Abstract

Microvessels are essential for tissue engineering and regeneration. In current methods, endothelial cells are usually cultured in commercially available media and form a monolayer of cell sheets on stiff substrates and a tubular structure when cultured with soft hydrogels. To mimic the microvessels in vivo, researchers usually coculture the endothelial cells and pericytes from different adult tissues or derived from pluripotent stem cells in a three-dimensional hydrogel. However, there is a challenge for these models to reflect tissue-specific characteristics due to the vascular heterogeneity throughout the body. Here, we established a culture model for expanding adult tubular microvessels on stiff substrates with endothelial cells and pericytes derived from the same tissue. We isolated microvessels from adult rat subcutaneous soft connective tissue and cultured them on regular plastic dishes. We performed a series of screenings and formulated a custom-made medium (Medium-X), containing mainly antioxidants and three small molecules, Chir99021, A83-01, and Y27632. Medium-X significantly promoted adult microvessel growth while maintaining their characteristic tubular morphology up to 8 weeks in vitro, contrary to the monolayer of endothelial cell sheets in the commercially available medium EGM2MV. Transcriptomic analysis showed that Medium-X maintained the tubular morphology of microvessels by promoting angiogenesis and vascular remodeling while suppressing oxidation and lipid metabolic pathways. The model presented in this study can be applied to other organs for expanding organ-specific microvessels for tissue engineering and vascular regeneration.