The rat neuroblastoma B104 cell line, which originated in the central nervous system, was able to proliferate in the absence of serum in synthetic medium supplemented with insulin, transferrin, progesterone, selenium, and putrescine. When added individually, each supplement had little or no effect; however, in combination there was a marked synergistic effect on cell number. The cells attained the same saturation density in this medium as in medium with 10% fetal calf serum added. More extensive process formation was observed in the supplemented medium, and other differentiated properties were retained as well. Synthetic media generally require serum supplementation to support the proliferation or survival of cultured cells. The inclusion of serum, however, may significantly affect experimental reproducibility, because batch variations occur as a result of differences in donor age, sex, nutrition, and physiological state even in pooled serum samples. In addition, the complex undefined nature of serum is a complication when assessing the effect(s) of regulatory agents, such as hormones or neurotransmitters, on differentiated properties of nervous system cells in culture. This is particularly important for long-term studies, because if serum is deleted a substantial reduction of cell numbers, and in many cases complete cell death, may occur within hours or a few days.In order to circumvent these problems, several cell lines have been adapted to proliferate in serum-free media (1-4). How The B104 rat neuroblastoma, a cell line of central nervous system origin, exhibits many of the properties characteristic of differentiated neurons, such as generation of action potentials, synthesis of neurotransmitters, and presence of neurotransmitter receptors and the neuron-specific 14-3-2 protein (9, 10). Furthermore, B104 cells, like C1300 neuroblastoma cells (11, 12), respond to removal of serum by rapidly extending neurites, a phenomenon that has been correlated with the preceding neuronal properties (9).In this communication we report that B104 cells can proliferate in a serum-free synthetic medium supplemented with insulin, transferrin, progesterone, selenium, and putrescine (N2 medium). More extensive process formation is seen in N2 than in serum-supplemented medium, and other differentiated properties have been retained as well. METHODS AND MATERIALSCell Culture. The rat neuroblastoma B104 cell line was obtained from D. Schubert of the Salk Institute, La Jolla, CA. Stock cultures were maintained in a 1:1 mixture of Ham's F12 medium and the Dulbecco-Vogt modification of Eagle's medium (DME) supplemented with 5% (vol/vol) horse serum; 2.5% (vol/vol) fetal calf serum; 1.2 g of NaHCO3 per liter; 15 mM Hepes buffer; and 40 mg of penicillin, 8 mg of ampicillin, and 90 mg of streptomycin per liter. Triple-distilled water was used to prepare media. Stock cultures were grown in flasks (Falcon Plastics 3013, 25-cm2 surface area) in 4 ml of medium in a humidified atmosphere of 5% C02/95% air at 370C and subcultured every ...

Rat glial tumors, induced by injections of N-nitrosomethylurea, were plated and propagated in culture. Among a few cell strains obtained, one clone contains S-100 protein, which is unique to brain in vertebrates. Stationary-phase cultures contain approximately ten times more S-100 protein per cell than exponentially growing cells. When injected into newborn rats, cells producing S-100 grew as a glial tumor, which contained S-100 protein.

Epidermal growth factor (EGF) at 3 nM maximally inhibits the proliferation of A431 epidermoid carcinoma cells. We show that at lower concentrations, in the range of 3-100 pM, EGF has a mitogenic effect on A431 cells. In Epidermal growth factor (EGF) promotes the growth of many cell types in vitro (1-3) and inhibits proliferation of several cell types-e.g., GH4 rat pituitary tumor cells (4), A431 epidermoid carcinoma cells (5, 6), and certain human breast cancer cells (7). EGF initially binds to receptors homogeneously distributed on the cell surface. Subsequent events have been described by various investigators and include receptor phosphorylation, aggregation, internalization, and degradation in lysosomes (1). The mechanism by which these events induce DNA synthesis and cytokinesis is unknown.It has been found that at least 6-8 hr of EGF exposure are required to stimulate DNA synthesis (8). Das and Fox have suggested that EGF-induced internalization and degradation of the EGF receptor are rate-limiting factors for EGF-induced mitogenesis (9, 10), perhaps through production of a second messenger. Recent studies showed enhancement of EGF stimulation of DNA synthesis by amine compounds, which inhibited clustering of receptors in coated pits (11), and by phorbol esters, which reduced both the affinity of EGF receptors for EGF and its subsequent degradation (12)(13)(14). These results suggest that EGF stimulation of cell growth might only require the presence of EGF-EGF receptor complexes at the cell surface in contradiction to the above hypothesis.Shechter et al., on the other hand, suggested that the stimulatory effect of EGF might be mediated by small amounts of high-affinity EGF receptors, which remain at the cell surface for more than 8 hr when occupied by EGF (15). King and Cuatrecasas also have suggested that the accumulation of stable intracellular complexes between high-affinity receptors and EGF are involved in growth stimulation, but the role of these highaffinity receptors in mitogenesis remains unclear (16).A431 cells lend themselves to the study of EGF interactions with receptors because of their extremely high number of EGF receptors (1-3 x 10' per cell) (1,17,18

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