Properties of neurons from dissociated fetal rat brain in serum-free culture

Abstract

Because of the unknown constituents and varying composition of serum, its presence in media used in cell culture unavoidably compromises attempts to study cellular mechanisms of growth and differentiation. To overcome this, we have devised a serum-free, chemically defined medium which maintains primary cultures of fetal rat brain cells for more than 6 weeks. This medium allows expression of characteristic properties of neurons and prevents overgrowth of non-neuronal elements without use of antimitotic agents. Cells prepared and plated without exposure to serum attach in less than 20 min to poly-D-lysine substratum and begin to extend processes within 1 hr. After 2 days in culture, process-bearing cells can be divided into those with characteristic neuronal morphology, including long processes which generally branch at a distance from the perikaryon, and those having the appearance of glial cells with many short, thin processes which branch frequently near the cell body. The remaining non-neuronal cells are large and flat with few or no processes. The presence of neurons and astroglia was demonstrated by immunofluorescence detection of bound tetanus toxin as a neuron-specific surface marker, and glial fibrillary acidic protein as an astroglial marker. By the 3rd day in culture, many cells of neuronal morphology were able to generate action potentials in response to electrical stimulation. The ionic composition of the inward current changes from Ca2+ to predominantly Na+ by about 10 days in culture. The presence of synaptic vesicles and myelin was demonstrated by electron microscopy. The ability of dissociated cells from mammalian brain to grow in defined medium without serum and acquire selected properties of mature cells in vivo demonstrates the potential of this culture system for neurobiological studies at the cellular level.