A previously described serum-free, defined medium (G2 medium) containing transferrin, selenium, hydrocortisone, biotin, fibroblast growth factor (FGF) and fibronectin developed for the growth of human and rat derived glioma cells was investigated for its ability to support proliferation of astrocytes in primary cultures of neonatal rat cerebrum. These cells were able to grow in G2 medium. Enhanced proliferation and repeated subcultivation were obtained after adding insulin and/or epidermal growth factor (EGF) to the G2 medium at concentrations of 5 μg/ml and 10 ng/ml, respectively. In these modified media (called G4 and G5 medium) astrocytes showed a higher degree of morphological differentiation as compared to serum supplemented medium. Cell type specificity was determined by immunocytochemical staining of glial fibrillary acidic (GFA) protein, which could already be demonstrated 5 days after plating cells. G4 and G5 represent the first serum-free defined media in which astrocytes proliferate and differentiate without preceding or intermediate contact to serum supplemented medium. Modification of the culture substratum by adding hyaluronic acid and chondroitin sulfate A to G4 medium (G2 medium + insulin) enhanced proliferation of astroglial cells by a factor of about 1.5. In the presence of epidermal growth factor no response to the altered culture dish surface was observed and the addition of fibronectin, otherwise a stringent plating requirement, was no longer necessary.
A serum-free defined medium has been formulated that supports proliferation and morphologic differentiation of U-251 MGsp human and C6-2BD rat glioma cells. This defined medium consists of a basal medium supplemented with transferrin, fibroblast growth factor, hydrocortisone, selenium, biotin, and fibronectin (G2 medium). When U-251 cells were plated in G2 medium on poly-D-lysine precoated dishes, their growth rate was 77% and final cell density was 82% of serum-grown counterparts. The growth rate of C6 cells in G2 medium was 67% compared to cells cultured in serum supplemented medium. Although G2 medium supported the growth of human and rat glioma cells, LA-N-1 human neuroblastoma and WI-38 human fibroblast cells showed no increase in cell number when grown in G2 medium compared to basal medium. A similar formulation (G3 medium), lacking fibroblast growth factor and hydrocortisone, supported the proliferation of RN-22 rat schwannoma cells. Morphologic differences were observed between cells grown in the presence of serum and in defined media. All three glial cell lines changed from a flattened shape in serum supplemented medium to a more spherical appearance in defined medium. In addition, both U-251 and C6 cells developed numerous processes, some reaching several cell diameters in length. These defined media will facilitate studies of the growth and differentiation of glial-derived cells.
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