Modulation of epithelial cell proliferation in culture by dissolved oxygen

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Abstract
Modulation of epithelial cell proliferation by the dissolved oxygen concentration (PO2) of the growth medium was assessed with primary human foreskin epithelium and a continuous monkey kidney epithelial cell line (LLC-MK2). Direct measurement of the growth medium PO2 provides the first quantitative evaluation of epithelial cell proliferation as a function of PO2 provides the first quantitative evaluation of epithelial cell proliferation as a function of PO2. Sustained proliferation of LLC-MK2 cells occurs in serum-free medium equilibrated with a gas phase containing 18% or 30% O2 v/v. Mid-logarithmic phase cultures rapidly consume dissolved oxygen; this results in a 60–70 mm Hg decline in PO2 and leads to a stable growth medium PO2 between 70 and 100 mm Hg, well above anoxic values. In contrast, if culture medium is equilibrated with a gas phase containing 0% or 1% O2 v/v to yield a growth medium PO2 ∼ 20–40 mm Hg, proliferation of LLC-MK2 and primary foreskin epithelial cells is retarded, and LLC-MK2 cells use little dissolved oxygen. Gentle, continuous rocking to prevent diffusion gradient formation enhances proliferation slightly at the higher PO2, but neither periodic fluid renewals nor continued rocking stimulates cells retarded by a lowered oxygen concentration to resume proliferation. The data collectively demonstrate that epithelial cell proliferation requires a PO2 > 40 mm Hg, and threshold requirements are probably closer to 70 mm Hg. Glycolysis continues at a PO2 insufficient for proliferation, but more lactic acid accumulates in actively proliferating cultures than in cultures equilibrated with 0% oxygen. We conclude that epithelial cells in vitro both consume more oxygen and require a higher PO2 for continued proliferation, and that the oxygen requirement for epithelial cell proliferation exceeds that of a comparable population of fibroblasts for which low oxygen may enhance survival and proliferation.