Cell body area expansion is a key factor determining connectivity and diameter of on-chip vascular networks

This study aimed to identify the key determinants of vascular networks (VN) morphology, with a particular focus on the role of endothelial cell body area expansion. We introduced a quantitative metric, the cell body area expansion (CBE) score, to assess endothelial cell (EC) enlargement during the early stage of self-organizing vascular network formation. The CBE score positively correlated with both VN connectivity and vessel diameter. Notably, VNs formed in the absence of growth factors exhibited reduced connectivity accompanied by lower CBE scores, while protrusion formation remained unaffected. Additionally, comparative analysis of multiple EC types revealed that cells forming wider vascular branches consistently showed higher CBE score. A mathematical model further supported these findings, demonstrating that greater CBE leads to VNs with enhanced connectivity and larger branch diameters. STATEMENT OF SIGNIFICANCE: The on-chip vascular network, a luminal network structure formed by endothelial cells within a microfluidic device, has attracted significant attention as a promising platform for vascular disease modeling and coculture with spheroids and organoids. However, the complexity of the vascular formation process poses challenges in optimizing network formation. This study identifies cell body expansion (CBE) as a key determinant of vascular network morphology through time lapse observations and image analysis of the formation process. Furthermore, mathematical modelling provided consistent supporting results. These findings provide valuable insights into vascular network formation and serve as a framework for designing vascular networks with desired connectivity and diameter.