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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

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[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.

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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

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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.

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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.

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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)

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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

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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

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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.