The Goal of the Project Was to the Design of an Experimental Procedure for the Use Of

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Introduction

The goal of the project was to the design of an experimental procedure for the use of the Dunn chamber (Figure 1). The Dunn chamber is a glass microscope slide with two concentric wells and a bridge in between. Cells were grown on a glass coversilp in such a manner that cells would be only covering the inner well. The inner well contained

media and the outer well contained EGF media, so that cell migration from the inner well to the outer well could be observed through sequential images.

The goal of this project changed over the course of the year. This is because of the difficulties that we encountered in attempting to perform the Dunn direct-viewing chamber experiment. The original goal was to design an assay to test the redistribution of Epidermal Growth Factor (EGF) receptors in living cells migrating towards EGF. This experiment was divided into three phases. The first phase was to examine cells migrating towards EGF in the Dunn chamber, the second phase was to label the EGF receptors with Texas Red antibodies, and the third phase was to observe redistribution of EGF receptors fused with Green Fluorescence Protein (GFP).

The demands for our experiment were to obtain a single cell suspension, to grow cells on the coverslip, to develop a step-by-step procedure for the Dunn chamber experiment, to observe cell migration through video microscopy, and to observe redistribution of EGF receptors. Other demands of the experiment required growing a substantial amount of cells to be observed and obtaining clear images of cells through sequential imaging.

EGF is a protein found in normal functioning intestinal epithelial cells. Migration of these intestinal cells is important for the growth and preservation of the epithelial layer. EGF plays a major role in wound closure by stimulating epithelial restitution. The chemotactic reorganization of EGF receptors within a cell is being studied. This involves the sensing of growth factor concentration gradient.

When reformation of the mucosal epithelium is altered, a number of inflammatory bowel diseases (IBD) may be caused. These diseases, including ulcerative colitis, Crohn’s disease, and peptic ulcer disease, primarily affect the gastrointestinal tract. Ulcerative colitis is inflammation and ulcers in the top layer of the lining of the large intestine. Crohn’s disease is the inflammation deep in the lining of the small intestine. It may be caused by the body’s immune system over-responding to myobacteria. Peptic ulcers are caused when the mucosal barrier of the stomach is broken and the gastric wall is injured by its acidic contents or a bacterial infection. Currently, one to two million Americans suffer from IBD and it is most common in people between the ages of twenty and thirty. It affects men and women equally and 20% of the people with IBD have a blood relative also with IBD. It has been shown to be more prevalent in industrialized nations, possibly linked to higher animal fat intake.

The study of the behavior of cells and their receptors in the presence of EGF is significant because it may aide in the discovery of a cure for IBD. It is possible that people suffering from IBD have non-functioning or partially functioning signal transduction pathways in the EGF receptors of their epithelial cells. The work done in this experiment was a precursor for determining which part of the EGF receptor is necessary for its proper function.

Methodology

The base of a 200ml pipette tip was used to make a plating ring. Before plating the cells on the glass coverslip, it was coated with rat collagen for 24 hours. The Young Adult Mouse Colon (YAMC) cells which were plated by Dr. Wei Tong were growing in the 33°C incubator. These were in a 35x10mm tissue culture dish with 5% Fetal Bovine Serum (FBS) medium. In order to get a single cell suspension, the medium was removed with a vacuum under a biological safety hood. The cells were then washed twice with phosphate buffer solution (PBS) to remove the acid. After the PBS was removed, .5ml of trypsin was added to digest the cells. The dish was placed in the 37°C incubator for five minutes. Once the cells were suspended, 2ml of 0.5% FBS medium were added. The solution was pipetted up and down several times to ensure single cell suspension.

Both the coverslip and the ring were rinsed with PBS to neutralize the rat collagen. A dot was placed on the coverslip to mark the center of the Dunn Chamber. A new tissue culture dish was filled with about .75ml of 0.5% FBS medium. The coverslip was positioned in the dish so that the media was barely coming over the edges. The plating ring was centered over the dot on the coverslip. The ring was filled with 30ml of cell solution and left in the 37°C incubator for 8 to 9 hours.

The coverslip was examined under the Nikon TMS microscope at 10x magnification to ensure attachment of the cells. Once cells were plated, the coverslip was gently rinsed with 0.5% FBS medium. The concentric wells of the Dunn Chamber were filled with 30ml of 0.5% FBS medium in the inner well and 60ml in the outer well. The dot on the coverslip was aligned with the center of the Dunn chamber so that a small gap was left in the outer well (Fig. 1). Wax was heated to seal the first three edges of the coverslip onto the Dunn Chamber so that no medium could evaporate. The media in the outer well was absorbed using a kimwipe. Medium containing 10ng/ml EGF was used to fill the outer well and the fourth edge of the coverslip was sealed with wax.

To obtain sequential images, the Dunn chamber was then placed under the Nikon phase contrast video microscope. The 37°C incubator surrounding the microscope and the camera were turned on. The microscope was focused on the outer wall of the inner well to observe migration from the inner well to the outer well. The BioQuant program was set to take up to 100 sequential images with one image obtained every ten minutes to capture cells migrating towards EGF.

Results

The majority of the semester was spent attempting to plate cells in a small ring on the coverslip of the Dunn chamber. The parameters of the experiment were adjusted many times. The coverslip was left to soak in rat collagen for different amounts of time. Rat collagen is important because it provides a matrix to which the cells can adhere. We determined that cellular adhesion was best when coverslips were coated with collagen at least twenty-four hours. We also found that the cells could not survive if left in trypsin for too long. The optimal time for trysinization was found to be about three minutes. The length of time needed for cells to plate onto the coverslip was adjusted to achieve maximal attachment. This time was difficult to determine because when cells were not left on the coverslip long enough, only one or two cells would attach. However, when cells were left too long in the incubator, they all died. We found the optimal time to be eight hours. The amount of cell solution in the plating ring was also altered. At first, we used only enough solution to coat the surface of the coverslip. This did not provide enough cells to survive long enough to attach. We found the best amount to be 30ml of cell suspension. There had to be enough media in the culture dish where the cells were plating to provide a humid environment for cell growth. We found that too much media caused the coverslip to be unstable and the ring to slip out of place. The optimal cellular environment was determined to occur when there was media barely coming over the edges of the coverslip (approximately 1ml).

By altering these parameters, we were able to grow cells in the plating ring in the glass coverslip. Due to time spent determining the optimal cell plating conditions, we were not able to plate cells until the final week of the project. Figure 2 demonstrates cells on the coverslip. The cells that have attached have an elongated appearance, as can be seen in the upper right side of the picture. Figure 3 demonstrates cells in the Dunn chamber. Unfortunately, the cells detached before sequential images could be taken to observe their migration in the presence of EGF.

Conclusions and Recommendations

We obtained single cell suspensions, grew cells on the coverslip of the Dunn chamber, obtained a substantial amount of cells to be observed, and developed a step-by-step procedure for the Dunn chamber experiment. We were not able to meet our initial draft plan because of unforeseen difficulties with cell plating. Two demands that were not met were observing cell migration through video microscopy and observing the redistribution of the EGF receptors. One wish that was not met was obtaining satisfactory sequential images.

Because we were not able to complete the entire scope of this project, it is our hope that someone else will use our methodology for the Dunn chamber experiment. We suggest that the next people to perform this experiment use a plating ring that is slightly larger. This will provide a larger surface area for cell attachment, while still keeping cells only in the inner well of the Dunn chamber. There are many more directions that this research could take. Keeping the cells alive in the Dunn chamber for long enough to obtain sequential images will be the next challenge in this research. After this step, the EGF receptors can be labeled with Texas Red antibodies to observe the redistribution of the receptors in the presence of EGF. Once the behavior of the receptors has been observed, the receptor can be altered to find the location of possible dysfunction in the signal transduction pathway. This discovery will aid in understanding the pathogenesis of IBD.

References

Polk DB. “Epidermal Growth Factor Receptor-Stimulated Intestinal Epithelial Cell Migration Requires Phospholipase C Activity” Gastroenterology 114 (1998): 493-502.

Polk DB, Tong W. “Epidermal and Hepatocyte Growth Factors Stimulate Chemotaxis in an Intestinal Epithelial Line.” Grant Report

Zicha D, Dunn G, Jones G. “Analyzing Chemotaxis Using the Dunn Direct-Viewing Chamber.” Methods in Molecular Biology 75: 449-457.