Video_4_The Use of Live Cell Imaging and Automated Image Analysis to Assist With Determining Optimal Parameters for Angiogenic Assay in vitro.MP4

Testing angiogenic potential and function of cells in culture is important for the understanding of the mechanisms that can modulate angiogenesis, especially when discovering novel anti- or pro-angiogenic therapeutics. Commonly used angiogenic assays include tube formation, proliferation, migration, and wound healing, and although well-characterized, it is important that methodology is standardized and reproducible. Human endothelial progenitor cells (EPCs) are critical for post-natal vascular homeostasis and can be isolated from human peripheral blood. Endothelial colony forming cells (ECFCs) are a subset of EPCs and are of interest as a possible therapeutic target for hypoxic diseases such as kidney disease, as they have a high angiogenic potential. However, once ECFCs are identified in culture, the exact timing of passaging has not been well-described and the optimal conditions to perform angiogenic assays such as seeding density, growth media (GM) concentrations and end-points of these assays is widely varied in the literature. Here, we describe the process of isolating, culturing and passaging ECFCs from patients with end-stage renal disease (ESRD), aided by image analysis. We further describe optimal conditions, for human bladder endothelial cells (hBECs), challenged in angiogenic assays and confirm that cell density is a limiting factor in accurately detecting angiogenic parameters. Furthermore, we show that GM along is enough to alter the angiogenic potential of cells, seeded at the same density. Lastly, we report on the success of human ECFCs in angiogenic assays and describe the benefits of live-cell imaging combined with time-lapse microscopy for this type of investigation.