Project Summary/Abstract Liver disease is a pressing public health challenge, because unlike most other major killers deaths due to liver disease are rapidly rising rather than falling. Although liver transplantation prolongs survival, there is a growing number of patients in need of transplant, but donor supply has remained stagnant. To address this major medical problem, we are working to build artificial liver tissue that could serve as a bridge or alterative to organ transplant. A crucial remaining hurdle for developing artificial liver tissue is building the multiscale vasculature needed to support billions of densely packed hepatocytes. Novel approaches that address this challenge would transform liver research and therapy. Our recent work pushed the field closer to addressing this hurdle by introducing a breakthrough method for 3D printing volumetric vascular networks in artificial tissues. This advance was made possible by addition of photoabsorbers to stereolithography bioinks, which enabled millions of voxels to be patterned over many tissue layers. Yet, tissues produced with stereolithography remain incompletely vascularized and sparsely cellularized, with functional levels that still fall short of those needed for therapy. We have recently gained important clues towards addressing this challenge. First, we identified new photoabsorber formulations that substantively improve print resolution, providing a new route to volumetrically scaling a denser vasculature. Furthermore, we found that adding biological matrices to bioinks allows us “expand” vasculature and hepatocytes within printed tissues after implantation in the body to produce tissues with native density. These data lead us to hypothesize that dual-role bioinks that support both technical and biological modes of scale-up will facilitate generation of human liver tissue with volumetric vasculature that expands in vivo. Further, such tissue will have hepatic functional levels sufficient to therapeutically treat liver disease. To test these hypotheses, we established a team with synergistic expertise in liver and vascular tissue engineering, biomaterials and bioprinting, clinical liver surgery, clinical hepatology, liver cell biology, and liver metabolism. We will employ our expertise to develop vascularized bioprinted liver tissue that grows in the body. We will first formulate a new library of bioinks for projection stereolithography with improved print resolution and bioactivity, to facilitate both 3D printing and in vivo tissue engineering (Aim 1). We will then 3D print scaled vascular topologies that mimic liver vasculature and support hepatocyte engraftment (Aim 2). Finally, we will trigger hepatocyte expansion in the tissues to achieve hepatocyte density and functional levels sufficient to rescue mice with liver injury (Aim 3). The real power of this proposal lies in conflating bioprinting and biological modes of tissue scale-up, which will transform tissue engineering a...