Angiogenesis and vasculogenesis are essential neovascularisation processes. Various cell types and growth factors are involved, with vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 identified as key components. The PhD project “In vitro vasculogenesis in 3D” tested the effect of parameters such as support cells, matrix composition and physiological hypoxia on the morphology and aggregation of ECs in 3D collagen hydrogels. Different aggregation patterns were identified depending on the culture conditions tested, and these were found to reflect the different developmental pathways that ECs take to form different sized tubular structures. ECs formed contiguous sheets in collagen only hydrogels, analogous to the ‘wrapping’ pathway in development. In contrast, in co-cultures in 3D collagen-laminin cultures, end-to-end networks formed, mimicking cord hollowing and cell hollowing. A relationship between matrix composition, growth factors and VEGF receptor levels in 3D collagen hydrogels was shown for the first time in this study. Results showed a key linkage between integrin expression on ECs and their uptake of VEGF, regulated by VEGFR2, resulting in end-to-end network aggregation in HBMSC-HUVEC co-cultures. The effect of physiological hypoxia on EC aggregation was also tested by lowering the oxygen tension to 5% O2 using a controlled culture environment. Angiogenic growth factors were quantified using ELISA and their levels were correlated to EC morphological progression within 3D collagen hydrogels. Overall, the findings here showed how different parameters affected EC morphology and aggregation in 3D in vitro collagen hydrogels. The study provides an understanding of how these individual parameters influence EC morphology and show the mechanisms of how this is achieved in 3D in vitro
