Growth of a rat neuroblastoma cell line in serum-free supplemented medium.

Abstract

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 ...

Oligodendrocyte progenitor cells can act as cell of origin for experimental glioma

“…Functional analysis of the Ctv-a construct in OLI-neu cells The Ctv-a plasmid was transfected using FuGENE (Roche Diagnostics, Bromma, Sweden) into the CNP-positive immortalized oligodendroglial cell line OLI-neu (Jung et al, 1995) in passage 42 cultured in modified oligodendrocyte and oligodendrocyte precursor selective Sato media (Supplementary Materials and methods; Bottenstein and Sato, 1979) on plates coated with poly-L-lysine (Sigma-Aldrich, St Louis, MO, USA). Transfected cells were infected for 4 days with sterile filtered media from RCAS-eGFP-producing chicken fibroblasts (production of RCAS-derived retroviruses using DF-1 cells was described by .…”

“…The cell suspension was peletted, resuspended and seeded on plates. Neural/glial progenitor cells were plated on dishes coated with polyornithine and fibronectin in N2 media (Bottenstein and Sato, 1979) with addition of 10 ng/ml basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, NJ, USA). OPCs were plated in dishes coated with poly-L-lysine (Sigma-Aldrich) or polyornithine and fibronectin in Sato media with addition of 10 ng/ml bFGF and 10 ng/ml PDGF-AA (PeproTech).…”

Initiation of Oligodendrocyte Progenitor Cell Migration by a PDGF-A Activated Extracellular Regulated Kinase (ERK) Signaling Pathway

“…Approximately 7-10 days after plating, the OP cells and microglia were dislodged by shaking the flasks, followed by plating on non-coated tissue culture plastic for 25-40 min to allow the differential adhesion of microglial cells. The cell suspension was collected, concentrated and resuspended in Sato defined medium with high insulin [16].…”

Incorporation of sodium channel blocking and free radical scavenging activities into a single drug, AM‐36, results in profound inhibition of neuronal apoptosis

“…Stimulation was carried out for 1 h in N2-supplemented Neurobasal TM medium containing 100 u ml 71 penicillinstreptomycin, 0.25% bovine serum albumin, 83 mM D(+)-galactose, 16 mM ethanolamine, 6 mM L-carnitine, 0.4 mM biotin, 500 mM L-glutamine and 25.4 mM KCl (Bottenstein et al, 1979). Cultures were then washed and left for 16 ± 18 h in N2-supplemented Neurobasal TM medium.…”