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.