We have examined the growth of human diploid fibroblasts (WI-38 and IMR90) as a function of initial seeding density and oxygen tension. Cells at young and mid-passage levels were subcultivated in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 0.005, 0.01, 0.03, 0.1, 0.3, 1, and 2 X 10(4) cells/cm2. Flasks were equilibrated before and after seeding with 1 of 10 gas mixtures containing the desired oxygen tension (9-591 mm Hg) and placed in incubators that measure and maintain a preset oxygen tension. The partial pressure of oxygen (PO2) in media of all flasks was determined at harvest. Cells were shielded from light of wavelength less than 500 nm. Cell growth varied inversely with oxygen tension and seeding density. At 50 cells/cm2, growth was maximal at PO2 9 and 16 mm Hg. Growth was progressively inhibited as the oxygen tension was increased. The population doubling increase at 14 d was 8.6 for PO2 9 and 16 mm Hg, 5.8 for PO2 42 mm Hg, 3.8 for PO2 78 mm Hg, 3.8 for PO2 104 mm Hg, and 3 for PO2 138 mm Hg. As the seeding density was increased, the differences in growth at PO2 less than 140 mm Hg were progressively minimized, such that at seeding densities of 10(4) cells/cm2 there was little difference in the rate of exponential growth or the final saturation density of cells cultivated between PO2 9 and 96 mm Hg. At all seeding densities tested, growth was progressively inhibited when the PO2 was increased greater than 140 mm Hg. The seeding density dependence of oxygen's influence on cellular growth is not explained by oxygen consumption of higher density cultures. Oxygen acts directly on the cells and not by destroying some essential medium component. We have found that oxygen regulates the growth of human cells under pressures of oxygen physiologic to humans, and that oxygen toxicity contributes to the seeding density dependence of cellular growth commonly seen in cell culture.

This content is only available as a PDF.