PROJECT SUMMARY/ABSTRACT The ten peer-reviewed and NIH funded Collaborative Projects and the eight Service Projects from leading hyperpolarized MR sites, that are the scientific driving force behind the HMTRC, demonstrate a clear need for the improved HP probes and techniques, realistic pre-clinical models and correlative pathology and biology methodologies that will be provided by this TR&D project. TR&D2 builds on our significant experience in the DNP polarization/dissolution process, the development of new hyperpolarized molecular probe preparations, and in the development and testing of novel biologically relevant NMR-compatible pre-clinical cell/tissue culture and associated novel cell/tissue graft murine models. In the current funding cycle, we developed new 13C labeled HP probes of glycolysis, TCA cycle and cellular redox as well as multi-polarization approaches that provide simultaneous information on metabolism, perfusion, and extracellular pH (pHe). Through this renewal, we aim to greatly improve these HP probes and methods with an emphasis on increased sensitivity, robustness, cost-effectiveness, ease of dissemination, and translatability. This will be accomplished through the use of glassing agents, chemically optimized new radicals, and polarization methods. We will also investigate novel HP probes of pHe, ROS and immune response and to address new questions raised by the CP's (aim 1). In this renewal, we will also investigate new long-lived hyperpolarized probes and use of lower field MRI scanners in order to prolong the T1's of hyperpolarized probes. In the current project, novel 10 and 5 mm MR and PET compatible 3D cell and tissue culture bioreactors were micro-engineered, robustly and cost- effectively produced, and extensively tested and validated in cell and tissue culture studies performed in the CP's. Driven by the needs of the CP's, we now propose future bioreactor designs incorporating new biologic measurement capabilities, addition of novel cell culture constructs, development of a micro-bioreactor system, and extension of human cell & tissue bioreactor studies to novel murine models (aim 2). TR&D2 also made substantial progress developing and implementing procedures to provide correlative pathologic, biologic and imaging data critical for understanding and validating HP MR findings in the CP's. As requested by the EAC and CP's, these validation studies will be augmented by the addition of new metabolomics, enzyme activity staining assays, and PET/HP MR correlative studies (aim 3). While the HP MR probes, model systems and correlative methodologies have been specifically developed for the CP's through a highly productive push-pull approach, these technology developments also have significantly benefited the SP's and the HP MR community in general through extensive dissemination and training as will be continued and expanded through this renewal.