Cultures of rat astrocytes challenged with a steady supply of glutamate: new model to study flux distribution in the glutamate-glutamine cycle.

Glutamate metabolism in astrocytes was studied using an experimental setup that simulates the role of neurons (glutamate producers and glutamine consumers) by the addition of glutaminase to the culture medium. Thereby, a steady supply of glutamate was imposed at the expense of glutamine, and the stress intensity was manipulated by changing the glutaminase concentration. Glutamate supply rates in the range 8-23 nmol/min/mg protein were examined for periods of up to 48 h. When the glutamate supply rate exceeded the uptake rate of this amino acid, a transient increase in the extracellular concentration of glutamate was observed. In response to this stress, the fluxes through the glutamate transporter and glutamine synthetase were increased considerably, and the extracellular concentration of glutamate was eventually restored to a low level. The increased levels of glutamine synthetase were demonstrated by immunoblotting analysis. The effect on glutamate metabolism of the transaminase inhibitor, aminooxyacetic acid (AOAA), and of NH4Cl was also investigated. The supply of glutamate caused a concomitant reduction in the levels of phosphocreatine, phosphoethanolamine, and phosphocholine without affecting the ATP pool. Glutamine synthetase was shown to be is a key element in the control of glutamate metabolism in astrocytic cultures. The metabolic fate of glutamate depends greatly on the time of endurance to the challenge: in naive cells, glutamate was primarily metabolized through the transaminase pathway, while in well-adapted cells glutamate was converted almost exclusively through glutamine synthetase.

Culturing primary brain astrocytes under a fully controlled environment in a novel bioreactor.

We report the first approach for growth and maintenance of primary astrocytes on a fully controlled environment. For this purpose, cells were immobilized in Cytodex microcarriers and grown in a stirred tank bioreactor. The distribution of astrocytes at the microcarrier surface was visualized using confocal microscopy and glial fibrillary acidic protein (GFAP) labeling, a specific glial probe. Crucial bioreaction parameters such as agitation rate, microcarrier type, and concentration, as well as cell inoculum concentration were assessed. Cytodex 3 proved the best microcarrier for astrocyte growth, with the highest cell densities obtained for 6 g/l of Cytodex 3 using an inoculum of approx. 0.15 x 10(6) cells/ml in vessels operated at 60 rpm, using a refeed operational mode consisting of complete medium replacement every 5 days. Using such optimized conditions, cells were maintained in steady-state for approximately 24 days, allowing online monitoring and control of environmental variables such as temperature, pH, and O(2). To test further the advantages of this fully controlled system, astrocytes were also subjected to hypoxic stress for 5 hr; the cell number was not affected by hypoxia but the glycolytic flux was enhanced during the stress imposed. The culture system described is a novel tool to study brain cell metabolism, allowing sampling over time and the monitoring of cellular behavior through stressful conditions and during recovery.