title: Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests.
abstract: Here we present a multipotent neuronal progenitor cell line for toxicity testing as an alternative to primary cultures of mixed cell types from brain tissue. The v-myc immortalised C17.2 cell line, originally cloned from mouse cerebellar neural stem cells, were maintained as monolayer in cell culture dishes in DMEM supplemented with fetal calf serum, horse serum and antibiotics. Different media and exposure scenarios were used to induce differentiation. The optimal condition which generated mixed cultures of neurons and astrocytes included serum-free DMEM:F12 medium with N2 supplements, brain-derived neurotrophic factor and nerve growth factor. The medium was changed every 3rd or 4th day to fresh N2 medium with supplements. After 7 days, the culture contained two different morphological cell types, assumed to be neurons and glia cells. The presence of astrocytes and neurons in the culture was confirmed by RT-PCR and Western blot analyses, indicating increased mRNA and protein levels of the specific biomarkers glial fibrillary acidic protein (GFAP) and βIII-tubulin, respectively. Concomitantly, the expression of the neural progenitor cell marker nestin was down-regulated.
authors.affiliation: Department of Neurochemistry, Arrhenius Laboratory for Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
authors.forename: Jessica
authors.initials: J
authors.lastname: Lundqvist
authors.forename: Johanna
authors.initials: J
authors.lastname: El Andaloussi-Lilja
authors.forename: Christina
authors.initials: C
authors.lastname: Svensson
authors.forename: Helena
authors.initials: H
authors.lastname: Gustafsson Dorfh
authors.forename: Anna
authors.initials: A
authors.lastname: Forsby
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D005904:Glial Fibrillary Acidic Protein; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D064231:Nestin / Q000235:genetics / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods*; D014404:Tubulin / Q000235:genetics / Q000378:metabolism
chemical_list: D005904:Glial Fibrillary Acidic Protein; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D012333:RNA, Messenger; D014404:Tubulin; C462941:beta3 tubulin, mouse; C528900:glial fibrillary astrocytic protein, mouse