Analysis of the effects of cell spacing and liquid depth on nitric oxide and its oxidation products in cell cultures.

A series of reaction-diffusion models was developed to describe spatial and temporal variations in the concentrations of NO and related species (O(2)(-), peroxynitrite, and N(2)O(3)) in a cell culture configuration that is commonly used to study the toxicity and/or mutagenicity of NO. In the analysis, adherent cells that generate NO and O(2)(-) (e.g., macrophages) were assumed to be present in sub-monolayer quantities at the bottom of a culture dish, with a stagnant layer of culture medium separating them from the incubator gas. It was assumed also that the level of O(2)(-) production is lower than that of NO, consistent with the available data. These conditions were found to yield a distinctive spatial segregation among the extracellular reactions, which permits considerable simplifications in modeling events at both the macroscopic (culture medium depth) and microscopic (cell spacing) length scales. Whereas NO and N(2)O(3) were predicted to be present throughout the liquid medium, O(2)(-) and peroxynitrite were each confined to small regions near the cells. It was found that such systems reach a steady state in a small fraction of typical experimental times, so diffusional transients are unimportant. In contrast to the usual assumption made in experimental studies, NO loss to the headspace was found to be very significant. Thus, using the rate of accumulation of NO(2)(-) and NO(3)(-) (the nonvolatile end products of NO oxidation) to infer the cellular rate of NO production may significantly underestimate that rate. Moreover, it was found that varying the cell number density had very different influences on the relative exposures of the cells to NO, O(2)(-), peroxynitrite, and N(2)O(3). These results suggest that previously reported decreases in macrophage viability with increasing cell number density are more likely to be the result of exposure to NO and/or N(2)O(3) than to O(2)(-) or peroxynitrite.