Automated high-throughput live cell monitoring of endothelial cell migration

Abstract

Background: Cellular migration is important for physiological and pathological processes. As such, angiogenesis and regenerative wound healing are reliant on the promotion of distinct endothelial cell phenotypes exhibiting increased migratory capacity. Functional monitoring of these hallmark events in vitro is invaluable for discovering novel therapeutics. However, while respective methods are usually simple and economic, they often lack a high-throughput character or accurate analysis tools, which are essential for effective screening suitability. Experimental approach: We stained nuclei of confluent human umbilical vein endothelial cells with Hoechst33342 prior to induction of an artificial scratch wound. Treatments with various growth factors and several concentrations of nintedanib were performed to evaluate impacts on wound closure. Images were taken frequently over 24 h to achieve high time-resolution. We developed an ImageJ macro and a Python script for automated analysis of these image sets. Utilizing cell-free area measuring or cellular density evaluation, respectively, cellular migration behavior was assessed well-wise for each time point. Key results: We proved the functionality of our novel tools and identified pro-migratory effects of interleukin 1β as well as inhibitory actions of nintedanib. Hoechst33342 staining allowed for cell counting which could be excluded as a contributing factor to wound closure in our assay. Conclusion: We herein developed a cost-effective, high-throughput pipeline that allows to monitor endothelial cell migration in vitro. We believe that our protocol will significantly accelerate pre-clinical screenings not only for medications targeting angiogenic processes but also drug discovery research in a broad range of diseases with altered vascularization.