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;external&#8221; curved forceps at the midline and perform a blunt dissection by creating a &#8220;Y&#8221; shape (from the level of the thigh to the subcostal angle).
	<ol>
		<li>Rinse scissors with 70% ethanol and, then, cut the muscle, also creating a &#8220;Y&#8221; shape.</li>
	</ol>
	</li>
	<li>Find the left adrenal gland behind the stomach. 
	NOTE: The right adrenal can be found deeply behind the liver.
	<ol>
		<li>Use one pair of forceps to lift the stomach or liver and isolate the adrenals with another curved forceps.</li>
	</ol>
	</li>
	<li>After putting the adrenal glands into a sterile dish, carefully remove the adrenal capsule with fine forceps.</li>
	<li>With a delicate pair of scissors, cut the adrenals into small pieces with a little bit of serum-free Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)/F-12 medium.</li>
	<li>Prepare three different pore-sized Pasteur pipets and use the first Pasteur pipet to aspirate all the adrenal pieces into a 50 mL conical tube containing medium with collagenase II (~0.5&#8211;0.8 mg/mL).
	<ol>
		<li>Gently triturate the tissue pieces about 10x. Use the rest of the pipets and repeat the above steps until all the pieces become smaller. 
		NOTE: The different pore sizes of the Pasteur pipets can be created by heating them with fire. The first pipet is the original size (about 1 mm), the pore size of the second one is about 0.8 mm, and the third Pasteur pipet is about 0.5 mm. During digestion, the pieces of adrenal will become smaller. Thus, via the mechanical procedure, the adrenals can be digested more completed. Incubate the tube in a water bath (at 37 &#176;C) and shake it every 5 min, 4x in total.</li>
	</ol>
	</li>
	<li>After the 20 min incubation, add fresh and cool medium (sixfold volume; for example, 5 mL of tissue + 25 mL of cool DMEM/F-12 medium) containing 2.5% fetal bovine serum (FBS) and 5% horse serum to stop the enzyme effect.</li>
	<li>Collect the cell pellets by centrifugation at 800 x g&#160;for 10 min.</li>
	<li>Add an appropriate volume of medium to suspend the cells. 
	NOTE: Each gland can make about ~3 x 105 to ~4 x 105 cells.</li>
	<li>Plate the cells in desired plates or dishes with growth medium (1:1 [v/v] mixture of DMEM and Ham&#8217;s F-12 medium, supplemented with 25 mM HEPES and 1% penicillin and streptomycin) overnight (day 0) at 37 &#176;C, in a humidified environment of 95% air and 5% CO2. 
	NOTE: Usually, we plate cells in 24-well plates (four glands/plate) for the corticosterone assay and in 35 mm dishes (one gland/approximately one to two dishes) for western blot analysis.</li>
	<li>Carefully wash the cells 2x with serum-free warm DMEM/F-12 medium (day 1). Replace the new growth medium.</li>
	<li>Maintain the cells at 37 &#176;C in a humidified environment of 95% air and 5% CO2 until day 3.</li>
</ol>

<ol>
	<li>Frith, C. H., Jones, T. C., Mohr, U., Hunt, R. D., Capen, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histology,+Adrenal+Gland,+Mouse.">Histology, Adrenal Gland, Mouse.</a> Endocrine System. Monographs on Pathology of Laboratory Animals. , 8-12 (1983).</li><li>Keegan, C. E., Hammer, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+organogenesis+of+the+adrenal+cortex.">Recent insights into organogenesis of the adrenal cortex.</a> Trends in Endocrinology Metabolism. 13 (5), 200-208 (2002).</li><li>Marieb, E., Wilhelm, P. B., Mallatt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chapter 17: The Endocrine System. , 558-581 (2017).</li><li>Chrousos, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hypothalamic-pituitary-adrenal+axis+and+immune-mediated+inflammation.">The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation.</a> New England Journal Medicine. 332 (20), 1351-1362 (1995).</li><li>Bornstein, S. R., Rutkowski, H., Vrezas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokines+and+steroidogenesis.">Cytokines and steroidogenesis.</a> Molecular and Cellular Endocrinology. (1-2), 135-141 (2004).</li><li>Bielohuby, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+analysis+of+the+mouse+adrenal+gland+from+weaning+to+adulthood:+time-+and+gender-dependent+alterations+of+cell+size+and+number+in+the+cortical+compartment.">Growth analysis of the mouse adrenal gland from weaning to adulthood: time- and gender-dependent alterations of cell size and number in the cortical compartment.</a> American Journal of Physiology-Endocrinology Metabolism. 293 (1), E139-E146 (2007).</li><li>van Weerden, W. M., Bierings, H. G., van Steenbrugge, G. J., de Jong, F. H., Schroder, F. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+responses+to+corticotrophin+and+cyclic+AMP+by+adult+rat+adrenocortical+cells+in+monolayer+culture.">Morphological responses to corticotrophin and cyclic AMP by adult rat adrenocortical cells in monolayer culture.</a> Journal of Endocrinology. 56 (3), 529-536 (1973).</li></ol>

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 condition is easy to control in the in vitro system. Hormones are likely to be affected by any stimulus. In vivo experiments usually result in a nonspecific reaction because animals are prone to distress. Second, the substances are not easy to monitor in the body but are liable in the cultured system. Third, the in vitro system reduces the variances between each independent experiment. Many scientific reports show a culture media of adrenocortical cells containing 10%&#8211;20% FBS12,13,14,15, whereas the protocol presented here uses 5% FBS and 2.5% horse serum to maintain the growth of adrenal cells for up to one week.
Steroidogenesis is started from the cleavage of free cholesterol by cytochrome P450 side-chain cleavage enzymes. As shown in Figure 1A,B, adrenal cells exhibit numerous and different sizes of lipid droplets, suggesting it is a good tool for the investigation of steroid biosynthesis and/or lipid biology (fasciculata cells have prominent and larger lipid droplets)1,3. The ADRP, also known as perilipin 2, is a lipid droplet surface protein which assists in the storage of neutral lipids within the lipid droplets16. Due to adrenocortical cells exhibiting an amount of lipid, the ADRP (or other lipid droplet surface proteins, like perilipin) can be used to examine whether the tested agents affect hormone synthesis16.
The growth and function of adrenal glands are tightly controlled by hypothalamic corticotropic-releasing hormone and pituitary ACTH, neuropeptides, neurotransmitters, and growth factors, etc.17. Figure 1C reveals that the corticosterone production was significantly induced by ACTH treatment in a concentration-dependent fashion. Moreover, ACTH at 1 pM and 1 nM stimulated the corticosterone production about 30- and 70-fold, respectively. It has been demonstrated that day-3 cultured primary adrenal cells exhibit a better response to ACTH stimulation15. This is also the reason why day-3 culture was used in the present study. In addition, the measurement of corticosterone by ELISA kit is also a simple and easy tool for research. Therefore, the primary cultured rat adrenal cells not only provide the platform for studying adrenal steroidogenesis but may also apply to the investigation of inflammatory-related diseases.
The critical steps in the present protocol are the digestion of the adrenals and control of contamination. Therefore, control of the digestion time, physical suction force, and observation of the tissue digestion level are very important for cell survival. Furthermore, the method of cutting the skin and isolating the adrenals is also significant. Always immerse all the instruments in 70% ethanol to prevent any contamination by animal fur.In order to obtain more cells, the suction frequency should be controlled less than ten times during each suction. Also, if the condition of the tissues is not good for further steps, the incubation in collagenase-containing medium should not be longer than 30 min.
Because the adrenal glands of rats are small, the glomerulosa layer might be lost during the peeling of the adrenal capsule. It has been demonstrated that decapsulation resulted in the removal of the connective tissue capsule and the entire zona glomerulosa18. The adrenal glands of male rats are smaller than those of female rats. Additionally, the lipid droplets appear to be larger in 11 week-old female or male mice6. Regarding the available cells, female rats may be a good choice for experiments.

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 phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde. After washing three times with PBS, the cells were blocked and permeabilized with 0.1% Triton X-100 in 5% nonfat milk. The primary ADRP antibody was diluted (1:50) in 5% nonfat milk and was incubated overnight. After three times of PBS washing, the cells were incubated with goat anti-rabbit Alexa Fluor 488 secondary antibodies (1:100). After repeating the washing steps, the cells were mounted with ProLong Gold Antifade mounting medium and were examined under the fluorescence microscope. Moreover, the hormone-producing ability of the rat adrenal cells could be determined by corticosterone assay. Stimulation of ACTH on rat adrenal cells could serve as a positive control (Figure 1C). The collected media were diluted and assayed for corticosterone content, according to the procedure described by Chen et al.11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59016/59016fig1.jpg" /> 
Figure 1: The characterization of adrenal cells and the corticosterone response to ACTH treatment. (A) Phase-contrast picture of day-3 primary cultured rat adrenal cells. (B) ADRP staining is shown. (C) Day-3 cultured rat adrenal cells were treated with ACTH (1 pM and 1 nM) for 6 h. The media were collected and stored in a -80 &#176;C refrigerator until use. Corticosterone levels were measured by enzyme-linked immunosorbent assay (ELISA). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59016/59016fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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8.0K Views.  Kaohsiung Medical University. 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.

Video: Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

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Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

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Primary Culture of Adult Rat Heart Myocytes

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Isolation and Primary Culture of Rat Hepatic Cells

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Viability Assays for Cells in Culture

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells