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Abstract The use of oxygen by cells is an essential aspect of cell metabolism and a reliable indicator of viable and functional cells. Here, we report partial pressure oxygen (pO₂) mapping of live cells as a reliable indicator of viable and metabolically active cells. For pO₂imaging, we utilized trityl OX071-based pulse electron paramagnetic resonance oxygen imaging (EPROI), in combination with a 25 mT EPROI instrument, JIVA-25™, that provides 3D oxygen maps with high spatial, temporal, and pO₂resolution. To perform oxygen imaging in an environment-controlled apparatus, we developed a novel multi-well-plate incubator-resonator (MWIR) system that could accommodate 3 strips from a 96-well strip-well plate and image the middle 12 wells noninvasively and simultaneously. The MWIR system was able to keep a controlled environment (temperature at 37 °C, relative humidity between 70%–100%, and a controlled gas flow) during oxygen imaging and could keep cells alive for up to 24 h of measurement, providing a rare previously unseen longitudinal perspective of 3D cell metabolic activities. The robustness of MWIR was tested using an adherent cell line (HEK-293 cells), a nonadherent cell line (Jurkat cells), a cell-biomaterial construct (Jurkat cells seeded in a hydrogel), and a negative control (dead HEK-293 cells). For the first time, we demonstrated that oxygen concentration in a multi-well plate seeded with live cells reduces exponentially with the increase in cell seeding density, even if the cells are exposed to incubator-like gas conditions. For the first time, we demonstrate that 3D, longitudinal oxygen imaging can be used to assess cells seeded in a hydrogel. These results demonstrate that MWIR-based EPROI is a versatile and robust method that can be utilized to observe the cell metabolic activity nondestructively, longitudinally, and in 3D. This approach may be useful for characterizing cell therapies, tissue-engineered medical products, and other advanced therapeutics. 

Abstract Inadequate oxygenation is a major challenge in cell encapsulation, a therapy which holds potential to treat many diseases including type I diabetes. In such systems, cellular oxygen (O 2 ) delivery is limited to slow passive diffusion from transplantation sites through the poorly O 2 -soluble encapsulating matrix, usually a hydrogel. This constrains the maximum permitted distance between the encapsulated cells and host site to within a few hundred micrometers to ensure cellular function. Inspired by the natural gas-phase tracheal O 2 delivery system of insects, we present herein the design of a biomimetic scaffold featuring internal continuous air channels endowed with 10,000-fold higher O 2 diffusivity than hydrogels. We incorporate the scaffold into a bulk hydrogel containing cells, which facilitates rapid O 2 transport through the whole system to cells several millimeters away from the device-host boundary. A computational model, validated by in vitro analysis, predicts that cells and islets maintain high viability even in a thick (6.6 mm) device. Finally, the therapeutic potential of the device is demonstrated through the correction of diabetes in immunocompetent mice using rat islets for over 6 months. 

Cell encapsulation represents a promising therapeutic strategy for many hormone-deficient diseases such as type 1 diabetes (T1D). However, adequate oxygenation of the encapsulated cells remains a challenge, especially in the poorly oxygenated subcutaneous site. Here, we present an encapsulation system that generates oxygen (O2) for the cells from their own waste product, carbon dioxide (CO2), in a self-regulated (i.e., “inverse breathing”) way. We leveraged a gas-solid (CO2–lithium peroxide) reaction that was completely separated from the aqueous cellular environment by a gas permeable membrane. O2 measurements and imaging validated CO2-responsive O2 release, which improved cell survival in hypoxic conditions. Simulation-guided optimization yielded a device that restored normoglycemia of immunocompetent diabetic mice for over 3 months. Furthermore, functional islets were observed in scaled-up device implants in minipigs retrieved after 2 months. This inverse breathing device provides a potential system to support long-term cell function in the clinically attractive subcutaneous site.