An Automated Quantification Tool for Angiogenic Sprouting From Endothelial Spheroids

Abstract

The process of sprouting angiogenesis can be measured in vitro using endothelial cells in sprouting assays such as the fibrin bead assay and the spheroid-based assay. While the technical aspects of these sprouting assays have been well-optimized, the analysis aspects have been limited to manual methods, which can be time-consuming and difficult to reproduce. Here, we developed an automated analysis tool called AQuTAS to quantify sprouting parameters from the spheroid-based sprouting assay. We trained and validated the algorithm on two subsets of data, and tested its sensitivity by measuring changes in sprouting parameters over a range of concentrations of pro- and antiangiogenic compounds. Our results demonstrate that the algorithm detects known differences in sprouting parameters in endothelial spheroids treated with pro- and antiangiogenic compounds. Moreover, it is sensitive to biological changes that are ≥40%. Among the five quantified parameters, cumulative sprout length is likely the most discriminative parameter for measuring differences in sprouting behavior because it had the highest effect size (>1.5 Cohen’s d). In summary, we have generated an automated tool that quantifies sprouting parameters from the spheroid-based assay in a reproducible and sensitive manner.

NRP1 interacts with endoglin and VEGFR2 to modulate VEGF signaling and endothelial cell sprouting

“…Spheroid sprouting assay. Sprouting of MEECs was measured using a modified protocol previously reported 95,96 . Briefly, MEEC +/+ or MEEC -/were cultured 24 h in 60 mm dishes to reach 70−80% confluence.…”

Parathyroid-on-a-chip simulating parathyroid hormone secretion in response to calcium concentration

“…In the parathyroid-on-achip, the possibility of engraftment of parathyroid organoids was confirmed by measuring the branches of organoids because a vascular sprout indeed becomes a capillary. 47,48 The vascular endothelium was formed between the two separate compartments of the parathyroid-on-chip as shown in Fig. 5(a and b), and the parathyroid organoids were ladened in fibrin gel as shown in Fig.…”

A novel 3D spheroid model of rheumatoid arthritis synovial tissue incorporating fibroblasts, endothelial cells, and macrophages

“…Of note, diverse quantitative approaches have been developed to quantify cell migration in 3D models, particularly to describe angiogenesis. The ImageJ plugin "Sprout morphology" and the Matlab-based algorithm "AQuTAS" were both created to assess angiogenic sprouting from EC spheroids (34,35). Comparably to EC sprouting, FLS have previously been documented to extend and retract a so-called dendritic network of extensions in collagen matrices (36).…”