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Question ID: WS-105
Submitted by: Kenna Shaw
July 6, 2011

Cell culture, the propagation of cells in artificial environment conducive to growth, has become one of the major tools in life sciences. Human cells grown in laboratory, subjected to various (single or multiple) perturbations, are serving well in elucidating physico-biochemical mechanisms of response in investigations of physiology and biochemistry. In addition, cell strains and cell lines have extensively been used to identify molecular markers of disease, and recent advances in cell culture have facilitated propagating stem cells for clinical and research purposes. In the laboratory, the cells are usually cultured in a medium containing serum that provides required nutrients for cell growth, 5% CO2 to maintain pH and a temperature of 37oC to mimic the physiological conditions of cells in vivo. However, the cells in culture are mostly subjected to 95% air, i.e., to a high concentration of oxygen (~20%), and high pressure (~152 mmHg). Oxygen status of different regions of an organ is different, and the levels and the requirement of oxygen (~1to10%; physioxia), and pressure (~8-100 mmHg) in each organ are different in the human body. Therefore, in terms of oxygen level, the culture conditions do not mimic the oxygen pressure in vivo. Cells cannot survive without oxygen because of its essential role in generation of ATP during oxidative phosphorylation, but high levels of oxygen (~20%, hyperoxia) are toxic through the production of reactive oxygen species (ROS) and peroxides that damage macromolecules, including proteins, lipids and DNA, and through depletion of active thiols. Reactive oxygen species attack guanine bases in DNA and form 8-hydroxydeoxyguanosine, which can cause transverse mutations. Wide regional variations in tissue oxygenation, low cellular oxygenation (~0.2%, hypoxia) and lack of oxygen (≤0.1%, anoxia) due to abnormal microcirculation in microenvironment are frequently observed in tumors. Although the cells under hypoxic and reoxygenation conditions remain replication competent, the intermittent and continuous oxygen deprivation was shown to deferentially affect cellular processes. Hypoxia and anoxia reduce transcriptional activity, transcription initiation and transcript elongation, while increasing the RNA stability to compensate. Hypoxia induces hypoxia inducible factors (HIF-1α and HIF-2α), and the HIF-1α was identified to play crucial role in carcinogenesis and tumor progression. In addition, hypoxia differentially affects methylation status of several genes, thereby altering the expression of both coding and non-coding RNAs. Thus, the oxygen concentration in the cellular environment could modulate highly dynamic and interconnected but finely regulated biomolecular networks, which drive biological processes, from the gene level to the proteome expression. Most of the tumors are hypoxic, but, currently, the cell lines are cultured in hyperoxic conditions and are used in downstream analysis (in transcriptomics, proteomics, in understanding changes in signal transduction pathways, and in identifying biomarkers). In fact, I feel, the high attrition rate of several drugs during drug development could be due to our current PD/PK assays, and pre-clinical Tox assays involving hyperoxic cultures. Drug absorption, availability, distribution, oxidation, metabolism and elimination are different in hypoxic and hyperoxic environments. Handling of biospecimens in hypoxic conditions, pre-culture processing of tissues in media at atmospheric oxygen levels (hyperoxia), and culturing cells originated from physioxic and hypoxic tissues in a hyperoxic environment may have contributed to generation of data that are spurious, and mislead our understandings of molecular networks, signal transduction pathways and pathophysiology. Most of the studies that identified biomarkers utilized cell lines cultured at hyperoxic conditions, even though the cells in tissues never reach to that level of oxygen. Current culture practices may have permanently altered the molecular profiles in cultured cells and therefore may not represent in vivo conditions. Unfortunately, currently, stem cells for research and regenerative medicine are cultured, and in vitro fertilization is being done at atmospheric oxygen. We may know the consequences from these manipulations, particularly with iPS cells, 15-20 years later. In fact, there are reports that do suggest higher health problems, including retinoblastoma, diabetes and developmental disorders in IVF kids. Therefore, there is a need for new cell strains and cell lines (co-cultures and 3D cultures) that survive and grow in physioxia and hypoxia and are optimized throughout the process (from the time the biospecimen is collected from patient to propagation and analysis) to represent physiological conditions and potentially maintain in vivo genotype and phenotype. A simple titration of oxygen levels in existing cell strain or cell line culture may not serve the purpose, since the oxygen effects are differential and in some cases irreversible. The concept behind this question is whether cell lines and other models, if treated at closer-to physiologic conditions, would be better models/and more apt for discovery and study of cancer biomarkers vs. whether what we study are mostly artifacts- consequences of the culture environment. Development of better tools for discovery using resources that better represent physiology and how to approach the development and testing of these tools is the core of this concept. -From Rao Divi/Kenna Shaw

Average Score: 5.0 (3 evaluations) Provocativeness - 4.5 Novelty - 4.5 Public Health Significance - 5.0 Feasibility - 4.5

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