Adrenal cells in tissue culture

This report provides a detailed analysis of the methods employed in our laboratory for the growth and maintenance of functional monolayer cultures of murine adrenal tumor cells. Radiochemical analysis of the products of incubation of these cells with 3H-pregnenolone, 14C-progesterone, and 14C-sodium acetate has confirmed that the predominant steroids secreted into the medium are 20α dihydroprogesterone and 11β hydroxy, 20α dihydroprogesterone. Chemical and enzymatic procedures for the synthesis of the latter compound and other 20α hydroxylated steroids are described. A fluorometric assay has been developed that permits precise quantitation of steroid output within several minutes' incubation. Consistent Steroidogenic responses to ACTH ranging as high as ten times the basal output have been observed. There is a linear log-dose response to ACTH (or Synacthen, a β1–24 peptide fragment of ACTH) ranging from 10 pg/ml to 1000 pg/ml. A variety of other peptide hormones were found to be inactive. The response to ACTH proceeds linearly from 10 min to 24 hr. These cells provide a viable and highly reproducible system for the in vitro study of ACTH action.