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J Neurosci Methods. 2011 Apr 15;197(1):118-27. doi: 10.1016/j.jneumeth.2011.02.011. Epub 2011 Feb 21.

Influence of rat substrain and growth conditions on the characteristics of primary cultures of adult rat spinal cord astrocytes.

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1
Department of Neuroscience, Karolinska Institutet, SE-17177 Stockholm, Sweden. simone.codeluppi@ki.se

Abstract

Primary astrocyte cell cultures have become a valuable tool for studies of signaling pathways that regulate astrocyte physiology, reactivity, and function; however, differences in culture preparation affect data reproducibility. The aim of this work was to define optimal conditions for obtaining primary astrocytes from adult rat spinal cord with an expression profile most similar to adult human spinal cord astrocytes. Hence, we examined whether different Sprague-Dawley substrains and culture conditions affect astrocyte culture quality. Medium supplemented with fetal bovine serum from three sources (Sigma, Gibco, Hyclone) or a medium with defined composition (AM medium) was used to culture astrocytes isolated from spinal cords of adult Harlan and Charles River Spraque-Dawley rats. Purity was significantly different between cultures established in media with different sera. No microglia were detected in AM or Hyclone cultures. Gene expression was also affected, with AM cultures expressing the highest level of glutamine synthetase, connexin-43, and glutamate transporter-1. Interestingly, cell response to starvation was substrain dependent. Charles River-derived cultures responded the least, while astrocytes derived from Harlan rats showed a greater decrease in Gfap and glutamine synthetase, suggesting a more quiescent phenotype. Human and Harlan astrocytes cultured in AM media responded similarly to starvation. Taken together, this study shows that rat substrain and growth medium composition affect purity, expression profile and response to starvation of primary astrocytes suggesting that cultures of Harlan rats in AM media have optimal astrocyte characteristics, purity, and similarity to human astrocytes.

PMID:
21345349
DOI:
10.1016/j.jneumeth.2011.02.011
[Indexed for MEDLINE]

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