Flow-dependent differentiation of cultured adrenal cells under different stimuli.

It still remains unclear how the functional organisation of the adrenal cortex arises. One aim of this study was to create a setup which allows for the establishment of a concentration gradient in vitro. This was achieved by a continuous flow of medium through the culture flask which caused differences in glucose and cortisol concentrations as well as in pH values between the sites of inflow and outflow of medium. Using real-time polymerase chain reaction, we found that a continuous supply of 1 ml medium per hour significantly increased the expression of MC2R, CYP11B1 and CYP17A1 genes of NCI-H295R cells in the distal area of the flask as compared with the proximal part. The expression of the AT1R showed a reverse regulation. The addition of dexamethasone to the medium led to an increase in gene expression of MC2R while AT1R was downregulated. Moreover, we detected a higher expression of CYP11B2 and a decreased expression of CYP11B1 when endothelial cell-conditioned medium (ECCM) was added to the inflow. Our experiments show that a directed medium delivery system creates different gradients and affects the functional differentiation of the NCI-H295R cells. Also, our results emphasise that products of endothelial cells have additional effects on the differentiation of the cultured adrenal cortical cells. Our results are in support that the regulation of the adrenal zonation is possible through different concentration gradients.