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27
CHAPTER 3
BUILDING AND DEVELOPMENT OF SHEAR STRESS
APPARATUS
3.1 OVERVIEW
Vascular endothelial cells line the inner surface of blood vessels
and serve as a selective barrier between the blood and other tissues and
organs. The endothelium is a metabolically active monolayer and is
constantly exposed to various biochemical and biomechanical stimuli. As
blood flows, the vascular endothelial cells are constantly subjected to physical
forces, which regulate important physiological and pathological blood vessel
responses. Changes in blood flow, generates altered hemodynamic forces
responsible for acute vessel tone regulation, development of blood vessel
structure, as well as chronic remodeling and generation of blood vessels. The
complex interaction of shear stress, derived by the flow of blood and the
vascular endothelium is a topic of interest for many researchers.
3.2 BASICS OF SHEAR STRESS STUDY
Many studies suggest that shear stress has varied effects on the
endothelium, based on the magnitude of shear stress, which in turn determines
the physiology or pathology of the cardiovascular system (Chien et al 1998;
Davies 1984; Davies 1995; Malek et al 1999; Nerem et al 1998; Resnick and
Gimbrone 1995; Dewey et al 1981). Since it is not feasible to carry out the
studies in animal model, a clear understanding of the effects of shear stress on
cellular metabolism is important for optimal design and operation of
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mammalian cells in vitro under shear stress. This problem may be best
approached by studying the cells by biochemical and biophysical approach
under conditions of characterized shear stress. We have developed a flow
apparatus capable of subjecting cultured cells to physiological range of shear
stress for long time periods based on the prototype previously standardized by
Frangos et al (1988). By applying shear stress to the cells, our understanding
of how the shear stress stimuli signals the cellular machinery will be a key
determinant in our attempts to mark diagnostic or therapeutic targets for
cardiovascular diseases.
In the present Chapter, the building and development of the flow
apparatus for the study of the response of cultured anchorage-dependent cells
to fluid shear stress is described in detail.
An in vitro apparatus has been developed to assess the dynamic
response of endothelial cultures to physiological range fluid shear stress. One
of the first parallel plate flow chamber described to study the effects of
in vitro shear stress on mammalian endothelium was described by Frangos et
al (1988). The flow system is similar in operation to previously standardized
(Frangos et al 1988; Lawrence et al 1987) or any commercial parallel plate
flow apparatus, capable of producing physiological and pathological levels of
laminar shear stress. The experimental parameters studied include: cell
remodeling, migration (wound healing), cytoskeletal reorganization, ring-
formation and biochemical parameters like nitric oxide production and eNOS
localization and phosphorylation.
3.3 PRINCIPLES OF PARALLEL PLATE FLOW APPARATUS
Endothelial shear stress (ESS) is the tangential stress derived from
the friction between the flowing blood and the endothelial surface of the
vessel wall, which is expressed in units of force / unit area (N/m2 or Pascal
29
[Pa] or dyne/cm2; 1 N/m
2 =1 Pa = 10 dyne/cm
2) (Nichols et al 2005; Slager
et al 2005). Inside a blood vessel, ESS is measured as the product of the blood
viscosity (µ) and the spatial gradient of blood velocity at the wall (dv/dy)
(Equation 3.1):
ESS = µ X dv/dy (3.1)
where dv is change in flow velocity unit and dy is change in unit of radial
distance from the wall. The spatial gradient of blood velocity describes how
fast the blood velocity increases from areas at the arterial wall toward areas at
the center of the lumen (dv/dy). Physiologically, the shear rate decreases at
the center of the lumen and gradually increases toward the wall.
The magnitude of the shear stress on the cell monolayer in the flow
chamber may be calculated using the momentum balance for a Newtonian
fluid (Equation 3.2):
= 6Qµ/bh2
(3.2)
where Q is the flow rate (cm3/s); µ is the viscosity (0.01 dynes); h is the
channel height (0. 019 cm); b is the slit width, (2.1 cm); and is the wall
shear stress (dyn/cm2).
The mean delay time of medium in the flow chamber and the
tubing between reservoirs for the experiments performed ranged from
5-30 seconds. The flow rate was controlled by adjusting the relative distance
between the flow distributor chamber to the parallel flow apparatus, by
changing the length of the overflow manifold tubing.
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Table 3.1 Shear stress magnitudes under various flow rates and mean
distance from flow regulator
Sl.NoShear stress
(dynes/ cm2)
Distance (in cms) at which
the flow chamber placed
(from flow regulator)
1 1 18
2 5 39
3 10 57
4 15 75
5 20 98
6 25 110
Reynolds number is an important factor to determine whether the
flow will be laminar or turbulent for a given geometry. For low Re values
blood flow is laminar, whereas for high Re values (typically, above 2,000)
blood flow is turbulent.
The Reynolds number of the flow through the chamber is given by:
Re = Uh /µ = Q /µb (3.3)
where U is the characteristic or mass average flow velocity; is the density of
the medium; and µ is the viscosity of the medium.
For the range of shear stresses used in the present study, the
Reynolds number varied from 0 to 20, indicating that fluid flow through the
chamber was laminar. Because of the large aspect ratio (b/h) and low
Reynolds number found in the flow chamber, the above equation is valid for
nearly the entire monolayer surface.
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3.4 CONSTRUCTION OF PARALLEL PLATE FLOW
APPARATUS
In order to study the effects of fluid shear stress on endothelial cell
structure and function, it was imperative to build an in vitro system with a
specific range shear stress magnitude. We have developed an apparatus that
utilizes the principle of parallel plate flow, based on the model of Frangos
et al (1988) (Figure 3.1). The apparatus consists of two reservoirs - upper
buffer chamber and lower reservoir, situated one above the other, with a
parallel-plate flow chamber positioned in between (Figure 3.1).
The hydrostatic pressure head created by the distance between the
upper and lower reservoirs drives the flow through the chamber. Continuous
pumping of culture medium from the lower to upper reservoirs by a peristaltic
pump maintains the pressure head at excess rates than the flow towards the
parallel plate chamber. The excess drains down the glass overflow manifold.
The upper and lower reservoirs were glass made, while the interconnecting
tubing was of Tygon (0.125- in. o.d., Saint Gobain), Silicone tubing was used
in the section through the roller pump and it joins the reservoirs to the
manifold and tubing.
The flow chamber consists of a machine-milled polycarbonate
plate, a rectangular teflon (0.020-cms) gasket, and the glass slide (75 x 38
mm) with the attached endothelial cell monolayer (Figure 3.1). These were
held together by a steel chamber. The polycarbonate plate has two manifolds
– an entry port and an exit port, through which medium enters and exits the
channel. The entry port is larger than the exit port and serves as a bubble trap.
A valve present opposite the entry port allows the removal of the bubbles.
32
Figure 3.1 Parallel plate flow apparatus
A. Schematic diagram of the apparatus. B. Parallel plate flow chamber C.
Cartoon showing cells plated on glass cover slip over the flow chamber.
3.5 APPLICATION OF SHEAR STRESS ON CELLS
3.5.1 Preparation of Cover Glasses with Cells
Shear stress was induced in the fluid contained between a stationary
plate and the cells plated on the cover glass, both separated by a washer. The
cell suspension was plated onto glass slides (75 x 38 mm). The glass slides
were pretreated with 0.5M NaOH for 3 hours and rinsed with distilled water,
thereby enhancing cell adhesion by conferring a charge on the glass surface.
The slides were then dried and sterilized in an autoclave and UV
consequently. The cells were seeded between 1x105- 1x10
6 cells per slide as
33
per experimental requirement. Cultures became confluent after 12hrs and flow
loop experiments were run for 30min.
3.5.2 Running of Shear Stress Apparatus
All the loop parts of the shear stress apparatus were washed and
rinsed in deionized water, oven-dried and then autoclaved. The whole set up
of flow loop apparatus was run initially with autoclaved double distilled
water. Medium was added to the top reservoir (100 ml), filling the bottom
reservoir as well, and flooding the chamber. The whole flow apparatus was
run with the media prior to the experiments with the cell sample. Then the
slide with the cultured cells was gently inverted over the flooded flow
chamber, and clamped. Care must be taken to avoid any air bubbles in the
flow channel. During an experiment, the flow apparatus was placed on a 37oC
hot plate.
3.5.3 Trouble Shooting
Problem had been faced initially to see through that the cells stuck
firmly on the shear plate. The cells were scrapped off the surface of the cover
glasses on application of shear stress. This problem had been addressed by
using a 3hr NaOH treatment over the cover glasses as mentioned before. The
second major problem we faced was the sterility of the Shear Stress
apparatus. A continuous flow of cell culture media through the flow chamber
resulted in frequent contamination of the cells. Finally, we had to miniaturize
the shear stress apparatus by reducing the dimensions of the chambers, to
make it fully autoclavable and also to reduce the volume of media used in the
buffer chamber.
34
3.6 DEVELOPMENT AND VALIDATION OF EIGHT
CHANNEL SHEAR STRESS APPARATUS
3.6.1 Parallel Eight Channel Flow Apparatus
The typical Parallel plate apparatus allows running a single set of
experiments. As it is time consuming to run several single runs of shear stress
one after the other for several combinations of treatments, we had modified
the flow apparatus to accommodate eight channels, which will allow to run
eight parallel experiments of shear stress simultaneously. This provided us not
only to run several parallel experiments under shear stress with different
treatments of inducers and inhibitors, but also strengthening the consistency
of the data for different cell based assays and biochemical assays as well
(Figure 3.2).
Figure 3.2 Parallel plate eight channel apparatus
Cartoon showing shear stress apparatus with parallel eight channel flow
apparatus.
35
3.6.2 Validation of Parallel Eight Channel Flow Apparatus
The eight channel flow apparatus had been validated and checked
for the consistency using endothelial function assays, prior to running the
main experiments. Firstly we have checked the consistency of the shear stress
magnitude coming from each of the parallel plate flow apparatus of the eight
channels set up. We have calculated the flow rates of all the channels and
thereby the shear stress (dyn/cm2), which was very much similar without
much variation. Next we have performed the endothelial wound healing
migration assay, cellular extensions assay and ring formation assay using a
low shear stress magnitude (5 dyn/cm2) and a physiological range of shear
stress (15 dyn/cm2) (Figure 3.3). We compared the data with available
experimental data from previously reported work (Frangos et al 1988;
Lawrence et al 1987).
Figure 3.3 (Continued)
36
Figure 3.3 Parallel plate flow apparatus and validation in EC
A. Cartoon showing shear stress apparatus with parallel eight channel flow
apparatus with adjustable shear magnitude. B. Graph showing comparison
of NO production at 5 dynes/cm2
and 15 dynes/cm2 at different time
intervals C. Graphical representation of wound healing assay for EC
migration under 0, 5 and 10 dynes/cm2 Shear stress at 5min and 15min time
points D. Cellular extensions under Static controls and shear stress were
analyzed and graphically represented above. The values obtained from
100cells in 3 sets of individual experiments E. Graph showing tube
formation in Static controls and time dependent shear-induced ring
formation.
3.7 LIVE CELL IMAGING SHEAR STRESS APPARATUS
The size and thickness of the plate used to apply shear stress has
been a limitation for the image processing. Moreover the flexibility in
maintaining the contamination free conditions and chemical treatments had
been a problem. This problem has been addressed by formulating a custom
made shear apparatus, which can fit a cover slip measured 24 x 60mm, readily
37
available in market. We have coupled the flow apparatus with an inverted
fluorescence microscope to track the changes in the cellular localization of
proteins using GFP. Further we standardized the modified shear apparatus
model for the smooth and proper run to give different magnitudes of shear
stress and also for measuring NO production and protein trafficking in live
cells under shear stress. We performed live cell NO production using NO
fluorescent probe DAF-2DA. The endothelial were treated with two
magnitudes of shear stress - a low shear stress (5 dyn/cm2) and a physiological
range of shear stress (15 dyn/cm2) (Figure 3.4).
Figure 3.5 Live cell flow apparatus
Image showing flow apparatus attached with fluorescent microscope
attached with a image capture set up.
3.8 CONCLUSION
The in vitro flow apparatus provides a simple and cost effective
method for exposing anchorage-dependent cells to laminar shear stress and
has several advantages over other devices used to evaluate the effect of
mechanical stress on cell function. The system can be upgraded to create
38
turbulence as well. Furthermore, the flow chamber can be mounted on an
inverted microscope, allowing for continuous visualization using video
microscopy. The flow system is well-suited for analysis of the effects of shear
stress on the metabolism of attached cells.