The rat neuroblastoma B104 cell line, derived from the central nervous system, can proliferate in serum-free synthetic medium supplemented with insulin, transferrin, progesterone, selenium, and putrescine (N2 medium). When added individually, these supplements have little effect, but in combination, they synergistically promote cell growth. The cells reach the same saturation density as in medium with 10% fetal calf serum. In N2 medium, more extensive process formation is observed, along with other differentiated properties. Synthetic media typically require serum for cell proliferation, but serum can affect experimental reproducibility due to batch variations. Serum also complicates the assessment of regulatory agents on nervous system cells. To avoid these issues, several cell lines have been adapted to grow in serum-free media, but adaptation is time-consuming and often results in loss of physiological properties. An alternative approach is to supplement serum-free media with hormones and growth factors, allowing cells to continue growing without lag. The B104 cells, like other neuroblastoma cells, respond to serum removal by extending neurites, a phenomenon linked to their neuronal properties. In this study, B104 cells were shown to proliferate in N2 medium. The growth of B104 cells in N2 was compared to serum-supplemented medium, and the results showed that N2 supported similar growth and differentiation. The study also found that selenium was cytotoxic at concentrations above 30 nM, while progesterone at 20 nM stimulated growth, and testosterone and β-estradiol were inhibitory. The morphology of B104 cells in N2 was different from those in serum-supplemented medium, with more extended neurites. The findings suggest that serum-free medium supplemented with specific factors can support the growth and differentiation of neuroblastoma cells. This has important implications for neurobiological research, as it allows for more controlled studies of neuronal cells. The study also highlights the importance of understanding the role of various factors in cell growth and differentiation.The rat neuroblastoma B104 cell line, derived from the central nervous system, can proliferate in serum-free synthetic medium supplemented with insulin, transferrin, progesterone, selenium, and putrescine (N2 medium). When added individually, these supplements have little effect, but in combination, they synergistically promote cell growth. The cells reach the same saturation density as in medium with 10% fetal calf serum. In N2 medium, more extensive process formation is observed, along with other differentiated properties. Synthetic media typically require serum for cell proliferation, but serum can affect experimental reproducibility due to batch variations. Serum also complicates the assessment of regulatory agents on nervous system cells. To avoid these issues, several cell lines have been adapted to grow in serum-free media, but adaptation is time-consuming and often results in loss of physiological properties. An alternative approach is to supplement serum-free media with hormones and growth factors, allowing cells to continue growing without lag. The B104 cells, like other neuroblastoma cells, respond to serum removal by extending neurites, a phenomenon linked to their neuronal properties. In this study, B104 cells were shown to proliferate in N2 medium. The growth of B104 cells in N2 was compared to serum-supplemented medium, and the results showed that N2 supported similar growth and differentiation. The study also found that selenium was cytotoxic at concentrations above 30 nM, while progesterone at 20 nM stimulated growth, and testosterone and β-estradiol were inhibitory. The morphology of B104 cells in N2 was different from those in serum-supplemented medium, with more extended neurites. The findings suggest that serum-free medium supplemented with specific factors can support the growth and differentiation of neuroblastoma cells. This has important implications for neurobiological research, as it allows for more controlled studies of neuronal cells. The study also highlights the importance of understanding the role of various factors in cell growth and differentiation.