Endothelial layers cultured on an aligned fibrin matrix exhibit enhanced barrier integrity

Abstract

The vascular endothelium is important in trafficking cells and molecules across the interface. Microphysiological systems (MPS) mimicking their barrier functions have demonstrated various utilities, including drug permeability tests and cell transmigration. However, conventional approaches for constructing endothelial layers in MPS mainly rely on seeding cells on porous membranes coated with extracellular matrix molecules that are sparsely dispersed, which do not represent their inherent microenvironmental characteristics, resulting in immature endothelial barrier functions. Here, we report that endothelial layers cultured on an aligned fibrin matrix (aFM) exhibit enhanced barrier integrity and more tightly regulate the transmigration of metastatic cancer cells. Human platelet-poor plasma (PPP), inherently containing fibrinogen, was flowed through a microfluidic MPS device separated vertically by 8 μm polyester track-etched (PETE) membranes at a flow rate that induced a shear stress of 0.01 dyne per cm². This resulted in the forming of an integrated fibrin fiber matrix through calcium-mediated crosslinking, with the fibers oriented along the flow direction. Human umbilical vein endothelial cells (HUVECs) cultured on aFM exhibited elongated and directional alignment of the cells, resulting in approximately up to a 2.5-fold increase in adherens and tight junction protein expression, along with a more than 30% reduction in permeability compared to those cultured on isotropically oriented fibrin matrix (iFM) or conventional fibronectin (FN)-coated PETE membranes. The co-culture with metastatic human breast cancer cells (MDA-MB-231) significantly compromised VE-cadherin of endothelial layers formed on conventional FN or iFM membranes, while endothelial cells (ECs) cultured on aFM maintained VE-cadherin junctional integrity even with the presence of MDA-MB-231 cells. Consequently, the number of MDA-MB-231 cells transmigrated through the endothelial layers on FN or iFM membranes was significantly higher than those observed in aFM due to the leaky EC layers damaged by the metastatic cancer cells. Thus, our approach to creating an anisotropic ECM microenvironment in MPS devices provides versatile utility for studying barrier functions by modulating mechanical cues and relevant gene expression in barrier-forming cells.