Alterations in fatty acid composition of murine keratinocytes with in vitro cultivation

The availability of methods for the in vitro cultivation of keratinocytes has spawned numerous studies utilizing these systems to analyze epidermal biochemical pathways, e.g., eicosanoid production. To determine whether these culture systems are indeed valid models for studies of eicosanoid products, we analyzed the fatty acid (FA) composition, especially of eicosanoid precursors linoleic acid (LA) and arachidonic acid (AA), of cultured and noncultured mouse keratinocytes. Neonatal mouse epidermal keratinocytes were cultivated in Dulbecco's modification of Eagle's medium + 10% fetal calf serum (FCS). Lipids of the cultivated cells, as well as noncultivated keratinocytes and whole epidermis were extracted in CHCl3:MeOH (2:1) and lipid classes separated by thin-layer chromatography. The FA composition of the total lipid extract as well as of the phospholipid class was determined by gas-liquid chromatography of FA methyl esters. There was a gradual decrease in the LA levels in the cultured cells; by day 5 of culture the cells demonstrated a 4-fold (p less than 0.001) decrease in LA as compared to either noncultured cells or whole epidermis. Levels of AA, on the other hand, remained unchanged during culture. Analysis of the FCS used in the culture medium revealed that the level of LA was 4-fold lower than that of normal mouse serum. Since LA is an essential FA which is not synthesized by the cell, the decreased LA in cultured cells probably results from the paucity of this FA in the FCS-containing culture medium. These studies indicate that keratinocytes cultivated in FCS-containing medium demonstrate profound alterations in levels of LA. Hence, in vitro keratinocyte studies dependent on cellular polyunsaturated FA substrates should be interpreted with caution. The relationship of altered cellular levels of LA on keratinocyte differentiation remains to be determined.