Optimization of primary endothelial culture methods and assessment of cell signaling pathways in the context of inflammation

摘要

Tissue engineering is a potentially valuable tool for clinical treatment of diseases where host tissues or organs need to be replaced. Progression of engineering metabolically complex organs and tissues has been severely limited by the lack of established, functional vasculature. The thesis work described herein focused on methods of establishing and studying specific endothelial cell types in vitro for potential applications in establishing functional microvascular architecture. To achieve these objectives, a model system of primary liver sinusoidal endothelial cells (LSEC) was initially studied due to the high metabolic requirements of the liver, as well as the unique phenotype that they possess. We were able to demonstrate that free fatty acids were able to rescue LSEC in culture, promote proliferation, and maintain their differentiated phenotype. Our work with lipid supplementation in serum-free conditions provides flexibility in engineering liver tissue with a functional vasculature comprised with relevant endothelial types encountered in vivo. Following up our work with LSEC, we explored the human dermal microvascular endothelial cell (HDMVEC) system to understand the signaling mechanisms involved in sprouting angiogenesis. Engineered tissues that are implanted will require integration with host vasculature. We established a method to collect large signaling data sets from a physiologically relevant in vitro culture system of HDMVEC that permitted angiogenic sprouting. We were able to find statistically significant data regarding how angiostatic cues like Platelet Factor 4 can modulate angiogenesis signaling pathways. Our results from working with both types of endothelial cell systems provide insight into potential methods for establishing specialized microvasculature for engineered tissues, both in propagation of differentiated endothelial cells in vitro and promotion of tissue/organ survival following their implantation.