Angiogenesis Analyzer for ImageJ — A comparative morphometric analysis of “Endothelial Tube Formation Assay” and “Fibrin Bead Assay”

Angiogenesis assays based on in vitro capillary-like growth of endothelial cells (EC) are widely used, either to evaluate the effect of anti- and pro-angiogenesis drugs of interest, or to test and compare the functional capacities of various types of EC and progenitor cells. Among the different methods applied to study angiogenesis, the most commonly used is the "Endothelial Tube Formation Assay" (ETFA). In suitable culture conditions, EC form two-dimensional (2D) branched structures that can lead to a meshed pseudo-capillary network. An alternative approach to ETFA is the "Fibrin Bead Assay" (FBA), based on the use of Cytodex 3 microspheres, which promote the growth of 3D capillary-like patterns from coated EC, suitable for high throughput in vitro angiogenesis studies. The analytical evaluation of these two widely used assays still remains challenging in terms of observation method and image analysis. We previously developed the "Angiogenesis Analyzer" for ImageJ (AA), a tool allowing analysis of ETFA-derived images, according to characteristics of the pseudo-capillary networks. In this work, we developed and implemented a new algorithm for AA able to recognize microspheres and to analyze the attached capillary-like structures from the FBA model. Such a method is presented for the first time in fully automated mode and using non-destructive image acquisition. We detailed these two algorithms and used the new AA version to compare both methods (i.e. ETFA and FBA) in their efficiency, accuracy and statistical relevance to model angiogenesis patterns of Human Umbilical Vein EC (HUVEC). Although the two methods do not assess the same biological step, our data suggest that they display specific and complementary information on the angiogenesis processes analysis.