Effects of Epidermal Growth Factor Presentation on Cellular Behavior Grant Karlsson Ellifson, Chloe S. Kim, M.S., Kristyn S. Masters, Ph.D., Pamela K. Kreeger, Ph.D. Introduction Results/Data Discussion Annually, 20 billion dollars are spent on chronic wound healing issues1, and 5-7 million Americans suffer from chronic skin wounds. In many of these chronic wounds, there are decreased levels of epidermal growth factor2. Epidermal growth factor has been shown to effect cell behavior in a variety of different ways. Many conclusions can be drawn from the data I collected. Some of the most important conclusions that can be made are that the presentation of EGF affects cells in different ways. This can clearly be seen in the differences in cell area even when the total amount of EGF is the same. Also, variability in the combinations of EGF Presentation result in variability in cell behavior. Another important conclusion that can be drawn from the data is the EGF affects cells differently depending on whether or not they are in the bulk or on the edge. Based on the data I collected, it appears that the cell area increases more for the edge cells, and that non edge cells exposed to high levels of EGF tend to proliferate more than their counterparts on the wounded edge. Experimental Design Epidermal growth factor (EGF) is a growth factor that stimulates cell proliferation, growth, and migration. In the human body, EGF has two main presentation schemes: immobilized, and soluble3. For the experiment conducted, immobilized EGF was linked the the bottom of a 24 well plate using the cross linker sulpho-SANPAH. while the soluble EGF was simply disolved into the DMEM used as a media. These graphs show the results of computing the area of cells relative to their location and presentation of EGF. The bars with the same colors represent and equal amount of EGF being presented. The data here shows that the cells on the “wounded edge” tended to expand more than the cells in the bulk. Also, the presence of both immobilized and soluble EGF in Combination on the “wounded edge” resulted in an increased cell area. EGF EGF EGF EGF EGF EGF EGF EGF Immobilized Soluble Sulpho-SANPAH, Sulpho-SANPAH, In the images to the below, different wells were treated with the immobilized EGF or no EGF, and either secondary (Fluorescent), or both primary (anti-EGF) and secondary. They were then imaged. The brighter the image, the higher the concentration of EGF. The brighter image for the High Immobilized Primary and Secondary well validates that the cross linking process results in EGF present on the bottom of the plates we use for migration assays References 1. Germansky M, Ginsburg P. Professional practice-clinical practice outcomes; standards of care; clinical pathways: 3358: Chronic wound healing in the home: A wound care pilot program. J Wound Ostomy Contin Nurs 2009; 36: S43. 2. C. M. Dipersio, Double Duty for Rac1 in Epidermal Wound Healing SCI STKE 2007, pg33 (2007) 3. Nimni M. Polypeptide growth factors: targeted delivery systems. Biomaterials 1997; 1201-25. Control Low Immobilized Low Soluble Control High Immobilized High Soluble Control Secondary Control Primary and Secondary High Immobilized Secondary These graphs show the results of calculating the percent proliferation of cells based on their location and presentation of EGF. The bars with the same colors represent equal amounts of EGF with variable presentation. The pictures below the graphs are there as visual evidence that the different levels of proliferation exist. The green cells were stained with Click it EDU which stains proliferating cells while the cells that are blue are the cells that only were stained by the DAPI stain which gives the user a total cell count. High Immobilized Primary and Secondary For my experiments, HaCat cells were seeded to be confluent around IBidi devices on plates with varying levels, and presentation schemes of EGF. After the trials were completed, the cells were analyzed for their proliferation levels and their cell area based on if they on the edge of the cell mass or if they were in the bulk. The cells were imaged, and their data was placed into excel graphs shown in the results section