Project Abstract: Lockett, Matthew Ryen Despite the importance gradients play in regulating tissue formation, structure, and homeostasis there are few analytical tools capable of generating tissue-like environments with experimentally defined oxygen gradi- ents. The tools that are available have not been widely adopted by tissue culture laboratories because they require specialized equipment and engineering expertise to setup, maintain, and analyze. To enable the study of oxygen’s role in directing responses at the cellular, tissue, and organ level new culture platforms are need- ed. My laboratory has been developing a paper-based culture platform for the last four years. This platform is unlike any other. Employing simple technological solutions, we can readily generate 3D cultures with defined extracellular environments, regardless of cell type or tissue structure. The level of experimental control afford- ed by the paper culture platform, makes them a powerful enabling technology to probe cellular responses in well-defined extracellular environments This MIRA application builds upon our prior successes and continues to innovate the paper platform. In par- ticular, we will leverage our ability to generate defined extracellular gradients to study the role of oxygen in regulating cellular phenotype, modulating protein expression and activity, and promoting directed movement. The diffusion-dominated gradients formed in our platform are similar to those that form in healthy tissue, and we are able to generate both healthy tissue environments as well as those resulting from ischemia or poor vascularization. In particular we will: 1) Develop tools to characterize the gradients that form in the paper cul- tures, focusing on oxygen, pH, glucose, and lactate. Through the use of luminescent extracellular, intracellular, and transcription-based sensors we will determine the design rules to generate gradients on demand. Such design rules will help others interested in studying particular aspects of the tissue microenvironment. 2) Evalu- ate differences between immotile and highly invasive cells. These datasets will shed light on the microenvi- ronment’s role—in particular oxygen gradients—in promoting varied cellular responses that result in move- ment, drug resistance, etc. 3) Generate tissue-like co-cultures with physiologically relevant oxygen gradients. By generating liver and breast lumen models, we will be able to experimentally determine how oxygen gradi- ents lead to zonation (liver) and regulate hormone receptor activity (breast). 4) Develop a platform to screen multiple 3D tissue structures in parallel. The ability to generate and evaluate many tissue structures in parallel will greatly improve screening processes to assess liver toxicity and identify potential endocrine disruptors.