Limitations to oxygen diffusion and equilibration in in vitro cell exposure systems in hyperoxia and hypoxia

Allen, Corrie B.; Schneider, Barbara; White, Carl W.

doi:10.1152/ajplung.2001.281.4.l1021

Cited by 126 publications

(111 citation statements)

References 16 publications

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“…An important difference between the in vitro and in vivo studies is the use of 95% O 2 in contrast to 75% O 2 . This oxygen level was selected for these experiments to address concerns of the relative insolubility of oxygen in the culture medium (34). BHT-QM has been demonstrated to be directly pneumotoxic in BALB/C mouse lungs (35).…”

Section: Discussionmentioning

confidence: 99%

β-Catenin in the Fibroproliferative Response to Acute Lung Injury

Douglas

Valle

Winn

et al. 2006

Am J Respir Cell Mol Biol

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Resolution of alveolar epithelial/capillary membrane damage after acute lung injury requires coordinated and effective tissue repair to reestablish a functional alveolar epithelial/capillary membrane barrier. We hypothesized that signaling pathways important in lung alveolar bud ontogeny are activated in the recovery and remodeling phases after profound oxidant stress lung injury in a murine model. To test this, we characterized the expression of noncanonical ␤-catenin pathway proteins E-cadherin, integrin-linked kinase-1, and ␤-catenin in mice undergoing normoxic recovery after exposure to butylated hydroxytoluene (BHT, ionol) and concomitant sublethal (75% O 2 ) hyperoxia. Mice developed early acute lung injury with subsequent inflammation, collagen deposition, interstitial cellular proliferation, and lung architectural distortion. Reduced E-cadherin expression after 6 d of BHT and hyperoxia was accompanied by enhanced expression and nuclear localization of ␤-catenin and increased integrin-linked kinase-1 expression during subsequent normoxic recovery. This resulted in increased expression of the cotranscriptional regulators TCF-1 and -3 and cyclin D1. Proliferation of murine lung epithelial-12 cells in vitro after 8 h of treatment with BHT quinone-methide and hyperoxia and 48 h of normoxic recovery was enhanced 2.7-fold compared with vehicle-treated control mice at the same time point. BHT/hyperoxia-exposed mice treated with the pan-caspase inhibitor z-ASP had increased acute lung injury and reduced survival despite the presence of TUNEL-positive cells, suggesting enhanced lung cell necrosis. ␤-Catenin expression was reduced in z-ASP-co-treated lungs after BHT/hyperoxia. The noncanonical cadherin-␤-catenin axis is associated with fibroproliferative repair after BHT/hyperoxia exposure and may regulate epithelial proliferation and lung matrix remodeling and repair in response to lung injury. Keywords: ␤-catenin; E-cadherin; lung injury repair; cell junctions; caspase Resolution of alveolar epithelial/capillary membrane damage after acute lung injury (ALI) requires coordinated and effective tissue reconstruction to reestablish a functional barrier. In particular, restoration of the normal air space architecture requires reconstitution of denuded type I alveolar epithelial cells that undergo apoptotic and necrotic death during the exudative phase of ALI. Coordination of extracellular matrix turnover and regeneration of type II epithelial cells is important in reestablishing surfactant production and ion transport (1). Orderly re-epithelialization suppresses fibroblast proliferation and matrix deposition after ALI (1, 2). However, resolution of ALI can result in disordered repair of the alveolus, characterized by impaired fibrinolysis of the in-

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Section: Discussionmentioning

confidence: 99%

β-Catenin in the Fibroproliferative Response to Acute Lung Injury

Douglas

Valle

Winn

et al. 2006

Am J Respir Cell Mol Biol

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“…Researchers do not accept such an inaccurate value for other parameters such as pH, glucose etc., yet, surprisingly, they ignore the importance of controlling for oxygen, which has been known for many years to be toxic. 34 It should be noted that equilibration of culture media to the oxygen level in a specific gassing mixture can take up to 3 h; 35 this can be avoided if the gas mixture is in direct contact with the cell monolayer, which can be achieved if cells are grown in oxygen-permeable culture flasks (www.coylab.com). Since % oxygen is more physiologically meaningful than mmHg, it is proposed that % oxygen is a better unit for reporting oxygen levels as it more adequately illustrates the relatively low, but clearly normal, oxygen levels in many tissues; it also better highlights the particularly low levels of oxygen found in tumours.…”

Section: Normoxia and Physoxiamentioning

confidence: 99%

Defining normoxia, physoxia and hypoxia in tumours—implications for treatment response

McKeown

2014

BJR

803

712

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Tumour hypoxia is increasingly recognized as a major deleterious factor in cancer therapies, as it compromises treatment and drives malignant progression. This review seeks to clarify the oxygen levels that are pertinent to this issue. It is argued that normoxia (20% oxygen) is an extremely poor comparator for "physoxia", i.e. the much lower levels of oxygen universally found in normal tissues, which averages about 5% oxygen, and ranges from about 3% to 7.4%. Importantly, it should be recognized that the median oxygenation in untreated tumours is significantly much lower, falling between approximately 0.3% and 4.2% oxygen, with most tumours exhibiting median oxygen levels ,2%. This is partially dependent on the tissue of origin, and it is notable that many prostate and pancreatic tumours are profoundly hypoxic. In addition, therapy can induce even further, often unrecognized, changes in tumour oxygenation that may vary longitudinally, increasing or decreasing during treatment in ways that are not always predictable. Studies that fail to take cognizance of the actual physiological levels of oxygen in tissues (approximately 5%) and tumours (approximately 1%) may fail to identify the real circumstances driving tumour response to treatment and/or malignant progression. This can be of particular importance in genetic studies in vitro when comparison to human tumours is required.

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“…However, the time needed to reach equilibrium of O 2 partial pressure between the chamber atmosphere and the culture medium could take several hours (Allen et al, 2001). Another challenge of using a hypoxic chamber is that O 2 diffusion from the air to the cell culture media occurs rapidly once the culture plates are removed from the hypoxic chamber.…”

Section: Introductionmentioning

confidence: 99%

Enzyme-immobilized hydrogels to create hypoxia for in vitro cancer cell culture

Dawes

König

Lin

2017

Journal of Biotechnology

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Hypoxia is a critical condition governing many aspects of cellular fate processes. The most common practice in hypoxic cell culture is to maintain cells in an incubator with controlled gas inlet (i.e., hypoxic chamber). Here, we describe the design and characterization of enzymeimmobilized hydrogels to create solution hypoxia under ambient conditions for in vitro cancer cell culture. Specifically, glucose oxidase (GOX) was acrylated and co-polymerized with poly(ethylene glycol)-diacrylate (PEGDA) through photopolymerization to form GOXimmobilized PEG-based hydrogels. We first evaluated the effect of soluble GOX on inducing solution hypoxia (O 2 < 5%) and found that both unmodified and acrylated GOX could sustain hypoxia for at least 24 hours even under ambient air condition with constant oxygen diffusion from the air-liquid interface. However, soluble GOX gradually lost its ability to sustain hypoxia after 24 hours due to the loss of enzyme activity over time. On the other hand, GOXimmobilized hydrogels were able to create hypoxia within the hydrogel for at least 120 hours, potentially due to enhanced protein stabilization by enzyme 'PEGylation' and immobilization. As a proof-of-concept, this GOX-immobilized hydrogel system was used to create hypoxia for in vitro culture of Molm14 (acute myeloid leukemia (AML) cell line) and Huh7 (hepatocellular carcinoma (HCC) cell line). Cells cultured in the presence of GOX-immobilized hydrogels remained viable for at least 24 hours. The expression of hypoxia associated genes, including carbonic anhydrase 9 (CA9) and lysyl oxidase (LOX), were significantly upregulated in cells cultured with GOX-immobilized hydrogels. These results have demonstrated the potential of using enzyme-immobilized hydrogels to create hypoxic environment for in vitro cancer cell culture.

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Limitations to oxygen diffusion and equilibration in in vitro cell exposure systems in hyperoxia and hypoxia

Cited by 126 publications

References 16 publications

β-Catenin in the Fibroproliferative Response to Acute Lung Injury

β-Catenin in the Fibroproliferative Response to Acute Lung Injury

Defining normoxia, physoxia and hypoxia in tumours—implications for treatment response

Enzyme-immobilized hydrogels to create hypoxia for in vitro cancer cell culture

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