectromagnetic flow meter installed in the downstreamof the tube with cells.
Microscope
The behavior of cells was observed through the tube wallby a phase contrast microscope (Figs. 10 & 11). The
wall of the tube was thin enough to keep transparency
for observation at the inner surface with an optical
microscope.

Fig. 13: The longitudinal axes of endothelial cells tilted
toward the direction of the stream line (from left toright).

4. DISCUSSION

7. REFERENCES

The glass tube is popular to be used for cell culture
because of its hydrophile property. The arterial wall
repeatedly deforms with pulsatile flow [6].
Thedeformation generates both tensile stress and shear stressin the endothelial cell. The compliant tube of silicone
rubber is preferred to the rigid tube of glass to simulatemechanical condition of vessel wall. Both the sputter
etching and the collagen coating were effective tochange the property of silicone rubber to be used for cellculture.

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[6] Shigehiro Hashimoto, Shirou Manabe, Kazuhiro
Ikegami, Yoshinori Murashige, Chiaki Miyamoto,
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HirojiImamura,"Measurementofcyclicmicrodeformation of arterial wall with pulsatileflow", SPIE Proceedings, Vol. 4609, 2002.
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Several studies have been performed about the difference
between pulsatile and non-pulsatile flows [7, 8]. The
manufactured micro diaphragm pump system wasavailable for long-term test of pulsatile flow in theincubator.
5. CONCLUSIONSThe experimental results show that the longitudinal axesof the cells tilt toward the flow direction and that
pulsatile flow may have an advantage for migration ofcells.
6. ACKNOWLEDGEMENTS
This work was supported in part by a Grant-in-Aid for
Scientific Research from the Japanese Ministry ofEducation, Culture, Sports, Science and Technology.