Name: Establishment of Zone-Enriched Primary Cultures from the Mouse Adrenal Cortex (Video) | JoVE Journal
Uploaded: 2026-05-08T14:20:00.000+00:00
Duration: 7 min 43 s
Description: Learn to establish zone-enriched primary cultures from mouse adrenal cortex for studying zona glomerulosa and zona fasciculata cell biology. This method enables detailed investigation of adrenal zone-specific signaling and hormone production.

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This work was supported by FAPESP (S&#227;o Paulo Research Foundation, Brazil), project number 2024/14165&#8211;6 (BEPE &#8211; JLK), FAPESP: 2020/02988&#8211;7 (C.F.P.L), and support by 2R01DK123694 (to DTB) and funds from the Division of Endocrinology at Boston Children&#8217;s Hospital (DTB).

All experiments were conducted in accordance with protocols approved by the Boston Children&#8217;s Hospital Institutional Animal Care and Use Committee (IACUC) under approval number 00002611. A complete list of all materials used in this protocol, including reagents, consumables, and equipment, is provided in the Table of Materials, along with the corresponding manufacturers and catalog numbers to ensure reproducibility.
Primary cultures of mouse adrenal zona glomerulosa and zona fasciculata cells 
The methods used to establish primary cultures of adult male mouse wild-type (Mus musculus, C57BL/6) adrenocortical cells in this study were adapted from a procedure originally developed for Sprague-Dawley rats28. To confirm the segregation and identity of zona glomerulosa and zona fasciculata cell populations, a lineage-tracing model was employed using Cyp11b2tm1.1(cre)Brlt/+ (also known as-Cre) mice1,2,3,4 and Gt(ROSA)26Sortm4(ACTB-tdTomato-EGFP) Luo/J (also known as ROSAmT/mG) mice29.
Preparation before the procedure 
Materials 
Two sets of forceps and scissors for superficial incisions and another set for incision of the abdominal cavity (Supplementary Figure 1A). Warmed the culture medium and PBS (37 &#176;C). Prepared 2 dishes (P60) with DMEM/F-12. Prepared 3 dishes (P60) with warmed PBS to wash the glands in sequence (bath 1, 2, and 3) (Supplementary Figures 1B, 1&#8211;3), and 1 dish to keep the adrenal in solution (Supplementary Figures 1B, 4). Prepared 2 dishes (P60) with sterile filter paper on the surface (Supplementary Figures 1C, 1&#8211;2). Prepared 2 P60 dishes with 500 &#181;L of digestion solution and labeled them for the glomerulosa (zG) or the fasciculata (zF) (Supplementary Figures 1D, zG, zF). Prepared 2 tubes, labeled zG and zF, and added 4.5 mL of digestion solution.
Solutions 
To make the digestion solution, collagenase type I (final concentration 4 mg/mL) and DNase I (final concentration 0.04 mg/mL) were dissolved in 5 mL of pure DMEM/F-12 medium. Filtered the solution into a 50 mL tube using a syringe and a 0.22 &#181;M filter. The digestion solution should be prepared fresh each time.
Animals 
To obtain primary cultures enriched for mouse adrenal zona glomerulosa and zona fasciculata cells, use 10&#8211;15 young adult male mice, approximately 6&#8211;7 weeks of age. Females have slightly larger adrenal glands than males, and either sex can be used with this protocol. However, in this study, only males were used to avoid potential interference from the X-zone present in young females, ensuring accurate zonal separation. Mice were euthanized by CO&#8322; inhalation in accordance with institutional animal care guidelines. Death was confirmed by the absence of respiration and lack of reflex response. Adrenal glands were dissected immediately, within 3&#8211;5 min after confirmation of death, to preserve tissue viability.
Position and exposure of the abdomen 
The mouse was placed in a dorsal decubitus position on a sterile flat surface (Figure 1A). The animal&#8217;s fur was disinfected with 70% alcohol. Forceps and scissors were used for the cutaneous incisions, and the second set for the abdominal cavity incision. An initial incision in the inguinal region of the abdomen was made, extending laterally towards the thorax on both sides to expose the peritoneum (Figure 1B). The skin was carefully retracted, followed by the abdominal musculature, to expose the abdominal cavity (Figure 1C). The initial incision should be superficial to avoid damage to the internal organs. Advance to the muscle layer with a cautious incision, then expose the abdominal cavity. Avoid rupturing blood vessels, as bleeding can make it difficult to locate the adrenal glands and delay collection. Change surgical instruments to minimize microbial contamination.
Identification and collection of the left adrenal gland 
The left adrenal gland is located medially and superior to the left kidney in the retroperitoneum, with an oval shape and pink coloration, surrounded by white adipose tissue (Figures 1D&#8211;G). While carefully displacing adjacent organs with blunt forceps to improve visualization, use scissors to remove most of the adipose tissue using short, controlled cuts, isolating the adrenal gland. Avoid directly grasping the adrenal gland to prevent damage to its capsule. The gland was placed immediately after isolation in a P60 culture dish with 10 mL of pre-warmed DMEM/F-12 at 37 &#176;C (Figure 1F).
Identification and collection of the right adrenal gland 
The right adrenal gland is located laterally and superior to the right kidney in the retroperitoneum, with an oval shape and pink coloration (Figures 1E&#8211;H). Identifying this adrenal gland may be more challenging due to its proximity to other organs, such as the liver and ascending colon, as well as nearby blood vessels. While carefully displacing adjacent organs with blunt forceps to improve visualization, use scissors to remove most of the adipose tissue using short, controlled cuts, isolating the adrenal gland, while taking care not to damage the capsule. The gland was placed immediately after isolation in a P60 culture dish with 10 mL of pre-warmed DMEM/F-12 at 37 &#176;C. The adrenals are combined in the same dish (Figure 1F). During the procedure, it is essential to handle the major blood vessels in the area with care to avoid accidental cuts, which can cause bleeding and complicate the collection process. The main vessels to be cautious of include the renal veins and renal arteries. Collected the adrenal gland with some adherent adipose tissue. This facilitates the handling of the gland and protects against capsule damage and contamination (Figure 1I).
Work in a sterile laminar flow hood 
Before transferring the glands to the laminar flow hood, the dish was wiped clean with 70% alcohol. The adrenals were washed with PBS three times at room temperature (25 &#176;C) (in dishes 1, 2, and 3 and collected in dish 4) (Supplementary Figure 1B). The filter paper helps with fat adhesion and ensures better results. During the preparation of multiple glands, the separated tissues should be maintained in pre-warmed DMEM to preserve viability. The adrenal gland was grasped by the adherent adipose tissue (Figures 1I&#8211;J), and the fat was carefully dissected on the filter paper (Supplementary Figure 1C, dish 1), using fine forceps and controlled movements to avoid damaging the capsule. The expected result is a smooth, intact gland free of excess adipose and connective tissue, as shown in Figure 1K. The adrenal glands were transferred carefully to fresh filter paper (Supplementary Figure 1C, dish 2) to facilitate precise microdissection. The capsule was gently pinched with fine forceps, and using the scalpel blade, a radial incision was made in the gland without completely transecting the tissue.
The inner contents of the cortex were gently exposed by expelling them outward through the incision through the application of slight mechanical pressure with the forceps. The capsule and the zG remained connected to the tip of the forceps, while the inner expelled glandular material corresponded to the zF and medulla. Enzymatic digestion should be initiated only after all glands have been processed (Figure 1L), due to mechanical separation of the outer and inner zones.
Digestion 
Two tubes were labeled as OF (Outer Fraction: capsule and zG) or IF (Inner Fraction: zF and medulla) (Figure 1M). A total of 4.5 mL of pre-warmed digestion solution (37 &#176;C) was added, and the OF and IF were transferred to the appropriate tubes. The samples were incubated at 37 &#176;C in a humidified incubator with 5% CO&#8322; for 30 min, and the tubes were gently swirled every 10 min, by manually inverting them 2&#8211;3 times to ensure uniform exposure to the enzyme solution. The time required for digestion may vary depending on the quality, activity, and storage conditions of the collagenase. After 30 min, the tissue was dissociated by pipetting up and down 40 times with slow, controlled strokes, using a 5 mL serological pipette to generate a single cell slurry. After dissociation of the tissue, ideally, no visible fragments should remain, and the medium should appear cloudy. If fragments are still present, an additional 15-min digestion may be considered.
Washing and plating 
Following the digestion, the cells were washed by adding 45 mL of DMEM/F-12 to remove residual enzyme (Figure 1 M). Samples were centrifuged at 150 &#215; g for 12 min at room temperature (25 &#176;C). Following centrifugation, a small and compact pellet with a slightly pinkish to pale yellow appearance is expected, while the supernatant should remain clear and free of visible particulates. The supernatant was carefully removed and discarded, taking care not to disturb the pellet. The pellet was resuspended in pre-warmed (37 &#176;C) culture medium according to the final volume required for plating (see below). An optimal plating density of approximately 70% was maintained to ensure culture viability and vigor. The cell number was estimated using a Neubauer counting chamber prior to plating, and the number of cells seeded was adjusted according to the culture dish area to achieve the desired confluency. Plate the zG-enriched outer fraction (OF) cells at a density of approximately one animal per well (typically yielding ~1.5&#8211;2.5 &#215; 104 cells) and the zF-enriched inner fraction (IF) cells at ~0.63 animals per well (typically yielding ~2.5&#8211;4.0 &#215; 104 cells) in a 48-well plate.
Cell culture maintenance 
Cells were cultured in the Culture Medium described above and maintained under standard conditions in a humidified incubator at 37 &#176;C with 5% CO&#8322;. After 24 h of culture, the medium was replaced to remove debris and any residual blood cells. This step promotes optimal growth. The plate was carefully aspirated, and fresh pre-warmed (37 &#176;C) medium was added. Wait at least 48 h before assessing the cells in functional experiments. It&#8217;s recommended to wait three days before performing functional experiments to ensure the cultures are well-established. Extended culture beyond this period may require trypsinization and passaging, which can lead to cell selection and loss of primary culture characteristics.
Data analysis and supporting procedures 
Preparation of primary adrenal cell cultures 
Primary cultures of whole adrenal glands were established following the same procedures for tissue collection, digestion, washing, and plating, and were used as standard controls for molecular and functional assays.
Gene expression analysis 
To purify total RNA from OF and IF cells, the cultured cells were homogenized in TRIReagent using the RNA kit, according to the manufacturer&#8217;s protocol. RNA integrity and concentration were evaluated by using spectrometry. Further processing of total RNA involved reverse transcription into cDNA using the high-capacity cDNA reverse transcription kit. Gene expression analysis was performed by real-time quantitative PCR (qPCR) using the thermocycler. Technical duplicates were used to control for variability. The TaqMan Universal PCR Master Mix and mouse TaqMan primers were used (Table 1). A cycle threshold (CT) value was selected within the linear amplification range for each sample run in duplicate. It was normalized by endogenous control genes &#946;-actin and ribosomal protein 18S (Rps18). The relative expression levels were calculated using the 2&#8211;&#916;&#916;Ct method30. The data from three different experiments are presented as mean &#177; standard deviation (SD). We performed three independent experiments.
Stimulation of aldosterone and corticosterone secretion in primary ZG and ZF cultures 
Primary cultures of zona glomerulosa (ZG) and zona fasciculata (ZF) cells were established and maintained at 37 &#176;C in a humidified atmosphere containing 5% CO&#8322;. Approximately 5 &#215; 104 cells were plated per well in a 48-well plate using complete growth medium. After four days of continuous culture, basal secretion measurements reflected cumulative hormone production over this time period. For stimulation assays, the medium was aspirated, and the cells were washed once with phosphate-buffered saline (PBS) to remove residual factors. Subsequently, 200 &#181;L of fresh medium was added to each well, and the following assays represent acute stimulated secretion within a defined time window. For aldosterone stimulation assays, the culture medium was supplemented with angiotensin II (Ang II, 10 nM), potassium (K+), as potassium chloride (KCl, 10 mM), and ACTH, (10 nM). Sodium chloride (NaCl, 10 mM) was used as the vehicle control. For corticosterone stimulation assays, the same procedures were followed, but the medium was supplemented with ACTH (10 nM), forskolin (10 &#181;M), or NaCl (10 mM) as a vehicle control. After 6 h of incubation at 37 &#176;C and 5% CO&#8322;, the culture supernatants were collected and stored at &#8722;80 &#176;C for subsequent hormone quantification, as described previously31. The remaining adherent cells were lysed for protein extraction, and hormone levels were normalized to the total protein content of the corresponding lysate.
Protein extraction and normalization of aldosterone and corticosterone measurements 
Protein extraction was performed using RIPA lysis and extraction buffer, supplemented with 1x halt protease inhibitor cocktail. Cells were resuspended by pipetting up and down 15 times using a P200 pipette tip and subsequently sonicated using a probe sonicator. Protein concentration was determined with the BCA protein assay kit according to the manufacturer&#8217;s instructions.
Aldosterone measurement by radioimmunoassay (RIA) 
The concentration of aldosterone in cell culture supernatants was determined by following the manufacturer&#8217;s protocol. In brief, 50 &#181;L of supernatant and 150 &#181;L of 1% BSA in PBS, together with 500 &#181;L of the aldosterone radioactive tracer, were added to aldosterone antibody-coated tubes. After mixing, the tubes were incubated at room temperature (25 &#176;C) for 18 h. Then, the incubation mixture was decanted, and the radioactivity in the tubes was counted using a Cobra II auto-gamma counter31. Aldosterone levels were normalized to total protein, as previously described.
Corticosterone measurement 
Corticosterone concentrations in cell culture supernatants were measured using a competitive enzyme-linked immunosorbent assay (ELISA) kit specific for mouse and rat corticosterone, following the manufacturer&#8217;s protocol. Corticosterone levels were normalized to total protein, as previously described32.
Immunostaining 
Immunofluorescence was performed on both paraffin-embedded adrenal sections and cultured cells grown on pre-treated glass coverslips coated with Poly-L-lysine. For all samples, blocking was performed with 5% normal goat serum (NGS) in PBS, and nuclei were counterstained with DAPI (4&#8242;, 6-diamidino-2-phenylindole, 1:1000). Slides and coverslips were washed three times for 5 min each with 0.1% Tween-20 in PBS between incubation steps. After staining, samples were mounted with Antifade Mountant and stored in the dark until imaging. Tissue sections: Adrenal paraffin sections were deparaffinized in xylene, then rinsed through a graded ethanol series (100%, 95%, 70%), and finally rinsed in PBS. Antigen retrieval was performed in Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA, 0.05% Tween-20, pH 9.0) by heating at 95 &#176;C for 20 min. After cooling to room temperature, sections were washed in PBS and blocked with 5% NGS in PBS for 1 h. Sections were incubated overnight at 4 &#176;C with the following primary antibodies diluted 1:100 in 5% NGS in PBS: mouse anti-Dab2, chicken anti-GFP, and rabbit anti-RFP. The following secondary antibodies were used (1:300): Alexa Fluor 594 goat anti-mouse IgG, Alexa Fluor 594 goat anti-chicken IgY, and Alexa Fluor 647 goat anti-rabbit IgG.
Cultured cells: Immunofluorescence on coverslips was performed four days after plating. Cells were fixed with pre-warmed 4% paraformaldehyde (PFA) at 37 &#176;C for 15 min and washed three times in PBS. Blocking was performed using 5% NGS in PBS for 1 h at room temperature. Coverslips were incubated overnight at 4 &#176;C with mouse anti-Dab2 (1:100 in 5% NGS/PBS), followed by Alexa Fluor 488 goat anti-mouse (1:500).
Image acquisition: Brightfield and fluorescence images (mGFP and mTomato) of cultured cells were acquired using a microscope system. The mGFP fluorescence was captured using a filter set for green fluorescence (excitation: 470 nM, emission: 525 nM), and mTomato fluorescence was captured using a filter set for red fluorescence (excitation: 585 nM, emission: 624 nM).
Software and statistical analysis 
Visual summaries, schematic diagrams, and figures were created using Adobe Illustrator 2023 (Adobe Inc., https://www.adobe.com/products/illustrator.html) and BioRender (https://www.biorender.com), accessed in May 2025. Data were analyzed using GraphPad Prism version 10.0. Available at: https://www.graphpad.com. All experiments were performed at least in triplicate and independently repeated to ensure reproducibility of the results. For comparisons between two groups, Student&#8217;s t-test (paired or unpaired, as appropriate) was used. For comparisons involving three or more groups, analysis of variance (ANOVA) followed by the appropriate post hoc test was performed to identify significant differences between groups. Results are presented as mean &#177; SD. A p-value of less than 0.05 was considered statistically significant.

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<li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+global+double-fluorescent+Cre+reporter+mouse%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45 (9), 593-605 (2007).</li>
<li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2-%CE%94%CE%94CT+method%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.</a> Methods. 25 (4), 402-408 (2001).</li>
<li>Berber, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rho%2FROCK+signaling+and+%CE%B1-catenin+mediate+%CE%B2-catenin-driven+hyperplasia+in+the+adrenal+cortex+via+adherens+junctions%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Rho/ROCK signaling and α-catenin mediate β-catenin-driven hyperplasia in the adrenal cortex via adherens junctions.</a> J. Clin. Invest. , (2026).</li>
<li>Smith, T. T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Forced+treadmill+running+reduces+systemic+inflammation+yet+worsens+upper+limb+discomfort+in+a+rat+model+of+work-related+musculoskeletal+disorders%5BTitle%5D)+AND+%22BMC+Musculoskelet.+Disord%22%5BJournal%5D)">Forced treadmill running reduces systemic inflammation yet worsens upper limb discomfort in a rat model of work-related musculoskeletal disorders.</a> BMC Musculoskelet. Disord. 21 (1), 57(2020).</li>
<li>Kremer, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+matrix+protein+signatures+of+the+outer+and+inner+zones+of+the+rat+adrenal+cortex%5BTitle%5D)+AND+%22J.+Proteome+Res%22%5BJournal%5D)">Extracellular matrix protein signatures of the outer and inner zones of the rat adrenal cortex.</a> J. Proteome Res. 23 (8), 3418-3432 (2024).</li>
<li>Pignatti, E., Leng, S., Carlone, D. L., Breault, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+zonation+and+homeostasis+in+the+adrenal+cortex%5BTitle%5D)+AND+%22Mol.+Cell.+Endocrinol%22%5BJournal%5D)">Regulation of zonation and homeostasis in the adrenal cortex.</a> Mol. Cell. Endocrinol. 441, 146-155 (2017).</li>
<li>Pignatti, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beta-catenin+causes+adrenal+hyperplasia+by+blocking+zonal+transdifferentiation%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Beta-catenin causes adrenal hyperplasia by blocking zonal transdifferentiation.</a> Cell Rep. 31 (3), 107524(2020).</li>
<li>Romero, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disabled-2+is+expressed+in+the+adrenal+zona+glomerulosa+and+is+involved+in+aldosterone+secretion%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">Disabled-2 is expressed in the adrenal zona glomerulosa and is involved in aldosterone secretion.</a> Endocrinology. 148 (6), 2644-2652 (2007).</li>
<li>Sahut-Barnola, I., Lefrancois-Martinez, A. M., Jean, C., Veyssiere, G., Martinez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adrenal+tumorigenesis+targeted+by+the+corticotropin-regulated+promoter+of+the+aldo-keto+reductase+AKR1B7+gene+in+transgenic+mice%5BTitle%5D)+AND+%22Endocr.+Res%22%5BJournal%5D)">Adrenal tumorigenesis targeted by the corticotropin-regulated promoter of the aldo-keto reductase AKR1B7 gene in transgenic mice.</a> Endocr. Res. 26 (4), 885-898 (2000).</li>
<li>Vidal, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+adrenal+capsule+is+a+signaling+center+controlling+cell+renewal+and+zonation+through+Rspo3%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">The adrenal capsule is a signaling center controlling cell renewal and zonation through Rspo3.</a> Genes Dev. 30 (12), 1389-1394 (2016).</li>
<li>Dörner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GLI1%2B+progenitor+cells+in+the+adrenal+capsule+of+the+adult+mouse+give+rise+to+heterotopic+gonadal-like+tissue%5BTitle%5D)+AND+%22Mol.+Cell.+Endocrinol%22%5BJournal%5D)">GLI1+ progenitor cells in the adrenal capsule of the adult mouse give rise to heterotopic gonadal-like tissue.</a> Mol. Cell. Endocrinol. 441, 164-175 (2017).</li>
<li>Guo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D+culture+system+for+human+adrenal+glands+that+uses+a+sequential+processing+medium+to+facilitate+cortical-medullary+cell+development%5BTitle%5D)+AND+%22Clin.+Lab%22%5BJournal%5D)">3D culture system for human adrenal glands that uses a sequential processing medium to facilitate cortical-medullary cell development.</a> Clin. Lab. 70 (12), (2024).</li>
<li>Friedrich, L., Schuster, M., Rubin de Celis, M. F., Berger, I., Bornstein, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+in+vitro+cultivation+of+adrenal+cells+from+mice%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Isolation and in vitro cultivation of adrenal cells from mice.</a> STAR Protoc. 2 (4), 100999(2021).</li>
<li>Mattos, G. E., Jacysyn, J. F., Amarante-Mendes, G. P., Lotfi, C. F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+effect+of+FGF2%2C+synthetic+peptides+1%E2%80%9328+N-POMC+and+ACTH+on+proliferation+in+rat+adrenal+cell+primary+cultures%5BTitle%5D)+AND+%22Cell+Tissue+Res%22%5BJournal%5D)">Comparative effect of FGF2, synthetic peptides 1–28 N-POMC and ACTH on proliferation in rat adrenal cell primary cultures.</a> Cell Tissue Res. 345 (3), 343-356 (2011).</li>
<li>Gazdar, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+and+characterization+of+a+human+adrenocortical+carcinoma+cell+line+that+expresses+multiple+pathways+of+steroid+biosynthesis%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis.</a> Cancer Res. 50 (17), 5488-5496 (1990).</li>
<li>Wang, T., Rainey, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+adrenocortical+carcinoma+cell+lines%5BTitle%5D)+AND+%22Mol.+Cell.+Endocrinol%22%5BJournal%5D)">Human adrenocortical carcinoma cell lines.</a> Mol. Cell. Endocrinol. 351 (1), 58-65 (2012).</li>
</ol>

The authors declare that they have no competing financial interests related to this study.

In this study, a refined protocol was presented that generates primary mouse adrenal cell cultures enriched for zG (OF) and zF (IF) cells, which faithfully recapitulate the zonal identity and steroidogenic function of each zone. This protocol should enable detailed investigation of the cellular and molecular biology of the adrenal cortex in a zone-specific context, supporting the study of proteins, signaling pathways, and mechanisms involved in adrenal cortex development, differentiation, function, and renewal, with redu...

A thin mesenchymal capsule encapsulates the mouse adrenal gland, which consists of two main compartments: the outer cortex and the inner medulla. The adrenal cortex of the adult male mouse is further divided into distinct zones, the zona glomerulosa (zG) and the zona fasciculata (zF), each with unique morphological and functional characteristics1,2,3,4. The primary function of the adrenal cortex is the production of steroid hormones, including mineralocorticoids (aldosterone) and glucocorticoids (corticosterone in mice)5. The zG is the outermost and most compact region and consists of ovoid cells organized in rosette structures that express aldosterone synthase (CYP11B2), allowing the production of aldosterone2,3,5,6,7. Secretion of aldosterone is primarily regulated by the renin-angiotensin-aldosterone system (RAAS), potassium, and to a lesser extent ACTH5,8. The zF is composed of larger cells arranged in radial cords/fascicles, constitutes the majority (~85%) of the cortex, and is regulated primarily by ACTH2,3,5,9. Multiple signaling pathways orchestrate adrenal gland development, maintenance, and function by regulating progenitor cell dynamics, zonation, and steroidogenic activity. Among these, SF-1 (Steroidogenic Factor-1, Nr5a1) is a master regulator essential for adrenocortical specification, proliferation, and hormone production10. Sonic hedgehog (Shh) signaling, active in the zG, promotes the expansion of progenitor cells and supports cortical renewal in the zG11,12,13.
Wnt signaling, particularly through the canonical &#946;-catenin pathway, is crucial for adrenal zonation and cortical integrity. In the mouse, normal adrenocortical size and zonation are regulated by a gradient of WNT signaling activity extending from the capsule to the zF. This gradient is supported by localized expression of WNT ligands and potentiators, mainly WNT4 in the zG, and WNT2B and R-spondins (RSPO1 and/or RSPO3) in the capsule7,14,15,16,17,18,19,20,21,22. The adrenal cortex is maintained by the centripetal flow of cells from proliferative progenitor cells located in the subcapsular/outer zG region, which give rise to differentiated zG cells that migrate inward toward the zF. As cells transition from the zG to the zF, they undergo changes in gene expression and function referred to as trans-differentiation. This model has been strongly supported by lineage-tracing studies using the ASCre/+ transgenic mouse line, which specifically targets aldosterone-producing cells in the zG23,24,25.
Several in vitro approaches have been used to study adrenal biology based on the dissociation of the whole adrenal gland26,27,28. For example, a study described dissociation of the adrenal cortex for electrophysiological and patch-clamp analyses27, and another study employed dissociated adrenal cortical cells to investigate signaling mechanisms related to adrenal physiology and blood pressure regulation26. While these methodologies have provided important functional insights, they rely on cells from whole adrenal preparations rather than on the separation of cortical zones prior to cell culture.
Given the complexity and histological diversity of the adrenal gland, understanding the distinct roles of its cellular compartments is essential for elucidating the mechanisms underlying adrenal function. In this study, primary mouse adrenocortical cell cultures enriched for either zG or zF cells were established. A major advantage of this method is the generation of zG-enriched cell cultures, enabling experimental modulation of signaling pathways and environmental factors to better investigate its specific role in adrenal cortex biology. This includes the study of progenitor cell maintenance, the mechanisms underlying zonal plasticity and trans-differentiation toward the zF, and the modeling of extracellular matrix composition. In parallel, this method generates highly enriched zF cell cultures, providing a complementary platform for zone-specific functional and signaling studies. Together, these features provide a robust, experimentally controlled system for advancing understanding of adrenal biology.

<table><tbody><tr><td>Conical Tube (50 mL )</td><td>Corning (Corning, NY, USA)</td><td>352070</td><td>Sterile</td></tr><tr><td>0.22 &amp;micro;m Syringe Filter</td><td>MilliporeSigma (Burlington, MA, USA)</td><td>SLGV033RS</td><td>Digestion solution filtration</td></tr><tr><td>10 mL Disposable Syringe</td><td>BD Plastipak (Franklin Lakes, NJ, USA)</td><td>300912</td><td>Sterile</td></tr><tr><td>5 mL Serological Pipette</td><td>Corning (Corning, NY, USA)</td><td>4487</td><td>Sterile</td></tr><tr><td>10 mL Serological Pipette</td><td>Corning (Corning, NY, USA)</td><td>4488</td><td>Sterile</td></tr><tr><td>Filtered Pipette Tips (P200)</td><td>Axygen (Union City, CA, USA)</td><td>TF-200-R-S</td><td>Sterile</td></tr><tr><td>Filtered Pipette Tips (P1000)</td><td>Axygen (Union City, CA, USA)</td><td>TF-1000-R-S</td><td>Sterile</td></tr><tr><td>P60 Culture Dish</td><td>Corning (Corning, NY, USA)</td><td>430166</td><td>Tissue culture treated</td></tr><tr><td>48-well Plate</td><td>Corning (Corning, NY, USA)</td><td>3527</td><td>Tissue culture treated</td></tr><tr><td>Sterile Filter Paper</td><td>N/A</td><td>N/A</td><td>Fat removal and dissection support</td></tr><tr><td>DMEM/F-12 Medium</td><td>Gibco, Thermo Fisher Scientific (Waltham, MA, USA)</td><td>11320033</td><td>1:1 formulation</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco, Thermo Fisher Scientific (Waltham, MA, USA)</td><td>10082147</td><td>Heat-inactivated</td></tr><tr><td>ITS Supplement</td><td>Gibco, Thermo Fisher Scientific (Waltham, MA, USA)</td><td>41400045</td><td>100X</td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco, Thermo Fisher Scientific (Waltham, MA, USA)</td><td>35050061</td><td>100X</td></tr><tr><td>Penicillin-Streptomycin (100X)</td><td>Gibco, Thermo Fisher Scientific (Waltham, MA, USA)</td><td>15140122</td><td>Antibiotic</td></tr><tr><td>PBS 1X</td><td>Gibco, Thermo Fisher Scientific (Waltham, MA, USA)</td><td>10010023</td><td>pH 7.4</td></tr><tr><td>Collagenase Type I</td><td>Gibco, Thermo Fisher Scientific (Waltham, MA, USA)</td><td>17100-017</td><td>Tissue digestion</td></tr><tr><td>DNase I</td><td>Sigma-Aldrich (St. Louis, MO, USA)</td><td>DN25-1</td><td>Tissue digestion</td></tr><tr><td>Angiotensin II (Ang II)</td><td>Sigma-Aldrich (St. Louis, MO, USA)</td><td>A9525</td><td>Hormone stimulation</td></tr><tr><td>Adrenocorticotropic Hormone (ACTH)</td><td>Sigma-Aldrich (St. Louis, MO, USA)</td><td>A0298</td><td>Hormone stimulation</td></tr><tr><td>Forskolin</td><td>Sigma-Aldrich (St. Louis, MO, USA)</td><td>F6886</td><td>cAMP stimulation</td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma-Aldrich (St. Louis, MO, USA)</td><td>P9541</td><td>Aldosterone stimulation</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma-Aldrich (St. Louis, MO, USA)</td><td>S7653</td><td>Vehicle control</td></tr><tr><td>Small Surgical Scissors</td><td>Fine Science Tools (Foster City, CA, USA)</td><td>14060-09</td><td>Dissection</td></tr><tr><td>Scalpel with Disposable Blade</td><td>Fine Science Tools (Foster City, CA, USA)</td><td>10003-12</td><td>Microdissection</td></tr><tr><td>Fine Forceps</td><td>Fine Science Tools (Foster City, CA, USA)</td><td>11252-00</td><td>Dissection</td></tr><tr><td>Curved Forceps</td><td>Fine Science Tools (Foster City, CA, USA)</td><td>11251-10</td><td>Dissection</td></tr><tr><td>Laminar Flow Hood</td><td>Thermo Fisher Scientific (Waltham, MA, USA)</td><td>N/A</td><td>Sterile tissue processing</td></tr><tr><td>Incubator</td><td>Thermo Fisher Scientific (Waltham, MA, USA)</td><td>N/A</td><td>Cell culture</td></tr><tr><td>Centrifuge</td><td>Eppendorf (Hamburg, Germany)</td><td>N/A</td><td>Cell pelleting</td></tr><tr><td>Neubauer Counting Chamber</td><td>Hausser Scientific (Horsham, PA, USA)</td><td>1490</td><td>Cell counting</td></tr><tr><td>RIPA Lysis and Extraction Buffer</td><td>Thermo Fisher Scientific (Waltham, MA, USA)</td><td>89901</td><td>Protein extraction</td></tr><tr><td>Halt Protease Inhibitor Cocktail</td><td>Thermo Fisher Scientific (Waltham, MA, USA)</td><td>87786</td><td>Protein extraction</td></tr><tr><td>Fisherbrand Model 120 Sonic Dismembrator</td><td>Thermo Fisher Scientific (Waltham, MA, USA)</td><td>FB120</td><td>Sonication</td></tr><tr><td>RIA System</td><td>Tecan (M&amp;auml;nnedorf, Switzerland)</td><td>MG13051</td><td>Hormone quantification</td></tr><tr><td>Cobra II Auto-Gamma Counter</td><td>Berthold Technologies (Bad Wildbad, Germany)</td><td>N/A</td><td>Radioactivity measurement</td></tr><tr><td>Corticosterone ELISA Kit (96-well)</td><td>Thermo Fisher Scientific (Waltham, MA, USA)</td><td>55-CORMS-E01</td><td>Hormone measurement</td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Thermo Fisher Scientific (Waltham, MA, USA)</td><td>P36930</td><td>Mounting medium</td></tr><tr><td>Mouse anti-Dab2</td><td>BD Biosciences (San Jose, CA, USA)</td><td>610464</td><td>Primary antibody</td></tr><tr><td>Chicken anti-GFP</td><td>Aves Labs (Tigard, OR, USA)</td><td>GFP-1020</td><td>Primary antibody</td></tr><tr><td>Rabbit anti-RFP</td><td>Rockland Immunochemicals (Limerick, PA, USA)</td><td>600-401-379</td><td>Primary antibody</td></tr><tr><td>Alexa Fluor 594 Goat anti-Mouse</td><td>Invitrogen (Carlsbad, CA, USA)</td><td>A-11005</td><td>Secondary antibody</td></tr><tr><td>Alexa Fluor 594 Goat anti-Chicken</td><td>Invitrogen (Carlsbad, CA, USA)</td><td>A-11042</td><td>Secondary antibody</td></tr><tr><td>Alexa Fluor 647 Goat anti-Rabbit</td><td>Invitrogen (Carlsbad, CA, USA)</td><td>A-21244</td><td>Secondary antibody</td></tr><tr><td>Alexa Fluor 488 Goat anti-Mouse</td><td>Thermo Fisher Scientific (Waltham, MA, USA)</td><td>A-11001</td><td>Secondary antibody</td></tr><tr><td>EVOS Microscope System</td><td>Thermo Fisher Scientific (Waltham, MA, USA)</td><td>N/A</td><td>Imaging</td></tr><tr><td>TRI Reagent</td><td>Sigma-Aldrich (St. Louis, MO, USA)</td><td>T9424</td><td>RNA extraction</td></tr><tr><td>Direct-zol RNA Kit</td><td>Zymo Research (Irvine, CA, USA)</td><td>R2050</td><td>RNA purification</td></tr><tr><td>NanoDrop ND-1000</td><td>Thermo Fisher Scientific (Waltham, MA, USA)</td><td>ND-1000</td><td>RNA quantification</td></tr><tr><td>TaqMan Universal PCR Master Mix</td><td>Applied Biosystems (Foster City, CA, USA)</td><td>4304437</td><td>qPCR</td></tr><tr><td>QuantStudio 6 Flex Thermocycler</td><td>Life Technologies (Carlsbad, CA, USA)</td><td>N/A</td><td>qPCR</td></tr></tbody></table>

establishment of zone-enriched primary cultures from the mouse adrenal cortex

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/&#946;-catenin pathway markers (Wnt4, Lef1). In contrast, zF-enriched cultures exhibited higher expression of zF markers (Cyp11b1 and Akr1b7) and lower levels of Wnt/&#946;-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.

Establishment and characterization of zone-enriched primary adrenal cell cultures 
Primary adrenal cell cultures were successfully established from the OF, enriched for zG cells, and the IF, enriched for zF cells. Representative images of the cultured cells are shown in Figure 2A (OF–zG) and Figure 2B (IF–zF). To assess enrichment of zG cells and non-zG cells in these cultures, the ASCre/+:: ROSA<s...

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.

The mouse adrenal gland is encapsulated by a mesenchymal cell layer (capsule) and contains an underlying cortex organized into distinct concentric zones ...

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.

establishment-zone-enriched-primary-cultures-from-mouse-adrenal

Research

JoVE Journal

Biology

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Mouse Adrenal Chromaffin Cell Isolation

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice.  The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Culture of Mouse Neural Stem Cell Precursors

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

Establishing Primary Adult Fibroblast Cultures From Rodents

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

In this video, we describe the method to obtain a pure preparation of astrocytes from newly born mice.

Astrocyte Isolation: A Method to Obtain Pure Preparation of Mouse Cortical Astrocytes

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Isolation and Culture of Mouse Cortical Astrocytes

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

Learn to establish zone-enriched primary cultures from mouse adrenal cortex for studying zona glomerulosa and zona fasciculata cell biology. This method enables detailed investigation of adrenal zone-specific signaling and hormone production.

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

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