Supraphysiological Oxygen Levels in Mammalian Cell Culture: Current State and Future Perspectives

Ricardo Alva; Georgina L. Gardner; Ping Liang; Jeffrey A. Stuart

doi:10.3390/cells11193123

Supraphysiological Oxygen Levels in Mammalian Cell Culture: Current State and Future Perspectives

Alva, Ricardo;Gardner, Georgina L.;Liang, Ping;Stuart, Jeffrey A. 2022-10-04 00:00:00 Review Supraphysiological Oxygen Levels in Mammalian Cell Culture: Current State and Future Perspectives Ricardo Alva *, Georgina L. Gardner, Ping Liang and Jeffrey A. Stuart Department of Biological Sciences, Brock University, St. Catharines, ON L2S 3A1, Canada * Correspondence: [email protected] or [email protected] Abstract: Most conventional incubators used in cell culture do not regulate O2 levels, making the headspace O2 concentration ~18%. In contrast, most human tissues are exposed to 2–6% O2 (physi- oxia) in vivo. Accumulating evidence has shown that such hyperoxic conditions in standard cell culture practices affect a variety of biological processes. In this review, we discuss how supraphys- iological O2 levels affect reactive oxygen species (ROS) metabolism and redox homeostasis, gene expression, replicative lifespan, cellular respiration, and mitochondrial dynamics. Furthermore, we present evidence demonstrating how hyperoxic cell culture conditions fail to recapitulate the phys- iological and pathological behavior of tissues in vivo, including cases of how O 2 alters the cellular response to drugs, hormones, and toxicants. We conclude that maintaining physioxia in cell culture is imperative in order to better replicate in vivo-like tissue physiology and pathology, and to avoid artifacts in research involving cell culture. Keywords: oxygen; physioxia; hyperoxia; ROS; oxidative stress; gene expression; senescence; me- tabolism; mitochondrial dynamics; drug response Citation: Alva, R.; Gardner, G.L.; Liang, P.; Stuart, J.A. Supraphysiological Oxygen Levels 1. Introduction in Mammalian Cell Culture: Current At sea level, dry atmosphere is composed of ~21% O2. Incubators used in standard State and Future Perspectives. Cells cell culture practices regulate headspace CO2 and temperature, but the vast majority do 2022, 11, 3123. https://doi.org/ 10.3390/cells11193123 not actively regulate O2. Although this is usually referred to in the literature as 20% or 21% O2, in fact, the headspace O2 in a humid cell culture incubator maintained at 5% CO2 Academic Editor: Friedrich Jung typically equilibrates to ~18% in the absence of O2 regulation. The overwhelming majority of in vitro studies have been performed in these conditions, and, as such, 18–21% O2 is Received: 10 September 2022 commonly termed ‘normoxia’ in the literature [1]. While atmospheric O2 may be a Accepted: 30 September 2022 normoxic condition for an entire organism, the same is not true for mammalian cells in Published: 4 October 2022 vivo. O2 levels in most mammalian tissues range from 2–6% [2], i.e., three- to nine-times lower than standard cell culture normoxia. In this review, we will use the term ‘physioxia’ Publisher’s Note: MDPI stays neu- [3] to describe O2 levels in this 2–6% range, and ‘normoxia’ for the hyperoxic (18%) con- tral with regard to jurisdictional ditions of standard cell culture. We are, thus, interested here in the effects of O2 in the claims in published maps and institu- range between physioxia and normoxia, that is, the ‘physio-normoxic’ range. It is im- tional affiliations. portant to note that, although most authors refer to O2 levels in experiments where cells are grown in humidified cell culture incubators with 5% CO2 as being either 20% or 21% O2, these numbers are incorrect. We have used the more accurate 18% throughout this text, though we recognize that this too will depend on elevation above sea level. Copyright: © 2022 by the authors. Li- The appreciation that standard cell culture conditions are in fact hyperoxic, com- censee MDPI, Basel, Switzerland. pared to what mammalian cells encounter in vivo has led to efforts to replicate physio- This article is an open access article distributed under the terms and con- logical O2 conditions (physioxia) in vitro. In this review, we present our current under- ditions of the Creative Commons At- standing of how cellular processes are affected by O2 in the range from physioxia to tribution (CC BY) license (http://crea- normoxia, including reactive oxygen species (ROS) generation and clearance, gene ex- tivecommons.org/licenses/by/4.0/). pression, proliferation, replicative lifespan, bioenergetics, and mitochondrial function. In Cells 2022, 11, 3123. https://doi.org/10.3390/cells11193123 www.mdpi.com/journal/cells Cells 2022, 11, 3123 2 of 21 addition, we discuss how cells cultured in normoxia exhibit phenotypes that fall short of reproducing tissue physiology and pathology in vivo. In this light, we consider recent findings showing that cells respond to drugs, hormones, and toxicants differently when cultured in physioxia, compared to 18% O2. We finalize by commenting on solutions to these challenges and future research directions. 2. Oxygen and Redox Homeostasis The different types of ROS, their generation, and their clearance have been exten- sively investigated and reviewed elsewhere [4–6]. Thus, we will mainly focus on how ROS metabolism is affected in the physio-normoxic range. − − Superoxide anion (O2 ) is formed by single electron reduction of O2. O2 is rapidly dismuted into hydrogen peroxide (H2O2) by superoxide dismutases (SOD) 1–3 [7]. Due to its neutral charge and diffusivity, H2O2 is able to cross membranes and participate in sig- naling processes by oxidizing cysteine residues in key signaling proteins that act as redox- sensitive molecular switches [8]. H2O2 is transformed into water by catalase, glutathione 2+ peroxidase (GPx), thioredoxin (Txn), and peroxiredoxin (Prdx) [9]. In the presence of Fe , H2O2 is converted into the highly reactive hydroxyl radical (HO), which indiscriminately oxidizes lipids, proteins, and DNA [10]. Although ROS have well-characterized roles in cellular physiology (reviewed in [11]), overproduction of ROS can be detrimental, which is why it is important to study ROS production rates from different sources and under- stand the factors that regulate each. There are multiple sources of ROS in mammalian cells, with the most studied being mitochondrial enzymes (e.g., respiratory complexes I and III) and NADPH oxidases (NOX). The KM values of the complex I flavin mononucleotide (FMN) site and complex III Qo site toward O2 are 0.019% and 0.199%, respectively [12], meaning that in the 2–18% O2 range, these enzymes are likely saturated and that little difference would thus be expected in mitochondrial ROS (mROS) production of cells grown at 18% O2 versus physioxia. Nonetheless, some studies have reported higher ROS levels at 18% O2, using mitochon- dria-targeted ROS indicators such as MitoSOX and dihydrorhodamine 123 [13–15], sug- gesting increased mROS production at normoxia (18% O2). On the other hand, NOX isoforms have higher KM values. For example, NOX4 has a reported KM of ~18% O2 [16], indicating that the reaction rate is highly sensitive to O2 concentration in the 2–18% range. Accordingly, production of H2O2 in C2C12, PC-3, SH-SY5Y, HeLa, MCF-7, and mouse em- bryonic fibroblasts (MEFs) was found to be significantly higher at 18% O 2 than at 5% O2 [17]. Pharmacological inhibition of NOX1/4 abrogated this effect, confirming the im- portance of NOX enzymes in ROS production at O2 levels higher than physiological con- ditions. A thorough discussion of how O2-consuming reactions are impacted by O2 tension in cell culture is provided in [18]. Indeed, elevated ROS production at supraphysiological O2 levels has been demonstrated by numerous studies in multiple cell types [13–15,19– 21]. In addition, significantly lower reduced glutathione (GSH) levels have been observed in cells grown at 18% O2 [20,22,23]. Thus, there is ample evidence that ROS production is greater in normoxia, compared to in physioxia, often compromising the antioxidant de- fenses of cells. Disruption of the balance between ROS production and antioxidant defenses results in oxidative stress, characterized by damage to lipids, proteins, and DNA. Timpano et al. reported that U87MG, HRPTEC, HEK293, and MCF-7 cells grown at 18% O2 exhibit more DNA strand breaks than their counterparts cultured in physioxia, as determined by comet assay [24]. Further, increased levels of 8-oxodeoxyguanine and 8-oxoguanine were ob- served in all cell lines but MCF-7, indicating oxidative damage to the nitrogen bases of DNA and RNA in these cells grown at 18% O2. Protein oxidation was observed in rat my- oblast precursor cells (MPCs). Protein carbonyl levels, one of the most common bi- omarkers of protein oxidation, were higher at 18% O2, compared to 3% O2, in old MPCs only, with no effects observed in cells from young mice [25]. Human mesenchymal stem cells (hMSCs) also showed increased protein carbonyl content and DNA strand breaks Cells 2022, 11, 3123 3 of 21 when cultured in ambient air [19]. In addition, hMSCs grown at 18% O2 had higher levels of malonyl aldehyde (MDA), which is an end-product and biomarker of lipid peroxida- tion. Elevated MDA levels were also detected in human dental pulp stem cells (hDPSCs) grown at 18% O2 for 7 days, compared to cells grown at 3% O2, which was prevented with Trolox administration, a water-soluble vitamin E analog [15]. Finally, levels of both pro- tein carbonyls and 4-hydroxynonenal (4-HNE), another lipid peroxidation end-product, were found to be higher in mouse cardiac fibroblasts cultured at 21% O2, compared to 3% O2 [13]. To conclude, there is abundant evidence that the hyperoxic environment of standard cell culture disrupts redox homeostasis by increasing the production of ROS, which re- sults in oxidative damage to biomolecules and organelles. Biomolecule oxidation can, in turn, result in the dysregulation of signaling pathways and cellular processes, such as gene expression, proliferation, senescence, differentiation, metabolism, and response to drugs and other bioactive molecules. 3. Intracellular Oxygen and ROS Sensing Discovered in the early 1990s, the hypoxia-inducible factor (HIF) is regarded as the master intracellular O2 sensor in response to hypoxia [26,27]. HIF is a heterodimer com- posed of one α and one β subunit. While there are three isoforms of HIF-α (HIF-1α, HIF- 2α, and HIF-3α, encoded by the genes HIF1A, HIF2A, and HIF3A, respectively), only one HIF-β protein has been described (reviewed in [28]). When translocated to the nucleus, HIF-α partners with the constitutively expressed HIF-β, binds to the hypoxia response element (HRE) of DNA and initiates transcription of target genes. The transcriptional ac- tivation of HIF allows the cell to adapt to low O2 tensions, by mediating expression of genes involved in glycolysis and energy metabolism, angiogenesis, cell migration, prolif- eration, and survival, among other processes [29]. Although HIF-1α and HIF-2α have unique gene targets, both are critical for regulating the expression of genes necessary for cell adaptation to hypoxia [30] (reviewed in [31]). In normoxia, prolyl hydroxylases (PHD) use molecular O2 as substrate to introduce hydroxyl groups in two highly conserved prolyl residues of HIF-α. Hydroxylated HIF-α is then recognized by the Von Hippel- Lindau tumor suppressor protein (pVHL), which leads to its ubiquitination and subse- quent degradation by the proteosome [32]. In contrast, a low O2 concentration in hypoxia blunts PHD activity and allows for the stabilization of HIF-α and its translocation to the nucleus (Figure 1A) [33]. It is important to note that nearly all studies on HIF-1/2 activity have been conducted as comparisons between normoxia (18%) and hypoxia (≤1% O2). Fu- ture studies should be conducted comparing physioxia and hypoxia. While HIF activation has long been considered solely as a cellular response to hy- poxia, a few studies have also suggested its role in physiological O2 conditions. Given that the KM values of PHD isoforms are slightly above atmospheric O2 level [34], the enzymatic rate of HIF-α hydroxylation is highly sensitive to O2 levels in the physio-normoxic range. Bracken et al. investigated the stabilization of HIF-1/2α in six cell lines, HeLa, 293T, Cos- 1, PC-12, CACO2, and HepG2, and they observed cell-type specific patterns [35]. For ex- ample, HIF-1/2α were barely detectable in HeLa cells at 5% O2 but upregulated at 2% O2. On the other hand, 293T cells had higher HIF-1/2α levels at 5% O2. These results likely reflect different sensitivities of distinct cell types to O2 levels [35]. In another study, Yan et al. detected HIF-1α localization to the nuclei of primary hepatocytes at 5% O2 via immu- nofluorescence [36]. More recently, detectable protein levels of HIF-1α and HIF-2α were observed in patient-derived melanoma cells cultured at 6% O2, supporting the notion that HIF is partially active at physiologically relevant O2 tensions [37]. Moreover, in this study, increased expression of HIF targets was sustained in cells grown at 6% O2 for at least 3 weeks, further suggesting a role for HIF activity at physioxia rather than it being a mere consequence of an acute reduction of O2 tension while performing the experiments. Stuart et al. have hypothesized that transcriptional activity of HIF-1/2 at low O2 tensions within a physiological range (2–6%) helps to maintain the production of ROS and reactive Cells 2022, 11, 3123 4 of 21 nitrogen species (RNS), which are necessary for many physiological processes [38]. This hypothesis is based on the fact that several ROS-producing enzymes are induced by HIF- 1/2 (reviewed in [18]). In contrast to direct oxygen-sensing mechanisms in hypoxia, the cellular response to hyperoxia is regulated by molecular switches that are sensitive to H2O2. Perhaps the most important and best described is the pathway regulated by the nuclear factor erythroid 2– related factor 2 (Nrf2). The molecular mechanisms regulating Nrf2 degradation, stabiliza- tion, and transcriptional activity have been thoroughly reviewed in [39–41]. Under un- stressed conditions, Nrf2 is bound to Kelch-like ECH-associated protein 1 (Keap1), which facilitates ubiquitination and proteasomal degradation of Nrf2. Keap1 acts as an intracel- lular ROS sensor, with multiple cysteine residues identified as redox-sensitive regulators. In conditions of oxidative stress, such as hyperoxia, ROS-mediated oxidation of specific cysteine residues inhibits Keap1 binding to Nrf2, leading to Nrf2 stabilization, transloca- tion to the nucleus, and binding to the antioxidant response element (ARE) of DNA (Fig- ure 1B) [42]. In addition to oxidants, the regulatory cysteine residues of Keap1 are also sensitive to electrophiles that readily react with thiol groups [40]. Such electrophiles can originate from both exogenous and endogenous sources. In the latter case, for instance, the lipid peroxidation end-product 4-HNE reacts with specific cysteine residues of Keap1, promoting Nrf2 stabilization and transcriptional activity [43,44]. Nrf2 induces the expres- sion of genes involved in ROS and xenobiotic detoxification, GSH biosynthesis and regen- eration, NADPH regeneration, and heme and iron metabolism [45]. It is important to note that Nrf2 is part of the cellular response to oxidative and elec- trophilic stresses, which may be caused by a variety of insults, not just hyperoxia. Never- theless, hyperoxia is a well-characterized condition of increased ROS production, in which the role of Nrf2 has been demonstrated. Indeed, activation of Nrf2 in severe hyperoxia (>60% O2) has been extensively studied (reviewed in [46]). Its role in perceived hyperoxia at ambient air (~18% O2) in vitro, on the other hand, has been less studied. However, a handful of studies have reported upregulation of Nrf2 in cells cultured in normoxia, com- pared to cells grown in physioxia. Epithelial cells from mouse mammary tumors pro- cessed under ambient air had increased levels of Nrf2 and decreased levels of Keap1, com- pared to tumors under physioxia (3% O2) [47]. In accordance, upregulation of known Nrf2 targets, such as SOD1, thioredoxin reductase, and NAD(P)H quinone dehydrogenase (NQO1), has been observed in normoxia versus physioxia in different cell types [15,23,24]. Due to the induction of antioxidant and cytoprotective enzymes in normoxia (~18% O 2), some studies have shown that cells adapted to these conditions may be preconditioned and, thus, resistant to pro-oxidant insults (e.g., drugs and toxins). This shall be further addressed in Section 9. Moreover, mechanisms of enhanced Nrf2-dependent gene tran- scription in normoxia may go beyond increased Keap1 oxidation or downregulation. Chapple et al. demonstrated nuclear accumulation and DNA binding of Nrf2 in human endothelial cells grown in physioxia [48]. Instead, Bach1, another Nrf2 repressor, was found to be upregulated in physioxia, thus interfering with expression of Nrf2 targets. Inhibition of Nrf2 transcriptional activity was abrogated by re-exposure to ambient air or Bach1 silencing. This provides a novel mechanism for Nrf2-mediated regulation of gene expression induced by O2 in the physio-normoxic range. Other ROS-activated pathways known to be affected in hyperoxia include those me- diated by the nuclear factor kappa B (NF-κB) and mitogen activated protein kinases (MAPKs) (reviewed in [46]), but their regulation in physioxia versus atmospheric O2 has been scarcely investigated. Carrera et al. did observe increased phosphorylation of extra- cellular signal-regulated protein kinase (ERK1/2), in HCT116 and U2OS cells grown at 5% O2 in the absence of any stress [49]. They showed that the constitutive activation of this pro-survival pathway affects their response to proapoptotic drugs (see Section 9). Indeed, differential regulation of O2-sensitive and redox-sensitive signaling molecules and tran- scription factors leads to the alteration of multiple biological processes, as shall be further discussed throughout this review. Additional research is required to fully elucidate the Cells 2022, 11, 3123 5 of 21 roles and molecular mechanisms of HIF, Nrf2, and other pathways in the cellular adapta- tion to supraphysiological O2 in different cell types. Figure 1. Pathways for intracellular oxygen/redox status sensing by HIF-1/2 and Nrf2. (A) Low O2 levels reduce the rate of HIF-1/2α hydroxylation, which leads to molecular recognition by the Von Hippel-Lindau tumor suppressor protein (pVHL) and subsequent proteasomal degradation. Stabi- lized HIF-1/2α binds HIF-β and translocates to the nucleus, where it binds the hypoxia response element (HRE) in DNA, inducing expression of glycolytic, proliferative, and pro-survival genes, among others. (B) High O2 levels promote increased reactive oxygen species (ROS) production. ROS-mediated oxidation of Keap1 releases Nrf2 preventing its proteasomal degradation. Nrf2 then translocates to the nucleus, heterodimerizes with small musculo-aponeurotic fibrosarcoma (sMaf), and binds to the antioxidant response element (ARE), inducing expression of genes involved in ROS and xenobiotic detoxification. Created with BioRender.com (accessed on 14 July 2022). 4. Effects of Oxygen and ROS on Gene Expression Differential regulation of O2/ROS-sensitive transcription factors in the physio- normoxic range affects gene expression, as we have recently shown in an RNA-seq-based study with LNCaP, Huh-7, PC-3, and SH-SY5Y cells. These cell lines were cultured at ei- ther 5% or 18% O2 for two weeks, prior to mRNA isolation and sequencing. The O2 level significantly affected the abundance of thousands of transcripts in all cell lines. Surpris- ingly, there was limited overlap in the specific transcripts affected, with >80% of the dif- ferentially expressed genes being cell-line specific [50]. Interestingly, a handful of HIF tar- gets were found to be upregulated at 5% O2 in LNCaP and Huh-7 cells, further confirming a role for HIF activation under physiological O2 conditions. Similar patterns were found by Duś-Szachniewicz et al. at the proteome level [51]. They analyzed proteomic changes in three diffuse large B-cell lymphoma cell lines grown at 1% O2 (hypoxia), 5% O2 (physi- oxia), and 18% O2 (normoxia). Importantly, proteomes from cells grown in hypoxia and physioxia cells shared more similarities, compared to the proteome of cells grown in at- mospheric O2. In agreement with our study, this latter study showed that the effects of Cells 2022, 11, 3123 6 of 21 physioxia versus normoxia were largely cell-type specific, even when the three cell lines are from the same type of cancer [51]. Mechanisms of translation are also regulated by O2 (reviewed in [52,53]). Hypoxia prevents the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the 7- methylguanosine cap at the 5′ end of mRNA molecules, which is the initial step of protein synthesis. In 2012, Uniacke et al. demonstrated that, under hypoxic conditions, HIF-2α forms a complex with eIF4E2 (eIF4E homologue) and the RNA binding motif protein 4 (RBM4), which then recognizes and binds to the 5′ cap of specific mRNA molecules, al- lowing initiation of translation independent of eIF4E [54]. Years later, the same research group found that, while eIF4E is the dominant cap-binding protein in normoxia (18% O2) and eIF4E2 is the dominant cap-binding protein in hypoxia, both are active at physioxia (here, 3–12%) and simultaneously act to initiate the translation of distinct classes of mRNA molecules [55]. These results demonstrate that differential expression of proteins in phys- ioxia versus normoxia is not only regulated at the transcriptional level but also at the translational level. In addition to transcriptional and translational regulation, O2-dependent mecha- nisms mediate gene expression through epigenetic modifications. Processes such as his- tone methylation and acetylation, DNA methylation, and chromatin organization are known to be affected by hypoxia and oxidative stress (reviewed in [56,57]). The roles of these mechanisms in the physio-normoxic range have been investigated less often, alt- hough a handful of studies have reported different epigenetic responses to O2 in a variety of cells, with a special focus on stem cells and cancer cells. Lengner et al. found that culturing human embryonic stem cells (hESCs) in physioxia prevents chromosome X inactivation that is otherwise observed in normoxia, which, in turn, preserves the pluripotency of hESCs [58]. This was associated with the methylation of the XIST promoter region, while chromosome X inactivation was associated with the demethylation and transcriptional activity of XIST. In another study, Xie et al. observed an increased methylation pattern and irreversible silencing of the DLK1-DIO3 gene cluster in cultured hESCs after 20 passages in normoxia [59]. Interestingly, culture of hESCs in 5% O2 preserved expression of the DLK1-DIO3 cluster. The DLK1-DIO3 cluster has been associated with pluripotency in mouse induced pluripotent stem cells and may, thus, be a biomarker for the epigenetic stability of hESCs. Further, acetylation of histone protein H3 at lysine residues 9 and 27 (H3K9ac and H3K27ac, respectively) was elevated in human pluripotent stem cells (hPSCs) grown at 5% O2, while trimethylation of H3 lysine 27 was reduced [60]. These epigenetic changes are associated with a more open chromatin struc- ture and enhanced gene expression. RNA-seq and functional enrichment analysis also re- vealed increased expression of genes involved in H3K27 demethylation at 5% O2, while increased methyltransferase and cell cycle activities were observed at 18% O2. The ten-eleven translocation (TET) family of proteins catalyzes the O2-dependent hy- droxylation of 5-methylcytosine (5mC) in DNA, which results in demethylation and con- comitant increase in gene expression. Thienpont et al. investigated the effect of tumor hy- poxia (0.5% O2) on the expression profiling and activities of TET isoforms, TET1, TET2, and TET3 [61]. The mRNA levels of TET paralogues were increased at a variable degree among a range of cell lines exposed to hypoxia, with neuroblastoma cell lines SH-SY5Y and SK-N-Be2c being the most strongly affected. Chromatin immune precipitation fol- lowed by sequencing (ChIP–seq) confirmed the binding of HIF near the promoters of up- regulated TET genes. However, the activity of TET enzymes was actually reduced in hy- poxic human and mouse tumor cells, which is not surprising, since these enzymes use O2 as substrate. Reduced TET activity was then correlated with decreased hydroxylated 5mC (5hmC) and concomitant hypermethylation. Although these landmark results provided a mechanism for epigenetic-mediated repression of tumor suppressor genes in hypoxia, no changes in TET activity were observed at 2–5% O2. In a different study, however, the ex- pression of the three TET paralogues was increased in HepG2 cells at both 5% and 1% O2, compared to 18% O2 [62]. Upregulation of these enzymes at 1% O2 was blunted by HIF- Cells 2022, 11, 3123 7 of 21 1α knockdown, although this was not tested at 5% O2. The activity of TET enzymes was not measured in this study. In stem cells, on the other hand, a different trend in the regu- lation of TET genes has been observed. TET1 mRNA and protein levels were decreased in bone-marrow-derived hMSCs grown at 2% O2, compared to atmospheric O2 [63]. Expres- sion of DNA methyltransferase 3B (DNMT3B) was also reduced at 2% O2. As a result, global 5hmC and 5mC levels were also decreased at physioxia. In a subsequent study by the same lab, DNMT3B, DNM3L, TET1, and TET3 were similarly downregulated in hPSCs cultured at 2% O2, compared to cells grown at 18% O2 [64]. This was accompanied by enhanced proliferation, metabolic activity, and stemness attributes. Interestingly, both studies showed downregulation of HIF1A and upregulation of HIF2A (in hMSCs and hPSCs, respectively) cultured at physioxia. Another mechanism of epigenetic regulation of gene expression involves a variety of non-coding RNA molecules, such as microRNA (miRNA), long non-coding RNA (lncRNA), and small nucleolar RNA (snoRNA). Johnston et al. measured the abundance of miRNA molecules in MCF-7 cells across a range of O2 pressures, from 18% O2 to 1% O2 [65]. Among the most strongly affected miRNAs, miR-675, miR-1293, miR-7974, miR-653, and miR-140 were found upregulated at 18% O2, compared to 4% O2 (physioxia). On the other hand, miR-758, miR-335, miR-1185-1, miR-1185-2, and miR-889 were upregulated at 4% O2, compared to 18% O2. These miRNA molecules have been implicated in processes such as apoptosis, proliferation, metastasis, and tumor suppression. In the study by Xie et al. where silencing of the DLK1-DIO3 cluster was observed in hESCs grown at 18% O2 after 20 passages, decreased expression of several non-coding RNA molecules was shown, including the lncRNA MEG3 and the snoRNA SNORD114-3 [59]. The expression of these molecules was rescued in cells cultured at 5% O2. MEG3 is known to inhibit tumor prolif- eration by interacting with p53. Accordingly, silencing of MEG3 was correlated with re- duced DNA-damage-induced apoptosis in hESCs and their differentiated hepatocyte-like cells grown at ambient O2 tension. Using microarray and bioinformatic analyses, Shi et al. identified 47 lncRNAs and 14 miRNAs differentially expressed in hDPSCs cultured at 18% O2 and 3% O2, revealing a complex gene network between the non-coding RNAs and mRNAs [66]. Further, functional enrichment analysis revealed biological processes, such as the HIF-1 pathway, the Wnt pathway, carbon metabolism, and glutathione metabolism, to be affected by O2, among other processes. In summary, broad epigenetic responses to physiological O2 levels have been re- ported, with striking differences observed depending on the cell type. These responses include DNA methylation and hydroxylation, histone acetylation and methylation, and altered expression of non-coding RNAs. Future research should be directed to fully char- acterize the specific mechanisms governing these cell-type-specific responses. 5. Oxygen, Proliferation, and Senescence The first observation made about the impact of culturing cells at O2 tensions above physiological conditions was a decrease in their proliferation rate. In 1958, Cooper et al. reported that lowering O2 tension below normoxia increased the growth rate of rabbit embryo kidney cells [67]. A later study found that lowering O2 from 18% to 10% O2 in- creases the replicative lifespan (i.e., Hayflick limit) of WI-38 fibroblasts [68]. Similar ob- servations were made by Balin et al. [69]. Perhaps expectedly, immortalized cells are not as sensitive to the cytostatic effect of atmospheric O2, as Falanga and Kirsner showed that single-seeded human fibrosarcoma and immortalized 3T3 cells could proliferate without difficulty in standard O2 conditions (18% O2), in contrast to primary dermal fibroblasts [70]. Subsequent studies focused on the molecular mechanisms of the antiproliferative ef- fects of atmospheric O2. Hyperoxia was established to be an inducer of replicative senes- cence, which is a process initiated by oxidative DNA damage and telomere shortening and mediated by p53- and p21-dependent cell cycle arrest [71]. Culture of MEFs at 18% O2 resulted in DNA mutations and senescence. G:C to T:A transversions, a signature mutation caused by DNA oxidation, were observed in Cells 2022, 11, 3123 8 of 21 hyperoxic cells. Senescence was not observed in MEFs grown at 3% O2 [72]. Similar obser- vations were made by Parrinello et al., showing induction of senescence and DNA strand breaks in MEFs grown in 18% O2 [73]. Cardiac fibroblasts from adult murine ventricle cultured at 10% and 18% O2 exhibited greater ROS production and cell cycle arrest at G2/M, compared with cells at 3% O2 [13]. Induction of p21 and decreased telomerase ac- tivity were also detected in hyperoxia. Fibroblasts lacking p21 were resistant to the effects of higher O2 tension. Overexpression of p21 was confirmed at both mRNA and protein levels in muscle precursor cells (MPCs) exposed to 18% O2, compared to 5% O2, along with increased p53 transcriptional activity [74]. Estrada et al. reported that telomere shortening rate per division cycle is 24% higher in hMSCs cultured in normoxia, compared to in phys- ioxia (here, 3% O2) [19]. Another study reported increased p38 phosphorylation, an up- stream regulator of p21, along with concomitant p21 overexpression in hDPSCs cultured in 18% O2. Elevated HO-1 and NQO-1 protein levels were also detected, suggesting Nrf2 activation [15]. More recently, Bon-Mathier et al. demonstrated that primary neonatal mouse cardiomyocytes have a higher proliferative capacity when cultured at 3% O 2, which was associated with a dedifferentiation of cardiomyocytes at physioxia [75]. Recent evidence has linked the Wnt/β-catenin signaling pathway to the mechanisms regulating the response to O2 at physioxia/hyperoxia. Stabilized β-catenin and upregula- tion of Wnt gene targets were observed in fetal mouse neural stem cells (NSCs) at physi- oxia, promoting proliferation and reducing spontaneous differentiation of NSCs [76]. These effects were sustained even in HIF-1α–deficient cells, indicating that the O2-sensi- tive mechanism of Wnt activation is independent of HIF. Further research is required to understand the involvement and mechanisms of Wnt in O2 sensing at physiological O2 tensions. In conclusion, there is ample evidence showing that hyperoxic cell culture induces senescence in a variety of cells by promoting oxidative DNA damage and activating p53 and p21. Collectively, these results emphasize the importance of culturing primary cells at physioxia, in order for them to retain their proliferative capacity. More evidence regard- ing the link between O2 and cell differentiation is discussed in the next section. 6. Oxygen and Cell Differentiation The transcriptional program controlling stemness and differentiation of embryonic stem cells is in part regulated by HIF. For example, the expression of OCT4, a critical tran- scription factor in mediating stemness and pluripotency of embryonic stem cells, is regu- lated by HIF-2α [77]. Westfall et al. showed that expression of known HIF-1/2α and OCT4 targets was markedly increased in human embryonic cell lines at 4% O2, compared to at 18% O2 [78]. The mechanisms governing hypoxia and stemness are reviewed in [79]. In accordance, lowering O2 to 5% increased the proliferation and differentiation of human macrophage progenitor cells [80,81]. Fehrer et al. found that while culturing hMSCs at 3% O2 extends their proliferative lifespan and inhibits senescence, their capacity of differentiating into osteoblasts and adipocytes was blunted at this O2 tension [82]. Sim- ilar mechanisms seem to affect osteoblast differentiation. Culture of bone-marrow-iso- lated adult multilineage-inducible cells at 3% O2 increased their proliferation but blocked their differentiation into osteoblasts, which is otherwise present when cultured at 18% O2 [83]. mRNA levels of OCT4, HIF1A, and TERT (telomerase reverse transcriptase) were up- regulated at 3% O2. In contrast, lower O2 levels have been found to promote chondrogen- esis, which is in agreement with the fact that cartilage is avascular and in hypoxic envi- ronment [84–87] (reviewed in [88]). Studer et al. reported that culture of mesencephalic precursor cells at 3% O2 enhances both their proliferation and differentiation into dopa- minergic neurons [89]. Recent evidence has also shown that culture of stem cells at phys- ioxia improves their therapeutic potential for clinical application [90,91]. The relevance of O2 levels for stem cell biology and their application in regenerative medicine is reviewed in detail in [92]. Cells 2022, 11, 3123 9 of 21 Immortalized cell lines are widely used in research as surrogates of primary cells, which are often less accessible than the former to the research community. A study found that the ability of the monocyte-derived cell line THP-1 to differentiate into macrophage- like-cells is affected by O2 tension. The metabolic activity and rate of drug-induced differ- entiation of THP-1 cells were enhanced at 5% O2, compared to 18%, with the primary mac- rophage-like functions of the differentiated cells being affected by O2 as well [93]. In summary, the O2 levels in culture have a profound impact on the differentiation capacity and stemness of cells, with specific effects varying among cell types, likely ac- cording to their oxygenation status in vivo. 7. Oxygen, Cell Bioenergetics, and Mitochondrial Dynamics Since most cellular O2 is consumed by mitochondria during respiration and ROS pro- duction, it is unsurprising that broad oxygen-dependent effects on cellular metabolism and mitochondrial function been reported in a variety of cells. Estrada et al. showed that culturing hMSCs at 18% O2 enhances oxidative phosphorylation (OXPHOS) and decreases glycolysis, which, in turn, leads to higher rates of ROS production and oxidative-stress- related damage. In this study, they found a decreased oxygen consumption rate (OCR)/ex- tracellular acidification rate (ECAR) ratio in hMSCs cultured at 3% O2, compared to at 18% O2, which was associated with increased expression of HIF-1/2 targets that encode en- zymes involved in glucose metabolism, such as pyruvate dehydrogenase kinase, phos- phofructokinase, and lactate dehydrogenase [19]. Enhanced activity of these enzymes in- creases the glycolytic rate and diverts pyruvate from entering the TCA cycle, while pro- moting its reduction to lactate in order to regenerate reduced nicotinamide dinucleotide (NAD ). In agreement with these results, Lees et al. found that hPSCs cultured in 18% O2 have an increased OCR and ATP production rate, compared to cells cultured at physioxia [60]. Although no significant differences were observed in basal ECAR, which is a proxy for glycolytic rate, RNA-seq revealed increased expression of genes related to glycolysis in cells grown at 5% O2. Using C glucose labeling, they also observed increased glycolytic intermediary metabolites (e.g., fructose-1,6-biphosphate) and lactate. In primary human corneal endothelial cells, no effect on the basal OCR was observed at 2.5% O 2, compared to atmospheric O2 tension. In turn, basal ECAR was increased at 2.5% O2 [94]. This result is in accordance with the ability of the corneal endothelium to rely on glycolysis to meet the energy requirements of the cornea under lower oxygen availability. In another study, Zhu et al. demonstrated that culture of rat primary cortical neurons at physioxia improves mitochondrial function, as shown by increased mitochondrial membrane potential, mito- chondrial dehydrogenase activity, and ATP levels at 5% O2, compared to at 18% O2 [21]. Although these outcomes suggest enhanced mitochondrial respiration, glucose uptake and consumption rates were also found to be higher at 5% O2. Further, Zhu et al. observed reduced glucose oxidation, measured by CO2 production, along with higher lactate lev- els in neurons cultured at 5% O2. AMPK phosphorylation was also found to be higher at physioxia. As such, many of the metabolic effects of culturing neurons at 5% O2 were abol- ished by pharmacological inhibition of AMPK. A handful of other studies have reported opposite patterns, suggesting higher OXPHOS rates at physioxia. This could be explained by the fact that hyperoxia-induced ROS directly inactivate mitochondrial enzymes such as aconitase, pyruvate dehydrogen- ase complex, and respiratory complexes I and II (reviewed by Alva et al. in [95]). For ex- ample, MPCs isolated from old mice have a higher basal and maximal OCR at 3% O2, compared to 18% O2. In fact, when old MPCs are cultured at 3% O2, they have the same OCR as that of MPCs from young mice, which show no difference in OCR between the two O2 concentrations [25]. Old MPCs grown at hyperoxia seemed to compensate by up- regulating glycolytic metabolism, as the ECAR measured was higher than that of old MPCs grown at physioxia and young MPCs at either O2 tension, which, again, did not seem to be affected much by O2. In addition to age, sex has also been shown to influence the bioenergetic response to different oxygen concentrations. Experiments with primary Cells 2022, 11, 3123 10 of 21 rat cortical astrocytes have uncovered sex differences in their bioenergetic profile when cultured at 3% O2. In contrast, no differences in bioenergetic parameters were found in astrocytes grown at 18% O2, denoting how physiological phenomena can be masked by culturing cells at nonphysiological oxygen tensions [96]. Studies with immortalized cell lines have shown similar trends. Timpano et al. stud- ied the effects of O2 tension in the metabolism of four human cell lines, U87MG, HRPTEC, HEK293, and MCF-7. In general, cells had a lower mitochondrial metabolic activity when cultured at 12% O2 or higher, compared to at physioxia, as measured by fluorometric de- tection of resaruzin reduction [24]. Similarly, Moradi et al. investigated the effect of O2 concentration in cellular respiration using four human cancer cell lines. MCF-7, Huh-7 and LNCaP cells had a lower basal OCR at 18% O2, compared to at 5% O2, while no dif- ference was observed in SaOS2 [97]. Maximal OCR was also lower at 18% O2 in all cell lines, except LNCaP, which had a greater maximal OCR at 18%. Collectively, these results indicate that physiological O2 levels seem to promote mitochondrial metabolism, although no effects on glycolytic rate (ECAR) were observed in this study. The same study by Moradi et al. also revealed differential effects of O2 on mitochon- drial network dynamics, which were analyzed using the MiNA tool [98]. Normoxia was associated with an increased mitochondrial footprint, indicative of mitochondrial abun- dance, in Huh-7 and SaOS2 cells, but had the opposite effect on LNCaP cells. In addition, the mean network size, which is indicative of the number of network branches, was lower in LNCaP and SaOS2 cells grown at 18% O2, while no significant difference was observed in MCF-7 and Huh-7. In the study by Timpano et al., a variety of effects in mitochondrial fusion and morphology of U87MG, HRPTEC, HEK293, and MCF-7 cells cultured in dif- ferent O2 levels were measured. U87MG and HEK293 cells displayed a more globular mi- tochondrial morphology, when cultured at atmospheric O2, compared to cells cultured at physioxia, while HRPTEC cells showed no difference in mitochondrial morphology, and MCF-7 had a more globular mitochondrial morphology at 5% O2 than at 18% O2. In a different report, mitochondria in primary neurons were found to be longer and have a more elongated morphology at 5% and 2% O2 levels, compared to cells kept at atmos- pheric O2 [99]. Accordingly, mitochondrial network parameters, such as network size, mi- tochondrial perimeter, and mitochondrial fraction, were higher with decreasing O2 con- centration. In summary, O2 conditions affect mitochondrial respiration, glycolysis, mitochon- drial morphology, and mitochondrial network dynamics (Table 1). Although, in some cases, the trend would seem to indicate that OXPHOS and highly fused mitochondrial networks are favored under physioxia, these are not widespread observations. This is likely due to the unique metabolic profiles of each type of cell, causing cells to react dif- ferently when challenged with varying O2 tensions. In any case, the effects of environmen- tal O2 on metabolic and mitochondrial characteristics of cells can no longer be neglected and must be considered when conducting experiments that target the mitochondria and cellular metabolism. Table 1. Effects of O2 tension (between 2–18% O2) on cellular metabolism, mitochondrial abundance, morphology, and network dynamics. Cell Types Experimental Conditions Methods Outcomes Reference Metabolic Effects  OCR/ECAR ratio at 18% O2 Seahorse XF analysis, hMSCs/18% or 3% O2  expression HIF targets involved [19] microarray, RT-PCR in glucose metabolism at 3% O2  OCR/ECAR ratio at 18% O2 Stem cells Seahorse XF analysis,  glycolytic intermediates hPSCs/18% or 5% O2 NMR spectroscopy, RNA- at 3% O2 [60] seq, RT-PCR  expression HIF targets involved in glucose metabolism at 3% O2 Cells 2022, 11, 3123 11 of 21  OCR at 18% O2 MPCs from old mice/18% or 3% O2 Seahorse XF analysis [25]  ECAR at 18% O2 hCEnCs/18% or 2.5% O2 Seahorse XF analysis  ECAR at 2.5% O2 [94]  glucose uptake at 5% O2 Rat primary cortical neurons/18% or ATP bioluminescent as-  glucose oxidation at 5% O2 Primary differen- [21] 5% O2 say, LSC, lactate assay  lactate levels at 5% O2 tiated cells  metabolic activity at 18% O2, HRPTEC/18–3% O2 Resazurin assay compared to 15% and 12% O2  metabolic activity at 18% O2, U87MG/18–3% O2 Resazurin assay [24] compared to 8–3% O2  metabolic activity at 18% O2, MCF-7/18–3% O2 Resazurin assay compared to 8% O2  basal and maximal OCR MCF-7/18% or 5% O2 Seahorse XF analysis Cancer/immortal- at 18% O2 ized cells  basal OCR at 18% O2 LNCaP/18% or 5% O2 Seahorse XF analysis  maximal OCR at 18% O2 [97]  basal and maximal OCR Huh-7/18% or 5% O2 Seahorse XF analysis at 18% O2 SaOS2/18% or 5% O2 Seahorse XF analysis  maximal OCR at 18% O2 Effects on mitochondrial morphology, abundance, and dynamics Globular-shaped mitochondria at 18% O2 (versus elongated at 2% Primary differen- rat primary neurons/18%, 5%, or 2% TEM and confocal micros- and 5% O2) [99] tiated cells O2 copy; Image J  mitochondrial network size, mi- tochondrial fraction, and mito- chondrial perimeter at 18% O2 Confocal microscopy; U87MG/18–3% O2 Rounder mitochondria at 18% O2 Volocity Confocal microscopy; HEK293/18–3% O2 Rounder mitochondria at 18% O2 [24] Volocity Confocal microscopy; Elongated mitochondria MCF-7/18–3% O2 Volocity at 18% O2 Cancer/immortal-  mitochondrial footprint Confocal microscopy; ized cells LNCaP/18% or 5% O2 at 18% O2 MiNA  mean network size 18% O2 Confocal microscopy;  mitochondrial footprint Huh-7/18% or 5% O2 [97] MiNA at 18% O2  mitochondrial footprint Confocal microscopy; SaOS2/18% or 5% O2 at 18% O2 MiNA  mean network size 18% O2 Abbreviations: ECAR, extracellular acidification rate; hCEnCs, human corneal endothelial cells; hMSCs, human mesenchymal stem cells; hPSC, human pluripotent stem cells; HRPTEC, human renal proximal tubule epithelial cells; LSC, liquid scintillation counting; MiNA, mitochondrial network analysis; NMR, nuclear magnetic resonance; OCR, oxygen consumption rate; TEM, trans- mission electron microscopy; XF, extracellular flux. 8. Modeling Tissue Physiology and Pathology in Physioxia One of the goals of cell culture is to mimic the behavior of cells and tissues in vivo, in both their physiological and pathological states. In this regard, physiological O2 condi- tions have been shown to facilitate the maintenance of in vivo-like properties of cells cul- tured in vitro. For instance, when compared to cells grown in physioxia, microvascular endothelial cells isolated from the foreskins of neonates cultured in normoxia exhibit a differential expression of proteins related to the functionality of endothelial cells, such as type IV collagen, platelet endothelial cell adhesion molecule-1 (PECAM-1), and von Wil- lebrand factor. In addition, vascular channel formation induced by collagen gels was Cells 2022, 11, 3123 12 of 21 increased in 18% O2 [100]. Piossek et al. showed that co-culture of human renal proximal tubular epithelial cells with human fibroblasts at physioxia improves their functional properties, as measured by epithelial barrier integrity, expression of membrane transport- ers, and transport of cations and anions [101]. Another study showed that hyperoxia pro- motes hepatocyte dedifferentiation in vitro, confirmed by the observation that mouse hepatocytes cultured in normoxia (18% O2) undergo epithelial-to-mesenchymal transi- tion, obtain fibroblast-like morphology, and show impaired hepatic functions. These ef- fects were prevented by culturing hepatocytes at 5% O2, which show increased glycogen stores, increased LDL-uptake ability, and sustained expression of cytochrome P450 en- zymes, all important characteristics of hepatocytes in vivo [102]. 2+ 2+ Intracellular Ca concentration ([Ca ]i) is critical in mediating a wide variety of pro- cesses, in both physiological and pathological conditions. Keeley et al. have recently 2+ shown that culturing endothelial cells at atmospheric O2 levels impairs Ca handling. Us- ing human umbilical vascular endothelial cells (HUVECs) cultured at 5% O2, they re- ported a negative feedback mechanism mediated by protein phosphatase 2A that regu- 2+ lates Ca –dependent NO biosynthesis in response to histamine, which is not observed at 2+ atmospheric O2 [103]. Interestingly, although they observed a similar intracellular Ca 2+ peak in response to histamine at both O2 conditions, [Ca ]i at the plateau phase was re- 2+ duced in cells cultured in 5% O2, suggesting enhanced cytosolic Ca removal mechanisms. Similar results were obtained when HUVECs were treated with ATP and ionomycin. These observations were further characterized in a subsequent study by the same lab. The 2+ difference in plateau [Ca ]i upon stimulation with histamine at 5% and 18% O2 was absent when cells were treated with cyclopiazonic acid, an inhibitor of the sarco/endoplasmic 2+ reticulum Ca –ATPase (SERCA). Moreover, agonist-stimulated phosphorylation of phos- pholamban, an endogenous negative regulator of SERCA, was higher at 5% O2 than at ambient air. Consequently, enhanced SERCA activity at physioxia blunted ionomycin-in- 2+ duced Ca overload and cell death, compared to the 18% O2 group [104]. Taken together, 2+ these results suggest that hyperoxic conditions in cell culture dysregulate Ca handling by reducing SERCA activity, which may artificially increase the sensitivity of cells to cy- 2+ 2+ totoxic stimuli that target intracellular Ca . Regulation of additional cytosolic Ca influx and efflux mechanisms at 5% and 18% O2 should be investigated. Hypoxia/reoxygenation (H/R) is the in vitro model of ischemia/reperfusion injury that takes place in pathologies such as strokes and myocardial infarctions. It involves ex- posing cells to hypoxia (≤ 1% O2) for a sustained period, usually coupled with nutrient deprivation, followed by placing them back to normoxic conditions. Researchers have hy- pothesized that oxidative stress and cell injury are artefactually exacerbated at the rela- tively hyperoxic reoxygenation conditions (at least an 18-fold pO2 increase, compared to hypoxia) used in most studies and that such stress may be less severe at physioxia (~5 - fold pO2 increase, compared to hypoxia). Warpisnki et al. reported that H/R did not seem to increase the generation of mitochondrial and extramitochondrial O2 at physioxia. Con- sequently, no protective effects of sulforaphane pretreatment were observed at 5% O 2, compared to at ambient air [105]. Similarly, Danilov and Fiskum cultured rat cortical as- trocytes at 7% or 18% O2 and exposed them to both hypoxia and glucose deprivation and then to reoxygenation in their respective O2 tensions [106]. Decreased cell death and lower levels of protein nitration and DNA oxidation were detected during reoxygenation at 7% O2, again confirming that hyperoxic reoxygenation under ambient air exacerbates cell in- jury in H/R models. In summary, keeping physiological O2 conditions in cell culture will provide us a better understanding of the physiological and pathological processes of cells and, in turn, allow us to conduct more reliable and relevant experiments to study the effects of different stimuli on cell physiology in vitro. In the next section, we will discuss how the cellular response to drugs, hormones, and toxicants is dependent on O2 tension, leading to arti- facts caused by supraphysiological O2 levels. Cells 2022, 11, 3123 13 of 21 9. Cellular Response to Drugs, Hormones, and Toxicants In vitro studies have provided us invaluable knowledge about the cellular effects and mechanisms of endogenous and exogenous substances, such as drugs, hormones, pollu- tants, toxins, and nanoparticles. However, experimental findings from cell culture often translate poorly in vivo. Recent evidence has shown that culturing cells in supraphysio- logical O2 levels can lead to an overestimation or underestimation of the potency of bio- logically active compounds or even demonstrate opposing effects. For example, resvera- trol acts as a prooxidant in C2C12 and PC-3 cells grown in low glucose DMEM at 18% O2, as shown by an increased H2O2 efflux rate. However, this effect is absent in both cell lines when cultured at 5% O2 [107]. Yan et al. reported that the hepatotoxic effect of acetamino- phen was less pronounced at physioxia, compared to atmospheric O2 [36]. On the other hand, the cytotoxic effects of copper oxide nanoparticles (CuO NPs) were diminished in normoxia, likely due to a preconditioning to oxidant insults in the hyperoxic environment of standard cell culture [108]. Similarly, the antiproliferative and cytotoxic effect of teri- flunomide on the colon cancer cell lines SW480 and SW620 was significantly enhanced with decreasing O2 concentration [109]. Table 2 summarizes the findings in the literature on biologically active substances that have been shown to elicit different cellular re- sponses at physioxia versus standard O2 conditions. Indeed, these observations highlight the need for utilizing physiologically representative O2 levels in cell culture, to improve the predictive value of in vitro pharmacological and toxicological studies. Table 2. Differential cellular responses to drugs, hormones, and toxicants at atmospheric O2 versus physioxia. Molecule Mechanism Conditions Outcomes Reference Drugs ROS scavenger, PC-3 and C2C12 cells Differential H2O2 production, proliferation, and resveratrol [107] multiple targets 18% or 5% O2 mitochondrial network dynamics bEnd.3 cells ROS scavenger, Attenuated reoxygenation-induced ROS produc- sulforaphane 18% or 5% O2 [105] multiple targets tion at 18% O2 but not at 5% O2 H/R human neonatal fore- ROS scavenger, GSH depletion and loss of type I quercetin skin fibroblasts [110] multiple targets cells at 18% O2 but not at 4% O2 18% or 4% O2 HCT116, IMR90, U2OS, DNA intercalat- doxorubicin and MCF-7 cells  apoptosis at 18% O2 [49] ing agent 18% O2 or 5% O2 mouse hepatocytes  hepatotoxicity at 18% O2 [36] 18%, 10%, or 5% O2  mROS and RNS production at 18% O2 acetaminophen COX inhibitor  hepatotoxicity at 18% O2 HepG2 cells differential regulation of [111] 18%, 8%, or 3% O2 phase I and II enzymes DNA cross-link- HepG2 cells cyclophosphamide  hepatotoxicity at 18% O2 [111] ing agent 18%, 8%, or 3% O2 pyrimidine syn- SW480 and SW620 cells  proapoptotic effect at 18% O2 teriflunomide thesis [109] 18% or 10% O2  antiproliferative effect at 18% O2 inhibitor DNA synthesis SW480 and SW620 cells oxaliplatin  antiproliferative effect at 18% O2 [109] inhibitor 18% or 10% O2 mouse mammary microtubule sta- paclitaxel tumors  cytotoxicity at 18% O2 [47] bilizer 18% or 3–5% O2 mouse mammary alpelisib PI3K inhibitor tumors  cytotoxicity at 18% O2 [47] 18% or 3–5% O2 Cells 2022, 11, 3123 14 of 21 mouse mammary erlotinib EGFR inhibitor tumors  cytotoxicity at 18% O2 [47] 18% or 3–5% O2 patient-derived  Ki-67-positive cells at 18% O2 V600 BRAF vemurafenib melanoma cells  reduction of VEGF, PCG-1α, [37] inhibitor 18% or 6% O2 and SLC7A11 levels at 18% O2 patient-derived  Ki-67-positive cells at 18% O2 MEK1/2 trametinib melanoma cells  reduction of VEGF, PCG-1α, [37] inhibitor 18% or 6% O2 and SLC7A11 levels at 18% O2 topoisomerase U87MG cells camptothecin  cytotoxicity at 18% O2 [112] inhibitor 18% O2 or 9% O2 RAW 264.7 cells  expression of Nrf2 targets and antioxidant re- dimethyl fumarate Nrf2 inducer [20] 18% O2 or 5% O2 sponse Hs68 and HaCaT keratolytic, Differential regulation of skin barrier glycolic acid cells [113] antioxidant and dermal network-related genes 18% or 2% O2 Hs68 and HaCaT keratolytic, Differential regulation of skin barrier gluconolactone cells [113] antioxidant and dermal network-related genes 18% or 2% O2 keratolytic, Hs68 and HaCaT Differential regulation of skin barrier salicylic acid AMPK cells [113] and dermal network-related genes activator 18% or 2% O2 Hormones C2C12 cells Differential H2O2 production, metabolism, and 17β-estradiol ER antagonist [114] 18% O2 or 5% O2 mitochondrial network dynamics Toxicants RAW 264.7 cells LPS TLR4 agonist  production of inflammatory mediators [20] 18% O2 or 5% O2  cytotoxicity at 18% O2 complex I SH-SY5Y cells No inhibition of ATP synthesis rotenone [115] inhibitor 18% O2 or 5% O2 with 0.2 µM rotenone at 18% O2 (with effects observed at 5%) DNA and differentiated acrolein protein adduct H9c2 cells  cytotoxicity at 18% O2 [116] inducer 18% O2 and 5% O2 DNA adduct in- HepG2 cells aflatoxin B  hepatotoxicity at 18% O2 [111] ducer 18%, 8%, or 3% O2 Other V. baccifera Prooxidant, HepG2 cells  cytotoxicity at 18% O2 [117] leaf extract cytotoxic 18% O2 or 8% O2 Prooxidant, gen- A549 cells  NP-induced oxidative stress at 18% O2 CuO NPs otoxic, [108] 18% O2 or 13% O2  cytotoxicity at 18% O2 cytotoxic V600 Abbreviations: AMPK, AMP-dependent kinase; BRAF , B-raf (mutated); COX, cyclooxygenase; EGFR, epidermal growth factor receptor; ER, estrogen receptor; GSH, reduced glutathione; H/R, hypoxia/reoxygenation; LPS, lipopolysaccharide; MEK1/2, MAPK/ERK kinase 1/2; NPs, nanoparti- cles; PI3K, phosphatidylinositol 3-kinase; ROS, reactive oxygen species; mROS, mitochondrial ROS; RNS, reactive nitrogen species, TLR4, toll-like receptor 4. 10. Conclusions and Future Directions An increasing amount of evidence shows that culturing cells in the hyperoxic envi- ronment of ambient air disturbs their redox homeostasis, alters gene expression, inhibits proliferation, impairs differentiation, impacts energy metabolism, and alters the cellular response to drugs (illustrated in Figure 2). The potential artifacts caused by growing cells at nonphysiological O2 levels underline the need for revisiting previous results from cell- Cells 2022, 11, 3123 15 of 21 culture-based research, particularly the ones intimately related with oxygen and oxidative stress, including, but not limited to, studies using hypoxia and hyperoxia models. Figure 2. Cellular mechanisms differentially regulated at physioxia (2–6% O2) versus normoxia (18% O2). In physioxia (A), activation of HIF may drive the induction of genes that regulate glucose me- tabolism, survival, proliferation, differentiation, and other processes. In many cases, mitochondria have a more elongated morphology and form highly fused networks. In normoxia (B), increased ROS production by NADPH oxidases (NOX) and the electron transport chain (ETC) leads to oxida- tive stress, characterized by DNA and lipid oxidation, and promotes activation of a variety of path- ways such as p53 and Nrf2, which, in turn, results in the induction of antioxidant genes and senes- cence. Further, in many instances, mitochondria show a more globular morphology and increased fission of mitochondrial networks. These cellular mechanisms result in nonphysiological pheno- types and differential responses to bioactive molecules. Created with BioRender.com (accessed on 26 September 2022). Additional research should be conducted to further characterize the mechanisms of altered cellular responses to O2 at 18% O2, compared to at physioxia, with a focus on elu- cidating the molecular basis of the response in this range and its specificity between Cells 2022, 11, 3123 16 of 21 different cell types. In particular, future research on oxidative post-translational modifi- cations through the use of redox proteomics, in conjunction with other “omics” ap- proaches, will be important. In addition, more research should be conducted to delineate the role of HIF at physiological O2 levels. While HIF stabilization/activation reported by some studies could well be the result of an acute reduction in O2 tension, the detection of HIF targets in a handful of cell types adapted to physioxia for at least two weeks [37,50,118] suggests a role for HIF activity at physioxia. In this respect, it is also critical to keep in mind that physioxia is a range rather than a set value, as it varies depending on the tissue. For example, while 3–6% O2 would be appropriate for cells like hepatocytes, cardiomyocytes, neurons, and enterocytes, the same would not be true for alveolar epi- thelial cells, which are exposed to ~13% O2 in vivo. On the other hand, most tumors exhibit median O2 levels below 2% [3]. As such, O2 tensions relevant to the specific tissue being studied should be utilized. Moreover, it is important to consider potential differences in pericellular O2 levels (in the liquid phase) versus the incubator headspace O2 (gaseous phase). Pericellular O2 levels depend on many factors, such as the O2 consumption rate of each cell type, the cell seeding density, the material of the cell culture vessel, and the vol- ume of the media in the vessel, to mention a few [119]. These and other considerations are elegantly discussed in the seminal review by Keeley and Mann [2]. The findings discussed in this review highlight the need to incorporate incubators capable of regulating O2 concentration into our research facilities. While the elevated cost of commercially available O2-regulating incubators (> USD 10,000) may seem like a barrier to implement physioxia in cell culture workflows, our lab has recently developed a port- able, home-made incubator with a total production cost of < USD 1000, which effectively regulates O2, CO2, and temperature [120]. We also demonstrated its ability to maintain both the headspace and pericellular levels at 5% O2 for several weeks. Similarly, Marchus et al. developed an O2-regulating incubator with a cost of ~USD 800 [121], showing that implementing physiological O2 conditions into routine cell culture does not need to come at a great expense. Finally, additional considerations should be made to recreate a more physiologically representative environment in cell culture, such as using physiological culture media (e.g., Plasmax, HPLM), 3D culture, and co-culture (see [1]). Implementing physiological cell culture conditions will improve the quality and validity of studies conducted in vitro. Author Contributions: Conceptualization, R.A.; writing—original draft preparation, R.A.; writ- ing—review and editing, R.A., G.L.G., P.L., and J.A.S. All authors have read and agreed to the pub- lished version of the manuscript. Funding: P.L. and J.A.S. are funded by the Natural Sciences and Engineering Research Council (NSERC) of Canada Discovery Grant (RGPIN 2017-06785 and RGPIN 2020-05274, respectively). R.A. is supported by a Mitacs Globalink Graduate Fellowship. Acknowledgments: We thank Olivia R.M. Bagshaw for her exceptionally helpful comments and suggestions. The graphical abstract was created using BioRender.com, via a subscription by J.A.S. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Abbas, M.; Moradi, F.; Hu, W.; Regudo, K.L.; Osborne, M.; Pettipas, J.; Atallah, D.S.; Hachem, R.; Ott-Peron, N.; Stuart, J.A. Vertebrate cell culture as an experimental approach – limitations and solutions. Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol. 2021, 254, 110570, https://doi.org/10.1016/j.cbpb.2021.110570. 2. Keeley, T.P.; Mann, G.E. Defining Physiological Normoxia for Improved Translation of Cell Physiology to Animal Models and Humans. Physiol. Rev. 2019, 99, 161–234, https://doi.org/10.1152/physrev.00041.2017. 3. McKeown, S.R. Defining normoxia, physoxia and hypoxia in tumours—implications for treatment response. Br. J. Ra- diol. 2014, 87, 20130676, https://doi.org/10.1259/bjr.20130676. 4. Brand, M.D. The sites and topology of mitochondrial superoxide production. Exp. Gerontol. 2010, 45, 466–472, https://doi.org/10.1016/j.exger.2010.01.003. Cells 2022, 11, 3123 17 of 21 5. Alfadda, A.A.; Sallam, R.M. Reactive Oxygen Species in Health and Disease. J. Biomed. Biotechnol. 2012, 2012, 1–14, https://doi.org/10.1155/2012/936486. 6. Zorov, D.B.; Juhaszova, M.; Sollott, S.J. Mitochondrial Reactive Oxygen Species (ROS) and ROS-Induced ROS Release. Physiol. Rev. 2014, 94, 909–950. https://doi.org/10.1152/physrev.00026.2013. 7. Fukai, T.; Ushio-Fukai, M. Superoxide Dismutases: Role in Redox Signaling, Vascular Function, and Diseases. Antioxidants Re- dox Signal. 2011, 15, 1583–1606, https://doi.org/10.1089/ars.2011.3999. 8. Sies, H. Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress. Redox Biol. 2017, 11, 613–619, https://doi.org/10.1016/j.redox.2016.12.035. 9. Kurutas, E.B. The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: Cur- rent state. Nutr. J. 2016, 15, 71, doi:10.1186/s12937-016-0186-5. 10. Pham-Huy, L.A.; He, H.; Pham-Huy, C. Free Radicals, Antioxidants in Disease and Health. Int. J. Biomed. Sci. IJBS 2008, 4, 89. 11. Sies, H.; Belousov, V.V.; Chandel, N.S.; Davies, M.J.; Jones, D.P.; Mann, G.E.; Murphy, M.P.; Yamamoto, M.; Winterbourn, C. Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology. Nat. Rev. Mol. Cell Biol. 2022, 23, 499– 515, https://doi.org/10.1038/s41580-022-00456-z. 12. Hoffman, D.L.; Brookes, P.S. Oxygen Sensitivity of Mitochondrial Reactive Oxygen Species Generation Depends on Metabolic Conditions. J. Biol. Chem. 2009, 284, 16236–16245, https://doi.org/10.1074/jbc.m809512200. 13. Roy, S.; Khanna, S.; Bickerstaff, A.A.; Subramanian, S.V.; Atalay, M.; Bierl, M.; Pendyala, S.; Levy, D.; Sharma, N.; Venojar vi, M.; et al. Oxygen Sensing by Primary Cardiac Fibroblasts. Circ. Res. 2003, 92, 264–271, https://doi.org/10.1161/01.res.0000056770.30922.e6. 14. Nasto, L.A.; Robinson, A.R.; Ngo, K.; Clauson, C.L.; Dong, Q.; Croix, C.S.; Sowa, G.; Pola, E.; Robbins, P.D.; Kang, J.; et a l. Mitochondrial-derived reactive oxygen species (ROS) play a causal role in aging-related intervertebral disc degeneration. J. Orthop. Res. 2013, 31, 1150–1157, https://doi.org/10.1002/jor.22320. 15. El Alami, M.; Viña-Almunia, J.; Gambini, J.; Mas-Bargues, C.; Siow, R.C.; Peñarrocha, M.; Mann, G.E.; Borrás, C.; Viña, J. Acti- vation of p38, p21, and NRF-2 Mediates Decreased Proliferation of Human Dental Pulp Stem Cells Cultured under 21% O2. Stem Cell Rep. 2014, 3, 566–573, https://doi.org/10.1016/j.stemcr.2014.08.002. 16. Nisimoto, Y.; Diebold, B.A.; Cosentino-Gomes, D.; Lambeth, J.D. Nox4: A Hydrogen Peroxide-Generating Oxygen Sensor. Bio- chemistry 2014, 53, 5111–5120, https://doi.org/10.1021/bi500331y. 17. Maddalena, L.A.; Selim, S.M.; Fonseca, J.; Messner, H.; McGowan, S.; Stuart, J.A. Hydrogen peroxide production is affected by oxygen levels in mammalian cell culture. Biochem. Biophys. Res. Commun. 2017, 493, 246–251, https://doi.org/10.1016/j.bbrc.2017.09.037. 18. Stuart, J.A.; Fonseca, J.; Moradi, F.; Cunningham, C.; Seliman, B.; Worsfold, C.R.; Dolan, S.; Abando, J.; Maddalena, L.A. Ho w Supraphysiological Oxygen Levels in Standard Cell Culture Affect Oxygen-Consuming Reactions. Oxidative Med. Cell. Lon- gev. 2018, 2018, 1–13, https://doi.org/10.1155/2018/8238459. 19. Estrada, J.C.; Albo, C.; Benguría, A.; Dopazo, A.; López-Romero, P.; Carrera-Quintanar, L.; Roche, E.; Clemente, E.P.; A Enríquez, J.; Bernad, A.; et al. Culture of human mesenchymal stem cells at low oxygen tension improves growth and genetic stability by activating glycolysis. Cell Death Differ. 2011, 19, 743–755, https://doi.org/10.1038/cdd.2011.172. 20. Haas, B.; Chrusciel, S.; Fayad-Kobeissi, S.; Dubois-Randé, J.-L.; Azuaje, F.; Boczkowski, J.; Motterlini, R.; Foresti, R. Permanent Culture of Macrophages at Physiological Oxygen Attenuates the Antioxidant and Immunomodulatory Properties of Dimethyl Fumarate. J. Cell. Physiol. 2014, 230, 1128–1138, https://doi.org/10.1002/jcp.24844. 21. Zhu, J.; Aja, S.; Kim, E.-K.; Park, M.J.; Ramamurthy, S.; Jia, J.; Hu, X.; Geng, P.; Ronnett, G.V. Physiological oxygen level is critical for modeling neuronal metabolism in vitro. J. Neurosci. Res. 2011, 90, 422–434, https://doi.org/10.1002/jnr.22765. 22. Atkuri, K.R.; Herzenberg, L.A.; Niemi, A.-K.; Cowan, T. Importance of culturing primary lymphocytes at physiological oxygen levels. Proc. Natl. Acad. Sci. 2007, 104, 4547–4552, https://doi.org/10.1073/pnas.0611732104. 23. Ferguson, D.C.; Smerdon, G.R.; Harries, L.W.; Dodd, N.J.; Murphy, M.P.; Curnow, A.; Winyard, P.G. Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia. Free Radic. Biol. Med. 2018, 126, 322–333, https://doi.org/10.1016/j.freeradbiomed.2018.08.025. 24. Timpano, S.; Guild, B.D.; Specker, E.J.; Melanson, G.; Medeiros, P.J.; Sproul, S.L.J.; Uniacke, J. Physioxic human cell culture improves viability, metabolism, and mitochondrial morphology while reducing DNA damage. FASEB J. 2019, 33, 5716–5728, https://doi.org/10.1096/fj.201802279r. 25. Konigsberg, M.; Pérez, V.; Ríos, C.; Liu, Y.; Lee, S.; Shi, Y.; Van Remmen, H. Effect of oxygen tension on bioenergetics and proteostasis in young and old myoblast precursor cells. Redox Biol. 2013, 1, 475–482, https://doi.org/10.1016/j.redox.2013.09.004. 26. Semenza, G.L.; Wang, G.L. A Nuclear Factor Induced by Hypoxia via De Novo Protein Synthesis Binds to the Human Erythro- poietin Gene Enhancer at a Site Required for Transcriptional Activation. Mol. Cell Biol. 1992, 12, 5447–5454. 27. Wang, G.L.; Jiang, B.-H.; Rue, E.A.; Semenza, G.L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer reg- ulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA 1995, 92, 5510–5514, doi:10.1073/pnas.92.12.5510. 28. Lee, P.; Chandel, N.S.; Simon, M.C. Cellular adaptation to hypoxia through hypoxia inducible factors and beyond. Nat. Rev. Mol. Cell Biol. 2020, 21, 268–283, https://doi.org/10.1038/s41580-020-0227-y. 29. Dengler, V.L.; Galbraith, M.; Espinosa, J.M. Transcriptional regulation by hypoxia inducible factors. Crit. Rev. Biochem. Mol. Biol. 2014, 49, 1–15, https://doi.org/10.3109/10409238.2013.838205. Cells 2022, 11, 3123 18 of 21 30. Hu, C.-J.; Wang, L.-Y.; Chodosh, L.A.; Keith, B.; Simon, M.C. Differential Roles of Hypoxia-Inducible Factor 1α (HIF-1α) and HIF-2α in Hypoxic Gene Regulation. Mol. Cell. Biol. 2003, 23, 9361–9374, https://doi.org/10.1128/mcb.23.24.9361-9374.2003. 31. Loboda, A.; Jozkowicz, A.; Dulak, J. HIF-1 and HIF-2 transcription factors — Similar but not identical. Mol. Cells 2010, 29, 435– 442, https://doi.org/10.1007/s10059-010-0067-2. 32. Maxwell, P.H.; Wiesener, M.S.; Chang, G.-W.; Clifford, S.C.; Vaux, E.C.; Cockman, M.E.; Wykoff, C.C.; Pugh, C.W.; Maher, E.R.; Ratcliffe, P.J. The Tumour Suppressor Protein VHL Targets Hypoxia-Inducible Factors for Oxygen-Dependent Pro-teolysis. Nature 1999, 399, 271–275. 33. Metzen, E.; Ratcliffe, P.J.; Ratcliffe, E.M. HIF hydroxylation and cellular oxygen sensing. Biol. Chem. 2004, 385, 223–230, https://doi.org/10.1515/bc.2004.016. 34. Hirsilä, M.; Koivunen, P.; Günzler, V.; Kivirikko, K.I.; Myllyharju, J. Characterization of the Human Prolyl 4-Hydroxylases That Modify the Hypoxia-inducible Factor. J. Biol. Chem. 2003, 278, 30772–30780, https://doi.org/10.1074/jbc.m304982200. 35. Bracken, C.P.; Fedele, A.O.; Linke, S.; Balrak, W.; Lisy, K.; Whitelaw, M.L.; Peet, D.J. Cell-specific Regulation of Hypoxia-induc- ible Factor (HIF)-1α and HIF-2α Stabilization and Transactivation in a Graded Oxygen Environment. J. Biol. Chem. 2006, 281, 22575–22585, https://doi.org/10.1074/jbc.m600288200. 36. Yan, H.-M.; Ramachandran, A.; Bajt, M.L.; Lemasters, J.J.; Jaeschke, H. The Oxygen Tension Modulates Acetaminophen-Induced Mitochondrial Oxidant Stress and Cell Injury in Cultured Hepatocytes. Toxicol. Sci. 2010, 117, 515–523, https://doi.org/10.1093/toxsci/kfq208. 37. Osrodek, M.; Hartman, M.L.; Czyz, M. Physiologically Relevant Oxygen Concentration (6% O2) as an Important Component of the Microenvironment Impacting Melanoma Phenotype and Melanoma Response to Targeted Therapeutics In Vitro. Int. J. Mol. Sci. 2019, 20, 4203, https://doi.org/10.3390/ijms20174203. 38. Stuart, J.A.; Aibueku, O.; Bagshaw, O.; Moradi, F. Hypoxia inducible factors as mediators of reactive oxygen/nitrogen species homeostasis in physiological normoxia. Med Hypotheses 2019, 129, 109249, https://doi.org/10.1016/j.mehy.2019.109249. 39. Suzuki, T.; Yamamoto, M. Molecular basis of the Keap1–Nrf2 system. Free Radic. Biol. Med. 2015, 88, 93–100, https://doi.org/10.1016/j.freeradbiomed.2015.06.006. 40. Yamamoto, M.; Kensler, T.W.; Motohashi, H. The KEAP1-NRF2 System: a Thiol-Based Sensor-Effector Apparatus for Maintain- ing Redox Homeostasis. Physiol. Rev. 2018, 98, 1169–1203, doi:10.1152/physrev.00023.2017. 41. Suzuki, T.; Muramatsu, A.; Saito, R.; Iso, T.; Shibata, T.; Kuwata, K.; Kawaguchi, S.-I.; Iwawaki, T.; Adachi, S.; Suda, H.; et al. Molecular Mechanism of Cellular Oxidative Stress Sensing by Keap1. Cell Rep. 2019, 28, 746–758.e4, https://doi.org/10.1016/j.celrep.2019.06.047. 42. Baird, L.; Yamamoto, M. The molecular mechanisms regulating the KEAP1-NRF2 pathway. Mol. Cell. Biol. 2020, 40, e00099-20, doi:10.1128/mcb.00099-20. 43. Chen, Z.-H.; Saito, Y.; Yoshida, Y.; Sekine, A.; Noguchi, N.; Niki, E. 4-Hydroxynonenal Induces Adaptive Response and En- hances PC12 Cell Tolerance Primarily through Induction of Thioredoxin Reductase 1 via Activation of Nrf2. J. Biol. Chem. 2005, 280, 41921–41927, https://doi.org/10.1074/jbc.m508556200. 44. McMahon, M.; Lamont, D.J.; Beattie, K.A.; Hayes, J.D. Keap1 perceives stress via three sensors for the endogenous signaling molecules nitric oxide, zinc, and alkenals. Proc. Natl. Acad. Sci. USA 2010, 107, 18838–18843, https://doi.org/10.1073/pnas.1007387107. 45. Tonelli, C.; Chio, I.I.C.; Tuveson, D.A. Transcriptional Regulation by Nrf2. Antioxid. Redox Signal. 2018, 29, 1727–1745, doi:10.1089/ars.2017.7342. 46. Alva, R.; Mirza, M.; Baiton, A.; Lazuran, L.; Samokysh, L.; Bobinski, A.; Cowan, C.; Jaimon, A.; Obioru , D.; Al Makhoul, T.; et al. Oxygen toxicity: cellular mechanisms in normobaric hyperoxia. Cell Biol. Toxicol. 2022, 1–33, https://doi.org/10.1007/s10565- 022-09773-7. 47. Kumar, B.; Adebayo, A.K.; Prasad, M.; Capitano, M.L.; Wang, R.; Bhat-Nakshatri, P.; Anjanappa, M.; Simpson, E.; Chen, D.; Liu, Y.; et al. Tumor collection/processing under physioxia uncovers highly relevant signaling networks and drug sensitivity. Sci. Adv. 2022, 8, https://doi.org/10.1126/sciadv.abh3375. 48. Chapple, S.J.; Keeley, T.P.; Mastronicola, D.; Arno, M.; Vizcay-Barrena, G.; Fleck, R.; Siow, R.C.; Mann, G.E. Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxy- gen levels. Free Radic. Biol. Med. 2015, 92, 152–162, https://doi.org/10.1016/j.freeradbiomed.2015.12.013. 49. Carrera, S.; de Verdier, P.J.; Khan, Z.; Zhao, B.; Mahale, A.; Bowman, K.J.; Zainol, M.; Jones, G.D.D.; Lee, S.W.; Aaronson, S.A.; et al. Protection of Cells in Physiological Oxygen Tensions against DNA Damage-induced Apoptosis. J. Biol. Chem. 2010, 285, 13658–13665, https://doi.org/10.1074/jbc.m109.062562. 50. Alva, R.; Moradi, F.; Liang, P.; Stuart, J.A. Culture of Cancer Cells at Physiological Oxygen Levels Affects Gene Expression in a Cell-Type Specific Manner. Preprints (Basel) 2022, 2022080497, doi:10.20944/preprints202208.0497.v1. 51. Duś-Szachniewicz, K.; Gdesz-Birula, K.; Zduniak, K.; Wiśniewski, J. Proteomic-Based Analysis of Hypoxia- and Physioxia-Re- sponsive Proteins and Pathways in Diffuse Large B-Cell Lymphoma. Cells 2021, 10, 2025, https://doi.org/10.3390/cells10082025. 52. Chee, N.T.; Lohse, I.; Brothers, S.P. mRNA-to-protein translation in hypoxia. Mol. Cancer 2019, 18, 1–13, https://doi.org/10.1186/s12943-019-0968-4. 53. Ivanova, I.G.; Park, C.V.; Kenneth, N.S. Translating the Hypoxic Response—The Role of HIF Protein Translation in the Cellular Response to Low Oxygen. Cells 2019, 8, 114, https://doi.org/10.3390/cells8020114. Cells 2022, 11, 3123 19 of 21 54. Uniacke, J.; Holterman, C.E.; Lachance, G.; Franovic, A.; Jacob, M.D.; Fabian, M.R.; Payette, J.; Holcik, M.; Pause, A.; Lee, S. An oxygen-regulated switch in the protein synthesis machinery. Nature 2012, 486, 126–129, doi:10.1038/nature11055. 55. Timpano, S.; Uniacke, J. Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Dis- tinct mRNAs for Translation. J. Biol. Chem. 2016, 291, 10772–10782, https://doi.org/10.1074/jbc.m116.717363. 56. Batie, M.; Rocha, S. Gene transcription and chromatin regulation in hypoxia. Biochem. Soc. Trans. 2020, 48, 1121–1128, https://doi.org/10.1042/bst20191106. 57. Guillaumet-Adkins, A.; Yañez, Y.; Peris-Diaz, M.D.; Calabria, I.; Palanca-Ballester, C.; Sandoval, J. Epigenetics and Oxidative Stress in Aging. Oxidative Med. Cell. Longev. 2017, 2017, 1–8, https://doi.org/10.1155/2017/9175806. 58. Lengner, C.; Gimelbrant, A.A.; Erwin, J.; Cheng, A.; Guenther, M.G.; Welstead, G.G.; Alagappan, R.; Frampton, G.M.; Xu, P.; Muffat, J.; et al. Derivation of Pre-X Inactivation Human Embryonic Stem Cells under Physiological Oxygen Concentra- tions. Cell 2010, 141, 872–883, https://doi.org/10.1016/j.cell.2010.04.010. 59. Xie, P.; Sun, Y.; Ouyang, Q.; Hu, L.; Tan, Y.; Zhou, X.; Xiong, B.; Zhang, Q.; Yuan, D.; Pan, Y.; et al. Physiological Oxygen Prevents Frequent Silencing of the DLK1-DIO3 Cluster during Human Embryonic Stem Cells Culture. Stem Cells 2013, 32, 391– 401, https://doi.org/10.1002/stem.1558. 60. Lees, J.G.; Cliff, T.S.; Gammilonghi, A.; Ryall, J.G.; Dalton, S.; Gardner, D.K.; Harvey, A.J. Oxygen Regulates Human Pluripotent Stem Cell Metabolic Flux.. Stem Cells Int. 2019, 2019, 8195614–17, https://doi.org/10.1155/2019/8195614. 61. Thienpont, B.; Steinbacher, J.; Zhao, H.; D’Anna, F.; Kuchnio, A.; Ploumakis, A.; Hermans, E. Tumour hypoxia causes DNA hypermethylation by reducing TET activity. Nature 2016, 537, 63–68, https://doi.org/10.1038/nature19081. 62. Lin, G.; Sun, W.; Yang, Z.; Guo, J.; Liu, H.; Liang, J. Hypoxia induces the expression of TET enzymes in HepG2 cells. Oncol. Lett. 2017, 14, 6457–6462, https://doi.org/10.3892/ol.2017.7063. 63. Dogan, F.; Aljumaily, R.; Kitchen, M.; Forsyth, N. DNMT3B Is an Oxygen-Sensitive De Novo Methylase in Human Mesenchy- mal Stem Cells. Cells 2021, 10, 1032, https://doi.org/10.3390/cells10051032. 64. Dogan, F.; Aljumaily, R.M.K.; Kitchen, M.; Forsyth, N.R. Physoxia Influences Global and Gene-Specific Methylation in Pluripo- tent Stem Cells. Int. J. Mol. Sci. 2022, 23, 5854, https://doi.org/10.3390/ijms23105854. 65. Johnston, H.; Dickinson, P.; Ivens, A.; Buck, A.H.; Levine, R.D.; Remacle, F.; Campbell, C.J. Intracellular redox potential is cor- related with miRNA expression in MCF7 cells under hypoxic conditions. Proc. Natl. Acad. Sci. 2019, 116, 19753–19759, https://doi.org/10.1073/pnas.1909455116. 66. Shi, R.; Yang, H.; Lin, X.; Cao, Y.; Zhang, C.; Fan, Z.; Hou, B. Analysis of the characteristics and expression profiles of coding and noncoding RNAs of human dental pulp stem cells in hypoxic conditions. Stem Cell Res. Ther. 2019, 10, 89, https://doi.org/10.1186/s13287-019-1192-2. 67. Cooper, P.D.; Burt, A.M.; Wilson, J.N. Critical Effect of Oxygen Tension on Rate of Growth of Animal Cells in Continuous Suspended Culture. Nature 1958, 182, 1508–1509, https://doi.org/10.1038/1821508b0. 68. Packer, L.; Fuehr, K. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature 1977, 267, 423–425, https://doi.org/10.1038/267423a0. 69. Balin, A.K.; Fisher, A.J.; Carter, D.M. Oxygen modulates growth of human cells at physiologic partial pressures.. J. Exp. Med. 1984, 160, 152–166, https://doi.org/10.1084/jem.160.1.152. 70. Falanga, V.; Kirsner, T.S. Low Oxygen Stimulates Proliferation of Fibroblasts Seeded as Single Cells. J. Cell Physiol. 1993, 154, 71. McHugh, D.; Gil, J. Senescence and aging: Causes, consequences, and therapeutic avenues. J. Cell Biol. 2017, 217, 65–77, https://doi.org/10.1083/jcb.201708092. 72. Busuttil, R.A.; Rubio, M.; Dollé, M.E.T.; Campisi, J.; Vijg, J. Oxygen accelerates the accumulation of mutations during the senes- cence and immortalization of murine cells in culture. Aging Cell 2003, 2, 287–294, https://doi.org/10.1046/j.1474- 9728.2003.00066.x. 73. Parrinello, S.; Samper, E.; Krtolica, A.; Goldstein, J.; Melov, S.; Campisi, J. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat. Cell Biol. 2003, 5, 741–747, https://doi.org/10.1038/ncb1024. 74. Lees, S.J.; Childs, T.E.; Booth, F.W. P21 Cip1 Expression Is Increased in Ambient Oxygen, Compared to Estimated Physi-ological (5%) Levels in Rat Muscle Precursor Cell Culture. Cell Prolif. 2008, 41, 193–207, doi:10.1111/j.1365-2184.2008.00512. 75. Bon-Mathier, A.-C.; Rignault-Clerc, S.; Bielmann, C.; Rosenblatt-Velin, N. Oxygen as a key regulator of cardiomyocyte prolifer- ation: New results about cell culture conditions!. Biochim. et Biophys. Acta 2019, 1867, 118460, https://doi.org/10.1016/j.bbamcr.2019.03.007. 76. Braunschweig, L.; Meyer, A.K.; Wagenführ, L.; Storch, A. Oxygen regulates proliferation of neural stem cells through Wnt/β - catenin signalling. Mol. Cell. Neurosci. 2015, 67, 84–92, https://doi.org/10.1016/j.mcn.2015.06.006. 77. Covello, K.L.; Kehler, J.; Yu, H.; Gordan, J.D.; Arsham, A.M.; Hu, C.-J.; Labosky, P.A.; Simon, M.C.; Keith, B. HIF-2α regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth. Genes Dev. 2006, 20, 557–570, https://doi.org/10.1101/gad.1399906. 78. Westfall, S.D.; Sachdev, S.; Das, P.; Hearne, L.B.; Hannink, M.; Roberts, R.M.; Ezashi, T. Identification of Oxygen -Sensitive Tran- scriptional Programs in Human Embryonic Stem Cells. Stem Cells Dev. 2008, 17, 869–882, https://doi.org/10.1089/scd.2007.0240. 79. Keith, B.; Simon, M.C. Hypoxia-Inducible Factors, Stem Cells, and Cancer. Cell 2007, 129, 465–472, https://doi.org/10.1016/j.cell.2007.04.019. Cells 2022, 11, 3123 20 of 21 80. Broxmeyer, H.E.; Cooper, S.; Gabigbad, T. The Effects of Oxidizing Species Derived from Molecular Oxygen on the Pro-lifera- tion In Vitro of Human Granulocyte-Macrophage Progenitor Cells. Ann. NY Acad. Sci. 1989, 554, 177–184. 81. Broxmeyer, H.E.; Cooper, S.; Lu, L.; Miller, M.E.; Langefeld, C.D.; Ralph, P. Enhanced Stimulation of Human Bone Marrow Macrophage Colony Formation In Vitro by Recombinant Human Macrophage Colony-Stimulating Factor in Agarose Medium and at Low Oxygen Tension. Blood 1990, 76, 323–329. 82. Fehrer, C.; Brunauer, R.; Laschober, G.; Unterluggauer, H.; Reitinger, S.; Kloss, F.; Gülly, C.; Gaßner, R.; Lepperdinger, G. Re- duced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan. Aging Cell 2007, 6, 745–757, https://doi.org/10.1111/j.1474-9726.2007.00336.x. 83. D'Ippolito, G.; Diabira, S.; Howard, G.A.; Roos, B.A.; Schiller, P.C. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells. Bone 2006, 39, 513–522, https://doi.org/10.1016/j.bone.2006.02.061. 84. Mizuno, S.; Glowacki, J. Low Oxygen Tension Enhances Chondroinduction by Demineralized Bone Matrix in Human Dermal Fibroblasts in vitro. Cells Tissues Organs 2005, 180, 151–158, https://doi.org/10.1159/000088243. 85. Betre, H.; Ong, S.R.; Guilak, F.; Chilkoti, A.; Fermor, B.; Setton, L.A. Chondrocytic differentiation of human adipose-derived adult stem cells in elastin-like polypeptide. Biomaterials 2006, 27, 91–99, https://doi.org/10.1016/j.biomaterials.2005.05.071. 86. Khan, W.S.; Adesida, A.B.; E Hardingham, T. Hypoxic conditions increase hypoxia-inducible transcription factor 2α and en- hance chondrogenesis in stem cells from the infrapatellar fat pad of osteoarthritis patients. Arthritis Res. Ther. 2007, 9, R55–9, https://doi.org/10.1186/ar2211. 87. Dennis, J.E.; Whitney, G.A.; Rai, J.; Fernandes, R.J.; Kean, T.J. Physioxia Stimulates Extracellular Matrix Deposition and Increases Mechanical Properties of Human Chondrocyte-Derived Tissue-Engineered Cartilage. Front. Bioeng. Biotechnol. 2020, 8, https://doi.org/10.3389/fbioe.2020.590743. 88. Pattappa, G.; Johnstone, B.; Zellner, J.; Docheva, D.; Angele, P. The Importance of Physioxia in Mesenchymal Stem Cell Chon- drogenesis and the Mechanisms Controlling Its Response. Int. J. Mol. Sci. 2019, 20, 484, https://doi.org/10.3390/ijms20030484. 89. Studer, L.; Csete, M.; Lee, S.-H.; Kabbani, N.; Walikonis, J.; Wold, B.; McKay, R. Enhanced Proliferation, Survival, and Dopa- minergic Differentiation of CNS Precursors in Lowered Oxygen. J. Neurosci. 2000, 20, 7377–7383, https://doi.org/10.1523/jneuro- sci.20-19-07377.2000. 90. Antebi, B.; Ii, L.A.R.; Walker, K.P.; Asher, A.M.; Kamucheka, R.M.; Alvarado, L.; Mohammadipoor, A.; Cancio, L.C. Short-term physiological hypoxia potentiates the therapeutic function of mesenchymal stem cells. Stem Cell Res. Ther. 2018, 9, 1–15, https://doi.org/10.1186/s13287-018-1007-x. 91. Chen, C.; Tang, Q.; Zhang, Y.; Yu, M.; Jing, W.; Tian, W. Physioxia: a more effective approach for culturing human adipose - derived stem cells for cell transplantation.. Stem Cell Res. Ther. 2018, 9, 148, https://doi.org/10.1186/s13287-018-0891-4. 92. Mas-Bargues, C.; Sanz-Ros, J.; Román-Domínguez, A.; Inglés, M.; Gimeno-Mallench, L.; El Alami, M.; Viña-Almunia, J.; Gam- bini, J.; Viña, J.; Borrás, C. Relevance of Oxygen Concentration in Stem Cell Culture for Regenerative Medicine. Int. J. Mol. Sci. 2019, 20, 1195, https://doi.org/10.3390/ijms20051195. 93. Grodzki, A.C.G.; Giulivi, C.; Lein, P.J. Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line. PLoS ONE 2013, 8, e54926, https://doi.org/10.1371/journal.pone.0054926. 94. Patel, S.P.; Gonzalez, B.C.; Paone, N.; Mueller, C.; Floss, J.C.; Sousa, M.E.; Shi, M.Y. Effect of Physiological Oxygen on Pr imary Human Corneal Endothelial Cell Cultures. Transl. Vis. Sci. Technol. 2022, 11, 33–33, https://doi.org/10.1167/tvst.11.2.33. 95. Alva, R.; Abbas, M.; Bagshaw, O.R.; Moffatt, C.; Gardner, G.; Stuart, J.A. Mitochondrial Oxygen Toxicity. In Mitochondrial Intoxication; de Oliveira, M.R., Ed. Academic Press, Amsterdam, Netherlands, in press ISBN 9780323884624. 96. Jaber, S.M.; Bordt, E.A.; Bhatt, N.M.; Lewis, D.M.; Gerecht, S.; Fiskum, G.; Polster, B.M. Sex differences in the mitochondri al bioenergetics of astrocytes but not microglia at a physiologically relevant brain oxygen tension. Neurochem. Int. 2018, 117, 82– 90, https://doi.org/10.1016/j.neuint.2017.09.003. 97. Moradi, F.; Moffatt, C.; Stuart, J. The Effect of Oxygen and Micronutrient Composition of Cell Growth Media on Cancer Cell Bioenergetics and Mitochondrial Networks. Biomolecules 2021, 11, 1177, https://doi.org/10.3390/biom11081177. 98. Valente, A.J.; Maddalena, L.A.; Robb, E.L.; Moradi, F.; Stuart, J.A. A simple ImageJ macro tool for analyzing mitochondrial network morphology in mammalian cell culture. Acta Histochem. 2017, 119, 315–326, https://doi.org/10.1016/j.acthis.2017.03.001. 99. Tiede, L.M.; A Cook, E.; Morsey, B.; Fox, H.S. Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well as responses to HIV viroproteins. Cell Death Dis. 2011, 2, e246–e246, https://doi.org/10.1038/cddis.2011.128. 100. Zhou, L.; Dosanjh, A.; Chen, H.; Karasek, M. Divergent Effects of Extracellular Oxygen on the Growth, Morphology, and Func- tion of Human Skin Microvascular Endothelial Cells. J. Cell Physiol. 2000, 182, 134–140. 101. Piossek, F.; Beneke, S.; Schlichenmaier, N.; Mucic, G.; Drewitz, S.; Dietrich, D.R. Physiological oxygen and co-culture with hu- man fibroblasts facilitate in vivo-like properties in human renal proximal tubular epithelial cells. Chem. Interactions 2022, 361, https://doi.org/10.1016/j.cbi.2022.109959. 102. Guo, R.; Xu, X.; Lu, Y.; Xie, X. Physiological oxygen tension reduces hepatocyte dedifferentiation in in vitro culture. Sci. Rep. 2017, 7, 5923, https://doi.org/10.1038/s41598-017-06433-3. 2+ 103. Keeley, T.P.; Siow, R.C.M.; Jacob, R.; Mann, G.E. A PP2A-mediated feedback mechanism controls Ca -dependent NO synthe- sis under physiological oxygen. FASEB J. 2017, 31, 5172–5183, https://doi.org/10.1096/fj.201700211r. 2+ 104. Keeley, T.P.; Siow, R.C.M.; Jacob, R.; Mann, G.E. Reduced SERCA activity underlies dysregulation of Ca homeostasis under atmospheric O2levels. FASEB J. 2017, 32, 2531–2538, https://doi.org/10.1096/fj.201700685rrr. Cells 2022, 11, 3123 21 of 21 105. Warpsinski, G.; Smith, M.J.; Srivastava, S.; Keeley, T.P.; Siow, R.C.; Fraser, P.A.; Mann, G.E. Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation. Redox Biol. 2020, 37, 101708, https://doi.org/10.1016/j.redox.2020.101708. 106. Danilov, C.A.; Fiskum, G. Hyperoxia promotes astrocyte cell death after oxygen and glucose deprivation. Glia 2008, 56, 801– 808, https://doi.org/10.1002/glia.20655. 107. Fonseca, J.; Moradi, F.; Valente, A.J.F.; Stuart, J.A. Oxygen and Glucose Levels in Cell Culture Media Determine Resveratrol’s Effects on Growth, Hydrogen Peroxide Production, and Mitochondrial Dynamics. Antioxidants 2018, 7, 157, https://doi.org/10.3390/antiox7110157. 108. Kumar, A.; Dailey, L.A.; Swedrowska, M.; Siow, R.; Mann, G.E.; Vizcay-Barrena, G.; Arno, M.; Mudway, I.S.; Forbes, B. Quan- tifying the magnitude of the oxygen artefact inherent in culturing airway cells under atmospheric oxygen versus physiological levels. FEBS Lett. 2015, 590, 258–269, https://doi.org/10.1002/1873-3468.12026. 109. Otto-Ślusarczyk, D.; Graboń, W.; Mielczarek-Puta, M.; Chrzanowska, A. Teriflunomide – The common drug with underesti- mated oxygen - Dependent anticancer potential. Biochem. Biophys. Rep. 2021, 28, 101141, https://doi.org/10.1016/j.bbrep.2021.101141. 110. Alaluf, S.; Muir-Howie, H.; Hu, H.-L.; Evans, A.; Green, M.R.; Alaluf, S.; Muir-Howie, H.; Hu, H.-H.; Evans, A.; Green, M.R. Atmospheric Oxygen Accelerates the Induction of a Post-Mitotic Phenotype in Human Dermal Fibroblasts: The Key Pro-tective Role of Glutathione. Differentiation 2000, 66, 147–155. 111. DiProspero, T.J.; Dalrymple, E.; Lockett, M.R. Physiologically relevant oxygen tensions differentially regulate hepatotoxic r e- sponses in HepG2 cells. Toxicol. Vitr. 2021, 74, 105156, https://doi.org/10.1016/j.tiv.2021.105156. 112. Albert, I.; Hefti, M.; Luginbuehl, V. Physiological oxygen concentration alters glioma cell malignancy and responsiveness to photodynamic therapyin vitro. Neurol. Res. 2014, 36, 1001–1010, https://doi.org/10.1179/1743132814y.0000000401. 113. Lee, K.-H.; Kim, D.-Y.; Kim, W. Cultivation of human skin cells under physiological oxygen concentration modulates expression of skin significant genes and response to hydroxy acids. Biochem. Biophys. Res. Commun. 2021, 551, 161–167, https://doi.org/10.1016/j.bbrc.2021.02.113. 114. Moradi, F.; Fiocchetti, M.; Marino, M.; Moffatt, C.; Stuart, J.A. Media composition and O2 levels determine effects of 17β -Estra- diol on mitochondrial bioenergetics and cellular reactive oxygen species. Am. J. Physiol. Physiol. 2021, https://doi.org/10.1152/aj- pcell.00080.2021. 115. Villeneuve, L.; Tiede, L.M.; Morsey, B.; Fox, H.S. Quantitative Proteomics Reveals Oxygen-Dependent Changes in Neuronal Mitochondria Affecting Function and Sensitivity to Rotenone. J. Proteome Res. 2013, 12, 4599–4606, https://doi.org/10.1021/pr400758d. 116. Coyle, J.P.; Rinaldi, R.J.; Johnson, G.T.; Bourgeois, M.M.; McCluskey, J.D.; Harbison, R.D. Reduced oxygen tension culturing conditionally alters toxicogenic response of differentiated H9c2 cardiomyoblasts to acrolein. Toxicol. Mech. Methods 2018, 28, 488–498, https://doi.org/10.1080/15376516.2018.1455785. 117. Trepiana, J.; Meijide, S.; Navarro, R.; Hernández, M.L.; Ruiz-Sanz, J.I.; Ruiz-Larrea, M.B. Influence of oxygen partial pressure on the characteristics of human hepatocarcinoma cells. Redox Biol. 2017, 12, 103–113, https://doi.org/10.1016/j.redox.2017.02.004. 118. Spyrou, J.; Gardner, D.K.; Harvey, A.J. Metabolomic and Transcriptional Analyses Reveal Atmospheric Oxygen During Human Induced Pluripotent Stem Cell Generation Impairs Metabolic Reprogramming. Stem Cells 2019, 37, 1042–1056, https://doi.org/10.1002/stem.3029. 119. Al-Ani, A.; Toms, D.; Kondro, D.; Thundathil, J.; Yu, Y.; Ungrin, M. Oxygenation in cell culture: Critical parameters for repro- ducibility are routinely not reported. PLoS ONE 2018, 13, e0204269, https://doi.org/10.1371/journal.pone.0204269. 120. Samokhin, P.; Gardner, G.L.; Moffatt, C.; Stuart, J.A. An Inexpensive Incubator for Mammalian Cell Culture Capable of Regu- lating O2, CO2, and Temperature. Oxygen 2022, 2, 22–30, https://doi.org/10.3390/oxygen2010003. 121. Marchus, C.R.N.; Knudson, J.A.; Morrison, A.E.; Strawn, I.K.; Hartman, A.J.; Shrestha, D.; Pancheri, N.M.; Glasgow, I.; Schie le, N.R. Low-Cost, Open-Source Cell Culture Chamber for Regulating Physiologic Oxygen Levels Specifications Table. HardwareX 2022, 11, e00253, doi:10.17605/OSF.IO/JVHDQ. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cells Multidisciplinary Digital Publishing Institute http://www.deepdyve.com/lp/multidisciplinary-digital-publishing-institute/supraphysiological-oxygen-levels-in-mammalian-cell-culture-current-wrl6mQIGb8

Loading next page...

References (122)

M. McMahon, D. Lamont, K. Beattie, J. Hayes (2010)
Keap1 perceives stress via three sensors for the endogenous signaling molecules nitric oxide, zinc, and alkenals
Proceedings of the National Academy of Sciences, 107
L. Studer, M. Csete, Sang-Hun Lee, N. Kabbani, Jean Walikonis, B. Wold, R. McKay (2000)
Enhanced Proliferation, Survival, and Dopaminergic Differentiation of CNS Precursors in Lowered Oxygen
The Journal of Neuroscience, 20
S. Parrinello, Enrique Samper, A. Krtolica, J. Goldstein, S. Melov, J. Campisi (2003)
Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts
Nature Cell Biology, 5
(2018)
Transcriptional Regulation by Nrf2
Antioxid. Redox Signal., 29
S. Westfall, S. Sachdev, P. Das, L. Hearne, M. Hannink, R. Roberts, T. Ezashi (2008)
Identification of oxygen-sensitive transcriptional programs in human embryonic stem cells.
Stem cells and development, 17 5
J. Stuart, J. Fonseca, Fereshteh Moradi, Cassandra Cunningham, Bishoy Seliman, Cydney Worsfold, Sarah Dolan, John Abando, Lucas Maddalena (2018)
How Supraphysiological Oxygen Levels in Standard Cell Culture Affect Oxygen-Consuming Reactions
Oxidative Medicine and Cellular Longevity, 2018
Sara Timpano, Brianna Guild, Erin Specker, Gaelan Melanson, Philip Medeiros, S. Sproul, J. Uniacke (2019)
Physioxic human cell culture improves viability, metabolism, and mitochondrial morphology while reducing DNA damage
The FASEB Journal, 33
L. Braunschweig, Anne Meyer, Lisa Wagenführ, A. Storch (2015)
Oxygen regulates proliferation of neural stem cells through Wnt/β-catenin signalling
Molecular and Cellular Neuroscience, 67
Pearl Lee, N. Chandel, M. Simon (2020)
Cellular adaptation to hypoxia through hypoxia inducible factors and beyond
Nature Reviews Molecular Cell Biology, 21
A. Bon-Mathier, Stéphanie Rignault-Clerc, C. Bielmann, N. Rosenblatt-Velin (2020)
Oxygen as a key regulator of cardiomyocyte proliferation: New results about cell culture conditions!
Biochimica et biophysica acta. Molecular cell research
Ricardo Alva, Fereshteh Moradi, Ping Liang, J. Stuart (2022)
Culture of Cancer Cells at Physiological Oxygen Levels Affects Gene Expression in a Cell-Type Specific Manner
Biomolecules, 12
S. Mckeown (2014)
Defining normoxia, physoxia and hypoxia in tumours-implications for treatment response.
The British journal of radiology, 87 1035
V. Falanga, R. Kirsner (1993)
Low oxygen stimulates proliferation of fibroblasts seeded as single cells
Journal of Cellular Physiology, 154
A. Grodzki, C. Giulivi, P. Lein (2013)
Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line
PLoS ONE, 8
J. Dennis, G. Whitney, J. Rai, R. Fernandes, T. Kean (2020)
Physioxia Stimulates Extracellular Matrix Deposition and Increases Mechanical Properties of Human Chondrocyte-Derived Tissue-Engineered Cartilage
Frontiers in Bioengineering and Biotechnology, 8
J. Stuart, Odosa Aibueku, Olivia Bagshaw, Fereshteh Moradi (2019)
Hypoxia inducible factors as mediators of reactive oxygen/nitrogen species homeostasis in physiological normoxia.
Medical hypotheses, 129
Mitochondrial Oxygen Toxicity
W. Khan, A. Adesida, T. Hardingham (2007)
Hypoxic conditions increase hypoxia-inducible transcription factor 2α and enhance chondrogenesis in stem cells from the infrapatellar fat pad of osteoarthritis patients
Arthritis Research & Therapy, 9
Guofu Lin, Wenyu Sun, Zhi Yang, Jinshuai Guo, Hai-yang Liu, Jian Liang (2017)
Hypoxia induces the expression of TET enzymes in HepG2 cells
Oncology Letters, 14
Zhi-Hua Chen, Yoshiro Saito, Y. Yoshida, Azusa Sekine, N. Noguchi, E. Niki (2005)
4-Hydroxynonenal Induces Adaptive Response and Enhances PC12 Cell Tolerance Primarily through Induction of Thioredoxin Reductase 1 via Activation of Nrf2*
Journal of Biological Chemistry, 280
Daniel Ferguson, G. Smerdon, L. Harries, N. Dodd, M. Murphy, A. Curnow, P. Winyard (2018)
Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia
Free Radical Biology and Medicine, 126
David Hoffman, P. Brookes (2009)
Oxygen Sensitivity of Mitochondrial Reactive Oxygen Species Generation Depends on Metabolic Conditions
The Journal of Biological Chemistry, 284
M. Alami, José Viña-Almunia, J. Gambini, C. Mas-Bargues, R. Siow, M. Peñarrocha, G. Mann, C. Borrás, J. Viña (2014)
Activation of p38, p21, and NRF-2 Mediates Decreased Proliferation of Human Dental Pulp Stem Cells Cultured under 21% O2
Stem Cell Reports, 3
A. Loboda, A. Józkowicz, J. Dulak (2010)
HIF-1 and HIF-2 transcription factors — Similar but not identical
Molecules and Cells, 29
Sara Timpano, J. Uniacke (2016)
Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation*
The Journal of Biological Chemistry, 291
K. Covello, James Kehler, Hongwei Yu, J. Gordan, A. Arsham, Cheng-Jun Hu, P. Labosky, M. Simon, B. Keith (2006)
HIF-2 regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth
I. Ivanova, Catherine Park, Niall Kenneth (2019)
Translating the Hypoxic Response—The Role of HIF Protein Translation in the Cellular Response to Low Oxygen
Cells, 8
B. Kumar, Adedeji Adebayo, M. Prasad, M. Capitano, Ruizhong Wang, P. Bhat-Nakshatri, M. Anjanappa, Edward Simpson, Duojiao Chen, Yunlong Liu, J. Schilder, Austyn Colter, Calli Maguire, C. Temm, G. Sandusky, E. Doud, A. Wijeratne, A. Mosley, H. Broxmeyer, H. Nakshatri (2022)
Tumor collection/processing under physioxia uncovers highly relevant signaling networks and drug sensitivity
Science Advances, 8
Felicitas Piossek, S. Beneke, Nadja Schlichenmaier, G. Mucić, Sabine Drewitz, D. Dietrich (2022)
Physiological oxygen and co-culture with human fibroblasts facilitate in vivo-like properties in human renal proximal tubular epithelial cells.
Chemico-biological interactions
Feresteh Moradi, Marco Fiocchetti, M. Marino, Christopher Moffatt, J. Stuart (2021)
Media composition and O2 levels determine effects of 17β-Estradiol on mitochondrial bioenergetics and cellular reactive oxygen species.
American journal of physiology. Cell physiology
C. Lengner, A. Gimelbrant, Jennifer Erwin, A. Cheng, M. Guenther, G. Welstead, R. Alagappan, G. Frampton, P. Xu, J. Muffat, S. Santagata, D. Powers, C. Barrett, R. Young, Jeannie Lee, R. Jaenisch, M. Mitalipova (2010)
Derivation of Pre-X Inactivation Human Embryonic Stem Cells under Physiological Oxygen Concentrations
Cell, 141
C. Mas-Bargues, J. Sanz-Ros, Aurora Román-Domínguez, M. Inglés, L. Gimeno-Mallench, M. Alami, José Viña-Almunia, J. Gambini, J. Viña, C. Borrás (2019)
Relevance of Oxygen Concentration in Stem Cell Culture for Regenerative Medicine
International Journal of Molecular Sciences, 20
A. Balin, A. Fisher, D. Carter (1984)
Oxygen modulates growth of human cells at physiologic partial pressures
The Journal of Experimental Medicine, 160
Marta Osrodek, Mariusz Hartman, M. Czyż (2019)
Physiologically Relevant Oxygen Concentration (6% O2) as an Important Component of the Microenvironment Impacting Melanoma Phenotype and Melanoma Response to Targeted Therapeutics In Vitro
International Journal of Molecular Sciences, 20
A. Alfadda, R. Sallam (2012)
Reactive Oxygen Species in Health and Disease
Journal of Biomedicine and Biotechnology, 2012
J. Lees, Timothy Cliff, A. Gammilonghi, J. Ryall, S. Dalton, D. Gardner, A. Harvey (2019)
Oxygen Regulates Human Pluripotent Stem Cell Metabolic Flux
Stem Cells International, 2019
Takafumi Suzuki, Masayuki Yamamoto (2015)
Molecular basis of the Keap1-Nrf2 system.
Free radical biology & medicine, 88 Pt B
P. Xie, Yi Sun, Ouyang Qi, Liang Hu, Yueqiu Tan, Xiaoying Zhou, B. Xiong, Qianjun Zhang, D. Yuan, Yi Pan, Tian-cheng Liu, P. Liang, G. Lu, G. Lin (2014)
Physiological Oxygen Prevents Frequent Silencing of the DLK1‐DIO3 Cluster during Human Embryonic Stem Cells Culture
STEM CELLS, 32
K. Atkuri, L. Herzenberg, A. Niemi, T. Cowan, L. Herzenberg (2007)
Importance of culturing primary lymphocytes at physiological oxygen levels
Proceedings of the National Academy of Sciences, 104
L. Tiede, L. Tiede, E. Cook, Brenda Morsey, H. Fox, H. Fox (2011)
Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well as responses to HIV viroproteins
Cell Death & Disease, 2
Fereshteh Moradi, Christopher Moffatt, J. Stuart (2021)
The Effect of Oxygen and Micronutrient Composition of Cell Growth Media on Cancer Cell Bioenergetics and Mitochondrial Networks
Biomolecules, 11
Y. Nisimoto, B. Diebold, Daniela Constentino-Gomes, J. Lambeth (2014)
Nox4: A Hydrogen Peroxide-Generating Oxygen Sensor
Biochemistry, 53
H. Sies, V. Belousov, N. Chandel, M. Davies, Dean Jones, G. Mann, M. Murphy, Masayuki Yamamoto, C. Winterbourn (2022)
Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology
Nature Reviews Molecular Cell Biology, 23
Camelia Danilov, G. Fiskum (2008)
Hyperoxia promotes astrocyte cell death after oxygen and glucose deprivation
Glia, 56
S. Chapple, Thomas Keeley, D. Mastronicola, M. Arno, G. Vizcay‐Barrena, R. Fleck, R. Siow, G. Mann (2016)
Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxygen levels.
Free radical biology & medicine, 92
Abhinav Kumar, L. Dailey, M. Swedrowska, R. Siow, G. Mann, G. Vizcay‐Barrena, M. Arno, I. Mudway, B. Forbes (2016)
Quantifying the magnitude of the oxygen artefact inherent in culturing airway cells under atmospheric oxygen versus physiological levels
FEBS Letters, 590
T. Fukai, M. Ushio-Fukai (2011)
Superoxide dismutases: role in redox signaling, vascular function, and diseases.
Antioxidants & redox signaling, 15 6
Low - Cost , Open - Source Cell Culture Chamber for Regulating Physiologic Oxygen Levels Specifications Table
C. Bracken, Anthony Fedele, S. Linke, Wiltiana Balrak, K. Lisy, M. Whitelaw, D. Peet (2006)
Cell-specific Regulation of Hypoxia-inducible Factor (HIF)-1α and HIF-2α Stabilization and Transactivation in a Graded Oxygen Environment*
Journal of Biological Chemistry, 281
Lisa Zhou, A. Dosanjh, Huosheng Chen, M. Karasek (2000)
Divergent effects of extracellular oxygen on the growth, morphology, and function of human skin microvascular endothelial cells
Journal of Cellular Physiology, 182
H. Broxmeyer, S. Cooper, Theodore Gabic (1989)
The Effects of Oxidizing Species Derived from Molecular Oxygen on the Proliferation In Vitro of Human Granulocyte‐Macrophage Progenitor Cells a
Annals of the New York Academy of Sciences, 554
James Spyrou, D. Gardner, A. Harvey (2019)
Metabolomic and Transcriptional Analyses Reveal Atmospheric Oxygen During Human Induced Pluripotent Stem Cell Generation Impairs Metabolic Reprogramming
STEM CELLS, 37
Chang Chen, Q. Tang, Yan Zhang, Mei Yu, W. Jing, W. Tian (2018)
Physioxia: a more effective approach for culturing human adipose-derived stem cells for cell transplantation
Stem Cell Research & Therapy, 9
D. Zorov, M. Juhaszova, S. Sollott (2014)
Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release.
Physiological reviews, 94 3
H. Sies (2017)
Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress☆
Redox Biology, 11
Samantha Carrera, P. Verdier, Zahid Khan, Bo Zhao, A. Mahale, K. Bowman, M. Zainol, George Jones, Sam Lee, S. Aaronson, S. Macip (2010)
Protection of Cells in Physiological Oxygen Tensions against DNA Damage-induced Apoptosis*
The Journal of Biological Chemistry, 285
Sangita Patel, Brayan Gonzalez, Nataliia Paone, C. Mueller, Jamie Floss, M. Sousa, Michael Shi (2022)
Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures
Translational Vision Science & Technology, 11
J. Estrada, C. Albó, A. Benguría, A. Dopazo, P. López-Romero, Lucrecia Carrera-Quintanar, E. Roche, E. Clemente, J. Enríquez, J. Enríquez, A. Bernad, Enrique Samper (2011)
Culture of human mesenchymal stem cells at low oxygen tension improves growth and genetic stability by activating glycolysis
Cell Death and Differentiation, 19
Ruitang Shi, Haoqing Yang, Xiao Lin, Yangyang Cao, Chen Zhang, Z. Fan, Benxiang Hou (2019)
Analysis of the characteristics and expression profiles of coding and noncoding RNAs of human dental pulp stem cells in hypoxic conditions
Stem Cell Research & Therapy, 10
Takafumi Suzuki, A. Muramatsu, Ryota Saito, T. Iso, T. Shibata, K. Kuwata, Shin-ichi Kawaguchi, T. Iwawaki, Saki Adachi, Hiromi Suda, Masanobu Morita, K. Uchida, Liam Baird, Masayuki Yamamoto (2019)
Molecular Mechanism of Cellular Oxidative Stress Sensing by Keap1.
Cell reports, 28 3
G. D’Ippolito, S. Diabira, G. Howard, B. Roos, P. Schiller (2006)
Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells.
Bone, 39 3
Nancy Chee, I. Lohse, S. Brothers (2019)
mRNA-to-protein translation in hypoxia
Molecular Cancer, 18
M. Konigsberg, V. Pérez, C. Rios, Y. Liu, S. Lee, Y. Shi, H. Remmen (2013)
Effect of oxygen tension on bioenergetics and proteostasis in young and old myoblast precursor cells☆
Redox Biology, 1
Cheng-Jun Hu, Liyi Wang, Lewis Chodosh, B. Keith, M. Simon (2003)
Differential Roles of Hypoxia-Inducible Factor 1α (HIF-1α) and HIF-2α in Hypoxic Gene Regulation
Molecular and Cellular Biology, 23
E. Kurutaş (2015)
The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state
Nutrition Journal, 15
Hui‐min Yan, A. Ramachandran, M. Bajt, J. Lemasters, H. Jaeschke (2010)
The oxygen tension modulates acetaminophen-induced mitochondrial oxidant stress and cell injury in cultured hepatocytes.
Toxicological sciences : an official journal of the Society of Toxicology, 117 2
S. Lees, T. Childs, Frank Booth (2008)
p21Cip1 expression is increased in ambient oxygen, compared to estimated physiological (5%) levels in rat muscle precursor cell culture
Cell Proliferation, 41
Helawe Betre, Shin Ong, F. Guilak, A. Chilkoti, B. Fermor, L. Setton (2006)
Chondrocytic differentiation of human adipose-derived adult stem cells in elastin-like polypeptide.
Biomaterials, 27 1
R. Guo, Xinxiu Xu, Yuting Lu, Xin Xie (2017)
Physiological oxygen tension reduces hepatocyte dedifferentiation in in vitro culture
Scientific Reports, 7
Amy Guillaumet-Adkins, Y. Yáñez, M. Peris-Díaz, I. Calabria, Cora Palanca-Ballester, J. Sandoval (2017)
Epigenetics and Oxidative Stress in Aging
Oxidative Medicine and Cellular Longevity, 2017
Masayuki Yamamoto, T. Kensler, H. Motohashi (2018)
The KEAP1-NRF2 System: a Thiol-Based Sensor-Effector Apparatus for Maintaining Redox Homeostasis.
Physiological reviews, 98 3
H. Broxmeyer, S. Cooper, L. Lu, M. Miller, C. Langefeld, P. Ralph (1990)
Enhanced stimulation of human bone marrow macrophage colony formation in vitro by recombinant human macrophage colony-stimulating factor in agarose medium and at low oxygen tension.
Blood, 76 2
Liam Baird, Masayuki Yamamoto (2020)
The Molecular Mechanisms Regulating the KEAP1-NRF2 Pathway
Molecular and Cellular Biology, 40
J. Mazumdar, V. Dondeti, M. Simon (2007)
Hypoxia-Inducible Factors, Stem Cells, and Cancer
Cell, 129
Christine Fehrer, R. Brunauer, G. Laschober, H. Unterluggauer, S. Reitinger, F. Kloss, C. Gülly, R. Gassner, G. Lepperdinger (2007)
Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan
Aging Cell, 6
Thomas Keeley, R. Siow, R. Jacob, G. Mann (2017)
A PP2A-mediated feedback mechanism controls Ca2+-dependent NO synthesis under physiological oxygen
The FASEB Journal, 31
Sashwati Roy, S. Khanna, A. Bickerstaff, S. Subramanian, M. Atalay, Michael Bierl, S. Pendyala, D. Levy, Nidhi Sharma, Mika Venojarvi, A. Strauch, C. Orosz, C. Sen (2003)
Oxygen Sensing by Primary Cardiac Fibroblasts: A Key Role of p21Waf1/Cip1/Sdi1
Circulation Research: Journal of the American Heart Association, 92
S. Mizuno, J. Glowacki (2005)
Low Oxygen Tension Enhances Chondroinduction by Demineralized Bone Matrix in Human Dermal Fibroblasts in vitro
Cells Tissues Organs, 180
Lien Pham-Huy, Hua-Dong He, C. Pham-Huy (2008)
Free Radicals, Antioxidants in Disease and Health
International Journal of Biomedical Science : IJBS, 4
P. Cooper, A. Burt, J. Wilson (1958)
Critical Effect of Oxygen Tension on Rate of Growth of Animal Cells in Continuous Suspended Culture
Nature, 182
K. Duś-Szachniewicz, Katarzyna Gdesz-Birula, K. Zduniak, J. Wiśniewski (2021)
Proteomic-Based Analysis of Hypoxia- and Physioxia-Responsive Proteins and Pathways in Diffuse Large B-Cell Lymphoma
Cells, 10
Mohammad Abbas, Fereshteh Moradi, Wenyu Hu, Katrina Regudo, M. Osborne, Josh Pettipas, Dena Atallah, Rauann Hachem, Nadine Ott-Peron, Nathan Webster, J. Stuart (2021)
Vertebrate cell culture as an experimental approach - limitations and solutions.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology
B. Thienpont, J. Steinbacher, Hui Zhao, F. D’Anna, Anna Kuchnio, Athanasios Ploumakis, B. Ghesquière, Laurien Dyck, B. Boeckx, L. Schoonjans, Els Hermans, F. Amant, V. Kristensen, Kian Koh, M. Mazzone, M. Coleman, T. Carell, P. Carmeliet, D. Lambrechts (2016)
Tumor hypoxia causes DNA hypermethylation by reducing TET activity
Nature, 537
Fatma Doğan, R. Aljumaily, M. Kitchen, N. Forsyth (2021)
DNMT3B Is an Oxygen-Sensitive De Novo Methylase in Human Mesenchymal Stem Cells
Cells, 10
Lucas Maddalena, Shehab Selim, J. Fonseca, H. Messner, S. McGowan, J. Stuart (2017)
Hydrogen peroxide production is affected by oxygen levels in mammalian cell culture.
Biochemical and biophysical research communications, 493 1
B. Haas, Sandra Chrusciel, Sarah Fayad-Kobeissi, J. Dubois-Randé, F. Azuaje, J. Boczkowski, R. Motterlini, R. Foresti (2015)
Permanent Culture of Macrophages at Physiological Oxygen Attenuates the Antioxidant and Immunomodulatory Properties of Dimethyl Fumarate
Journal of Cellular Physiology, 230
Simon Alaluf, H. Muir-Howie, Heng-Long Hu, Anita Evans, M. Green (2000)
Atmospheric oxygen accelerates the induction of a post-mitotic phenotype in human dermal fibroblasts: the key protective role of glutathione.
Differentiation; research in biological diversity, 66 2-3
L. Nasto, A. Robinson, Kevin Ngo, Cheryl Clauson, Qing Dong, C. Croix, G. Sowa, E. Pola, P. Robbins, James Kang, L. Niedernhofer, P. Wipf, N. Vo (2013)
Mitochondrial‐derived reactive oxygen species (ROS) play a causal role in aging‐related intervertebral disc degeneration
Journal of Orthopaedic Research, 31
P. Maxwell, M. Wiesener, G. Chang, S. Clifford, E. Vaux, M. Cockman, C. Wykoff, C. Pugh, E. Maher, P. Ratcliffe (1999)
The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis
Nature, 399
Lance Villeneuve, L. Tiede, B. Morsey, H. Fox (2013)
Quantitative proteomics reveals oxygen-dependent changes in neuronal mitochondria affecting function and sensitivity to rotenone.
Journal of proteome research, 12 10
Ina Albert, M. Hefti, V. Luginbuehl (2014)
Physiological oxygen concentration alters glioma cell malignancy and responsiveness to photodynamic therapy in vitro
Neurological Research, 36
Thomas Keeley, G. Mann (2019)
Defining Physiological Normoxia for Improved Translation of Cell Physiology to Animal Models and Humans.
Physiological reviews, 99 1
E. Metzen, P. Ratcliffe (2004)
HIF hydroxylation and cellular oxygen sensing
, 385
P. Samokhin, Georgina Gardner, Christopher Moffatt, J. Stuart (2022)
An Inexpensive Incubator for Mammalian Cell Culture Capable of Regulating O2, CO2, and Temperature
Oxygen
Hannah Johnston, P. Dickinson, A. Ivens, A. Buck, R. Levine, F. Remacle, C. Campbell (2019)
Intracellular redox potential is correlated with miRNA expression in MCF7 cells under hypoxic conditions
Proceedings of the National Academy of Sciences, 116
J. Coyle, Robert Rinaldi, G. Johnson, M. Bourgeois, J. McCluskey, R. Harbison (2018)
Reduced oxygen tension culturing conditionally alters toxicogenic response of differentiated H9c2 cardiomyoblasts to acrolein
Toxicology Mechanisms and Methods, 28
Thomas Keeley, R. Siow, R. Jacob, G. Mann (2017)
Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels
The FASEB Journal, 32
Guang-lei Wang, B. Jiang, E. Rue, G. Semenza (1995)
Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.
Proceedings of the National Academy of Sciences of the United States of America, 92 12
Jing Zhu, S. Aja, Eun-Kyoung Kim, M. Park, S. Ramamurthy, Junling Jia, X. Hu, Ping Geng, G. Ronnett (2012)
Physiological oxygen level is critical for modeling neuronal metabolism in vitro
Journal of Neuroscience Research, 90
Andrew Valente, Lucas Maddalena, Ellen Robb, Fereshteh Moradi, J. Stuart (2017)
A simple ImageJ macro tool for analyzing mitochondrial network morphology in mammalian cell culture.
Acta histochemica, 119 3
Fatma Doğan, Rakad Aljumaily, M. Kitchen, N. Forsyth (2022)
Physoxia Influences Global and Gene-Specific Methylation in Pluripotent Stem Cells
International Journal of Molecular Sciences, 23
G. Semenza, Guang-lei Wang (1992)
A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation
Molecular and Cellular Biology, 12
(2022)
Culture of Cancer Cells at Physiological Oxygen Levels Affects Gene Expression in a Cell-Type Specific Manner
Preprints (Basel)
Dagmara Otto-Ślusarczyk, W. Graboń, Magdalena Mielczarek-Puta, A. Chrzanowska (2021)
Teriflunomide – The common drug with underestimated oxygen - Dependent anticancer potential
Biochemistry and Biophysics Reports, 28
L. Packer, Kathy Fuehr (1977)
Low oxygen concentration extends the lifespan of cultured human diploid cells
Nature, 267
Thomas Diprospero, Erin Dalrymple, Matthew Lockett (2021)
Physiologically relevant oxygen tensions differentially regulate hepatotoxic responses in HepG2 cells.
Toxicology in vitro : an international journal published in association with BIBRA
B. Antebi, Luis Rodriguez, K. Walker, Amber Asher, R. Kamucheka, L. Alvarado, Arezoo Mohammadipoor, L. Cancio (2018)
Short-term physiological hypoxia potentiates the therapeutic function of mesenchymal stem cells
Stem Cell Research & Therapy, 9
V. Dengler, M. Galbraith, J. Espinosa (2014)
Transcriptional regulation by hypoxia inducible factors
Critical Reviews in Biochemistry and Molecular Biology, 49
M. Brand (2010)
The sites and topology of mitochondrial superoxide production
Experimental Gerontology, 45
G. Pattappa, B. Johnstone, J. Zellner, D. Docheva, P. Angele (2019)
The Importance of Physioxia in Mesenchymal Stem Cell Chondrogenesis and the Mechanisms Controlling Its Response
International Journal of Molecular Sciences, 20
Ricardo Alva, Maha Mirza, Adam Baiton, Lucas Lazuran, Lyuda Samokysh, Ava Bobinski, C. Cowan, Alvin Jaimon, Dede Obioru, Tala Makhoul, J. Stuart (2022)
Oxygen toxicity: cellular mechanisms in normobaric hyperoxia
Cell Biology and Toxicology, 39
A. Al-Ani, Derek Toms, D. Kondro, Jarin Thundathil, Yang Yu, M. Ungrin (2018)
Oxygenation in cell culture: Critical parameters for reproducibility are routinely not reported
PLoS ONE, 13
Maija Hirsilä, P. Koivunen, V. Günzler, K. Kivirikko, J. Myllyharju (2003)
Characterization of the Human Prolyl 4-Hydroxylases That Modify the Hypoxia-inducible Factor*
Journal of Biological Chemistry, 278
Gabriela Warpsinski, Matthew Smith, S. Srivastava, Thomas Keeley, R. Siow, P. Fraser, G. Mann (2020)
Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation
Redox Biology, 37
R. Busuttil, M. Rubio, M. Dollé, J. Campisi, J. Vijg (2003)
Oxygen accelerates the accumulation of mutations during the senescence and immortalization of murine cells in culture
Aging Cell, 2
S. Jaber, E. Bordt, Niraj Bhatt, Daniel Lewis, S. Gerecht, G. Fiskum, B. Polster (2017)
Sex differences in the mitochondrial bioenergetics of astrocytes but not microglia at a physiologically relevant brain oxygen tension
Neurochemistry International, 117
J. Trepiana, Susana Meijide, R. Navarro, M. Hernández, J. Ruiz-Sanz, M. Ruiz-Larrea (2017)
Influence of oxygen partial pressure on the characteristics of human hepatocarcinoma cells
Redox Biology, 12
J. Uniacke, C. Holterman, G. Lachance, A. Franovic, Mathieu Jacob, M. Fabian, Josianne Payette, M. Holcik, A. Pause, Stephen Lee (2012)
An oxygen-regulated switch in the protein synthesis machinery
Nature, 486
Domhnall McHugh, J. Gil (2018)
Senescence and aging: Causes, consequences, and therapeutic avenues
The Journal of Cell Biology, 217
J. Fonseca, Fereshteh Moradi, Andrew Valente, J. Stuart (2018)
Oxygen and Glucose Levels in Cell Culture Media Determine Resveratrol’s Effects on Growth, Hydrogen Peroxide Production, and Mitochondrial Dynamics
Antioxidants, 7
Kyung‐Ha Lee, Do-Yeon Kim, Wanil Kim (2021)
Cultivation of human skin cells under physiological oxygen concentration modulates expression of skin significant genes and response to hydroxy acids.
Biochemical and biophysical research communications, 551
M. Batie, S. Rocha (2020)
Gene transcription and chromatin regulation in hypoxia
Biochemical Society Transactions, 48

Publisher: Multidisciplinary Digital Publishing Institute
Copyright: © 1996-2022 MDPI (Basel, Switzerland) unless otherwise stated Disclaimer The statements, opinions and data contained in the journals are solely those of the individual authors and contributors and not of the publisher and the editor(s). MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Terms and Conditions Privacy Policy
ISSN: 2073-4409
DOI: 10.3390/cells11193123
Publisher site: See Article on Publisher Site

Abstract

Review Supraphysiological Oxygen Levels in Mammalian Cell Culture: Current State and Future Perspectives Ricardo Alva *, Georgina L. Gardner, Ping Liang and Jeffrey A. Stuart Department of Biological Sciences, Brock University, St. Catharines, ON L2S 3A1, Canada * Correspondence: [email protected] or [email protected] Abstract: Most conventional incubators used in cell culture do not regulate O2 levels, making the headspace O2 concentration ~18%. In contrast, most human tissues are exposed to 2–6% O2 (physi- oxia) in vivo. Accumulating evidence has shown that such hyperoxic conditions in standard cell culture practices affect a variety of biological processes. In this review, we discuss how supraphys- iological O2 levels affect reactive oxygen species (ROS) metabolism and redox homeostasis, gene expression, replicative lifespan, cellular respiration, and mitochondrial dynamics. Furthermore, we present evidence demonstrating how hyperoxic cell culture conditions fail to recapitulate the phys- iological and pathological behavior of tissues in vivo, including cases of how O 2 alters the cellular response to drugs, hormones, and toxicants. We conclude that maintaining physioxia in cell culture is imperative in order to better replicate in vivo-like tissue physiology and pathology, and to avoid artifacts in research involving cell culture. Keywords: oxygen; physioxia; hyperoxia; ROS; oxidative stress; gene expression; senescence; me- tabolism; mitochondrial dynamics; drug response Citation: Alva, R.; Gardner, G.L.; Liang, P.; Stuart, J.A. Supraphysiological Oxygen Levels 1. Introduction in Mammalian Cell Culture: Current At sea level, dry atmosphere is composed of ~21% O2. Incubators used in standard State and Future Perspectives. Cells cell culture practices regulate headspace CO2 and temperature, but the vast majority do 2022, 11, 3123. https://doi.org/ 10.3390/cells11193123 not actively regulate O2. Although this is usually referred to in the literature as 20% or 21% O2, in fact, the headspace O2 in a humid cell culture incubator maintained at 5% CO2 Academic Editor: Friedrich Jung typically equilibrates to ~18% in the absence of O2 regulation. The overwhelming majority of in vitro studies have been performed in these conditions, and, as such, 18–21% O2 is Received: 10 September 2022 commonly termed ‘normoxia’ in the literature [1]. While atmospheric O2 may be a Accepted: 30 September 2022 normoxic condition for an entire organism, the same is not true for mammalian cells in Published: 4 October 2022 vivo. O2 levels in most mammalian tissues range from 2–6% [2], i.e., three- to nine-times lower than standard cell culture normoxia. In this review, we will use the term ‘physioxia’ Publisher’s Note: MDPI stays neu- [3] to describe O2 levels in this 2–6% range, and ‘normoxia’ for the hyperoxic (18%) con- tral with regard to jurisdictional ditions of standard cell culture. We are, thus, interested here in the effects of O2 in the claims in published maps and institu- range between physioxia and normoxia, that is, the ‘physio-normoxic’ range. It is im- tional affiliations. portant to note that, although most authors refer to O2 levels in experiments where cells are grown in humidified cell culture incubators with 5% CO2 as being either 20% or 21% O2, these numbers are incorrect. We have used the more accurate 18% throughout this text, though we recognize that this too will depend on elevation above sea level. Copyright: © 2022 by the authors. Li- The appreciation that standard cell culture conditions are in fact hyperoxic, com- censee MDPI, Basel, Switzerland. pared to what mammalian cells encounter in vivo has led to efforts to replicate physio- This article is an open access article distributed under the terms and con- logical O2 conditions (physioxia) in vitro. In this review, we present our current under- ditions of the Creative Commons At- standing of how cellular processes are affected by O2 in the range from physioxia to tribution (CC BY) license (http://crea- normoxia, including reactive oxygen species (ROS) generation and clearance, gene ex- tivecommons.org/licenses/by/4.0/). pression, proliferation, replicative lifespan, bioenergetics, and mitochondrial function. In Cells 2022, 11, 3123. https://doi.org/10.3390/cells11193123 www.mdpi.com/journal/cells Cells 2022, 11, 3123 2 of 21 addition, we discuss how cells cultured in normoxia exhibit phenotypes that fall short of reproducing tissue physiology and pathology in vivo. In this light, we consider recent findings showing that cells respond to drugs, hormones, and toxicants differently when cultured in physioxia, compared to 18% O2. We finalize by commenting on solutions to these challenges and future research directions. 2. Oxygen and Redox Homeostasis The different types of ROS, their generation, and their clearance have been exten- sively investigated and reviewed elsewhere [4–6]. Thus, we will mainly focus on how ROS metabolism is affected in the physio-normoxic range. − − Superoxide anion (O2 ) is formed by single electron reduction of O2. O2 is rapidly dismuted into hydrogen peroxide (H2O2) by superoxide dismutases (SOD) 1–3 [7]. Due to its neutral charge and diffusivity, H2O2 is able to cross membranes and participate in sig- naling processes by oxidizing cysteine residues in key signaling proteins that act as redox- sensitive molecular switches [8]. H2O2 is transformed into water by catalase, glutathione 2+ peroxidase (GPx), thioredoxin (Txn), and peroxiredoxin (Prdx) [9]. In the presence of Fe , H2O2 is converted into the highly reactive hydroxyl radical (HO), which indiscriminately oxidizes lipids, proteins, and DNA [10]. Although ROS have well-characterized roles in cellular physiology (reviewed in [11]), overproduction of ROS can be detrimental, which is why it is important to study ROS production rates from different sources and under- stand the factors that regulate each. There are multiple sources of ROS in mammalian cells, with the most studied being mitochondrial enzymes (e.g., respiratory complexes I and III) and NADPH oxidases (NOX). The KM values of the complex I flavin mononucleotide (FMN) site and complex III Qo site toward O2 are 0.019% and 0.199%, respectively [12], meaning that in the 2–18% O2 range, these enzymes are likely saturated and that little difference would thus be expected in mitochondrial ROS (mROS) production of cells grown at 18% O2 versus physioxia. Nonetheless, some studies have reported higher ROS levels at 18% O2, using mitochon- dria-targeted ROS indicators such as MitoSOX and dihydrorhodamine 123 [13–15], sug- gesting increased mROS production at normoxia (18% O2). On the other hand, NOX isoforms have higher KM values. For example, NOX4 has a reported KM of ~18% O2 [16], indicating that the reaction rate is highly sensitive to O2 concentration in the 2–18% range. Accordingly, production of H2O2 in C2C12, PC-3, SH-SY5Y, HeLa, MCF-7, and mouse em- bryonic fibroblasts (MEFs) was found to be significantly higher at 18% O 2 than at 5% O2 [17]. Pharmacological inhibition of NOX1/4 abrogated this effect, confirming the im- portance of NOX enzymes in ROS production at O2 levels higher than physiological con- ditions. A thorough discussion of how O2-consuming reactions are impacted by O2 tension in cell culture is provided in [18]. Indeed, elevated ROS production at supraphysiological O2 levels has been demonstrated by numerous studies in multiple cell types [13–15,19– 21]. In addition, significantly lower reduced glutathione (GSH) levels have been observed in cells grown at 18% O2 [20,22,23]. Thus, there is ample evidence that ROS production is greater in normoxia, compared to in physioxia, often compromising the antioxidant de- fenses of cells. Disruption of the balance between ROS production and antioxidant defenses results in oxidative stress, characterized by damage to lipids, proteins, and DNA. Timpano et al. reported that U87MG, HRPTEC, HEK293, and MCF-7 cells grown at 18% O2 exhibit more DNA strand breaks than their counterparts cultured in physioxia, as determined by comet assay [24]. Further, increased levels of 8-oxodeoxyguanine and 8-oxoguanine were ob- served in all cell lines but MCF-7, indicating oxidative damage to the nitrogen bases of DNA and RNA in these cells grown at 18% O2. Protein oxidation was observed in rat my- oblast precursor cells (MPCs). Protein carbonyl levels, one of the most common bi- omarkers of protein oxidation, were higher at 18% O2, compared to 3% O2, in old MPCs only, with no effects observed in cells from young mice [25]. Human mesenchymal stem cells (hMSCs) also showed increased protein carbonyl content and DNA strand breaks Cells 2022, 11, 3123 3 of 21 when cultured in ambient air [19]. In addition, hMSCs grown at 18% O2 had higher levels of malonyl aldehyde (MDA), which is an end-product and biomarker of lipid peroxida- tion. Elevated MDA levels were also detected in human dental pulp stem cells (hDPSCs) grown at 18% O2 for 7 days, compared to cells grown at 3% O2, which was prevented with Trolox administration, a water-soluble vitamin E analog [15]. Finally, levels of both pro- tein carbonyls and 4-hydroxynonenal (4-HNE), another lipid peroxidation end-product, were found to be higher in mouse cardiac fibroblasts cultured at 21% O2, compared to 3% O2 [13]. To conclude, there is abundant evidence that the hyperoxic environment of standard cell culture disrupts redox homeostasis by increasing the production of ROS, which re- sults in oxidative damage to biomolecules and organelles. Biomolecule oxidation can, in turn, result in the dysregulation of signaling pathways and cellular processes, such as gene expression, proliferation, senescence, differentiation, metabolism, and response to drugs and other bioactive molecules. 3. Intracellular Oxygen and ROS Sensing Discovered in the early 1990s, the hypoxia-inducible factor (HIF) is regarded as the master intracellular O2 sensor in response to hypoxia [26,27]. HIF is a heterodimer com- posed of one α and one β subunit. While there are three isoforms of HIF-α (HIF-1α, HIF- 2α, and HIF-3α, encoded by the genes HIF1A, HIF2A, and HIF3A, respectively), only one HIF-β protein has been described (reviewed in [28]). When translocated to the nucleus, HIF-α partners with the constitutively expressed HIF-β, binds to the hypoxia response element (HRE) of DNA and initiates transcription of target genes. The transcriptional ac- tivation of HIF allows the cell to adapt to low O2 tensions, by mediating expression of genes involved in glycolysis and energy metabolism, angiogenesis, cell migration, prolif- eration, and survival, among other processes [29]. Although HIF-1α and HIF-2α have unique gene targets, both are critical for regulating the expression of genes necessary for cell adaptation to hypoxia [30] (reviewed in [31]). In normoxia, prolyl hydroxylases (PHD) use molecular O2 as substrate to introduce hydroxyl groups in two highly conserved prolyl residues of HIF-α. Hydroxylated HIF-α is then recognized by the Von Hippel- Lindau tumor suppressor protein (pVHL), which leads to its ubiquitination and subse- quent degradation by the proteosome [32]. In contrast, a low O2 concentration in hypoxia blunts PHD activity and allows for the stabilization of HIF-α and its translocation to the nucleus (Figure 1A) [33]. It is important to note that nearly all studies on HIF-1/2 activity have been conducted as comparisons between normoxia (18%) and hypoxia (≤1% O2). Fu- ture studies should be conducted comparing physioxia and hypoxia. While HIF activation has long been considered solely as a cellular response to hy- poxia, a few studies have also suggested its role in physiological O2 conditions. Given that the KM values of PHD isoforms are slightly above atmospheric O2 level [34], the enzymatic rate of HIF-α hydroxylation is highly sensitive to O2 levels in the physio-normoxic range. Bracken et al. investigated the stabilization of HIF-1/2α in six cell lines, HeLa, 293T, Cos- 1, PC-12, CACO2, and HepG2, and they observed cell-type specific patterns [35]. For ex- ample, HIF-1/2α were barely detectable in HeLa cells at 5% O2 but upregulated at 2% O2. On the other hand, 293T cells had higher HIF-1/2α levels at 5% O2. These results likely reflect different sensitivities of distinct cell types to O2 levels [35]. In another study, Yan et al. detected HIF-1α localization to the nuclei of primary hepatocytes at 5% O2 via immu- nofluorescence [36]. More recently, detectable protein levels of HIF-1α and HIF-2α were observed in patient-derived melanoma cells cultured at 6% O2, supporting the notion that HIF is partially active at physiologically relevant O2 tensions [37]. Moreover, in this study, increased expression of HIF targets was sustained in cells grown at 6% O2 for at least 3 weeks, further suggesting a role for HIF activity at physioxia rather than it being a mere consequence of an acute reduction of O2 tension while performing the experiments. Stuart et al. have hypothesized that transcriptional activity of HIF-1/2 at low O2 tensions within a physiological range (2–6%) helps to maintain the production of ROS and reactive Cells 2022, 11, 3123 4 of 21 nitrogen species (RNS), which are necessary for many physiological processes [38]. This hypothesis is based on the fact that several ROS-producing enzymes are induced by HIF- 1/2 (reviewed in [18]). In contrast to direct oxygen-sensing mechanisms in hypoxia, the cellular response to hyperoxia is regulated by molecular switches that are sensitive to H2O2. Perhaps the most important and best described is the pathway regulated by the nuclear factor erythroid 2– related factor 2 (Nrf2). The molecular mechanisms regulating Nrf2 degradation, stabiliza- tion, and transcriptional activity have been thoroughly reviewed in [39–41]. Under un- stressed conditions, Nrf2 is bound to Kelch-like ECH-associated protein 1 (Keap1), which facilitates ubiquitination and proteasomal degradation of Nrf2. Keap1 acts as an intracel- lular ROS sensor, with multiple cysteine residues identified as redox-sensitive regulators. In conditions of oxidative stress, such as hyperoxia, ROS-mediated oxidation of specific cysteine residues inhibits Keap1 binding to Nrf2, leading to Nrf2 stabilization, transloca- tion to the nucleus, and binding to the antioxidant response element (ARE) of DNA (Fig- ure 1B) [42]. In addition to oxidants, the regulatory cysteine residues of Keap1 are also sensitive to electrophiles that readily react with thiol groups [40]. Such electrophiles can originate from both exogenous and endogenous sources. In the latter case, for instance, the lipid peroxidation end-product 4-HNE reacts with specific cysteine residues of Keap1, promoting Nrf2 stabilization and transcriptional activity [43,44]. Nrf2 induces the expres- sion of genes involved in ROS and xenobiotic detoxification, GSH biosynthesis and regen- eration, NADPH regeneration, and heme and iron metabolism [45]. It is important to note that Nrf2 is part of the cellular response to oxidative and elec- trophilic stresses, which may be caused by a variety of insults, not just hyperoxia. Never- theless, hyperoxia is a well-characterized condition of increased ROS production, in which the role of Nrf2 has been demonstrated. Indeed, activation of Nrf2 in severe hyperoxia (>60% O2) has been extensively studied (reviewed in [46]). Its role in perceived hyperoxia at ambient air (~18% O2) in vitro, on the other hand, has been less studied. However, a handful of studies have reported upregulation of Nrf2 in cells cultured in normoxia, com- pared to cells grown in physioxia. Epithelial cells from mouse mammary tumors pro- cessed under ambient air had increased levels of Nrf2 and decreased levels of Keap1, com- pared to tumors under physioxia (3% O2) [47]. In accordance, upregulation of known Nrf2 targets, such as SOD1, thioredoxin reductase, and NAD(P)H quinone dehydrogenase (NQO1), has been observed in normoxia versus physioxia in different cell types [15,23,24]. Due to the induction of antioxidant and cytoprotective enzymes in normoxia (~18% O 2), some studies have shown that cells adapted to these conditions may be preconditioned and, thus, resistant to pro-oxidant insults (e.g., drugs and toxins). This shall be further addressed in Section 9. Moreover, mechanisms of enhanced Nrf2-dependent gene tran- scription in normoxia may go beyond increased Keap1 oxidation or downregulation. Chapple et al. demonstrated nuclear accumulation and DNA binding of Nrf2 in human endothelial cells grown in physioxia [48]. Instead, Bach1, another Nrf2 repressor, was found to be upregulated in physioxia, thus interfering with expression of Nrf2 targets. Inhibition of Nrf2 transcriptional activity was abrogated by re-exposure to ambient air or Bach1 silencing. This provides a novel mechanism for Nrf2-mediated regulation of gene expression induced by O2 in the physio-normoxic range. Other ROS-activated pathways known to be affected in hyperoxia include those me- diated by the nuclear factor kappa B (NF-κB) and mitogen activated protein kinases (MAPKs) (reviewed in [46]), but their regulation in physioxia versus atmospheric O2 has been scarcely investigated. Carrera et al. did observe increased phosphorylation of extra- cellular signal-regulated protein kinase (ERK1/2), in HCT116 and U2OS cells grown at 5% O2 in the absence of any stress [49]. They showed that the constitutive activation of this pro-survival pathway affects their response to proapoptotic drugs (see Section 9). Indeed, differential regulation of O2-sensitive and redox-sensitive signaling molecules and tran- scription factors leads to the alteration of multiple biological processes, as shall be further discussed throughout this review. Additional research is required to fully elucidate the Cells 2022, 11, 3123 5 of 21 roles and molecular mechanisms of HIF, Nrf2, and other pathways in the cellular adapta- tion to supraphysiological O2 in different cell types. Figure 1. Pathways for intracellular oxygen/redox status sensing by HIF-1/2 and Nrf2. (A) Low O2 levels reduce the rate of HIF-1/2α hydroxylation, which leads to molecular recognition by the Von Hippel-Lindau tumor suppressor protein (pVHL) and subsequent proteasomal degradation. Stabi- lized HIF-1/2α binds HIF-β and translocates to the nucleus, where it binds the hypoxia response element (HRE) in DNA, inducing expression of glycolytic, proliferative, and pro-survival genes, among others. (B) High O2 levels promote increased reactive oxygen species (ROS) production. ROS-mediated oxidation of Keap1 releases Nrf2 preventing its proteasomal degradation. Nrf2 then translocates to the nucleus, heterodimerizes with small musculo-aponeurotic fibrosarcoma (sMaf), and binds to the antioxidant response element (ARE), inducing expression of genes involved in ROS and xenobiotic detoxification. Created with BioRender.com (accessed on 14 July 2022). 4. Effects of Oxygen and ROS on Gene Expression Differential regulation of O2/ROS-sensitive transcription factors in the physio- normoxic range affects gene expression, as we have recently shown in an RNA-seq-based study with LNCaP, Huh-7, PC-3, and SH-SY5Y cells. These cell lines were cultured at ei- ther 5% or 18% O2 for two weeks, prior to mRNA isolation and sequencing. The O2 level significantly affected the abundance of thousands of transcripts in all cell lines. Surpris- ingly, there was limited overlap in the specific transcripts affected, with >80% of the dif- ferentially expressed genes being cell-line specific [50]. Interestingly, a handful of HIF tar- gets were found to be upregulated at 5% O2 in LNCaP and Huh-7 cells, further confirming a role for HIF activation under physiological O2 conditions. Similar patterns were found by Duś-Szachniewicz et al. at the proteome level [51]. They analyzed proteomic changes in three diffuse large B-cell lymphoma cell lines grown at 1% O2 (hypoxia), 5% O2 (physi- oxia), and 18% O2 (normoxia). Importantly, proteomes from cells grown in hypoxia and physioxia cells shared more similarities, compared to the proteome of cells grown in at- mospheric O2. In agreement with our study, this latter study showed that the effects of Cells 2022, 11, 3123 6 of 21 physioxia versus normoxia were largely cell-type specific, even when the three cell lines are from the same type of cancer [51]. Mechanisms of translation are also regulated by O2 (reviewed in [52,53]). Hypoxia prevents the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the 7- methylguanosine cap at the 5′ end of mRNA molecules, which is the initial step of protein synthesis. In 2012, Uniacke et al. demonstrated that, under hypoxic conditions, HIF-2α forms a complex with eIF4E2 (eIF4E homologue) and the RNA binding motif protein 4 (RBM4), which then recognizes and binds to the 5′ cap of specific mRNA molecules, al- lowing initiation of translation independent of eIF4E [54]. Years later, the same research group found that, while eIF4E is the dominant cap-binding protein in normoxia (18% O2) and eIF4E2 is the dominant cap-binding protein in hypoxia, both are active at physioxia (here, 3–12%) and simultaneously act to initiate the translation of distinct classes of mRNA molecules [55]. These results demonstrate that differential expression of proteins in phys- ioxia versus normoxia is not only regulated at the transcriptional level but also at the translational level. In addition to transcriptional and translational regulation, O2-dependent mecha- nisms mediate gene expression through epigenetic modifications. Processes such as his- tone methylation and acetylation, DNA methylation, and chromatin organization are known to be affected by hypoxia and oxidative stress (reviewed in [56,57]). The roles of these mechanisms in the physio-normoxic range have been investigated less often, alt- hough a handful of studies have reported different epigenetic responses to O2 in a variety of cells, with a special focus on stem cells and cancer cells. Lengner et al. found that culturing human embryonic stem cells (hESCs) in physioxia prevents chromosome X inactivation that is otherwise observed in normoxia, which, in turn, preserves the pluripotency of hESCs [58]. This was associated with the methylation of the XIST promoter region, while chromosome X inactivation was associated with the demethylation and transcriptional activity of XIST. In another study, Xie et al. observed an increased methylation pattern and irreversible silencing of the DLK1-DIO3 gene cluster in cultured hESCs after 20 passages in normoxia [59]. Interestingly, culture of hESCs in 5% O2 preserved expression of the DLK1-DIO3 cluster. The DLK1-DIO3 cluster has been associated with pluripotency in mouse induced pluripotent stem cells and may, thus, be a biomarker for the epigenetic stability of hESCs. Further, acetylation of histone protein H3 at lysine residues 9 and 27 (H3K9ac and H3K27ac, respectively) was elevated in human pluripotent stem cells (hPSCs) grown at 5% O2, while trimethylation of H3 lysine 27 was reduced [60]. These epigenetic changes are associated with a more open chromatin struc- ture and enhanced gene expression. RNA-seq and functional enrichment analysis also re- vealed increased expression of genes involved in H3K27 demethylation at 5% O2, while increased methyltransferase and cell cycle activities were observed at 18% O2. The ten-eleven translocation (TET) family of proteins catalyzes the O2-dependent hy- droxylation of 5-methylcytosine (5mC) in DNA, which results in demethylation and con- comitant increase in gene expression. Thienpont et al. investigated the effect of tumor hy- poxia (0.5% O2) on the expression profiling and activities of TET isoforms, TET1, TET2, and TET3 [61]. The mRNA levels of TET paralogues were increased at a variable degree among a range of cell lines exposed to hypoxia, with neuroblastoma cell lines SH-SY5Y and SK-N-Be2c being the most strongly affected. Chromatin immune precipitation fol- lowed by sequencing (ChIP–seq) confirmed the binding of HIF near the promoters of up- regulated TET genes. However, the activity of TET enzymes was actually reduced in hy- poxic human and mouse tumor cells, which is not surprising, since these enzymes use O2 as substrate. Reduced TET activity was then correlated with decreased hydroxylated 5mC (5hmC) and concomitant hypermethylation. Although these landmark results provided a mechanism for epigenetic-mediated repression of tumor suppressor genes in hypoxia, no changes in TET activity were observed at 2–5% O2. In a different study, however, the ex- pression of the three TET paralogues was increased in HepG2 cells at both 5% and 1% O2, compared to 18% O2 [62]. Upregulation of these enzymes at 1% O2 was blunted by HIF- Cells 2022, 11, 3123 7 of 21 1α knockdown, although this was not tested at 5% O2. The activity of TET enzymes was not measured in this study. In stem cells, on the other hand, a different trend in the regu- lation of TET genes has been observed. TET1 mRNA and protein levels were decreased in bone-marrow-derived hMSCs grown at 2% O2, compared to atmospheric O2 [63]. Expres- sion of DNA methyltransferase 3B (DNMT3B) was also reduced at 2% O2. As a result, global 5hmC and 5mC levels were also decreased at physioxia. In a subsequent study by the same lab, DNMT3B, DNM3L, TET1, and TET3 were similarly downregulated in hPSCs cultured at 2% O2, compared to cells grown at 18% O2 [64]. This was accompanied by enhanced proliferation, metabolic activity, and stemness attributes. Interestingly, both studies showed downregulation of HIF1A and upregulation of HIF2A (in hMSCs and hPSCs, respectively) cultured at physioxia. Another mechanism of epigenetic regulation of gene expression involves a variety of non-coding RNA molecules, such as microRNA (miRNA), long non-coding RNA (lncRNA), and small nucleolar RNA (snoRNA). Johnston et al. measured the abundance of miRNA molecules in MCF-7 cells across a range of O2 pressures, from 18% O2 to 1% O2 [65]. Among the most strongly affected miRNAs, miR-675, miR-1293, miR-7974, miR-653, and miR-140 were found upregulated at 18% O2, compared to 4% O2 (physioxia). On the other hand, miR-758, miR-335, miR-1185-1, miR-1185-2, and miR-889 were upregulated at 4% O2, compared to 18% O2. These miRNA molecules have been implicated in processes such as apoptosis, proliferation, metastasis, and tumor suppression. In the study by Xie et al. where silencing of the DLK1-DIO3 cluster was observed in hESCs grown at 18% O2 after 20 passages, decreased expression of several non-coding RNA molecules was shown, including the lncRNA MEG3 and the snoRNA SNORD114-3 [59]. The expression of these molecules was rescued in cells cultured at 5% O2. MEG3 is known to inhibit tumor prolif- eration by interacting with p53. Accordingly, silencing of MEG3 was correlated with re- duced DNA-damage-induced apoptosis in hESCs and their differentiated hepatocyte-like cells grown at ambient O2 tension. Using microarray and bioinformatic analyses, Shi et al. identified 47 lncRNAs and 14 miRNAs differentially expressed in hDPSCs cultured at 18% O2 and 3% O2, revealing a complex gene network between the non-coding RNAs and mRNAs [66]. Further, functional enrichment analysis revealed biological processes, such as the HIF-1 pathway, the Wnt pathway, carbon metabolism, and glutathione metabolism, to be affected by O2, among other processes. In summary, broad epigenetic responses to physiological O2 levels have been re- ported, with striking differences observed depending on the cell type. These responses include DNA methylation and hydroxylation, histone acetylation and methylation, and altered expression of non-coding RNAs. Future research should be directed to fully char- acterize the specific mechanisms governing these cell-type-specific responses. 5. Oxygen, Proliferation, and Senescence The first observation made about the impact of culturing cells at O2 tensions above physiological conditions was a decrease in their proliferation rate. In 1958, Cooper et al. reported that lowering O2 tension below normoxia increased the growth rate of rabbit embryo kidney cells [67]. A later study found that lowering O2 from 18% to 10% O2 in- creases the replicative lifespan (i.e., Hayflick limit) of WI-38 fibroblasts [68]. Similar ob- servations were made by Balin et al. [69]. Perhaps expectedly, immortalized cells are not as sensitive to the cytostatic effect of atmospheric O2, as Falanga and Kirsner showed that single-seeded human fibrosarcoma and immortalized 3T3 cells could proliferate without difficulty in standard O2 conditions (18% O2), in contrast to primary dermal fibroblasts [70]. Subsequent studies focused on the molecular mechanisms of the antiproliferative ef- fects of atmospheric O2. Hyperoxia was established to be an inducer of replicative senes- cence, which is a process initiated by oxidative DNA damage and telomere shortening and mediated by p53- and p21-dependent cell cycle arrest [71]. Culture of MEFs at 18% O2 resulted in DNA mutations and senescence. G:C to T:A transversions, a signature mutation caused by DNA oxidation, were observed in Cells 2022, 11, 3123 8 of 21 hyperoxic cells. Senescence was not observed in MEFs grown at 3% O2 [72]. Similar obser- vations were made by Parrinello et al., showing induction of senescence and DNA strand breaks in MEFs grown in 18% O2 [73]. Cardiac fibroblasts from adult murine ventricle cultured at 10% and 18% O2 exhibited greater ROS production and cell cycle arrest at G2/M, compared with cells at 3% O2 [13]. Induction of p21 and decreased telomerase ac- tivity were also detected in hyperoxia. Fibroblasts lacking p21 were resistant to the effects of higher O2 tension. Overexpression of p21 was confirmed at both mRNA and protein levels in muscle precursor cells (MPCs) exposed to 18% O2, compared to 5% O2, along with increased p53 transcriptional activity [74]. Estrada et al. reported that telomere shortening rate per division cycle is 24% higher in hMSCs cultured in normoxia, compared to in phys- ioxia (here, 3% O2) [19]. Another study reported increased p38 phosphorylation, an up- stream regulator of p21, along with concomitant p21 overexpression in hDPSCs cultured in 18% O2. Elevated HO-1 and NQO-1 protein levels were also detected, suggesting Nrf2 activation [15]. More recently, Bon-Mathier et al. demonstrated that primary neonatal mouse cardiomyocytes have a higher proliferative capacity when cultured at 3% O 2, which was associated with a dedifferentiation of cardiomyocytes at physioxia [75]. Recent evidence has linked the Wnt/β-catenin signaling pathway to the mechanisms regulating the response to O2 at physioxia/hyperoxia. Stabilized β-catenin and upregula- tion of Wnt gene targets were observed in fetal mouse neural stem cells (NSCs) at physi- oxia, promoting proliferation and reducing spontaneous differentiation of NSCs [76]. These effects were sustained even in HIF-1α–deficient cells, indicating that the O2-sensi- tive mechanism of Wnt activation is independent of HIF. Further research is required to understand the involvement and mechanisms of Wnt in O2 sensing at physiological O2 tensions. In conclusion, there is ample evidence showing that hyperoxic cell culture induces senescence in a variety of cells by promoting oxidative DNA damage and activating p53 and p21. Collectively, these results emphasize the importance of culturing primary cells at physioxia, in order for them to retain their proliferative capacity. More evidence regard- ing the link between O2 and cell differentiation is discussed in the next section. 6. Oxygen and Cell Differentiation The transcriptional program controlling stemness and differentiation of embryonic stem cells is in part regulated by HIF. For example, the expression of OCT4, a critical tran- scription factor in mediating stemness and pluripotency of embryonic stem cells, is regu- lated by HIF-2α [77]. Westfall et al. showed that expression of known HIF-1/2α and OCT4 targets was markedly increased in human embryonic cell lines at 4% O2, compared to at 18% O2 [78]. The mechanisms governing hypoxia and stemness are reviewed in [79]. In accordance, lowering O2 to 5% increased the proliferation and differentiation of human macrophage progenitor cells [80,81]. Fehrer et al. found that while culturing hMSCs at 3% O2 extends their proliferative lifespan and inhibits senescence, their capacity of differentiating into osteoblasts and adipocytes was blunted at this O2 tension [82]. Sim- ilar mechanisms seem to affect osteoblast differentiation. Culture of bone-marrow-iso- lated adult multilineage-inducible cells at 3% O2 increased their proliferation but blocked their differentiation into osteoblasts, which is otherwise present when cultured at 18% O2 [83]. mRNA levels of OCT4, HIF1A, and TERT (telomerase reverse transcriptase) were up- regulated at 3% O2. In contrast, lower O2 levels have been found to promote chondrogen- esis, which is in agreement with the fact that cartilage is avascular and in hypoxic envi- ronment [84–87] (reviewed in [88]). Studer et al. reported that culture of mesencephalic precursor cells at 3% O2 enhances both their proliferation and differentiation into dopa- minergic neurons [89]. Recent evidence has also shown that culture of stem cells at phys- ioxia improves their therapeutic potential for clinical application [90,91]. The relevance of O2 levels for stem cell biology and their application in regenerative medicine is reviewed in detail in [92]. Cells 2022, 11, 3123 9 of 21 Immortalized cell lines are widely used in research as surrogates of primary cells, which are often less accessible than the former to the research community. A study found that the ability of the monocyte-derived cell line THP-1 to differentiate into macrophage- like-cells is affected by O2 tension. The metabolic activity and rate of drug-induced differ- entiation of THP-1 cells were enhanced at 5% O2, compared to 18%, with the primary mac- rophage-like functions of the differentiated cells being affected by O2 as well [93]. In summary, the O2 levels in culture have a profound impact on the differentiation capacity and stemness of cells, with specific effects varying among cell types, likely ac- cording to their oxygenation status in vivo. 7. Oxygen, Cell Bioenergetics, and Mitochondrial Dynamics Since most cellular O2 is consumed by mitochondria during respiration and ROS pro- duction, it is unsurprising that broad oxygen-dependent effects on cellular metabolism and mitochondrial function been reported in a variety of cells. Estrada et al. showed that culturing hMSCs at 18% O2 enhances oxidative phosphorylation (OXPHOS) and decreases glycolysis, which, in turn, leads to higher rates of ROS production and oxidative-stress- related damage. In this study, they found a decreased oxygen consumption rate (OCR)/ex- tracellular acidification rate (ECAR) ratio in hMSCs cultured at 3% O2, compared to at 18% O2, which was associated with increased expression of HIF-1/2 targets that encode en- zymes involved in glucose metabolism, such as pyruvate dehydrogenase kinase, phos- phofructokinase, and lactate dehydrogenase [19]. Enhanced activity of these enzymes in- creases the glycolytic rate and diverts pyruvate from entering the TCA cycle, while pro- moting its reduction to lactate in order to regenerate reduced nicotinamide dinucleotide (NAD ). In agreement with these results, Lees et al. found that hPSCs cultured in 18% O2 have an increased OCR and ATP production rate, compared to cells cultured at physioxia [60]. Although no significant differences were observed in basal ECAR, which is a proxy for glycolytic rate, RNA-seq revealed increased expression of genes related to glycolysis in cells grown at 5% O2. Using C glucose labeling, they also observed increased glycolytic intermediary metabolites (e.g., fructose-1,6-biphosphate) and lactate. In primary human corneal endothelial cells, no effect on the basal OCR was observed at 2.5% O 2, compared to atmospheric O2 tension. In turn, basal ECAR was increased at 2.5% O2 [94]. This result is in accordance with the ability of the corneal endothelium to rely on glycolysis to meet the energy requirements of the cornea under lower oxygen availability. In another study, Zhu et al. demonstrated that culture of rat primary cortical neurons at physioxia improves mitochondrial function, as shown by increased mitochondrial membrane potential, mito- chondrial dehydrogenase activity, and ATP levels at 5% O2, compared to at 18% O2 [21]. Although these outcomes suggest enhanced mitochondrial respiration, glucose uptake and consumption rates were also found to be higher at 5% O2. Further, Zhu et al. observed reduced glucose oxidation, measured by CO2 production, along with higher lactate lev- els in neurons cultured at 5% O2. AMPK phosphorylation was also found to be higher at physioxia. As such, many of the metabolic effects of culturing neurons at 5% O2 were abol- ished by pharmacological inhibition of AMPK. A handful of other studies have reported opposite patterns, suggesting higher OXPHOS rates at physioxia. This could be explained by the fact that hyperoxia-induced ROS directly inactivate mitochondrial enzymes such as aconitase, pyruvate dehydrogen- ase complex, and respiratory complexes I and II (reviewed by Alva et al. in [95]). For ex- ample, MPCs isolated from old mice have a higher basal and maximal OCR at 3% O2, compared to 18% O2. In fact, when old MPCs are cultured at 3% O2, they have the same OCR as that of MPCs from young mice, which show no difference in OCR between the two O2 concentrations [25]. Old MPCs grown at hyperoxia seemed to compensate by up- regulating glycolytic metabolism, as the ECAR measured was higher than that of old MPCs grown at physioxia and young MPCs at either O2 tension, which, again, did not seem to be affected much by O2. In addition to age, sex has also been shown to influence the bioenergetic response to different oxygen concentrations. Experiments with primary Cells 2022, 11, 3123 10 of 21 rat cortical astrocytes have uncovered sex differences in their bioenergetic profile when cultured at 3% O2. In contrast, no differences in bioenergetic parameters were found in astrocytes grown at 18% O2, denoting how physiological phenomena can be masked by culturing cells at nonphysiological oxygen tensions [96]. Studies with immortalized cell lines have shown similar trends. Timpano et al. stud- ied the effects of O2 tension in the metabolism of four human cell lines, U87MG, HRPTEC, HEK293, and MCF-7. In general, cells had a lower mitochondrial metabolic activity when cultured at 12% O2 or higher, compared to at physioxia, as measured by fluorometric de- tection of resaruzin reduction [24]. Similarly, Moradi et al. investigated the effect of O2 concentration in cellular respiration using four human cancer cell lines. MCF-7, Huh-7 and LNCaP cells had a lower basal OCR at 18% O2, compared to at 5% O2, while no dif- ference was observed in SaOS2 [97]. Maximal OCR was also lower at 18% O2 in all cell lines, except LNCaP, which had a greater maximal OCR at 18%. Collectively, these results indicate that physiological O2 levels seem to promote mitochondrial metabolism, although no effects on glycolytic rate (ECAR) were observed in this study. The same study by Moradi et al. also revealed differential effects of O2 on mitochon- drial network dynamics, which were analyzed using the MiNA tool [98]. Normoxia was associated with an increased mitochondrial footprint, indicative of mitochondrial abun- dance, in Huh-7 and SaOS2 cells, but had the opposite effect on LNCaP cells. In addition, the mean network size, which is indicative of the number of network branches, was lower in LNCaP and SaOS2 cells grown at 18% O2, while no significant difference was observed in MCF-7 and Huh-7. In the study by Timpano et al., a variety of effects in mitochondrial fusion and morphology of U87MG, HRPTEC, HEK293, and MCF-7 cells cultured in dif- ferent O2 levels were measured. U87MG and HEK293 cells displayed a more globular mi- tochondrial morphology, when cultured at atmospheric O2, compared to cells cultured at physioxia, while HRPTEC cells showed no difference in mitochondrial morphology, and MCF-7 had a more globular mitochondrial morphology at 5% O2 than at 18% O2. In a different report, mitochondria in primary neurons were found to be longer and have a more elongated morphology at 5% and 2% O2 levels, compared to cells kept at atmos- pheric O2 [99]. Accordingly, mitochondrial network parameters, such as network size, mi- tochondrial perimeter, and mitochondrial fraction, were higher with decreasing O2 con- centration. In summary, O2 conditions affect mitochondrial respiration, glycolysis, mitochon- drial morphology, and mitochondrial network dynamics (Table 1). Although, in some cases, the trend would seem to indicate that OXPHOS and highly fused mitochondrial networks are favored under physioxia, these are not widespread observations. This is likely due to the unique metabolic profiles of each type of cell, causing cells to react dif- ferently when challenged with varying O2 tensions. In any case, the effects of environmen- tal O2 on metabolic and mitochondrial characteristics of cells can no longer be neglected and must be considered when conducting experiments that target the mitochondria and cellular metabolism. Table 1. Effects of O2 tension (between 2–18% O2) on cellular metabolism, mitochondrial abundance, morphology, and network dynamics. Cell Types Experimental Conditions Methods Outcomes Reference Metabolic Effects  OCR/ECAR ratio at 18% O2 Seahorse XF analysis, hMSCs/18% or 3% O2  expression HIF targets involved [19] microarray, RT-PCR in glucose metabolism at 3% O2  OCR/ECAR ratio at 18% O2 Stem cells Seahorse XF analysis,  glycolytic intermediates hPSCs/18% or 5% O2 NMR spectroscopy, RNA- at 3% O2 [60] seq, RT-PCR  expression HIF targets involved in glucose metabolism at 3% O2 Cells 2022, 11, 3123 11 of 21  OCR at 18% O2 MPCs from old mice/18% or 3% O2 Seahorse XF analysis [25]  ECAR at 18% O2 hCEnCs/18% or 2.5% O2 Seahorse XF analysis  ECAR at 2.5% O2 [94]  glucose uptake at 5% O2 Rat primary cortical neurons/18% or ATP bioluminescent as-  glucose oxidation at 5% O2 Primary differen- [21] 5% O2 say, LSC, lactate assay  lactate levels at 5% O2 tiated cells  metabolic activity at 18% O2, HRPTEC/18–3% O2 Resazurin assay compared to 15% and 12% O2  metabolic activity at 18% O2, U87MG/18–3% O2 Resazurin assay [24] compared to 8–3% O2  metabolic activity at 18% O2, MCF-7/18–3% O2 Resazurin assay compared to 8% O2  basal and maximal OCR MCF-7/18% or 5% O2 Seahorse XF analysis Cancer/immortal- at 18% O2 ized cells  basal OCR at 18% O2 LNCaP/18% or 5% O2 Seahorse XF analysis  maximal OCR at 18% O2 [97]  basal and maximal OCR Huh-7/18% or 5% O2 Seahorse XF analysis at 18% O2 SaOS2/18% or 5% O2 Seahorse XF analysis  maximal OCR at 18% O2 Effects on mitochondrial morphology, abundance, and dynamics Globular-shaped mitochondria at 18% O2 (versus elongated at 2% Primary differen- rat primary neurons/18%, 5%, or 2% TEM and confocal micros- and 5% O2) [99] tiated cells O2 copy; Image J  mitochondrial network size, mi- tochondrial fraction, and mito- chondrial perimeter at 18% O2 Confocal microscopy; U87MG/18–3% O2 Rounder mitochondria at 18% O2 Volocity Confocal microscopy; HEK293/18–3% O2 Rounder mitochondria at 18% O2 [24] Volocity Confocal microscopy; Elongated mitochondria MCF-7/18–3% O2 Volocity at 18% O2 Cancer/immortal-  mitochondrial footprint Confocal microscopy; ized cells LNCaP/18% or 5% O2 at 18% O2 MiNA  mean network size 18% O2 Confocal microscopy;  mitochondrial footprint Huh-7/18% or 5% O2 [97] MiNA at 18% O2  mitochondrial footprint Confocal microscopy; SaOS2/18% or 5% O2 at 18% O2 MiNA  mean network size 18% O2 Abbreviations: ECAR, extracellular acidification rate; hCEnCs, human corneal endothelial cells; hMSCs, human mesenchymal stem cells; hPSC, human pluripotent stem cells; HRPTEC, human renal proximal tubule epithelial cells; LSC, liquid scintillation counting; MiNA, mitochondrial network analysis; NMR, nuclear magnetic resonance; OCR, oxygen consumption rate; TEM, trans- mission electron microscopy; XF, extracellular flux. 8. Modeling Tissue Physiology and Pathology in Physioxia One of the goals of cell culture is to mimic the behavior of cells and tissues in vivo, in both their physiological and pathological states. In this regard, physiological O2 condi- tions have been shown to facilitate the maintenance of in vivo-like properties of cells cul- tured in vitro. For instance, when compared to cells grown in physioxia, microvascular endothelial cells isolated from the foreskins of neonates cultured in normoxia exhibit a differential expression of proteins related to the functionality of endothelial cells, such as type IV collagen, platelet endothelial cell adhesion molecule-1 (PECAM-1), and von Wil- lebrand factor. In addition, vascular channel formation induced by collagen gels was Cells 2022, 11, 3123 12 of 21 increased in 18% O2 [100]. Piossek et al. showed that co-culture of human renal proximal tubular epithelial cells with human fibroblasts at physioxia improves their functional properties, as measured by epithelial barrier integrity, expression of membrane transport- ers, and transport of cations and anions [101]. Another study showed that hyperoxia pro- motes hepatocyte dedifferentiation in vitro, confirmed by the observation that mouse hepatocytes cultured in normoxia (18% O2) undergo epithelial-to-mesenchymal transi- tion, obtain fibroblast-like morphology, and show impaired hepatic functions. These ef- fects were prevented by culturing hepatocytes at 5% O2, which show increased glycogen stores, increased LDL-uptake ability, and sustained expression of cytochrome P450 en- zymes, all important characteristics of hepatocytes in vivo [102]. 2+ 2+ Intracellular Ca concentration ([Ca ]i) is critical in mediating a wide variety of pro- cesses, in both physiological and pathological conditions. Keeley et al. have recently 2+ shown that culturing endothelial cells at atmospheric O2 levels impairs Ca handling. Us- ing human umbilical vascular endothelial cells (HUVECs) cultured at 5% O2, they re- ported a negative feedback mechanism mediated by protein phosphatase 2A that regu- 2+ lates Ca –dependent NO biosynthesis in response to histamine, which is not observed at 2+ atmospheric O2 [103]. Interestingly, although they observed a similar intracellular Ca 2+ peak in response to histamine at both O2 conditions, [Ca ]i at the plateau phase was re- 2+ duced in cells cultured in 5% O2, suggesting enhanced cytosolic Ca removal mechanisms. Similar results were obtained when HUVECs were treated with ATP and ionomycin. These observations were further characterized in a subsequent study by the same lab. The 2+ difference in plateau [Ca ]i upon stimulation with histamine at 5% and 18% O2 was absent when cells were treated with cyclopiazonic acid, an inhibitor of the sarco/endoplasmic 2+ reticulum Ca –ATPase (SERCA). Moreover, agonist-stimulated phosphorylation of phos- pholamban, an endogenous negative regulator of SERCA, was higher at 5% O2 than at ambient air. Consequently, enhanced SERCA activity at physioxia blunted ionomycin-in- 2+ duced Ca overload and cell death, compared to the 18% O2 group [104]. Taken together, 2+ these results suggest that hyperoxic conditions in cell culture dysregulate Ca handling by reducing SERCA activity, which may artificially increase the sensitivity of cells to cy- 2+ 2+ totoxic stimuli that target intracellular Ca . Regulation of additional cytosolic Ca influx and efflux mechanisms at 5% and 18% O2 should be investigated. Hypoxia/reoxygenation (H/R) is the in vitro model of ischemia/reperfusion injury that takes place in pathologies such as strokes and myocardial infarctions. It involves ex- posing cells to hypoxia (≤ 1% O2) for a sustained period, usually coupled with nutrient deprivation, followed by placing them back to normoxic conditions. Researchers have hy- pothesized that oxidative stress and cell injury are artefactually exacerbated at the rela- tively hyperoxic reoxygenation conditions (at least an 18-fold pO2 increase, compared to hypoxia) used in most studies and that such stress may be less severe at physioxia (~5 - fold pO2 increase, compared to hypoxia). Warpisnki et al. reported that H/R did not seem to increase the generation of mitochondrial and extramitochondrial O2 at physioxia. Con- sequently, no protective effects of sulforaphane pretreatment were observed at 5% O 2, compared to at ambient air [105]. Similarly, Danilov and Fiskum cultured rat cortical as- trocytes at 7% or 18% O2 and exposed them to both hypoxia and glucose deprivation and then to reoxygenation in their respective O2 tensions [106]. Decreased cell death and lower levels of protein nitration and DNA oxidation were detected during reoxygenation at 7% O2, again confirming that hyperoxic reoxygenation under ambient air exacerbates cell in- jury in H/R models. In summary, keeping physiological O2 conditions in cell culture will provide us a better understanding of the physiological and pathological processes of cells and, in turn, allow us to conduct more reliable and relevant experiments to study the effects of different stimuli on cell physiology in vitro. In the next section, we will discuss how the cellular response to drugs, hormones, and toxicants is dependent on O2 tension, leading to arti- facts caused by supraphysiological O2 levels. Cells 2022, 11, 3123 13 of 21 9. Cellular Response to Drugs, Hormones, and Toxicants In vitro studies have provided us invaluable knowledge about the cellular effects and mechanisms of endogenous and exogenous substances, such as drugs, hormones, pollu- tants, toxins, and nanoparticles. However, experimental findings from cell culture often translate poorly in vivo. Recent evidence has shown that culturing cells in supraphysio- logical O2 levels can lead to an overestimation or underestimation of the potency of bio- logically active compounds or even demonstrate opposing effects. For example, resvera- trol acts as a prooxidant in C2C12 and PC-3 cells grown in low glucose DMEM at 18% O2, as shown by an increased H2O2 efflux rate. However, this effect is absent in both cell lines when cultured at 5% O2 [107]. Yan et al. reported that the hepatotoxic effect of acetamino- phen was less pronounced at physioxia, compared to atmospheric O2 [36]. On the other hand, the cytotoxic effects of copper oxide nanoparticles (CuO NPs) were diminished in normoxia, likely due to a preconditioning to oxidant insults in the hyperoxic environment of standard cell culture [108]. Similarly, the antiproliferative and cytotoxic effect of teri- flunomide on the colon cancer cell lines SW480 and SW620 was significantly enhanced with decreasing O2 concentration [109]. Table 2 summarizes the findings in the literature on biologically active substances that have been shown to elicit different cellular re- sponses at physioxia versus standard O2 conditions. Indeed, these observations highlight the need for utilizing physiologically representative O2 levels in cell culture, to improve the predictive value of in vitro pharmacological and toxicological studies. Table 2. Differential cellular responses to drugs, hormones, and toxicants at atmospheric O2 versus physioxia. Molecule Mechanism Conditions Outcomes Reference Drugs ROS scavenger, PC-3 and C2C12 cells Differential H2O2 production, proliferation, and resveratrol [107] multiple targets 18% or 5% O2 mitochondrial network dynamics bEnd.3 cells ROS scavenger, Attenuated reoxygenation-induced ROS produc- sulforaphane 18% or 5% O2 [105] multiple targets tion at 18% O2 but not at 5% O2 H/R human neonatal fore- ROS scavenger, GSH depletion and loss of type I quercetin skin fibroblasts [110] multiple targets cells at 18% O2 but not at 4% O2 18% or 4% O2 HCT116, IMR90, U2OS, DNA intercalat- doxorubicin and MCF-7 cells  apoptosis at 18% O2 [49] ing agent 18% O2 or 5% O2 mouse hepatocytes  hepatotoxicity at 18% O2 [36] 18%, 10%, or 5% O2  mROS and RNS production at 18% O2 acetaminophen COX inhibitor  hepatotoxicity at 18% O2 HepG2 cells differential regulation of [111] 18%, 8%, or 3% O2 phase I and II enzymes DNA cross-link- HepG2 cells cyclophosphamide  hepatotoxicity at 18% O2 [111] ing agent 18%, 8%, or 3% O2 pyrimidine syn- SW480 and SW620 cells  proapoptotic effect at 18% O2 teriflunomide thesis [109] 18% or 10% O2  antiproliferative effect at 18% O2 inhibitor DNA synthesis SW480 and SW620 cells oxaliplatin  antiproliferative effect at 18% O2 [109] inhibitor 18% or 10% O2 mouse mammary microtubule sta- paclitaxel tumors  cytotoxicity at 18% O2 [47] bilizer 18% or 3–5% O2 mouse mammary alpelisib PI3K inhibitor tumors  cytotoxicity at 18% O2 [47] 18% or 3–5% O2 Cells 2022, 11, 3123 14 of 21 mouse mammary erlotinib EGFR inhibitor tumors  cytotoxicity at 18% O2 [47] 18% or 3–5% O2 patient-derived  Ki-67-positive cells at 18% O2 V600 BRAF vemurafenib melanoma cells  reduction of VEGF, PCG-1α, [37] inhibitor 18% or 6% O2 and SLC7A11 levels at 18% O2 patient-derived  Ki-67-positive cells at 18% O2 MEK1/2 trametinib melanoma cells  reduction of VEGF, PCG-1α, [37] inhibitor 18% or 6% O2 and SLC7A11 levels at 18% O2 topoisomerase U87MG cells camptothecin  cytotoxicity at 18% O2 [112] inhibitor 18% O2 or 9% O2 RAW 264.7 cells  expression of Nrf2 targets and antioxidant re- dimethyl fumarate Nrf2 inducer [20] 18% O2 or 5% O2 sponse Hs68 and HaCaT keratolytic, Differential regulation of skin barrier glycolic acid cells [113] antioxidant and dermal network-related genes 18% or 2% O2 Hs68 and HaCaT keratolytic, Differential regulation of skin barrier gluconolactone cells [113] antioxidant and dermal network-related genes 18% or 2% O2 keratolytic, Hs68 and HaCaT Differential regulation of skin barrier salicylic acid AMPK cells [113] and dermal network-related genes activator 18% or 2% O2 Hormones C2C12 cells Differential H2O2 production, metabolism, and 17β-estradiol ER antagonist [114] 18% O2 or 5% O2 mitochondrial network dynamics Toxicants RAW 264.7 cells LPS TLR4 agonist  production of inflammatory mediators [20] 18% O2 or 5% O2  cytotoxicity at 18% O2 complex I SH-SY5Y cells No inhibition of ATP synthesis rotenone [115] inhibitor 18% O2 or 5% O2 with 0.2 µM rotenone at 18% O2 (with effects observed at 5%) DNA and differentiated acrolein protein adduct H9c2 cells  cytotoxicity at 18% O2 [116] inducer 18% O2 and 5% O2 DNA adduct in- HepG2 cells aflatoxin B  hepatotoxicity at 18% O2 [111] ducer 18%, 8%, or 3% O2 Other V. baccifera Prooxidant, HepG2 cells  cytotoxicity at 18% O2 [117] leaf extract cytotoxic 18% O2 or 8% O2 Prooxidant, gen- A549 cells  NP-induced oxidative stress at 18% O2 CuO NPs otoxic, [108] 18% O2 or 13% O2  cytotoxicity at 18% O2 cytotoxic V600 Abbreviations: AMPK, AMP-dependent kinase; BRAF , B-raf (mutated); COX, cyclooxygenase; EGFR, epidermal growth factor receptor; ER, estrogen receptor; GSH, reduced glutathione; H/R, hypoxia/reoxygenation; LPS, lipopolysaccharide; MEK1/2, MAPK/ERK kinase 1/2; NPs, nanoparti- cles; PI3K, phosphatidylinositol 3-kinase; ROS, reactive oxygen species; mROS, mitochondrial ROS; RNS, reactive nitrogen species, TLR4, toll-like receptor 4. 10. Conclusions and Future Directions An increasing amount of evidence shows that culturing cells in the hyperoxic envi- ronment of ambient air disturbs their redox homeostasis, alters gene expression, inhibits proliferation, impairs differentiation, impacts energy metabolism, and alters the cellular response to drugs (illustrated in Figure 2). The potential artifacts caused by growing cells at nonphysiological O2 levels underline the need for revisiting previous results from cell- Cells 2022, 11, 3123 15 of 21 culture-based research, particularly the ones intimately related with oxygen and oxidative stress, including, but not limited to, studies using hypoxia and hyperoxia models. Figure 2. Cellular mechanisms differentially regulated at physioxia (2–6% O2) versus normoxia (18% O2). In physioxia (A), activation of HIF may drive the induction of genes that regulate glucose me- tabolism, survival, proliferation, differentiation, and other processes. In many cases, mitochondria have a more elongated morphology and form highly fused networks. In normoxia (B), increased ROS production by NADPH oxidases (NOX) and the electron transport chain (ETC) leads to oxida- tive stress, characterized by DNA and lipid oxidation, and promotes activation of a variety of path- ways such as p53 and Nrf2, which, in turn, results in the induction of antioxidant genes and senes- cence. Further, in many instances, mitochondria show a more globular morphology and increased fission of mitochondrial networks. These cellular mechanisms result in nonphysiological pheno- types and differential responses to bioactive molecules. Created with BioRender.com (accessed on 26 September 2022). Additional research should be conducted to further characterize the mechanisms of altered cellular responses to O2 at 18% O2, compared to at physioxia, with a focus on elu- cidating the molecular basis of the response in this range and its specificity between Cells 2022, 11, 3123 16 of 21 different cell types. In particular, future research on oxidative post-translational modifi- cations through the use of redox proteomics, in conjunction with other “omics” ap- proaches, will be important. In addition, more research should be conducted to delineate the role of HIF at physiological O2 levels. While HIF stabilization/activation reported by some studies could well be the result of an acute reduction in O2 tension, the detection of HIF targets in a handful of cell types adapted to physioxia for at least two weeks [37,50,118] suggests a role for HIF activity at physioxia. In this respect, it is also critical to keep in mind that physioxia is a range rather than a set value, as it varies depending on the tissue. For example, while 3–6% O2 would be appropriate for cells like hepatocytes, cardiomyocytes, neurons, and enterocytes, the same would not be true for alveolar epi- thelial cells, which are exposed to ~13% O2 in vivo. On the other hand, most tumors exhibit median O2 levels below 2% [3]. As such, O2 tensions relevant to the specific tissue being studied should be utilized. Moreover, it is important to consider potential differences in pericellular O2 levels (in the liquid phase) versus the incubator headspace O2 (gaseous phase). Pericellular O2 levels depend on many factors, such as the O2 consumption rate of each cell type, the cell seeding density, the material of the cell culture vessel, and the vol- ume of the media in the vessel, to mention a few [119]. These and other considerations are elegantly discussed in the seminal review by Keeley and Mann [2]. The findings discussed in this review highlight the need to incorporate incubators capable of regulating O2 concentration into our research facilities. While the elevated cost of commercially available O2-regulating incubators (> USD 10,000) may seem like a barrier to implement physioxia in cell culture workflows, our lab has recently developed a port- able, home-made incubator with a total production cost of < USD 1000, which effectively regulates O2, CO2, and temperature [120]. We also demonstrated its ability to maintain both the headspace and pericellular levels at 5% O2 for several weeks. Similarly, Marchus et al. developed an O2-regulating incubator with a cost of ~USD 800 [121], showing that implementing physiological O2 conditions into routine cell culture does not need to come at a great expense. Finally, additional considerations should be made to recreate a more physiologically representative environment in cell culture, such as using physiological culture media (e.g., Plasmax, HPLM), 3D culture, and co-culture (see [1]). Implementing physiological cell culture conditions will improve the quality and validity of studies conducted in vitro. Author Contributions: Conceptualization, R.A.; writing—original draft preparation, R.A.; writ- ing—review and editing, R.A., G.L.G., P.L., and J.A.S. All authors have read and agreed to the pub- lished version of the manuscript. Funding: P.L. and J.A.S. are funded by the Natural Sciences and Engineering Research Council (NSERC) of Canada Discovery Grant (RGPIN 2017-06785 and RGPIN 2020-05274, respectively). R.A. is supported by a Mitacs Globalink Graduate Fellowship. Acknowledgments: We thank Olivia R.M. Bagshaw for her exceptionally helpful comments and suggestions. The graphical abstract was created using BioRender.com, via a subscription by J.A.S. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Abbas, M.; Moradi, F.; Hu, W.; Regudo, K.L.; Osborne, M.; Pettipas, J.; Atallah, D.S.; Hachem, R.; Ott-Peron, N.; Stuart, J.A. Vertebrate cell culture as an experimental approach – limitations and solutions. Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol. 2021, 254, 110570, https://doi.org/10.1016/j.cbpb.2021.110570. 2. Keeley, T.P.; Mann, G.E. Defining Physiological Normoxia for Improved Translation of Cell Physiology to Animal Models and Humans. Physiol. Rev. 2019, 99, 161–234, https://doi.org/10.1152/physrev.00041.2017. 3. McKeown, S.R. Defining normoxia, physoxia and hypoxia in tumours—implications for treatment response. Br. J. Ra- diol. 2014, 87, 20130676, https://doi.org/10.1259/bjr.20130676. 4. Brand, M.D. The sites and topology of mitochondrial superoxide production. Exp. Gerontol. 2010, 45, 466–472, https://doi.org/10.1016/j.exger.2010.01.003. Cells 2022, 11, 3123 17 of 21 5. Alfadda, A.A.; Sallam, R.M. Reactive Oxygen Species in Health and Disease. J. Biomed. Biotechnol. 2012, 2012, 1–14, https://doi.org/10.1155/2012/936486. 6. Zorov, D.B.; Juhaszova, M.; Sollott, S.J. Mitochondrial Reactive Oxygen Species (ROS) and ROS-Induced ROS Release. Physiol. Rev. 2014, 94, 909–950. https://doi.org/10.1152/physrev.00026.2013. 7. Fukai, T.; Ushio-Fukai, M. Superoxide Dismutases: Role in Redox Signaling, Vascular Function, and Diseases. Antioxidants Re- dox Signal. 2011, 15, 1583–1606, https://doi.org/10.1089/ars.2011.3999. 8. Sies, H. Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress. Redox Biol. 2017, 11, 613–619, https://doi.org/10.1016/j.redox.2016.12.035. 9. Kurutas, E.B. The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: Cur- rent state. Nutr. J. 2016, 15, 71, doi:10.1186/s12937-016-0186-5. 10. Pham-Huy, L.A.; He, H.; Pham-Huy, C. Free Radicals, Antioxidants in Disease and Health. Int. J. Biomed. Sci. IJBS 2008, 4, 89. 11. Sies, H.; Belousov, V.V.; Chandel, N.S.; Davies, M.J.; Jones, D.P.; Mann, G.E.; Murphy, M.P.; Yamamoto, M.; Winterbourn, C. Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology. Nat. Rev. Mol. Cell Biol. 2022, 23, 499– 515, https://doi.org/10.1038/s41580-022-00456-z. 12. Hoffman, D.L.; Brookes, P.S. Oxygen Sensitivity of Mitochondrial Reactive Oxygen Species Generation Depends on Metabolic Conditions. J. Biol. Chem. 2009, 284, 16236–16245, https://doi.org/10.1074/jbc.m809512200. 13. Roy, S.; Khanna, S.; Bickerstaff, A.A.; Subramanian, S.V.; Atalay, M.; Bierl, M.; Pendyala, S.; Levy, D.; Sharma, N.; Venojar vi, M.; et al. Oxygen Sensing by Primary Cardiac Fibroblasts. Circ. Res. 2003, 92, 264–271, https://doi.org/10.1161/01.res.0000056770.30922.e6. 14. Nasto, L.A.; Robinson, A.R.; Ngo, K.; Clauson, C.L.; Dong, Q.; Croix, C.S.; Sowa, G.; Pola, E.; Robbins, P.D.; Kang, J.; et a l. Mitochondrial-derived reactive oxygen species (ROS) play a causal role in aging-related intervertebral disc degeneration. J. Orthop. Res. 2013, 31, 1150–1157, https://doi.org/10.1002/jor.22320. 15. El Alami, M.; Viña-Almunia, J.; Gambini, J.; Mas-Bargues, C.; Siow, R.C.; Peñarrocha, M.; Mann, G.E.; Borrás, C.; Viña, J. Acti- vation of p38, p21, and NRF-2 Mediates Decreased Proliferation of Human Dental Pulp Stem Cells Cultured under 21% O2. Stem Cell Rep. 2014, 3, 566–573, https://doi.org/10.1016/j.stemcr.2014.08.002. 16. Nisimoto, Y.; Diebold, B.A.; Cosentino-Gomes, D.; Lambeth, J.D. Nox4: A Hydrogen Peroxide-Generating Oxygen Sensor. Bio- chemistry 2014, 53, 5111–5120, https://doi.org/10.1021/bi500331y. 17. Maddalena, L.A.; Selim, S.M.; Fonseca, J.; Messner, H.; McGowan, S.; Stuart, J.A. Hydrogen peroxide production is affected by oxygen levels in mammalian cell culture. Biochem. Biophys. Res. Commun. 2017, 493, 246–251, https://doi.org/10.1016/j.bbrc.2017.09.037. 18. Stuart, J.A.; Fonseca, J.; Moradi, F.; Cunningham, C.; Seliman, B.; Worsfold, C.R.; Dolan, S.; Abando, J.; Maddalena, L.A. Ho w Supraphysiological Oxygen Levels in Standard Cell Culture Affect Oxygen-Consuming Reactions. Oxidative Med. Cell. Lon- gev. 2018, 2018, 1–13, https://doi.org/10.1155/2018/8238459. 19. Estrada, J.C.; Albo, C.; Benguría, A.; Dopazo, A.; López-Romero, P.; Carrera-Quintanar, L.; Roche, E.; Clemente, E.P.; A Enríquez, J.; Bernad, A.; et al. Culture of human mesenchymal stem cells at low oxygen tension improves growth and genetic stability by activating glycolysis. Cell Death Differ. 2011, 19, 743–755, https://doi.org/10.1038/cdd.2011.172. 20. Haas, B.; Chrusciel, S.; Fayad-Kobeissi, S.; Dubois-Randé, J.-L.; Azuaje, F.; Boczkowski, J.; Motterlini, R.; Foresti, R. Permanent Culture of Macrophages at Physiological Oxygen Attenuates the Antioxidant and Immunomodulatory Properties of Dimethyl Fumarate. J. Cell. Physiol. 2014, 230, 1128–1138, https://doi.org/10.1002/jcp.24844. 21. Zhu, J.; Aja, S.; Kim, E.-K.; Park, M.J.; Ramamurthy, S.; Jia, J.; Hu, X.; Geng, P.; Ronnett, G.V. Physiological oxygen level is critical for modeling neuronal metabolism in vitro. J. Neurosci. Res. 2011, 90, 422–434, https://doi.org/10.1002/jnr.22765. 22. Atkuri, K.R.; Herzenberg, L.A.; Niemi, A.-K.; Cowan, T. Importance of culturing primary lymphocytes at physiological oxygen levels. Proc. Natl. Acad. Sci. 2007, 104, 4547–4552, https://doi.org/10.1073/pnas.0611732104. 23. Ferguson, D.C.; Smerdon, G.R.; Harries, L.W.; Dodd, N.J.; Murphy, M.P.; Curnow, A.; Winyard, P.G. Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia. Free Radic. Biol. Med. 2018, 126, 322–333, https://doi.org/10.1016/j.freeradbiomed.2018.08.025. 24. Timpano, S.; Guild, B.D.; Specker, E.J.; Melanson, G.; Medeiros, P.J.; Sproul, S.L.J.; Uniacke, J. Physioxic human cell culture improves viability, metabolism, and mitochondrial morphology while reducing DNA damage. FASEB J. 2019, 33, 5716–5728, https://doi.org/10.1096/fj.201802279r. 25. Konigsberg, M.; Pérez, V.; Ríos, C.; Liu, Y.; Lee, S.; Shi, Y.; Van Remmen, H. Effect of oxygen tension on bioenergetics and proteostasis in young and old myoblast precursor cells. Redox Biol. 2013, 1, 475–482, https://doi.org/10.1016/j.redox.2013.09.004. 26. Semenza, G.L.; Wang, G.L. A Nuclear Factor Induced by Hypoxia via De Novo Protein Synthesis Binds to the Human Erythro- poietin Gene Enhancer at a Site Required for Transcriptional Activation. Mol. Cell Biol. 1992, 12, 5447–5454. 27. Wang, G.L.; Jiang, B.-H.; Rue, E.A.; Semenza, G.L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer reg- ulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA 1995, 92, 5510–5514, doi:10.1073/pnas.92.12.5510. 28. Lee, P.; Chandel, N.S.; Simon, M.C. Cellular adaptation to hypoxia through hypoxia inducible factors and beyond. Nat. Rev. Mol. Cell Biol. 2020, 21, 268–283, https://doi.org/10.1038/s41580-020-0227-y. 29. Dengler, V.L.; Galbraith, M.; Espinosa, J.M. Transcriptional regulation by hypoxia inducible factors. Crit. Rev. Biochem. Mol. Biol. 2014, 49, 1–15, https://doi.org/10.3109/10409238.2013.838205. Cells 2022, 11, 3123 18 of 21 30. Hu, C.-J.; Wang, L.-Y.; Chodosh, L.A.; Keith, B.; Simon, M.C. Differential Roles of Hypoxia-Inducible Factor 1α (HIF-1α) and HIF-2α in Hypoxic Gene Regulation. Mol. Cell. Biol. 2003, 23, 9361–9374, https://doi.org/10.1128/mcb.23.24.9361-9374.2003. 31. Loboda, A.; Jozkowicz, A.; Dulak, J. HIF-1 and HIF-2 transcription factors — Similar but not identical. Mol. Cells 2010, 29, 435– 442, https://doi.org/10.1007/s10059-010-0067-2. 32. Maxwell, P.H.; Wiesener, M.S.; Chang, G.-W.; Clifford, S.C.; Vaux, E.C.; Cockman, M.E.; Wykoff, C.C.; Pugh, C.W.; Maher, E.R.; Ratcliffe, P.J. The Tumour Suppressor Protein VHL Targets Hypoxia-Inducible Factors for Oxygen-Dependent Pro-teolysis. Nature 1999, 399, 271–275. 33. Metzen, E.; Ratcliffe, P.J.; Ratcliffe, E.M. HIF hydroxylation and cellular oxygen sensing. Biol. Chem. 2004, 385, 223–230, https://doi.org/10.1515/bc.2004.016. 34. Hirsilä, M.; Koivunen, P.; Günzler, V.; Kivirikko, K.I.; Myllyharju, J. Characterization of the Human Prolyl 4-Hydroxylases That Modify the Hypoxia-inducible Factor. J. Biol. Chem. 2003, 278, 30772–30780, https://doi.org/10.1074/jbc.m304982200. 35. Bracken, C.P.; Fedele, A.O.; Linke, S.; Balrak, W.; Lisy, K.; Whitelaw, M.L.; Peet, D.J. Cell-specific Regulation of Hypoxia-induc- ible Factor (HIF)-1α and HIF-2α Stabilization and Transactivation in a Graded Oxygen Environment. J. Biol. Chem. 2006, 281, 22575–22585, https://doi.org/10.1074/jbc.m600288200. 36. Yan, H.-M.; Ramachandran, A.; Bajt, M.L.; Lemasters, J.J.; Jaeschke, H. The Oxygen Tension Modulates Acetaminophen-Induced Mitochondrial Oxidant Stress and Cell Injury in Cultured Hepatocytes. Toxicol. Sci. 2010, 117, 515–523, https://doi.org/10.1093/toxsci/kfq208. 37. Osrodek, M.; Hartman, M.L.; Czyz, M. Physiologically Relevant Oxygen Concentration (6% O2) as an Important Component of the Microenvironment Impacting Melanoma Phenotype and Melanoma Response to Targeted Therapeutics In Vitro. Int. J. Mol. Sci. 2019, 20, 4203, https://doi.org/10.3390/ijms20174203. 38. Stuart, J.A.; Aibueku, O.; Bagshaw, O.; Moradi, F. Hypoxia inducible factors as mediators of reactive oxygen/nitrogen species homeostasis in physiological normoxia. Med Hypotheses 2019, 129, 109249, https://doi.org/10.1016/j.mehy.2019.109249. 39. Suzuki, T.; Yamamoto, M. Molecular basis of the Keap1–Nrf2 system. Free Radic. Biol. Med. 2015, 88, 93–100, https://doi.org/10.1016/j.freeradbiomed.2015.06.006. 40. Yamamoto, M.; Kensler, T.W.; Motohashi, H. The KEAP1-NRF2 System: a Thiol-Based Sensor-Effector Apparatus for Maintain- ing Redox Homeostasis. Physiol. Rev. 2018, 98, 1169–1203, doi:10.1152/physrev.00023.2017. 41. Suzuki, T.; Muramatsu, A.; Saito, R.; Iso, T.; Shibata, T.; Kuwata, K.; Kawaguchi, S.-I.; Iwawaki, T.; Adachi, S.; Suda, H.; et al. Molecular Mechanism of Cellular Oxidative Stress Sensing by Keap1. Cell Rep. 2019, 28, 746–758.e4, https://doi.org/10.1016/j.celrep.2019.06.047. 42. Baird, L.; Yamamoto, M. The molecular mechanisms regulating the KEAP1-NRF2 pathway. Mol. Cell. Biol. 2020, 40, e00099-20, doi:10.1128/mcb.00099-20. 43. Chen, Z.-H.; Saito, Y.; Yoshida, Y.; Sekine, A.; Noguchi, N.; Niki, E. 4-Hydroxynonenal Induces Adaptive Response and En- hances PC12 Cell Tolerance Primarily through Induction of Thioredoxin Reductase 1 via Activation of Nrf2. J. Biol. Chem. 2005, 280, 41921–41927, https://doi.org/10.1074/jbc.m508556200. 44. McMahon, M.; Lamont, D.J.; Beattie, K.A.; Hayes, J.D. Keap1 perceives stress via three sensors for the endogenous signaling molecules nitric oxide, zinc, and alkenals. Proc. Natl. Acad. Sci. USA 2010, 107, 18838–18843, https://doi.org/10.1073/pnas.1007387107. 45. Tonelli, C.; Chio, I.I.C.; Tuveson, D.A. Transcriptional Regulation by Nrf2. Antioxid. Redox Signal. 2018, 29, 1727–1745, doi:10.1089/ars.2017.7342. 46. Alva, R.; Mirza, M.; Baiton, A.; Lazuran, L.; Samokysh, L.; Bobinski, A.; Cowan, C.; Jaimon, A.; Obioru , D.; Al Makhoul, T.; et al. Oxygen toxicity: cellular mechanisms in normobaric hyperoxia. Cell Biol. Toxicol. 2022, 1–33, https://doi.org/10.1007/s10565- 022-09773-7. 47. Kumar, B.; Adebayo, A.K.; Prasad, M.; Capitano, M.L.; Wang, R.; Bhat-Nakshatri, P.; Anjanappa, M.; Simpson, E.; Chen, D.; Liu, Y.; et al. Tumor collection/processing under physioxia uncovers highly relevant signaling networks and drug sensitivity. Sci. Adv. 2022, 8, https://doi.org/10.1126/sciadv.abh3375. 48. Chapple, S.J.; Keeley, T.P.; Mastronicola, D.; Arno, M.; Vizcay-Barrena, G.; Fleck, R.; Siow, R.C.; Mann, G.E. Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxy- gen levels. Free Radic. Biol. Med. 2015, 92, 152–162, https://doi.org/10.1016/j.freeradbiomed.2015.12.013. 49. Carrera, S.; de Verdier, P.J.; Khan, Z.; Zhao, B.; Mahale, A.; Bowman, K.J.; Zainol, M.; Jones, G.D.D.; Lee, S.W.; Aaronson, S.A.; et al. Protection of Cells in Physiological Oxygen Tensions against DNA Damage-induced Apoptosis. J. Biol. Chem. 2010, 285, 13658–13665, https://doi.org/10.1074/jbc.m109.062562. 50. Alva, R.; Moradi, F.; Liang, P.; Stuart, J.A. Culture of Cancer Cells at Physiological Oxygen Levels Affects Gene Expression in a Cell-Type Specific Manner. Preprints (Basel) 2022, 2022080497, doi:10.20944/preprints202208.0497.v1. 51. Duś-Szachniewicz, K.; Gdesz-Birula, K.; Zduniak, K.; Wiśniewski, J. Proteomic-Based Analysis of Hypoxia- and Physioxia-Re- sponsive Proteins and Pathways in Diffuse Large B-Cell Lymphoma. Cells 2021, 10, 2025, https://doi.org/10.3390/cells10082025. 52. Chee, N.T.; Lohse, I.; Brothers, S.P. mRNA-to-protein translation in hypoxia. Mol. Cancer 2019, 18, 1–13, https://doi.org/10.1186/s12943-019-0968-4. 53. Ivanova, I.G.; Park, C.V.; Kenneth, N.S. Translating the Hypoxic Response—The Role of HIF Protein Translation in the Cellular Response to Low Oxygen. Cells 2019, 8, 114, https://doi.org/10.3390/cells8020114. Cells 2022, 11, 3123 19 of 21 54. Uniacke, J.; Holterman, C.E.; Lachance, G.; Franovic, A.; Jacob, M.D.; Fabian, M.R.; Payette, J.; Holcik, M.; Pause, A.; Lee, S. An oxygen-regulated switch in the protein synthesis machinery. Nature 2012, 486, 126–129, doi:10.1038/nature11055. 55. Timpano, S.; Uniacke, J. Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Dis- tinct mRNAs for Translation. J. Biol. Chem. 2016, 291, 10772–10782, https://doi.org/10.1074/jbc.m116.717363. 56. Batie, M.; Rocha, S. Gene transcription and chromatin regulation in hypoxia. Biochem. Soc. Trans. 2020, 48, 1121–1128, https://doi.org/10.1042/bst20191106. 57. Guillaumet-Adkins, A.; Yañez, Y.; Peris-Diaz, M.D.; Calabria, I.; Palanca-Ballester, C.; Sandoval, J. Epigenetics and Oxidative Stress in Aging. Oxidative Med. Cell. Longev. 2017, 2017, 1–8, https://doi.org/10.1155/2017/9175806. 58. Lengner, C.; Gimelbrant, A.A.; Erwin, J.; Cheng, A.; Guenther, M.G.; Welstead, G.G.; Alagappan, R.; Frampton, G.M.; Xu, P.; Muffat, J.; et al. Derivation of Pre-X Inactivation Human Embryonic Stem Cells under Physiological Oxygen Concentra- tions. Cell 2010, 141, 872–883, https://doi.org/10.1016/j.cell.2010.04.010. 59. Xie, P.; Sun, Y.; Ouyang, Q.; Hu, L.; Tan, Y.; Zhou, X.; Xiong, B.; Zhang, Q.; Yuan, D.; Pan, Y.; et al. Physiological Oxygen Prevents Frequent Silencing of the DLK1-DIO3 Cluster during Human Embryonic Stem Cells Culture. Stem Cells 2013, 32, 391– 401, https://doi.org/10.1002/stem.1558. 60. Lees, J.G.; Cliff, T.S.; Gammilonghi, A.; Ryall, J.G.; Dalton, S.; Gardner, D.K.; Harvey, A.J. Oxygen Regulates Human Pluripotent Stem Cell Metabolic Flux.. Stem Cells Int. 2019, 2019, 8195614–17, https://doi.org/10.1155/2019/8195614. 61. Thienpont, B.; Steinbacher, J.; Zhao, H.; D’Anna, F.; Kuchnio, A.; Ploumakis, A.; Hermans, E. Tumour hypoxia causes DNA hypermethylation by reducing TET activity. Nature 2016, 537, 63–68, https://doi.org/10.1038/nature19081. 62. Lin, G.; Sun, W.; Yang, Z.; Guo, J.; Liu, H.; Liang, J. Hypoxia induces the expression of TET enzymes in HepG2 cells. Oncol. Lett. 2017, 14, 6457–6462, https://doi.org/10.3892/ol.2017.7063. 63. Dogan, F.; Aljumaily, R.; Kitchen, M.; Forsyth, N. DNMT3B Is an Oxygen-Sensitive De Novo Methylase in Human Mesenchy- mal Stem Cells. Cells 2021, 10, 1032, https://doi.org/10.3390/cells10051032. 64. Dogan, F.; Aljumaily, R.M.K.; Kitchen, M.; Forsyth, N.R. Physoxia Influences Global and Gene-Specific Methylation in Pluripo- tent Stem Cells. Int. J. Mol. Sci. 2022, 23, 5854, https://doi.org/10.3390/ijms23105854. 65. Johnston, H.; Dickinson, P.; Ivens, A.; Buck, A.H.; Levine, R.D.; Remacle, F.; Campbell, C.J. Intracellular redox potential is cor- related with miRNA expression in MCF7 cells under hypoxic conditions. Proc. Natl. Acad. Sci. 2019, 116, 19753–19759, https://doi.org/10.1073/pnas.1909455116. 66. Shi, R.; Yang, H.; Lin, X.; Cao, Y.; Zhang, C.; Fan, Z.; Hou, B. Analysis of the characteristics and expression profiles of coding and noncoding RNAs of human dental pulp stem cells in hypoxic conditions. Stem Cell Res. Ther. 2019, 10, 89, https://doi.org/10.1186/s13287-019-1192-2. 67. Cooper, P.D.; Burt, A.M.; Wilson, J.N. Critical Effect of Oxygen Tension on Rate of Growth of Animal Cells in Continuous Suspended Culture. Nature 1958, 182, 1508–1509, https://doi.org/10.1038/1821508b0. 68. Packer, L.; Fuehr, K. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature 1977, 267, 423–425, https://doi.org/10.1038/267423a0. 69. Balin, A.K.; Fisher, A.J.; Carter, D.M. Oxygen modulates growth of human cells at physiologic partial pressures.. J. Exp. Med. 1984, 160, 152–166, https://doi.org/10.1084/jem.160.1.152. 70. Falanga, V.; Kirsner, T.S. Low Oxygen Stimulates Proliferation of Fibroblasts Seeded as Single Cells. J. Cell Physiol. 1993, 154, 71. McHugh, D.; Gil, J. Senescence and aging: Causes, consequences, and therapeutic avenues. J. Cell Biol. 2017, 217, 65–77, https://doi.org/10.1083/jcb.201708092. 72. Busuttil, R.A.; Rubio, M.; Dollé, M.E.T.; Campisi, J.; Vijg, J. Oxygen accelerates the accumulation of mutations during the senes- cence and immortalization of murine cells in culture. Aging Cell 2003, 2, 287–294, https://doi.org/10.1046/j.1474- 9728.2003.00066.x. 73. Parrinello, S.; Samper, E.; Krtolica, A.; Goldstein, J.; Melov, S.; Campisi, J. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat. Cell Biol. 2003, 5, 741–747, https://doi.org/10.1038/ncb1024. 74. Lees, S.J.; Childs, T.E.; Booth, F.W. P21 Cip1 Expression Is Increased in Ambient Oxygen, Compared to Estimated Physi-ological (5%) Levels in Rat Muscle Precursor Cell Culture. Cell Prolif. 2008, 41, 193–207, doi:10.1111/j.1365-2184.2008.00512. 75. Bon-Mathier, A.-C.; Rignault-Clerc, S.; Bielmann, C.; Rosenblatt-Velin, N. Oxygen as a key regulator of cardiomyocyte prolifer- ation: New results about cell culture conditions!. Biochim. et Biophys. Acta 2019, 1867, 118460, https://doi.org/10.1016/j.bbamcr.2019.03.007. 76. Braunschweig, L.; Meyer, A.K.; Wagenführ, L.; Storch, A. Oxygen regulates proliferation of neural stem cells through Wnt/β - catenin signalling. Mol. Cell. Neurosci. 2015, 67, 84–92, https://doi.org/10.1016/j.mcn.2015.06.006. 77. Covello, K.L.; Kehler, J.; Yu, H.; Gordan, J.D.; Arsham, A.M.; Hu, C.-J.; Labosky, P.A.; Simon, M.C.; Keith, B. HIF-2α regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth. Genes Dev. 2006, 20, 557–570, https://doi.org/10.1101/gad.1399906. 78. Westfall, S.D.; Sachdev, S.; Das, P.; Hearne, L.B.; Hannink, M.; Roberts, R.M.; Ezashi, T. Identification of Oxygen -Sensitive Tran- scriptional Programs in Human Embryonic Stem Cells. Stem Cells Dev. 2008, 17, 869–882, https://doi.org/10.1089/scd.2007.0240. 79. Keith, B.; Simon, M.C. Hypoxia-Inducible Factors, Stem Cells, and Cancer. Cell 2007, 129, 465–472, https://doi.org/10.1016/j.cell.2007.04.019. Cells 2022, 11, 3123 20 of 21 80. Broxmeyer, H.E.; Cooper, S.; Gabigbad, T. The Effects of Oxidizing Species Derived from Molecular Oxygen on the Pro-lifera- tion In Vitro of Human Granulocyte-Macrophage Progenitor Cells. Ann. NY Acad. Sci. 1989, 554, 177–184. 81. Broxmeyer, H.E.; Cooper, S.; Lu, L.; Miller, M.E.; Langefeld, C.D.; Ralph, P. Enhanced Stimulation of Human Bone Marrow Macrophage Colony Formation In Vitro by Recombinant Human Macrophage Colony-Stimulating Factor in Agarose Medium and at Low Oxygen Tension. Blood 1990, 76, 323–329. 82. Fehrer, C.; Brunauer, R.; Laschober, G.; Unterluggauer, H.; Reitinger, S.; Kloss, F.; Gülly, C.; Gaßner, R.; Lepperdinger, G. Re- duced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan. Aging Cell 2007, 6, 745–757, https://doi.org/10.1111/j.1474-9726.2007.00336.x. 83. D'Ippolito, G.; Diabira, S.; Howard, G.A.; Roos, B.A.; Schiller, P.C. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells. Bone 2006, 39, 513–522, https://doi.org/10.1016/j.bone.2006.02.061. 84. Mizuno, S.; Glowacki, J. Low Oxygen Tension Enhances Chondroinduction by Demineralized Bone Matrix in Human Dermal Fibroblasts in vitro. Cells Tissues Organs 2005, 180, 151–158, https://doi.org/10.1159/000088243. 85. Betre, H.; Ong, S.R.; Guilak, F.; Chilkoti, A.; Fermor, B.; Setton, L.A. Chondrocytic differentiation of human adipose-derived adult stem cells in elastin-like polypeptide. Biomaterials 2006, 27, 91–99, https://doi.org/10.1016/j.biomaterials.2005.05.071. 86. Khan, W.S.; Adesida, A.B.; E Hardingham, T. Hypoxic conditions increase hypoxia-inducible transcription factor 2α and en- hance chondrogenesis in stem cells from the infrapatellar fat pad of osteoarthritis patients. Arthritis Res. Ther. 2007, 9, R55–9, https://doi.org/10.1186/ar2211. 87. Dennis, J.E.; Whitney, G.A.; Rai, J.; Fernandes, R.J.; Kean, T.J. Physioxia Stimulates Extracellular Matrix Deposition and Increases Mechanical Properties of Human Chondrocyte-Derived Tissue-Engineered Cartilage. Front. Bioeng. Biotechnol. 2020, 8, https://doi.org/10.3389/fbioe.2020.590743. 88. Pattappa, G.; Johnstone, B.; Zellner, J.; Docheva, D.; Angele, P. The Importance of Physioxia in Mesenchymal Stem Cell Chon- drogenesis and the Mechanisms Controlling Its Response. Int. J. Mol. Sci. 2019, 20, 484, https://doi.org/10.3390/ijms20030484. 89. Studer, L.; Csete, M.; Lee, S.-H.; Kabbani, N.; Walikonis, J.; Wold, B.; McKay, R. Enhanced Proliferation, Survival, and Dopa- minergic Differentiation of CNS Precursors in Lowered Oxygen. J. Neurosci. 2000, 20, 7377–7383, https://doi.org/10.1523/jneuro- sci.20-19-07377.2000. 90. Antebi, B.; Ii, L.A.R.; Walker, K.P.; Asher, A.M.; Kamucheka, R.M.; Alvarado, L.; Mohammadipoor, A.; Cancio, L.C. Short-term physiological hypoxia potentiates the therapeutic function of mesenchymal stem cells. Stem Cell Res. Ther. 2018, 9, 1–15, https://doi.org/10.1186/s13287-018-1007-x. 91. Chen, C.; Tang, Q.; Zhang, Y.; Yu, M.; Jing, W.; Tian, W. Physioxia: a more effective approach for culturing human adipose - derived stem cells for cell transplantation.. Stem Cell Res. Ther. 2018, 9, 148, https://doi.org/10.1186/s13287-018-0891-4. 92. Mas-Bargues, C.; Sanz-Ros, J.; Román-Domínguez, A.; Inglés, M.; Gimeno-Mallench, L.; El Alami, M.; Viña-Almunia, J.; Gam- bini, J.; Viña, J.; Borrás, C. Relevance of Oxygen Concentration in Stem Cell Culture for Regenerative Medicine. Int. J. Mol. Sci. 2019, 20, 1195, https://doi.org/10.3390/ijms20051195. 93. Grodzki, A.C.G.; Giulivi, C.; Lein, P.J. Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line. PLoS ONE 2013, 8, e54926, https://doi.org/10.1371/journal.pone.0054926. 94. Patel, S.P.; Gonzalez, B.C.; Paone, N.; Mueller, C.; Floss, J.C.; Sousa, M.E.; Shi, M.Y. Effect of Physiological Oxygen on Pr imary Human Corneal Endothelial Cell Cultures. Transl. Vis. Sci. Technol. 2022, 11, 33–33, https://doi.org/10.1167/tvst.11.2.33. 95. Alva, R.; Abbas, M.; Bagshaw, O.R.; Moffatt, C.; Gardner, G.; Stuart, J.A. Mitochondrial Oxygen Toxicity. In Mitochondrial Intoxication; de Oliveira, M.R., Ed. Academic Press, Amsterdam, Netherlands, in press ISBN 9780323884624. 96. Jaber, S.M.; Bordt, E.A.; Bhatt, N.M.; Lewis, D.M.; Gerecht, S.; Fiskum, G.; Polster, B.M. Sex differences in the mitochondri al bioenergetics of astrocytes but not microglia at a physiologically relevant brain oxygen tension. Neurochem. Int. 2018, 117, 82– 90, https://doi.org/10.1016/j.neuint.2017.09.003. 97. Moradi, F.; Moffatt, C.; Stuart, J. The Effect of Oxygen and Micronutrient Composition of Cell Growth Media on Cancer Cell Bioenergetics and Mitochondrial Networks. Biomolecules 2021, 11, 1177, https://doi.org/10.3390/biom11081177. 98. Valente, A.J.; Maddalena, L.A.; Robb, E.L.; Moradi, F.; Stuart, J.A. A simple ImageJ macro tool for analyzing mitochondrial network morphology in mammalian cell culture. Acta Histochem. 2017, 119, 315–326, https://doi.org/10.1016/j.acthis.2017.03.001. 99. Tiede, L.M.; A Cook, E.; Morsey, B.; Fox, H.S. Oxygen matters: tissue culture oxygen levels affect mitochondrial function and structure as well as responses to HIV viroproteins. Cell Death Dis. 2011, 2, e246–e246, https://doi.org/10.1038/cddis.2011.128. 100. Zhou, L.; Dosanjh, A.; Chen, H.; Karasek, M. Divergent Effects of Extracellular Oxygen on the Growth, Morphology, and Func- tion of Human Skin Microvascular Endothelial Cells. J. Cell Physiol. 2000, 182, 134–140. 101. Piossek, F.; Beneke, S.; Schlichenmaier, N.; Mucic, G.; Drewitz, S.; Dietrich, D.R. Physiological oxygen and co-culture with hu- man fibroblasts facilitate in vivo-like properties in human renal proximal tubular epithelial cells. Chem. Interactions 2022, 361, https://doi.org/10.1016/j.cbi.2022.109959. 102. Guo, R.; Xu, X.; Lu, Y.; Xie, X. Physiological oxygen tension reduces hepatocyte dedifferentiation in in vitro culture. Sci. Rep. 2017, 7, 5923, https://doi.org/10.1038/s41598-017-06433-3. 2+ 103. Keeley, T.P.; Siow, R.C.M.; Jacob, R.; Mann, G.E. A PP2A-mediated feedback mechanism controls Ca -dependent NO synthe- sis under physiological oxygen. FASEB J. 2017, 31, 5172–5183, https://doi.org/10.1096/fj.201700211r. 2+ 104. Keeley, T.P.; Siow, R.C.M.; Jacob, R.; Mann, G.E. Reduced SERCA activity underlies dysregulation of Ca homeostasis under atmospheric O2levels. FASEB J. 2017, 32, 2531–2538, https://doi.org/10.1096/fj.201700685rrr. Cells 2022, 11, 3123 21 of 21 105. Warpsinski, G.; Smith, M.J.; Srivastava, S.; Keeley, T.P.; Siow, R.C.; Fraser, P.A.; Mann, G.E. Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation. Redox Biol. 2020, 37, 101708, https://doi.org/10.1016/j.redox.2020.101708. 106. Danilov, C.A.; Fiskum, G. Hyperoxia promotes astrocyte cell death after oxygen and glucose deprivation. Glia 2008, 56, 801– 808, https://doi.org/10.1002/glia.20655. 107. Fonseca, J.; Moradi, F.; Valente, A.J.F.; Stuart, J.A. Oxygen and Glucose Levels in Cell Culture Media Determine Resveratrol’s Effects on Growth, Hydrogen Peroxide Production, and Mitochondrial Dynamics. Antioxidants 2018, 7, 157, https://doi.org/10.3390/antiox7110157. 108. Kumar, A.; Dailey, L.A.; Swedrowska, M.; Siow, R.; Mann, G.E.; Vizcay-Barrena, G.; Arno, M.; Mudway, I.S.; Forbes, B. Quan- tifying the magnitude of the oxygen artefact inherent in culturing airway cells under atmospheric oxygen versus physiological levels. FEBS Lett. 2015, 590, 258–269, https://doi.org/10.1002/1873-3468.12026. 109. Otto-Ślusarczyk, D.; Graboń, W.; Mielczarek-Puta, M.; Chrzanowska, A. Teriflunomide – The common drug with underesti- mated oxygen - Dependent anticancer potential. Biochem. Biophys. Rep. 2021, 28, 101141, https://doi.org/10.1016/j.bbrep.2021.101141. 110. Alaluf, S.; Muir-Howie, H.; Hu, H.-L.; Evans, A.; Green, M.R.; Alaluf, S.; Muir-Howie, H.; Hu, H.-H.; Evans, A.; Green, M.R. Atmospheric Oxygen Accelerates the Induction of a Post-Mitotic Phenotype in Human Dermal Fibroblasts: The Key Pro-tective Role of Glutathione. Differentiation 2000, 66, 147–155. 111. DiProspero, T.J.; Dalrymple, E.; Lockett, M.R. Physiologically relevant oxygen tensions differentially regulate hepatotoxic r e- sponses in HepG2 cells. Toxicol. Vitr. 2021, 74, 105156, https://doi.org/10.1016/j.tiv.2021.105156. 112. Albert, I.; Hefti, M.; Luginbuehl, V. Physiological oxygen concentration alters glioma cell malignancy and responsiveness to photodynamic therapyin vitro. Neurol. Res. 2014, 36, 1001–1010, https://doi.org/10.1179/1743132814y.0000000401. 113. Lee, K.-H.; Kim, D.-Y.; Kim, W. Cultivation of human skin cells under physiological oxygen concentration modulates expression of skin significant genes and response to hydroxy acids. Biochem. Biophys. Res. Commun. 2021, 551, 161–167, https://doi.org/10.1016/j.bbrc.2021.02.113. 114. Moradi, F.; Fiocchetti, M.; Marino, M.; Moffatt, C.; Stuart, J.A. Media composition and O2 levels determine effects of 17β -Estra- diol on mitochondrial bioenergetics and cellular reactive oxygen species. Am. J. Physiol. Physiol. 2021, https://doi.org/10.1152/aj- pcell.00080.2021. 115. Villeneuve, L.; Tiede, L.M.; Morsey, B.; Fox, H.S. Quantitative Proteomics Reveals Oxygen-Dependent Changes in Neuronal Mitochondria Affecting Function and Sensitivity to Rotenone. J. Proteome Res. 2013, 12, 4599–4606, https://doi.org/10.1021/pr400758d. 116. Coyle, J.P.; Rinaldi, R.J.; Johnson, G.T.; Bourgeois, M.M.; McCluskey, J.D.; Harbison, R.D. Reduced oxygen tension culturing conditionally alters toxicogenic response of differentiated H9c2 cardiomyoblasts to acrolein. Toxicol. Mech. Methods 2018, 28, 488–498, https://doi.org/10.1080/15376516.2018.1455785. 117. Trepiana, J.; Meijide, S.; Navarro, R.; Hernández, M.L.; Ruiz-Sanz, J.I.; Ruiz-Larrea, M.B. Influence of oxygen partial pressure on the characteristics of human hepatocarcinoma cells. Redox Biol. 2017, 12, 103–113, https://doi.org/10.1016/j.redox.2017.02.004. 118. Spyrou, J.; Gardner, D.K.; Harvey, A.J. Metabolomic and Transcriptional Analyses Reveal Atmospheric Oxygen During Human Induced Pluripotent Stem Cell Generation Impairs Metabolic Reprogramming. Stem Cells 2019, 37, 1042–1056, https://doi.org/10.1002/stem.3029. 119. Al-Ani, A.; Toms, D.; Kondro, D.; Thundathil, J.; Yu, Y.; Ungrin, M. Oxygenation in cell culture: Critical parameters for repro- ducibility are routinely not reported. PLoS ONE 2018, 13, e0204269, https://doi.org/10.1371/journal.pone.0204269. 120. Samokhin, P.; Gardner, G.L.; Moffatt, C.; Stuart, J.A. An Inexpensive Incubator for Mammalian Cell Culture Capable of Regu- lating O2, CO2, and Temperature. Oxygen 2022, 2, 22–30, https://doi.org/10.3390/oxygen2010003. 121. Marchus, C.R.N.; Knudson, J.A.; Morrison, A.E.; Strawn, I.K.; Hartman, A.J.; Shrestha, D.; Pancheri, N.M.; Glasgow, I.; Schie le, N.R. Low-Cost, Open-Source Cell Culture Chamber for Regulating Physiologic Oxygen Levels Specifications Table. HardwareX 2022, 11, e00253, doi:10.17605/OSF.IO/JVHDQ.

Journal

Cells – Multidisciplinary Digital Publishing Institute

Published: Oct 4, 2022

Keywords: oxygen; physioxia; hyperoxia; ROS; oxidative stress; gene expression; senescence; metabolism; mitochondrial dynamics; drug response

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Supraphysiological Oxygen Levels in Mammalian Cell Culture: Current State and Future Perspectives

Supraphysiological Oxygen Levels in Mammalian Cell Culture: Current State and Future Perspectives

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Supraphysiological Oxygen Levels in Mammalian Cell Culture: Current State and Future Perspectives

Supraphysiological Oxygen Levels in Mammalian Cell Culture: Current State and Future Perspectives

References (122)

Abstract

Journal

Recommended Articles

There are no references for this article.

Our policy towards the use of cookies