Elucidating the role of disparities in the oxygen microenvironment in organoids and engineered tissues

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Local oxygen concentration is a primary driver of cell behaviour in vitro and in vivo, but is surprisingly difficult to assess and interpret even in simple cell culture systems. The role of oxygen in vivo has been extensively studied under normal conditions, and in disease states including myocardial and cerebral ischemia, wound healing and infection, although this is primarily at the tissue level. Considerably less is known about the hyper-local distribution of oxygen at the scale of the individual cell, how it affects their behaviour, and how oxygen delivery and consumption is impacted by microscopic structures such as epithelia, and extracellular matrices. In normal living tissues, blood vessels constantly constrict, relax, grow and are remodelled, while breathing and heart rates adjust continuously, and red blood cells are created and broken down - providing cells with a consistent and finely-tuned supply of oxygen. In contrast, cells in culture of the same type, in the same incubator, can experience oxygen levels ranging from far above to far below normal, due only to differences in density and medium volume. Just as water thrown on a fire provides a barrier between fuel and air, the medium in which most cell and tissue cultures are submerged significantly impacts their access to oxygen. Cell density, metabolic rate, and medium depth are critical variables - and these can all change over the course of an experiment. Cutting-edge research increasingly requires more accurate models of biological systems. 3D cultured tissues known as "organoids" are a powerful and popular tool for this purpose, however their complexity adds to both the challenge and importance of understanding the role of oxygen in their behaviour. The funding requested here will allow us to establish genetically-encoded oxygen reporter constructs, introduce them into diverse organoid types, calibrate them, and apply them to understand the role of oxygen in those organoids. We will generate very low-cost biological monitoring systems, and educate trainees in their use, and in the philosophy of research reproducibility that underlies them. Internationally, researchers in a wide range of disciplines have demonstrated the effectiveness of this strategy by adopting my approaches and "AggreWell" organoid manufacturing platform as standard methods. Our progress towards filling this knowledge gap will be broadly useful to this large and rapidly growing audience. This program is part of my long-term vision of making organoid culture systems widely accessible, consistent and reproducible, to accelerate research in the biological sciences around the world. In addition to publishing our findings, the novel tools we develop will be distributed globally, and benefit cell and tissue culture research (and the biomanufacturing industry being built upon it) in Canada and around the world, following in the footsteps of my previous successes at this level.