Abstract
Chromaffin cells (CCs) are an important source of catecholamines and growth factors. Due to this fact, different neurological pathological paradigms have been approached with transplantation of CCs. Drawbacks of these studies concern problems with poor graft survival and unpredictable levels of catecholamine production. Previous in vitro studies have revealed basal and evoked release from cultures of CCs. However no basal and evoked release of catecholamines from long-term cultures of CCs have been studied. The objectives of the present study were design and develop a biocompatible platform for long-term CCs cultures and a superfusion flow chamber for the recording of basal and evoked release of catecholamines from these cells. CCs were obtained from adult bovine adrenal glands and plated (300,000 cells; n = 10) in developed micro-culture dishes with a platinum sputter-coated base. Basal (steady-state) and electrically evoked (50Hz, 100--250mA/15s) release of catecholamines from CC cultures were recorded using fast cyclic voltammetry with carbon-fiber microelectrodes at different times after plating (20d, 40d, 60d and 90d). Cell counts at each time period were made using an inverted microscope. After at least 120d, CCs were fixed and marked for TH, DBH, PNMT, and their nuclei stained. Electroactive compounds were released from the cultures at all tested intervals, although a small decrease in the detection occurred at 2 months. Electrical stimulation of the cells resulted in an additional catecholamine release (nanomolar range), which was best at stimulation intensities between 2 and 2.5 mA. Confocal microscopy of labeled cultures revealed that most of the CCs were adrenergic and noradrenergic. This study shows that viable CC cultures can be maintained for long periods and are physiologically.