Mechanical and chemical control of barrier in engineered microvessels

The formation of thick tissue constructs is currently limited by the nutritional and metabolic demands of cells. To overcome this constraint, a variety of approaches have been undertaken to vascularize engineered tissues in vivo and in vitro. Many of these methods, however, still rely on some amount of self-assembly by cells to form perfusable networks, which may delay the implant's integration with the host's circulation. Moreover, while the field has focused heavily on engineering microvascular tissue, little interest has been paid to the 'maturation' of these constructs. The microvasculature throughout the body is extremely heterogeneous in terms of its morphology and physiology. Therefore, it is reasonable to expect that constructing microvessels with a tissue-specific physiology may not only optimize development and function of thick tissue constructs in the in vitro setting, but may also enhance survival and accelerate functional integration of the construct upon engraftment.;Presented at the beginning of this work are three detailed methods developed in our lab for forming microvascular tissue---one method produces single microvessels, the other two produce complex networks. Each procedure uses lithographic techniques to create pre-formed microfluidic channels within collagen; these channels are perfused and are lined by a monolayer of endothelial cells. This work also focuses on the roles of the chemical and mechanical microenvironments in regulating endothelial barrier function (a particularly well characterized aspect of microvascular physiology) and in promoting stability in engineered microvasculature. We added high concentrations of cyclic AMP to single microvessels composed of lymphatic endothelial cells---a cell-type that forms a necessarily weak barrier in vivo---and found that cAMP drastically enhanced (tightened) the barrier of these vessels. We also exposed single microvessels to different hemodynamic factors; this revealed that shear stress regulates the endothelial barrier in our constructs, while transmural pressure is required for vessel stability. Clearly, changes to the microenvironment affect the stability and function of engineered microvascular tissue and we intend these results to provide an initial guide for establishing designs that promote stability and tissue-specific barrier function in complex vascular networks.