Adaptation of C6 glioma cells to serum-free conditions leads to the expression of a mixed astrocyte-oligodendrocyte phenotype and increased production of neurite-promoting activity.
C6 cells were adapted to proliferate in defined culture medium to allow the study of long-term effects of serum-free growth conditions on their phenotypic antigen expression and production of neurite promoting factors (NPFs). Cultures were grown in either Ham's F-12 or supplemented Opti-MEM-I containing 15% heat-inactivated horse serum and 2.5% fetal calf serum (serum-containing) or in supplemented Opti-MEM-I alone (serum-free). Immunocytochemical and immunofluorescence techniques were used to determine the antigenic expression of A2B5, galactocerebroside (GalC), and glial fibrillary acidic protein (GFAP) in passage matched and sister cultures of serum and serum-free grown C6 cells. When C6 cells were grown under serum-containing conditions, two populations of cells were seen: young oligodendrocytes (A2B5+, GFAP-, GalC+), and mixed astrocyte-oligodendrocyte phenotype (A2B5+, GFAP+, GalC+). After adaptation of the C6 cells to serum-free conditions over 2-3 passages, only one population of cells was observed, the mixed astrocyte-oligodendrocyte phenotype. The serum-free conditions also resulted in greater staining of the C6 cells. Conditioned media from the two growth conditions were fractionated by ultrafiltration into two fractions: components > 50 kDa and components of 10-50 kDa. The amount of neurite promoting activity seen between the two culture conditions resulted in a 3-fold increase in NPF activity under serum-free conditions in the > 50 kDa fraction. The 10-50 kDa fraction only expressed NPF activity if obtained from the serum-grown C6 cells. This alteration in NPF activity appears to be the result of the phenotypical alteration of the C6 cells, and may suggest that the NPF activities from the two culture conditions may not be identical.
Coyle
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Department of Anesthesia, University of Cincinnati College of Medicine, Ohio 45267-0531, USA.
D001253:Astrocytes / Q000166:cytology*; D002455:Cell Division / Q000235:genetics; D016895:Culture Media, Serum-Free; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000502:physiology*; D005910:Glioma; D007150:Immunohistochemistry; D016501:Neurites / Q000502:physiology*; D009836:Oligodendroglia / Q000166:cytology*; D010641:Phenotype; D014407:Tumor Cells, Cultured / Q000502:physiology
D016895:Culture Media, Serum-Free