Establishment of Zone-Enriched Primary Cultures from the Mouse Adrenal Cortex
May 8th, 2026
A robust and reproducible approach to establish 2D primary cultures of zona glomerulosa- and zona fasciculata-enriched cells was developed for the mouse adrenal cortex. This system preserves key molecular and functional features of each zone, enabling focused investigation of signaling pathways, hormone production, and adrenal cortical cell behavior ex vivo.
My research investigated adrenal microenvironment regulation, focusing on ACM and Wnt signaling in zona glomerulosa and zona fasciculata differentiation, and tumor development. Existing methods cannot clearly distinguish adrenal zones. This protocol enrich ZG and ZF, enabling the study of different cell types.
This protocol can be applied to study adrenophysiology in ZG and ZF, including zonation and cell differentiation. To isolate the adrenal gland for zone-enriched primary culture, use 10 to 15 young adult male mice, approximately six to seven weeks of age. After euthanizing the mouse, place it in a dorsal decubitus position on a sterile flat surface and disinfect the animal fur with 70%alcohol.
Using scissors, make an initial incision in the inguinal region of the abdomen. Extend the incision laterally toward the thorax on both sides to expose the peritoneum. Retract the skin carefully.
Then gently retract the abdominal muscles to expose the cavity, avoiding blood vessel damage to maintain clear visualization of the adrenal glands. Change surgical instruments to minimize microbial contamination. Identify the left adrenal gland located medially and superior to the left kidney in the retroperitoneum with an oval shape and pink coloration surrounded by white adipose tissue.
Using blunt forceps, carefully displace the adjacent organs to improve visualization of the adrenal gland. Using scissors, remove most of the surrounding adipose tissue with short controlled cuts to isolate the adrenal gland. Avoid directly grasping the adrenal gland, to prevent damage to its capsule.
Immediately place the isolated adrenal gland into a P60 culture dish containing 10 milliliters of pre-warmed DMEM F-12 medium until all samples have been collected. Next, identify the right adrenal gland located lateral and superior to the right kidney in the retroperitoneum, noting that its proximity to the liver, ascending colon, and nearby blood vessels may make visualization more challenging. Carefully handle the renal veins and renal arteries to avoid accidental cuts and bleeding.
After isolating the right adrenal gland, using the same procedure as for the left adrenal gland, place both glands together in the same dish. Wipe the culture dish clean with 70%alcohol before transferring the glands to the laminar flow hood. Then, wash the adrenal glands three times by sequentially transferring them through dishes containing PBS at 25 degrees Celsius.
After the final wash, collect and maintain the separated tissues in a fourth dish containing pre-warmed DMEM to preserve viability during preparation of multiple glands. Transfer the adrenal glands to a filter paper for dissection. Next, using fine forceps, grasp the adrenal gland by the adherent adipose tissue.
Carefully dissect the fat on the filter paper with controlled movements to avoid damaging the capsule. Ensure the gland is smooth and intact, and free of excess adipose and connective tissue. Transfer the adrenal glands carefully onto fresh filter paper to facilitate precise micro dissection.
Using fine forceps, gently pinch the capsule of the adrenal gland. Using a scalpel blade, make a radial incision in the gland without completely transecting the tissue. Apply slight mechanical pressure to expel the inner contents of the cortex outward through the incision.
Expose the inner glandular material while keeping the capsule and zona glomerulosa connected to the tip of the forceps. Identify the expelled inner glandular material as the zona fasciculata and medulla. Initiate enzymatic digestion only after all glands have been processed, to ensure proper mechanical separation of the outer and inner zones.
After centrifugation, re-suspend the collected cells in culture medium. After establishing the adrenal cell culture, perform molecular characterization of cultured cells using gene expression analysis and immunostaining. Primary adrenal cell cultures were successfully established from the outer fraction enriched for zonaglomerulosis cells and the inner fraction enriched for zona fasciculata cells.
The lineage tracing mouse model enabled visualization of zona glomerulosa-specific mGFP expression driven by the Cyp11b2 promoter. mGFP expression was predominantly observed in zonaglomerulosa-enriched cultures while zona fasciculata-enriched cultures primarily expressed mTomato. Higher relative expression levels of Cyp11b2, Dab2, and Sonic hedgehog were observed in outer fraction cultures compared to inner fraction cultures.
Inner fraction cultures showed higher relative expression of Cyp11b1, and Akr1b7 compared to outer fraction cultures. Aldosterone secretion was higher in zonaglomerulosa-enriched cultures than in zona fasciculata-enriched cultures. In outer fraction cultures, aldosterone secretion further increased upon stimulation with angiotensin II, potassium, and adrenocorticotropic hormone.
Corticosterone secretion was higher in zona fasciculata-enriched cultures compared to zona glomerulosa-enriched cultures. Zona fasciculata-enriched cultures showed increased corticosterone secretion in response to adrenocorticotropic hormone and forskolin stimulation. This protocol enables the study of zone-specific physiology and differentiation, including hormone production, signaling pathway, and extracellular matrix.
Future studies can explore adrenal zonation and differentiation mechanisms, and using these cells to build a 3D model and study ECM interactions.
View the full transcript and gain access to thousands of scientific videos
Overview
This article presents a novel fractionation and primary culture protocol for isolating and studying zone-specific cells from the adult mouse adrenal cortex. By microdissecting adrenal glands and generating two-dimensional (2D) primary cultures enriched for either zona glomerulosa (zG) or zona fasciculata (zF) cells, the method enables detailed investigation of molecular, cellular, and functional characteristics unique to each adrenal zone.
Key Study Components
Area of Science
- Endocrinology
- Cell Biology
- Adrenal Physiology
Background
- The adrenal gland consists of a capsule, cortex (with zG and zF zones), and medulla.
- zG produces aldosterone; zF produces corticosterone; the medulla produces catecholamines.
- In vitro models preserving zone-specific features have been lacking.
- Understanding zone-specific signaling and function is critical for adrenal research.
Purpose of Study
- To develop a reproducible method for isolating and culturing zG- and zF-enriched adrenal cells.
- To validate the molecular and functional identity of these primary cultures.
- To provide a platform for zone-specific mechanistic studies of adrenal cortex biology.
Methods Used
- Microdissection of adult mouse adrenal glands to separate outer (zG-enriched) and inner (zF-enriched) fractions.
- Generation of 2D primary cultures from each fraction.
- Gene expression analysis for zone-specific and pathway markers.
- Functional assays measuring steroid hormone production in response to physiological stimuli.
Main Results
- zG-enriched cultures expressed high levels of zG markers (Cyp11b2, Dab2, Shh) and Wnt/β-catenin pathway genes.
- zF-enriched cultures showed higher expression of zF markers (Cyp11b1, Akr1b7) and lower Wnt/β-catenin activity.
- Both fractions expressed adrenocortical markers (Nr5a1, Star); zF-enriched cultures also contained medullary cells (tyrosine hydroxylase positive).
- zG cultures produced aldosterone (basal and stimulated by angiotensin II, potassium, ACTH); zF cultures produced corticosterone (basal and ACTH-stimulated).
Conclusions
- This protocol enables robust, zone-specific primary cultures from mouse adrenal cortex.
- The system preserves molecular and functional characteristics of zG and zF cells.
- It provides a valuable in vitro model for dissecting adrenal cortex signaling and physiology.