Respiration-induced Oxygen Gradients in Cultured Mammalian Cells

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Abstract
The effect of cell respiration on the availability of intracellular oxygen was investigated by comparing the radiosensitivities of respiring and non-respiring cells over a range of oxygen tensions. Monolayers of Chinese hamster V79 fibroblasts on glass Petri dishes were irradiated at respiration-inhibiting (4°C) and normal (37°C) cell-culturing temperatures. Desired extracellular oxygen concentrations were achieved by aspirating the culture medium above the cells prior to irradiation, leaving a residual thin film which prevented drying of the cells while allowing rapid equilibration with the overlying gas. Measurement of clonogenic survival revealed that, at equivalent extracellular oxygen concentrations, the cells irradiated at 37°C were less radiosensitive than those irradiated at 4°C. The difference in respiring and non-respiring cell radiosensitivity was dependent on cell shape, and decreased when the cells attached to the Petri dish surface were allowed to assume a flatter configuration. These results imply that at low extracellular oxygen tensions the oxygenation of critical cellular radiation target(s) is dependent on respiration and diffusion distance, as would be expected if oxygen gradients induced by respiration exist within and immediately around actively metabolizing cells.