Human, rat and bovine adrenal chromaffin cell isolation and culture
Human adrenal glands were obtained from organ donors from three hospitals in Madrid (12 de Octubre, Fundación Jiménez Díaz and Clínico San Carlos). Written consent was signed by the relatives of donors and the use of human tissue was approved by the Universidad Autónoma de Madrid´s institutional Ethics Committee and by the review boards of each of the hospitals. The age of the male donors was 68.7 ± 3.3 (n=3) years and 53.3 ± 10.7 (n=3) years old for the female donors. Human chromaffin cells were isolated and cultured according to procedures described by Hone and colleagues (2015) and were maintained in culture for up to 7 days. Bovine adrenal glands were obtained from a local abattoir, and chromaffin cells were isolated using the method reported by Moro and colleagues (1990).
Rat adrenal glands were harvested from male PD30-60 Sprague Dawley rats of six weeks old. All animal care and experimental procedures were approved by the Universidad Autónoma de Madrid´s institutional Ethics Committee. The total number of animals as well as their suffering was minimized according to the 3R initiative and the principles of ARRIVE. Adrenal medulla were dissected from the adrenal glands and cut into 5-7 pieces using fine iridectomy scissors. The medullary pieces were then incubated in 2 ml of a saline solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 5.6 mM D-glucose, and 5 mM HEPES; pH was adjusted to 7.4 with NaOH) containing 0.25% (vol/vol) Trypsin (Sigma, Aldrich) and 0.1% (wt/vol) collagenase type I for 60 min at 37o C. After enzymatic digestion, the cells were triturated with a glass Pasteur pipette to obtain a single cell suspension after which 8 ml of saline was added. The cells were centrifuged at 200×g for 10 min, the supernatant aspirated, and the cells suspended in 200-300 μl of culture medium composed of DMEM, 500 μM Glutamax, 10% FBS, penicillin/streptomycin and plated on glass coverslips that had been treated with poly-D-lysine. Cells were maintained in DMEM culture medium at 37o C in an incubator under an atmosphere of 95% air and 5% CO2.Half of the rat chromaffin cells coverslips were treated with nicotine 110 nM from one day after the culture was performed. Experiments were conducted within 4 days after isolation.