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.
