Culturing primary brain astrocytes under a fully controlled environment in a novel bioreactor
✍ Scribed by Sónia Sá Santos; Luís L. Fonseca; Miguel A.R. Monteiro; Manuel J.T. Carrondo; Paula M. Alves
- Publisher
- John Wiley and Sons
- Year
- 2004
- Tongue
- English
- Weight
- 150 KB
- Volume
- 79
- Category
- Article
- ISSN
- 0360-4012
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✦ Synopsis
Abstract
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 × 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. © 2004 Wiley‐Liss, Inc.