Fabrication of blood capillary microtissues without fibroblast support via growth factors and matrix stiffness modulation

Abstract

The cells co-culture approach, involving endothelial cells and supporting stromal cells, such as fibroblasts, is commonly used for engineering microvascular networks. While this approach effectively promotes vascular morphogenesis through paracrine signaling and matrix remodeling, it often leads to excessive fibroblast proliferation. This uncontrolled growth can disrupt the structural organization of the developing vasculature, making it challenging to achieve reproducible and physiologically relevant microtissue architectures. In this work, we introduce an alternative monoculture method that uses only endothelial cells (HUVECs) in a fibrin gel matrix. To promote the formation of structured capillary-like networks without stromal support, we optimized vasculogenesis by supplementing exogenous vascular endothelial growth factor (VEGF), fine-tuning matrix stiffness, and applying it in a hypoxic environment (1% O₂). This approach was also applied to brain microvascular endothelial cells (BMEC) and liver sinusoidal endothelial cells (SEC). This innovation addresses the limitations of traditional methods, overcomes rapid matrix degradation caused by fibroblast-mediated remodeling, identifies ∼2.56 kPa as the optimal stiffness for blood capillary growth, and demonstrates that capillary development is significantly enhanced at VEGF concentrations above 50 ng ml⁻¹.