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

Abstract

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.

Biochemical Gradients to Generate 3D Heterotypic‐Like Tissues with Isotropic and Anisotropic Architectures

“…The v) top and vi) bottom regions of the scaffolds were analyzed to identify the tubular segments created per region. vii) The tubular segments were identified in the microscopic images using Angiogenesis Analyzer tool for ImageJ (NIH, v. 1.52e, http://imagej.nih.gov/ij). These segments were quantified per area and their formation profile was traced at three time‐points, 3, 5, and 7 d. Data expressed as (mean ± SE).…”

“…Construct were imaged on top and bottom regions at day 5 and 7 by fluorescent microscopy. ImageJ/FIJI (NIH, v. 1.52e, http://imagej.nih.gov/ij) plugin Angiogenesis Analyzer was applied to evaluate the major segments formation. Bright field and fluorescent images were individually analyzed and their outcomes were combined in average to reduce potential artefacts occurring in each image type ( n = 5).…”

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis <em>In Vitro</em>

“…Typically, following overnight incubation, images of cell networks are captured at a single time point, resulting in a small number of images for analysis 11 12 13 . Therefore, analytic approaches have largely been developed and optimized for single time point assessments 10 14 . However, static analyses are simply insufficient at capturing the dynamic process of vessel formation and provide limited insight into potential mechanisms involved.…”

“…However, static analyses are simply insufficient at capturing the dynamic process of vessel formation and provide limited insight into potential mechanisms involved. Although increasing imaging frequency would likely provide the necessary data to identify formation kinetics, application of previously developed analytics to a multi-time point imaging approach would be inefficient and labor intensive 14 . Additionally, despite development of commercially available analyses, a pay-per-image fee renders this option cost-prohibitive for kinetic studies in which thousands of images are generated 15 .…”

A Circulating MicroRNA Profile in a Laser-Induced Mouse Model of Choroidal Neovascularization

“…Cells were counted with a CASY Cell Counter (OLS OMNI Life Science, Bremen, Germany) and 10,000 cells per well were seeded on the coated 96-well plate. Photos were taken after 5 h incubation at 37 • C, and the total length as well as the total segments length of the cellular network was determined using the Angiogenesis Analyzer [74] for ImageJ.…”