This work was supported by a grant from the National Science Council Taiwan (NSC102-2320-B-037-008-MY3) and a National Cheng-Kung University Hospital Research Grant (NCKUH-10102045).

All the procedures including animal subjects have been approved by Institutional Animal Care and Use Committee of Kaohsiung Medical University.
1. Experimental Procedure
<ol>
	<li>Sacrifice the adult (8&#160;to 12 week-old) female/male SD rats by CO2 euthanasia.</li>
	<li>Apply 70% ethanol and let it be absorbed into the skin, and then wipe the skin with a tissue paper.</li>
	<li>Put the rat on a clean plastic plate in the supine position.</li>
	<li>Lift the skin with &#8220;exter...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenal+glands+of+mouse+and+rat+do+not+synthesize+androgens.">Adrenal glands of mouse and rat do not synthesize androgens.</a> Life Sciences. 50 (12), 857-861 (1992).</li><li>Wang, T., Rainey, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adrenocortical+carcinoma+cell+lines.">Human adrenocortical carcinoma cell lines.</a> Molecular and Cellular Endocrinology. 351 (1), 58-65 (2012).</li><li>Payne, A. H., Hales, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+steroidogenic+enzymes+in+the+pathway+from+cholesterol+to+active+steroid+hormones.">Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones.</a> Endocrine Review. 25 (6), 947-970 (2004).</li><li>Ruggiero, C., Lalli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+ACTH+Signaling+on+Transcriptional+Regulation+of+Steroidogenic+Genes.">Impact of ACTH Signaling on Transcriptional Regulation of Steroidogenic Genes.</a> Frontiers in Endocrinology. 7, 24 (2016).</li><li>Chen, Y. C., Liang, Y. L., Huang, Y. L., Huang, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Toona+sinensis-stimulated+adrenal+steroidogenesis+in+primary+rat+adrenal+cells.">Mechanism of Toona sinensis-stimulated adrenal steroidogenesis in primary rat adrenal cells.</a> Journal of Functional Foods. 14 (2015), 318-323 (2015).</li><li>Rybak, S. M., Ramachandran, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+normal+rat+adrenocortical+cells.+I.+Culture+conditions+for+optimal+growth+and+function.">Primary culture of normal rat adrenocortical cells. I. Culture conditions for optimal growth and function.</a> In Vitro. 17 (7), 599-604 (1981).</li><li>Rybak, S. M., Ramachandran, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+normal+rat+adrenocortical+cells.+II.+Quantitation+of+steroid+dehydrogenase+stain.">Primary culture of normal rat adrenocortical cells. II. Quantitation of steroid dehydrogenase stain.</a> In Vitro. 17 (7), 605-611 (1981).</li><li>O'Hare, M. J., Neville, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+metabolism+by+adult+rat+adrenocortical+cells+in+monolayer+culture.">Steroid metabolism by adult rat adrenocortical cells in monolayer culture.</a> Journal of Endocrinology. 58 (3), 447-462 (1973).</li><li>Di Blasio, A. M., Fujii, D. K., Yamamoto, M., Martin, M. C., Jaffe, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+cell+proliferation+and+steroidogenesis+in+cultured+human+fetal+adrenal+cells+chronically+exposed+to+adrenocorticotropic+hormone:+rationalization+of+in+vitro+and+in+vivo+findings.">Maintenance of cell proliferation and steroidogenesis in cultured human fetal adrenal cells chronically exposed to adrenocorticotropic hormone: rationalization of in vitro and in vivo findings.</a> Biology of Reproduction. 42 (4), 683-691 (1990).</li><li>Brasaemle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thematic+review+series:+adipocyte+biology.+The+perilipin+family+of+structural+lipid+droplet+proteins:+stabilization+of+lipid+droplets+and+control+of+lipolysis.">Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis.</a> Journal of Lipid Research. 48 (12), 2547-2559 (2007).</li><li>Hoeflich, A., Bielohuby, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+adrenal+gland+growth:+signal+integration+by+extracellular+signal+regulated+kinases1/2.">Mechanisms of adrenal gland growth: signal integration by extracellular signal regulated kinases1/2.</a> Journal of Molecular Endocrinology. 42 (3), 191-203 (2009).</li><li>O'Hare, M. J., Neville, A. 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The authors have nothing to disclose.

Adrenal glands play a key role in environmental adaptation3. The hormone by which the adrenal cortex secretes can regulate and adjust physiological functions and homeostasis3. The in vivo&#160;model reflects the real physiological effects in the body. However, it is still influenced by many complex factors, causing unstable effects in experiments. In contrast, the in vitro cell model has advantages which makes it incomparable to the animal model. First, the environmental co...

The endocrine system is responsible for regulating physiological activities and homeostasis1. Adrenal glands located at the cranial pole of the kidneys are one of the major endocrine organs which secrete mineralocorticoids, glucocorticoids, and androgen2,3. There are two distinct parts of the adrenal gland: the cortex and the medulla. The adrenal cortex consists of three layers: the outer glomerulosa, the intermediate fasciculata, and the inner reticularis3. The zona glomerulosa is the original secreting site of aldosterone, a mineralocorticoid, which aids in reabsorbing sodium ion and water in the kidneys3. The zona fasciculata primarily produces a basal level of glucocorticoids in normal physiological conditions3. Corticosteroids in rodents and cortisol in humans act to assist the body in coping with stress by regulating blood glucose3. To some extent, they can inhibit inflammatory responses and regulate the immune system4,5. In contrast to other mammals, mice and rats do not have a functional zona reticularis due to the lack of a 17&#945;-hydroxylase expression in the adrenal gland6,7. Thus, adrenals from mice and rats are devoid of the secretion of adrenal C-19 steroids (cortisol and adrenal androgens).
Primary cultured adrenocortical cells have been shown to be useful for investigating mechanisms controlling the adrenal physiology8. Like other steroidogenic tissues, each adrenal zone synthesizes its steroids from free cholesterol9. Upon adrenocorticotropic hormone (ACTH) stimulation, the free cholesterol is released from breakdown lipid droplets and, thus, enhances steroid production in the fasciculata and reticularis10.
Many groups have attempted to establish stable cell lines from adrenocortical carcinomas. However, several concerns have limited the use of adrenocortical cell lines as in vitro models8. The steroid responses of cell lines may differ between passages, the lot number of serum, and the quality of the serum, etc. Moreover, commercial adrenal cell lines (Y1 and SW13) do not secrete corticosterone, making it more difficult to study adrenal steroidogenesis8. Using bovine and equine adrenals as ex vivo models may be a good choice, even though tissues from these markets may obscure some unknown risks. Compared to them, managing rat adrenal glands is easier and the source is relatively more stable and pathogen-free. Taken together, the present method described here could be used to investigate the related mechanism of corticosterone synthesis in rat adrenal fasciculata cells.

<table>
	<tbody>
		<tr>
			<td>female/ male, SD/Wistar rats (8-12 wk)</td>
			<td>BioLASCO</td>
			<td></td>
			<td>male or femal rats are suitable for experiments. Female&#39;s adrenal glands are larger than male rats, however, female rats own the oestrous cycles that may influence the experiments</td>
		</tr>
		<tr>
			<td>collagenase, type II</td>
			<td>Sigma</td>
			<td>C-6885</td>
			<td>isolation of adrenal cells</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td>12800-017</td>
			<td>powder media for adrenal cells</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>ThermoFisher</td>
			<td>21700-075</td>
			<td>powder media for adrenal cells</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>JT.baker</td>
			<td>4018</td>
			<td>buffer (powder)</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>JT.baker</td>
			<td>3506-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Hyclone</td>
			<td>16050014</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>SAFC</td>
			<td>12103C</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin (10,000 U)-streptomycin (10,000 ug/ml)</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td>antibiotics</td>
		</tr>
		<tr>
			<td>curved forceps</td>
			<td>BRAUN</td>
			<td>BD343R</td>
			<td></td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>BRAUN</td>
			<td>BD331R</td>
			<td></td>
		</tr>
		<tr>
			<td>scissor</td>
			<td>BRAUN</td>
			<td>BC224R</td>
			<td>cut rat skin and muscle</td>
		</tr>
		<tr>
			<td>delicate scissor</td>
			<td>BRAUN</td>
			<td>BC101R</td>
			<td>cut adrenal glands</td>
		</tr>
		<tr>
			<td>adipose-differentiation related protein</td>
			<td>Abcam</td>
			<td>ab108323</td>
			<td>antibody for lipid droplet associated protein</td>
		</tr>
		<tr>
			<td>corticosterone ELISA kit</td>
			<td>cayman</td>
			<td>500655</td>
			<td>assay for corticosterone synthesis</td>
		</tr>
		<tr>
			<td>inverted light microscopy</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescence microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>JT.baker</td>
			<td>X198-07</td>
			<td>for immunofluorescence staining</td>
		</tr>
		<tr>
			<td>goat anti-rabbit IgG Alexa 488 Fluor secondary antibody</td>
			<td>ThermoFisher</td>
			<td>A-11034</td>
			<td>for immunofluorescence staining</td>
		</tr>
		<tr>
			<td>anti-ADFP</td>
			<td>Abcam</td>
			<td>108323</td>
			<td>for immunofluorescence staining</td>
		</tr>
		<tr>
			<td>ProLong Gold antifade mountant</td>
			<td>ThermoFisher</td>
			<td>P36935</td>
			<td>for immunofluorescence staining</td>
		</tr>
	</tbody>
</table>

primary culture of rat adrenocortical cells and assays of steroidogenic functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

Using the procedure described here, primary cultured adrenal cells could be distinguished under a phase-contrast microscope (Figure 1A). To further confirm that the vesicles within the cytoplasm are lipid droplets, the immunofluorescence staining of adipose differentiation-related protein (ADRP) could be conducted on day-3 cultures (Figure 1B). Cells were grown on the glass coverslips. The cells were washed three times with phosp...

Watch this Scientific Journal Video about Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions at JoVE.com

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

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.

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

The presented protocol is very easy and it will help researchers prepare primary adrenal cultures. It provides an easy procedure for starting adrenal steroidogenesis. This technique may be applied on screening the stress-related diseases, all the potentials of the agent-inhibited inflammatory responses.Demonstrating the procedure will be Wei-Chang Lee, a Masters student from my laboratory. Begin by sterilizing euthanized SD rat using 70%ethanol and letting it be absorbed into the skin. After wiping the skin with the tissue paper, place the rat on a clean plastic plate in the supine position.Next, use external curved forceps to lift the skin at the midline. Use scissors to perform a blunt dissection of the skin by creating a Y shape from the level of the thigh to the subcostal angle. After rinsing the scissors with 70%ethanol, cut the muscle, also creating a Y shape.Locate the left adrenal gland behind the stomach in the right behind the liver. With a pair of standard forceps and a pair of curved forceps, lift the stomach and liver and then isolate the adrenals with another curved forceps and place them into a sterile dish. Use fine forceps to carefully move the adrenal capsule from each gland.Now, use a delicate pair of scissors to cut the adrenals into small pieces in enough amount of serum-free DMEM F-12 medium to cover the tissue. Prepare three different pour size pasteur pipettes by heating them over a burner. Use the first pipette with the largest opening to transfer all the adrenal pieces into a 50mL conical tube containing medium with Collagenase II.Gently Pipette the tissue pieces up and down about 10 times.Then incubate the tube in a water bath at 37 degrees Celsius for twenty minutes, and shake it every five minutes, four times in total. It is very critical to properly perform tissue dispersion during enzyme digestion. Use the second and then the smallest pour-size pipette to repeat pipetting and incubation until all the tissue pieces become smaller.Then add sixfold volume of bresh in four degrees Celsius cold medium to stop the enzymatic reaction. Then centrafuge at 800 times G for 10 minutes, and discard the supernatant both times. Add an appropriate volume of medium, and re-suspend the cells.Plant the cells in desired plates or dishes with grown medium overnight at 37 degrees Celsius in 95%humidity and 5%carbon dioxide. On the following day, carefully wash the cells twice with serum-free, warm DMEM F-12 medium. Add fresh growth medium and maintain the cells at 37 degrees Celsius and 95%humidity and 5%carbon dioxide until Day 3.Using this protocol, primary cultured rat adrenal cells were obtained. Day 3 adrenal cells were confirmed under a phase contrast microscope. Immunofluorescent staining of adipose differentiation related protein performed on day 3 cultures confirmed that vesicles within the cytoplasm are lipid droplets.After adrenocorticotropic hormone stimulation of rat adrenal cells, hormone-producing ability of the cells was determined by corticosterone assay, showing increased corticosterone release with higher ACTH concentration. This method can be applied to other primary culture systems, such as primary cardiac cultures, adrenal medulla cultures, et cetera. This technique may also help to investigate the interaction between adrenocortical cells and chromaffin cells.

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7.8K Views.  Kaohsiung Medical University. The presented protocol is very easy and it will help researchers prepare primary adrenal cultures. It provides an easy procedure for starting adrenal steroidogenesis. This technique may be applied on screening the stress-related diseases, all the potentials of the agent-inhibited inflammatory responses.Demonstrating the procedure will be Wei-Chang Lee, a Masters student from my laboratory. Begin by sterilizing euthanized SD rat using 70%ethanol and letting it be absorbed into the skin....