Since early 2000 some researchers have pointed out that the amount of Oxygen available for cultured stem cells could influence the cell differentiation process (1-3). The immediate interpretation in general publications was that oxygen could be "toxic" for stem cells (4). Time passed and this is not as dramatic as implied; however, it is important to understand that the majority of cultured cells are maintained in flasks, covered by a shallow layer of water based media, in contact with the regular atmosphere, which contains about 20% of oxygen. In these conditions the medium dissolves oxygen until saturation, but replaces it as fast as cells use it. Cells have, therefore, oxygen ad libitum, and probably are adapted to this environment in years of passages, showing a regular and well known behavior that, certainly, is not the same as in vivo, and probably is not the best in vitro.

Cell-based protein synthesis systems are largely used in industry, and cells grown in culture constitute one of the most powerful instruments in biology. Cell cultures in artificial environments need the supply of all elements required for normal cell anabolism and metabolism including oxygen. Aerobic cells, like mammalian cells, have relative high oxygen consumption during proliferative phases; therefore, a sufficient oxygen supply constitutes one of the main concerns in cell culture technology. However, the actual limits of this "sufficient" amount of oxygen is not well defined; therefore, the general option is to provide it in excess, compared with the oxygen partial pressures existing in living tissues. This option may help the achievement of certain goals, but would also introduce certain unwanted collateral effects.

Normal human and other mammalian cells maintained in culture environments show limited proliferative capacity. The degree to which the artificial culture environment influences a proliferative life span is not completely understood. It is well known that standard procedures used to establish and maintain cultures significantly affect the proliferative life spans of different cell types. For example, the proliferative life span of human fibroblasts, established either from fetal or adult skin and lung, is significantly affected by both the procedures used to establish cell cultures and the culture conditions. In this context it is notorious that fibroblasts proliferative life span is inversely related to environmental oxygen tension (5).
The low solubility of oxygen in aqueous solutions and the limited diffusion of oxygen through a boundary layer of gas above the medium (gas phase) are major barriers to rapid oxygen transport into the culture medium. Experiments conducted with classic polystyrene tissue culture flasks have shown that, although only a few minutes are required to reach equilibrium between the oxygen concentration in the environmental chamber and that of the flushing gas, about 3 hours are required to saturate the medium dissolved oxygen with the environmental oxygen (6). This delay suggests that cells in culture could have deficient oxygen supply. And because oxygen is required for fundamental metabolic pathways such as the aerobic production of ATP, cell culture devices are designed to provide abundant oxygen through different strategies. However, even in regular flasks, the levels of oxygen are much higher than in the natural environment of tissues. That is especially true in anchorage dependent cell cultures, where the ratio of medium to cells is enormous.
Although oxygen is vital for normal aerobic respiration and many metabolic pathways, classic TEM observations showed that some cell types, exposed to excessive oxygen, became enlarged and showed vacuolization, increased lysosomes, and reductions in both polysomes and endoplasmic reticulum. However, mitochondria did not show alterations (7). Molecular analyses demonstrate that these morphology alterations are just the expression of more profound cellular changes. Even moderate excess of oxygen can alter fundamental cell functions (8), cause cellular edema (9), decrease fluidity in the hydrophobic core of the plasma membranes, and decrease transmembrane transport of amines (10).

Importantly, O2 excess can induce DNA damage, increase the rate of telomere shortening over 500 bp per population doubling (90 bp per population doubling under appropriate oxygen concentration) (11), generate chromosomal instability, and produce chromatid and chromosome gaps and breaks (12, 13). Moreover, oxygen surplus can inhibit proliferation in G1, S and G2 phases of the cell cycle (11, 14, 15), and inhibit the expression of protein p21WAF/CIP1, an important regulator of cell cycle, which inhibits cell cycle progression at the G1/S interface, resulting in cell growth arrest (14).

It has been observed that too much oxygen in culture upregulates mRNA expression of several key molecules, such as the insulin-like growth factor (IGF) system, including IGF-binding protein 2 (IGFBP-2), type 2 IGF-receptor and IGF-II (15), and transforming growth factor-beta 1 (TGF-beta 1) (15, 16).

Moreover, temporary hyperoxia can induce apoptosis and necrosis in certain tumor and normal cells (17, 18). For example it has been shown that Mv1Lu pulmonary adenocarcinoma cells in the presence of an excess of oxygen inhibit their proliferation in S and G2 phase, reduce clonogenic survival, and increase intracellular redox (19). This effect on DNA integrity can be severely influenced by cell attachment to biological substrates but not to artificial substrates such as plastic or poly-D-lysine. For example, when cultured on Matrigel, the protective effects of substrate attachment can reduce the levels of alveolar epithelial cells apoptosis induced by excessive environmental oxygen.
Also, compared with cultures on plastic, alveolar epithelial cells cultured on laminin under oxygen excess, increase Bcl-2 to Interleukin-1beta-converting enzyme ratio of expression, show reduced DNA damage, and increased activation of extracellular signal-regulated kinase (ERK). These cells are also capable of restoring depleted glutathione levels, and increasing mitochondria viability (20).

Cells may also respond to excessive oxygen, by upregulating the expression of antioxidants such as thioredoxin (TRX) and thioredoxin reductase (TR), potent and important in antioxidant defense of cell growth and signal transduction processes (21). Likewise, upregulating the expression of Cu,Zn-superoxide dismutase, key enzyme in the cellular defense against free radical oxidation (12, 22). In other cell lines with low antioxidant enzyme levels, transient hyperoxia induces either great increases in glutathione (GSH) concentration (5), or decreases GSH levels by massive degradation. This increased degradation of GSH is a result of increased expression of gamma-glutamyltransferase (gamma-GT), an important enzyme for the uptake of precursor molecules for intracellular synthesis of GSH (23).

Using uptake and secretion evaluation procedures, it has also been shown that excessive oxygen augments leptin secretion on cultured pulmonary lipofibroblasts and decreases cellular triglyceride uptake, both signs of transdifferentiation into myofibroblasts (24). This concept of lipofibroblast to myofibroblasts oxygen promoted transdifferentiation is reinforced by the fact that, on those cells, the expression of parathyroid hormone-related protein receptor [PTHrPR]), and adipose differentiation related protein (ADRP) both decrease, and alpha smooth muscle actin (alphaSMA) expression increases (24).

Other genes respond in a functional programmed manner to oxygen excess. For example retinal endothelial cells and adrenal capillary endothelial cells exposed to hyperoxic culture conditions, produce higher levels of the vasoactive peptide endothelin-1 (ET-1)(25), which also influences cell apoptosis (26), and intracellular glucose transport (27).

Importantly, cultured monocytes exposed to different levels of hyperoxia increase the expression of inflammatory cytokines, such as IL-1b, IL-6, TNFalpha (28), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) (29). Likewise, increases the expression of adhesion molecules such as Mac-1/CD11b/CD18 (28).

Excess of oxygen augments in macrophages and other cells the protein production and activity of MMP-2, TIMP-2, and MT1-MMP (30). This may affect cell behavior when re-implanted in animal experiments where cell migration is a critical factor, such as tumor invasion and metastasis models. For example, using normal alveolar epithelial cell migration models it has been observed that in these normal cells the excess of oxygen induced higher expression of MMP-9 gelatinase, which correlated with significantly more migratory capacity through gelatin and acquired a temporary capacity for migration similar to lung cancer cell lines (31).

Moreover, excess of oxygen can also modulate the expression of cell adhesion molecules such as ICAM-1 and VCAM-1 on cells, influencing cell-cell interactions (32). This could be an important issue in tissue engineering protocols,

In summary, all these data show that mammalian cells are sensitive to variations of oxygen concentration. Specifically high levels of oxygen trigger responses that should be considered with different repercussions that depend on the cell culture goals. In any case oxygen should not be an uncontrolled variable in cell culture technology, on the contrary, a tight control on it would improve standardization and repetitive results.


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