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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.)

Recent advances in our understanding of hypoxia and hypoxia-mediated mechanisms shed light on the critical implications of the hypoxic stress on cellular behavior. However, tools emulating hypoxic conditions ( i.e. , low oxygen tensions) for research are limited and often suffer from major shortcomings, such as lack of reliability and off-target effects, and they usually fail to recapitulate the complexity of the tissue microenvironment. Fortunately, the field of biomaterials is constantly evolving and has a central role to play in the development of new technologies for conducting hypoxia-related research in several aspects of biomedical research, including tissue engineering, cancer modeling, and modern drug screening. In this perspective, we provide an overview of several strategies that have been investigated in the design and implementation of biomaterials for simulating or inducing hypoxic conditions-a prerequisite in the stabilization of hypoxia-inducible factor (HIF), a master regulator of the cellular responses to low oxygen. To this end, we discuss various advanced biomaterials, from those that integrate hypoxia-mimetic agents to artificially induce hypoxia-like responses, to those that deplete oxygen and consequently create either transient (<1 day) or sustained (>1 day) hypoxic conditions. We also aim to highlight the advantages and limitations of these emerging biomaterials for biomedical applications, with an emphasis on cancer research., Competing Interests: B. E. and M. K. disclose financial interests in O2M Technologies. All other authors have no conflicts of interest to disclose., (This journal is © The Royal Society of Chemistry.)

The intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the β-galactosidase (β-gal) enzyme and the anti-cytokeratin8 (K8) antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium)-tren-cholesterol (BGTC) (bis (guanidinium)-tren-cholesterol) combined to the colipid dioleoyl phosphatidylethanolamine (DOPE) (dioleoyl phosphatidylethanolamine) was shown to efficiently deliver the β-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP) associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/β-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP)-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies.