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Oxygen (O 2 )-controlled cell culture has been pivotal in studying mammalian mechanisms of O 2 sensing, regulation, and utilization. We posit, however, that O 2 -controlled cell culture is paradoxically not controlling O 2 . There is overwhelming evidence that the pericellular O 2 is lower than the surrounding gas phase due to cellular O 2 consumption. Standard hypoxic cell culture is at high risk of inducing pericellular anoxia. We discuss the implications of poor O 2 control for cellular O 2 regulation mechanisms, bioenergetics, and redox signaling. We also highlight the evidence of frequent under-oxygenation in standard (i.e., normoxic) cell culture. This issue has been largely overlooked because strategies to control pericellular O 2 have been lacking. Here, we propose a framework to control pericellular O 2 based on our recent investigation into the nature of the gas/pericellular O 2 gradient. Implementing this framework into standard practice will unlock quantitative O 2 control in vitro, improving our ability to understand the role of O 2 in biology., Competing Interests: Declaration of competing interests The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In oxygen (O 2 )-controlled cell culture, an indispensable tool in biological research, it is presumed that the incubator setpoint equals the O 2 tension experienced by cells (i.e., pericellular O 2 ). However, it is discovered that physioxic (5% O 2 ) and hypoxic (1% O 2 ) setpoints regularly induce anoxic (0% O 2 ) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O 2 consumption is the driving factor. RNA-seq analysis revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to cellular O 2 consumption. A reaction-diffusion model is developed to predict pericellular O 2 tension a priori, demonstrating that the effect of cellular O 2 consumption has the greatest impact in smaller volume culture vessels. By controlling pericellular O 2 tension in cell culture, it is found that hypoxia vs. anoxia induce distinct breast cancer transcriptomic and translational responses, including modulation of the hypoxia-inducible factor (HIF) pathway and metabolic reprogramming. Collectively, these findings indicate that breast cancer cells respond non-monotonically to low O 2 , suggesting that anoxic cell culture is not suitable for modeling hypoxia. Furthermore, it is shown that controlling atmospheric O 2 tension in cell culture incubators is insufficient to regulate O 2 in cell culture, thus introducing the concept of pericellular O 2 -controlled cell culture., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Cell culture, the process of growing cells in conditions that mimic those in the body, is a key technique in biomedical research. Oxygen is not controlled in conventional cell culture, although chambers that control oxygen in the surrounding gas phase are commercially available. In both cases, it is valuable to understand the pericellular oxygen tension (i.e., the oxygen concentration that cells experience) in cultures. Herein we describe a procedure for using commercial optical sensor spots to measure pericellular oxygen for adherent and suspension cultures. Spots are placed on surfaces on which cells are grown, and optical cables are attached to the outside of the cell culture vessels and connected to a computer. Associated software allows for the real-time monitoring of pericellular oxygen during cell culture experiments. This procedure enhances the reproducibility and control of cell culture., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Oxygen (O 2 ) tension plays a key role in tissue function and pathophysiology. O 2 -controlled cell culture, in which the O 2 concentration in an incubator's gas phase is controlled, is an indispensable tool to study the role of O 2 in vivo . For this technique, it is presumed that the incubator setpoint is equal to the O 2 tension that cells experience ( i.e ., pericellular O 2 ). We discovered that physioxic (5% O 2 ) and hypoxic (1% O 2 ) setpoints regularly induce anoxic (0.0% O 2 ) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O 2 consumption is the driving factor. RNA-seq revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to anoxic exposure followed by rapid reoxygenation. To better understand the relationship between incubator gas phase and pericellular O 2 tensions, we developed a reaction-diffusion model that predicts pericellular O 2 tension a priori . This model revealed that the effect of cellular O 2 consumption is greatest in smaller volume culture vessels ( e.g., 96-well plate). By controlling pericellular O 2 tension in cell culture, we discovered that MCF7 cells have stronger glycolytic and glutamine metabolism responses in anoxia vs . hypoxia. MCF7 also expressed higher levels of HIF2A , CD73 , NDUFA4L2 , etc . and lower levels of HIF1A , CA9 , VEGFA, etc . in response to hypoxia vs . anoxia. Proteomics revealed that 4T1 cells had an upregulated epithelial-to-mesenchymal transition (EMT) response and downregulated reactive oxygen species (ROS) management, glycolysis, and fatty acid metabolism pathways in hypoxia vs . anoxia. Collectively, these results reveal that breast cancer cells respond non-monotonically to low O 2 , suggesting that anoxic cell culture is not suitable to model hypoxia. We demonstrate that controlling atmospheric O 2 tension in cell culture incubators is insufficient to control O 2 in cell culture and introduce the concept of pericellular O 2 -controlled cell culture ., Competing Interests: Competing Interests The authors declare no conflicts of interest.

Hypoxia is a major factor shaping the immune landscape, and several cancer models have been developed to emulate hypoxic tumors. However, to date, they still have several limitations, such as the lack of reproducibility, inadequate biophysical cues, limited immune cell infiltration, and poor oxygen (O 2 ) control, leading to non-pathophysiological tumor responses. Therefore, it is essential to develop better cancer models that mimic key features of the tumor extracellular matrix and recreate tumor-associated hypoxia while allowing cell infiltration and cancer-immune cell interactions. Herein, hypoxia-inducing cryogels (HICs) have been engineered using hyaluronic acid (HA) to fabricate three-dimensional microtissues and model a hypoxic tumor microenvironment. Specifically, tumor cell-laden HICs have been designed to deplete O 2 locally and induce long-standing hypoxia. HICs promoted changes in hypoxia-responsive gene expression and phenotype, a metabolic adaptation to anaerobic glycolysis, and chemotherapy resistance. Additionally, HIC-supported tumor models induced dendritic cell (DC) inhibition, revealing a phenotypic change in the plasmacytoid DC (pDC) subset and an impaired conventional DC (cDC) response in hypoxia. Lastly, our HIC-based melanoma model induced CD8 + T cell inhibition, a condition associated with the downregulation of pro-inflammatory cytokine secretion, increased expression of immunomodulatory factors, and decreased degranulation and cytotoxic capacity of T cells. Overall, these data suggest that HICs can be used as a tool to model solid-like tumor microenvironments and has great potential to deepen our understanding of cancer-immune cell relationship in low O 2 conditions and may pave the way for developing more effective therapies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Hypoxia, an important feature of solid tumors, is a major factor shaping the immune landscape, and several cancer models have been developed to emulate hypoxic tumors. However, to date, they still have several limitations, such as the lack of reproducibility, inadequate biophysical cues, limited immune cell infiltration, and poor oxygen (O 2 ) control, leading to non-pathophysiological tumor responses. As a result, it is essential to develop new and improved cancer models that mimic key features of the tumor extracellular matrix and recreate tumor-associated hypoxia while allowing cell infiltration and cancer-immune cell interactions. Herein, hypoxia-inducing cryogels (HICs) have been engineered using hyaluronic acid (HA) as macroporous scaffolds to fabricate three-dimensional microtissues and model a hypoxic tumor microenvironment. Specifically, tumor cell-laden HICs have been designed to deplete O 2 locally and induce long-standing hypoxia. This state of low oxygen tension, leading to HIF-1α stabilization in tumor cells, resulted in changes in hypoxia-responsive gene expression and phenotype, a metabolic adaptation to anaerobic glycolysis, and chemotherapy resistance. Additionally, HIC-supported tumor models induced dendritic cell (DC) inhibition, revealing a phenotypic change in plasmacytoid B220 + DC (pDC) subset and an impaired conventional B220 - DC (cDC) response in hypoxia. Lastly, our HIC-based melanoma model induced CD8+ T cell inhibition, a condition associated with the downregulation of pro-inflammatory cytokine secretion, increased expression of immunomodulatory factors, and decreased degranulation and cytotoxic capacity of T cells. Overall, these data suggest that HICs can be used as a tool to model solid-like tumor microenvironments and identify a phenotypic transition from cDC to pDC in hypoxia and the key contribution of HA in retaining cDC phenotype and inducing their hypoxia-mediated immunosuppression. This technology has great potential to deepen our understanding of the complex relationships between cancer and immune cells in low O 2 conditions and may pave the way for developing more effective therapies.