Growth and Function of Cultured Bovine Adrenocortical Cells in a Serum-Free Defined Medium*

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Abstract
A serum-free defined medium that supports cellular proliferation and the differentiated function of steroidogenesis has been developed for monolayer cultures of normal bovine adrenocortical cells. Cell cultures proliferate from a low cell density (1–2 × 103 cells/cm2) on a fibronectin-coated substratum in a medium composed of a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's Medium supplemented with 4 nM fibroblast growth factor, 2 nM insulin, 100 mU/ml thrombin, 10μg/ml low density lipoprotein, 100 μg/ml transferrin, 500 μg/ml fatty acid-free BSA, 100 μM ascorbic acid, 1 μM α-tocopherol, and 50 nM selenium. The cell cultures in the defined medium proliferate at the same growth rate as those in medium supplemented with serum and fibroblast growth factor. In the total absence of serum, the adrenocortical cells have been grown in the defined medium for three consecutive passages to the same final cell density. Cell proliferation in the defined medium was completely inhibited by ACTH, which previously has been shown to be antimitogenic for adrenocortical cell cultures in serum-supplemented medium. cell cultures grown in the defined medium demonstrated stimulated steroid production which required low density lipoprotein as the principal source of cholesterol. ACTH stimulated fluorogenic steroid production with a half-maximal effective concentration of 0.5 nM in cell cultures grown in the defined medium. As determined by high pressure liquid chromatography analysis of the steroid products, cells grown in the defined medium produce 17α-hydroxy-Δ4,3-ketosteroids (cortisol and deoxycortisol) characteristic of the zona fasciculata-reticularis. These studies demonstrate that using a fibronectin substratum, a defined mixture of growth factors and nutrients supports not only sustained cellular proliferation but also the full differentiated function of stimulated steroidogenesis in cultures of normal adrenocortical cells.