Fabrication of vascularized tissue constructs under chemically defined culture conditions

Abstract

Three-dimensional (3D) biofabrication techniques that enable the production of multicellular tissue equivalents for applications in basic biology, drug screening and regenerative medicne. Fabrication of these tissue constructs with in-built microvasculature enables recapitulation of the biological environment of the native tissues. Here, we present the fabrication of 3D vascularized tissue constructs containing microvascular networks using human embryonic stem cell (hESC)-derived endothelial cells (ECs) and pericytes encapsulated within a fibrin-based matrix and cultured under chemically defined conditions. Firstly, by manipulating the developmental signaling pathways under chemically defined culture conditions, hESCs were efficiently differentiated to hESC-ECs and hESC-pericytes through intermediate stages of lateral plate and paraxial mesoderm respectively. Next, encapsulation of these hESC-derived vascular cells within fibrin-based matrix and culture under chemically defined conditions, result in self-assembly of hESC-ECs into a network of microvessels within a period of 6-9 d. With the supporting influence of hESC-pericytes, the microvascular network with lumen was stable for at least 3 weeks. Quantification of the fractal dimensions of the microvascular networks demonstrate the increasing complexity of the vascular network with increasing endothelial cell densities. Dextran permeation studies in the presence or absence of vasodilating agent (histamine) showed the presence of hollow lumen, modulation of barrier properties of the microvasculature and its functional response to histamine. Hence, this versatile in vitro 3D model of vascularized constructs generated under chemically defined conditions is well suited to study early angiogenesis for in vitro drug testing applications and provide a clinically amenable, fundamental step towards fabrication of complex and functional tissues for regenerative applications in the future.