Journal of Undergraduate Research
Volume 2, Issue 1 - October 2000

Development of a Flow Chamber for Cell Adhesion Studies

Brian Lin

ABSTRACT

Hemodynamic flow environment plays a major role in the behavior and functions of cells. For example, it has been found to correlate to the onset of arteriosclerosis and arterogenesis (5, 6). Most in-vitro studies performed on vascular endothelial cells (ECs) consist of culturing the cells on either glass or plastic coverslips and then subjecting the cells to flow conditions. ECs are then viewed with the use of an inverted microscope. However, this method is not adequate for studying the dynamics of cell adhesion on opaque surfaces. Therefore, the objectives of this project are to build a flow system for testing anchorage dependent cell responses to shear, and to develop a method for studying the various morphological and physiological effects of flow on EC cultured on opaque biomaterials.

INTRODUCTION

Endothelial cells (ECs) form monolayers in vivo and line the vascular wall. The monolayer or endothelium provides a selective barrier between flowing blood components and the vessel walls. It interacts actively by serving as a signal transduction interface between blood and the underlying vessel wall. ECs provide a non-thrombogenic surface which allow for the diffusion of nutrients and the diffusion of gases (5).

In-vivo, endothelial cells at the arterial wall interface are acted upon by various mechanical stresses due to the flow of blood. The two main forces generated are the pressure force, which acts normal to the cells, and the shear stress, which acts tangentially. Both the pressure and shear stress are time varying due to the pulsatile nature of blood flow. The magnitude of stress caused by viscous forces is much lower than that caused by pressure, but vascular endothelial biology is more sensitive to shear stress effects. The average mean shear stress level on the EC's within a straight arterial section is between 8-12 dynes/cm2 (5).

There have been many in vitro experiments carried out in order to study the effects of shear stress and changes in flow on ECs. An overview of the important results is presented below.

Under static conditions, ECs display a cobblestone appearance. However, under flow conditions, vascular endothelial cells change their shape and orientation depending on the nature of the flow. ECs actively align and elongate in the axial direction of the flow, due to a reorganization of their cytoskeleton structure, mainly the F-actin filaments (3, 4).

Some of the more striking effects due to flow concern the synthesis and release of important biologically active molecules like nitric oxide (NO), a potent vasodilator. Shear stress is an important regulator of NO, from a shear level of 1 dyne/cm2 to 10 dynes/cm2, the percent release of NO increases by several orders of magnitude (1). Another vasodilator, prostaglandin I2 showed greater levels of secretion while under increased flow levels (2). However with endothelin-1 (ET-1), a vasoconstrictor, its release is increased under low shear conditions (5 dynes/cm2) and is decreased at higher shear stress. Therefore shear stress level has a differential effect on the secretion of ET-1 by ECs (5).

Studies have also been done to study the effects of flow on cell proliferation. It has been shown that with increased levels of shear cell, replication is decreased. They observed that the cells where inhibited from entering the S-phase of the cell replication. The S-phase is the time in which the ECs would replicate its DNA (6).

CURRENT DESIGN

A schematic of the proposed flow system is given in Figure 1, and a brief description of the major components is provided next. The flow system consists of an upper reservoir to store the culture medium (Dulbecco's modified Eagle's medium (DMEM) containing 25 mM Hepes buffer, 10-20% fetal bovine serum, and antibiotics). The tubing exiting out of the upper reservoir is wrapped with a 3ft. heating cord (Glascol Inc., Terre Haute, IN) to heat the media to 37 C. A LM-34 temperature sensor (Electronic Rainbow, Indianapolis, IN) is used to monitor the temperature of the fluid. The media then flows through a flow meter (Omega Ins., Stamford, CT) before entering the parallel plate flow chamber (Glyco-tec, Rockville, MD). Flow of the media is caused by a pressure drop due to a height difference between the upper and lower reservoirs. The outflowing media is stored in the lower reservoir which is connected to a peristaltic pump (Rainin Instrument Co.,Woburn, MA). The pump brings the fluid back up to the upper reservoir and the entire process is repeated. The media is diffused with a 95% air / 5% carbon dioxide mixture while being temporarily stored in the reservoirs.

Figure 1. Flow System Schematic

Parallel Plate Flow Chamber

The basis for using the parallel plate flow chamber is that the flow pattern generated between the plates is well understood and the shear stress at the wall is easily calculated. For a laminar, incompressible, Newtonian flow, the wall shear stress is given by:

tw = 6mQ / a2 b

where a and b are, respectively, the height and width of the chamber, m is the fluid viscosity, and Q represents the volumetric flow rate. However, in order for this relation to hold true the entrance length must be must smaller than the entire length of the chamber. Also the height of the chamber must be much less than the overall width (b>>>a). Therefore this implies that the Reynolds number must be on the order of 100 or less (5).

The parallel plate flow chamber (GlycoTech Corporation, Rockville, Maryland) is depicted in Figure 2. The flow deck is made from cast acrylic, and can be adapted with a variety of inlet/outlet designs, and silicon rubber gaskets with different sizes.

Figure 2. Parallel Plate Flow Chamber

The top of our flow deck has three threaded holes; two are for connectors leading to the inlet and outlet of the flow deck, and the third is for the attachment of the vacuum pump to ensure a tight seal between the flow deck and the bottom of the 35mm culture dish.

Upper Reservoir

The upper reservoir consists of two Plexiglas cylinders placed one inside the other. The larger outer cylinder has an outside diameter of 45 mm and an inside diameter of 39 mm. The inner cylinder has an outside diameter of 26mm and an inside diameter of 22.5mm with a length shorter than the outer cylinder. The inner cylinder is first mounted centered with adhesive cement on a flat circular disc, which has the same diameter as the outer cylinder (45mm). Silicon sealant is used to prevent leaks. The outer reservoir is then mounted around the inner cylinder and onto the disc, bonded with adhesive cement, and sealed with silicon sealant. Another disc identical to the one used previously is bonded to the top of the larger cylinder and sealed.

A gas inlet allows the air/CO2 mixture into the upper reservoir while a gas outlet permits air to be released through a 0.2 micro filter. There is also another inlet from which media cycled from the lower reservoir. The media exits the reservoir through two outlets. One discharges fluid from the inner cylinder, while the other outlet allows the media to exit from the overflow cylinder.

Lower Reservoir

The lower reservoir consists of a 45mm (OD) and 39mm (ID) Plexiglas cylinder capped off on both ends with 45 mm diameter Plexiglas discs. The top part of the reservoir has four inlets; one inlet is for the outflow from the parallel plate flow chamber, the second is connected to the upper reservoir, the third is for the 95% air / 5% CO2 mixture, and the fourth is the gas outlet. The bottom of the reservoir has three inlet/outlets; one outlet leads to the roller pump that recirculate the medium to the upper reservoir, the second is the sample port for the infusion of certain chemicals into the system, it is capped with a 2mm filter to ensure sterility of fluids entering the system, and the third is a drain.

Heating Element and Thermocouple

Heating cords (Glas-Col Inc., Terre Haute, IN) are wrapped around the tubing between the upper reservoir and parallel plate flow chamber. A variable DC power supply is used to power the cord. The cord maintains the medium at 37 ºC.

A dual probe digital thermometer is used to measure the temperature of the medium entering the flow chamber. The kit uses National LM34 temperature sensors (Electronic Rainbow, Indianapolis, IN) to measure the temperature to within 1 ºF of accuracy.

pH Control/Gas Diffusion

In order to control pH levels of the medium a 95% air and 5% CO2 mixture is diffused into the medium. In our system, the air/carbon dioxide gas mixture is controlled by a Linde Flow Controller. The oxygen in the air allows the cells to live under aerobic conditions. The CO2 regulates the pH by providing a buffering system regulated by the chemical equation

CO2(g) + H2O þ¦ HCO3- + H3O+

The reversibility of the carbonic acid-bicarbonate conversion buffers the media by releasing or removing H3O+ ions from the solution depending on the pH.

Endothelial Cell Cultures

Many different types of EC from bovine to human umbilical cord are used in flow studies. Though the cells may vary, the culturing techniques are essentially the same. Usually the specific cells are acquired and then grown in tissue culture dishes. They are fed with a minimum essential medium along with antibiotics to prevent contamination. After 1-5 days, confluent cells are detached by exposure to a solution of trypsin-EDTA. The detached cells in suspension are then spun and the supernatant is discarded. The remaining cells are then remixed with medium at a 1:3 dilution. Then, the cells are plated on tissue culture dishes, which were sterilized with UV light. Experiments are then performed when the cells are nearly confluent but not quiescent. (2)

SUMMARY/DISCUSSION

A closed loop flow system with a parallel plate chamber for studying anchorage dependent cells has been built. To the best of our knowledge, morphological studies on endothelial cells have been done only on a transparent surface, because of the difficulty to observe the cells otherwise. We think that a possible solution to the visualization problem associated with the observation of EC cultured on opaque biomaterials would be the use of a reflected light microscope. Commonly used in the fields of geology and materials science, the reflected light microscope or sometimes referred to as the incident light microscope is used to study the surfaces of various objects. The microscope is unique because light is directed vertically through the objective and reflected back through the objective. Traditional image enhancement options such as polarized light, interference contrast and dark field illumination methods can also be used in conjunction with this type of microscope.

One problem associated with reflected light microscope is their limited working distance. Because the gap width in the present flow chamber is too large, this approach is not possible. The next phase of this project is to investigate this option by redesigning the flow chamber.

REFERENCES

Corson, M.A., N.L. James, S.E. Latta, R.M. Nerem, B.C. Berk and D.G. Harrison. 1996. Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress. Circ Res 79:984-991.
Frangos, J.A., S.G. Eskin, L.V. McIntire and C.L. Ives. 1985. Flow effects on prostacyclin production by cultured human endothelial cells. Science 227:1477-1479.
Ives, C.L., S.G. Eskin and L.V. McIntire. 1986. Mechanical effects on endothelial cell morphology: in vitro assessment. In Vitro Cell Dev Biol 22:500-507.
Levesque, M.J. and R.M. Nerem. 1985. The elongation and orientation of cultured endothelial cells in response to shear stress. J Biomech Eng 107:341-347.
Nerem, R.M., R.W. Alexander, D.C. Chappell, R.M. Medford, S.E. Varner and W.R. Taylor. 1998. The study of the influence of flow on vascular endothelial biology. Am J Med Sci 316:169-175.
Ziegler, T. and R.M. Nerem. 1994. Effect of flow on the process of endothelial cell division. Arterioscler Thromb 14:636-643.

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