Microelectrode measurements of pericellular PO₂ in erythropoietin- producing human hepatoma cell cultures

On the basis of Fick's law of gas diffusion, it has been proposed that cells in conventional monolayer cultures may be severely hypoxic. Because knowledge of the cellular O₂ availability is important for the interpretation of biochemical and toxicological cell culture work, microelectrode measurements of the pericellular PO₂ were carried out using the erythropoietin (Epo)-producing human hepatoma cell lines Hep G2 and Hep 3B as an in vitro model. In confluent hepatoma cultures grown in polystyrene dishes and incubated in air with 5% CO₂, the pericellular steady-state PO₂ was <1 mmHg. The rates of the production of immunoreactive Epo and lactate were high due to a misproportion between O₂ supply and O₂ requirements. Epo production decreased when shaken instead of static cultures were studied, or when the O₂ concentration in the gas atmosphere was increased gradually up to 95%. In cultures grown on gas-permeable supports, pericellular and gas PO₂ values were very similar, with increased Epo production at lowered PO₂. In agreement with mathematical models, our experimental data make PO₂ measurements desirable for studies of O₂-dependent biological functions in cell cultures.