7. PROJECT SUMMARY/ABSTRACT Tissue engineering through 3D printing is a promising approach to address the donor organ shortage that severely limits the impact of transplantation therapy. Two current challenges in tissue engineering are how to create a complex microvascular system with vessel segments of different caliber to provide effective graft perfusion and how to prevent tissue engineered grafts made from allogeneic cell sources from triggering immune-mediated rejection by the graft recipient. These problems are linked in that human endothelial cells (ECs), required for perfusion, are immunogenic and can trigger rejection. We propose to address these problems using synthetic skin as a model system. We have successfully developed bioinks containing human fibroblasts (FBs), endothelial cells (ECs) and pericytes (PCs) from a single donor source and all cultured under xeno-free conditions, within a matrix formed from human matrix proteins to create a papillary dermis with an epidermis formed from keratinocytes (KCs), also from the same donor source and also cultured under xeno- free conditions. In aim 1 we will optimize an additional bioink to create a collagen-dense reticular dermis as a deep layer, adding vascular smooth muscle cells to drive formation of larger caliber vessels, thereby forming a trilayered skin substitute. We will explore the use of delivering extracellular growth factors or intracellular gene modifiers to enhance microvessel formation after engraftment on immunodeficient mouse hosts and we will challenge engraftment by pharmacologically impairing wound healing. In aim 2, we will evaluate the immunogenicity of these bioengineered skin constructs by engraftment on to human immune system mice, using an established model as well as a new model with a more replete human immune system that has myeloid and natural killer cells in addition to alloreactive human effector memory T cells. Finally, we will apply state-of-the art genetic engineering or drug delivery approaches to reduce the immunogenicity of the constituent graft cells, e.g., by ablation of HLA antigen expression, assessing whether this needs to be performed on multiple cell populations. Our focus on skin as a target for generating a well perfused but non- immunogenic engineered tissue also addresses an unmet clinical need and our use of wholly human constituents to do so brings this closer to clinical translation. This project takes advantage of the synergistic expertise of the two multiple principal investigators who have worked together for over a decade. Successful completion of our aims will establish a prototype for developing a clinical therapeutic and will establish principles with broad implications for tissue engineering and regenerative medicine.