The geometrical control of micronetwork structures (μNSs) formed by endothelial cells is an important topic in tissue engineering, cell‐based assays, and fundamental biological studies. In this study, μNSs are formed using human umbilical vein endothelial cells (HUVECs) by the coculture of HUVECs and human mesenchymal stem cells (MSCs) confined in a honeycomb‐patterned poly‐l‐lactic acid film (honeycomb film (HCF)), which is a novel cell culture scaffold. The HCF is produced using the breath figure method, which uses condensed water droplets as pore templates. The confinement of the HUVECs and MSCs in the HCF along with the application of centrifugal force results in μNS formation when the pore size is more than 20 μm. Furthermore, μNS development is geometrically restricted by the hexagonally packed and connected pores in the horizontal direction of the HCF. Network density is also controlled by changing the seeding density of the HUVECs and MSCs. The threshold pore size indicates that μNSs can be formed spontaneously by using an HCF with a perfectly uniform porous structure. This result provides an important design guideline for the structure of porous cell culture scaffolds by applying a blood vessel model in vitro.

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In vitro vascular wall bilayer models for drug testing and disease modeling must emulate the physical and biological properties of healthy vascular tissue and its endothelial barrier function. Both endothelial cell (EC)‐vascular smooth muscle cell (SMC) interaction across the internal elastic lamina (IEL) and blood vessel stiffness impact endothelial barrier integrity. Polymeric porous track‐etched membranes (TEM) typically represent the IEL in laboratory vascular bilayer models. However, TEM stiffness exceeds that of diseased blood vessels, and the membrane pore architecture limits EC‐SMC interaction. The mechanical properties of compliant honeycomb film (HCF) membranes better simulate the Young's modulus of healthy blood vessels, and HCFs are thinner (4 vs. 10 μm) and more porous (57 vs. 6.5%) than TEMs. We compared endothelial barrier integrity in vascular wall bilayer models with human ECs and SMCs statically cultured on opposite sides of HCFs and TEMs (5 μm pores) for up to 12 days. Highly segregated localization of tight junction (ZO‐1) and adherens junction (VE‐cadherin) proteins and quiescent F‐actin cytoskeletons demonstrated superior and earlier maturation of interendothelial junctions. Quantifying barrier integrity based on transendothelial electrical resistance (TEER), membranes showed only minor but significant TEER differences despite enhanced junctional protein localization on HCF. Elongated ECs on HCF likely experienced greater paracellular diffusion than blocky ECs on TEM. Also, larger populations of plaques of connexin 43 subunit‐containing gap junctions suggested enhanced EC‐SMC communication across the more porous, thinner HCF. Compared with standard TEMs, engineered vascular wall bilayers cultured on HCFs better replicate physiologic endothelial barrier integrity.

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