Automatic image analysis method for quantification of tube formation by endothelial cells in vitro

Abstract

Early stages of tumor angiogenesis can be modeled by various in vitro cultures in which endothelial cells (ECs) form networks that are considered to mimic the vascularization of tumors in vivo. Image based quantification of EC culture model is a useful method for effective characterization of early stage in vitro vasculogenesis and the effects of pro and anti-angiogenesis reagents. We propose an image analysis method to quantify the EC tube formation in 2D cultures. The method segments images by high pass filtering in Fourier space, followed by thresholding and a skeletonization and pruning process to generate the binary skeleton image of the cell patterns in culture. Several quantities such as the network entropy (NE), the node number, total number of chords, total and average chord length were used to quantify the evolution of EC tubes. The automatic measurement of chord length was validated against manual measurement, achieving an R² value of 0.953, and was used to assay for tubal extension as a function of increasing VEGF concentration. Measurements of NE, node number, chord lengths were demonstrated on ECs network-like patterns in culture.