PROJECT SUMMARY For more than three decades, scientists have aspired to create engineered tissues that mimic the remarkable physiological and functional properties of natural tissues. Such tissues and organs can be used not only for transplantation to save lives but also for ex vivo tissue-on-a-chip approaches to test pharmaceuticals the response to pathogens. However, we still lack the ability to create tissues with three-dimensional tissue-mimetic vasculature. Without 3D, smooth branched, and multi-scale vascular networks, which facilitate nutrient/oxygen transport, engineered full-thickness tissues will not be fully functional due to cell death limited by diffusion. As such, biomimetic vasculature networks are very important for viable and clinically relevant tissue constructs. We propose an innovative approach to address the challenge of fabricating tissue constructs with biomimetic vasculature. Our approach involves 3D freeform ice printing of sacrificial vasculature templates and uses them to fabricate scaffolds for creating biomimetic vascularized tissue constructs. Our novel 3D freeform (as opposed to layer-by-layer) ice printing process uniquely enables fabricating ice templates that mimic the geometry of the actual vasculature, including complex shapes, circular cross-sections, and varying diameters and branched structures with smooth transitions. Our fundamental hypothesis is that our 3D ice printing technique enables the creation of tissue scaffolds with biomimetic 3D vasculature networks. In this R21 project, we aim to develop our 3D ice printing-based vascularized tissue fabrication platform and to demonstrate its feasibility by creating human-skin tissue-on-a-chip systems with 3D biomimetic vasculature that also in the future can be used for tissue implantation. Our preliminary results show our general ability to print multi-scale ice structures, fabricate porous scaffolds with vasculature conduits, and grow endothelial and fibroblasts cells on porous dissolvable scaffolds. The proposed studies aim to show the feasibility of our approach towards creating vascularized tissues. Our approach, based on our preliminary data and published experience, will involve two Specific Aims: Aim 1 will focus on the development of our 3D ice printing technique to reproducibly create defined biomimetic vasculature within porous tissue scaffolds. Aim 2 will demonstrate our approach for creating 3D vascularized tissue-on-a-chip constructs using the fabricated scaffolds and multiple cell systems. Our interdisciplinary project team combines complementary expertise and research infrastructure that directly addresses the proposed project. The PIs have a decade-long history of strong collaboration, including co-advised PhD students and multiple co-authored publications. We expect the results of this work will bring the ability to create tissue scaffolds with 3D biomimetic vasculature toward creating many different vascularized tissues and organs in the f...