Establishment of Zone-Enriched Primary Cultures from the Mouse Adrenal Cortex

The mouse adrenal gland is encapsulated by a mesenchymal cell layer (capsule) and contains an underlying cortex organized into distinct concentric zones with specialized endocrine functions: the zona glomerulosa (zG), which produces aldosterone, and the zona fasciculata (zF), which produces corticosterone. The adrenal medulla, located at the center of the gland, produces catecholamines. Mechanistic studies of cells from each adrenocortical zone have been limited by the absence of in vitro models that preserve their zone-specific molecular, cellular, and functional characteristics. To overcome this limitation, a fractionation approach was developed using microdissection of adult mouse adrenal glands. This method separates adrenal cells from male mice into a zG-enriched outer fraction (OF), containing capsule and zG cells, and a zF-enriched inner fraction (IF), which contains zF and medullary cells. These fractions were used to generate two-dimensional (2D) primary cultures enriched for either zG or zF cells. Gene expression analysis confirmed that the zG-enriched cultures express high levels of zG markers (Cyp11b2, Dab2, and Shh), along with increased Wnt/β-catenin pathway markers (Wnt4, Lef1). In contrast, zF-enriched cultures exhibited higher expression of zF markers (Cyp11b1 and Akr1b7) and lower levels of Wnt/β-catenin pathway markers, consistent with a zF transcriptional signature. Expression of steroidogenic markers (Nr5a1, Star) validated the adrenocortical origin of both cell fractions. Tyrosine hydroxylase indicates the presence of adrenal medullary cells in zF-enriched cultures. Functionally, zG-enriched cultures produced aldosterone under basal conditions and showed increased production in response to angiotensin II, potassium, and adrenocorticotropic hormone (ACTH). Conversely, zF-enriched cultures produced corticosterone at baseline and exhibited increased output in response to ACTH stimulation. Overall, this protocol represents a robust, reproducible, and biologically relevant in vitro model for studying adrenal cortex biology. This system enables zone-specific investigation of signaling pathways, molecular mechanisms, and physiological responses.