A robust vasculogenic microfluidic model using human immortalized endothelial cells and Thy1 positive fibroblasts.

Human umbilical vein endothelial cells (HUVECs) and stromal cells, such as human lung fibroblasts (FBs), have been widely used to generate functional microvascular networks (μVNs) in vitro. However, primary cells derived from different donors have batch-to-batch variations and limited lifespans when cultured in vitro, which hampers the reproducibility of μVN formation. Here, we immortalize HUVECs and FBs by exogenously expressing human telomerase reverse transcriptase (hTERT) to obtain stable endothelial cell and FB sources for μVN formation in vitro. Interestingly, we find that immortalized HUVECs can only form functional μVNs with immortalized FBs from earlier passages but not from later passages. Mechanistically, we show that Thy1 expression decreases in FBs from later passages. Compared to Thy1 negative FBs, Thy1 positive FBs express higher IGFBP2, IGFBP7, and SPARC, which are important for angiogenesis and lumen formation during vasculogenesis in 3D. Moreover, Thy1 negative FBs physically block microvessel openings, reducing the perfusability of μVNs. Finally, by culturing immortalized FBs on gelatin-coated surfaces in serum-free medium, we are able to maintain the majority of Thy1 positive immortalized FBs to support perfusable μVN formation. Overall, we establish stable cell sources for μVN formation and characterize the functions of Thy1 positive and negative FBs in vasculogenesis in vitro.