Culturing and characterization of astrocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss).
An access to brain cell cultures from fish would enable screening of possible neurotoxic chemicals contaminating the aquatic environment. In the present study, a protocol for a successful routine isolation and culturing of brain cells from juvenile rainbow trout was worked out. The coating material was shown to be of importance for cell proliferation. Cells grow better on a surface coated with laminin than on those coated with poly-L-lysine (PLL), poly-D-lysin (PDL) or poly-L-ornithine (PLO). The best cell growth was obtained on double-coated surfaces (PLL, PDL or PLO plus laminin). On such a culture substrate and with a seeding density of 1 x 10(7) cells/cm(2) confluence was obtained within 3-4 weeks at an incubation temperature of 18 degrees C. Approximately 95% of the cells were identified as astrocytes on the basis of a positive staining with antibodies against the astrocyte specific glial protein (GFAP). No oligodendrocytes or fibroblasts were identified in the cultures, and despite several efforts, neurons did not grow under the culture conditions used. When challenged with ligands known to awake a calcium transient in mammalian astrocytes, 44% of the cells responded to ATP with an increase in [Ca 2+](i), 38% to norepinephrine, 27% to 5-hydroxytryptamine, 7% to histamine and 6% to glutamate. Kainate, quisqualate and gamma-aminobutyric acid did not awake a calcium transient in the cells. Using a proper protocol, it is thus quite easy to get an almost pure culture of astrocyte, whereas neurones proved to very difficult to culture.
Fröjdö
E-M
EM
Department of Biology, Abo Akademi University, BioCity, Artillerigatan 6, FIN-20520, Turku/Abo, Finland.
Westerlund
J
J
Isomaa
B
B
D000818:Animals; D001253:Astrocytes / Q000166:cytology* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D017686:Oncorhynchus mykiss
D002118:Calcium