Quantitative analysis of human umbilical vein endothelial cell morphology and tubulogenesis.

Abstract

Primary human umbilical vein endothelial cells can grow as both a monolayer in culture and also as a capillary-like network making them an ideal model system in order to study vascular remodelling. Image-based analysis can allow assessment of cell morphology and motility but is dependent on accurate cell segmentation which requires high-contrast images not normally achievable without fluorescent markers. Here, ptychography is employed as a label-free image-based modality in order to extract quantitative metrics of morphology and tubulogenesis from cultured HUVECs over time in an automated multiwell assay. Phase-specific parameters of dry mass, optical thickness and sphericity were extracted and assessed alongside other metrics of cell number and shape. Tubulogenesis could be captured dynamically without any imaging artefacts from use of a basement membrane matrix and metrics of tube number, growth and branching exported alongside morphology metrics at early time-points. Utilising ptychography-based image analysis, all VEGF₁₆₅a isoforms studied, elicited a concentration-dependent effect on cell elongation and survival within a HUVEC monolayer. Pharmacologically relevant parameters of potency (EC₅₀) and efficacy were derived, exemplifying this label-free approach for the multiparameter and multiwell quantitative study of vascular remodelling in physiologically relevant cells at 37°C.