Substratum interactions modulate interplay between endothelial cell, epithelial cell, and fibroblast phenotype and immunomodulatory function.

Endothelial cells (ECs) sense and adapt to their environment, allowing them to display a range of functional phenotypes and promote vascular homeostasis across organ systems. However, many of these cues are lost when cells are cultured in vitro. This work explores how substratum interactions influence cellular phenotype. Culture conditions, specifically 2D culture on tissue culture polystyrene (TCP) versus 3D culture on collagen scaffolds, had a much greater effect on EC phenotype than did in vivo cell source. The 3D ECs responded to hypoxic gradients by inducing the expression of HIF1-a while 2D ECs underwent endothelial-to-mesenchymal transition. In comparing the effect of culture condition on EC phenotype and function to its effect on epithelial cells (EPs) and fibroblasts (FBs), it is evident that ECs are not simply vascular EPs but are unique in their response. For cell types like ECs, which are particularly responsive to their microenvironment, traditional culture on TCP is insufficient for revealing in vivo behavior. Further applying these findings, we found that culture-condition differentially affected the expression of immunomodulatory factors and in an in vivo model of allotransplantation, 3D culture reduced the cytotoxic response of host immune cells to all three cell types compared to 2D culture.