Development of a culture system for pure rat neurons: advantages of a sandwich technique.

Primary cell cultures were derived from the cerebral cortices of embryonic rats (E 17). Survival of the cultures under serum-free conditions was improved by creating a sandwich: a poly-D-lysine-coated coverslip with plated cells was placed upside down in plastic culture dishes. Neurite outgrowth was observed within three hours after plating, and a neuronal network was established after 24 hours. The viability of the neurons gradually decreased. However, the cells could be cultivated for up to 24 days. Under these conditions the contamination with non-neuronal cells was minimized to less than 5%, as evidenced by immunohistochemical methods using the well-established cell marker proteins: neuron-specific enolase (NSE) as neuronal marker, and vimentin and glial fibrillary acidic protein (GFAP) as astroglial markers. Returning the coverslip to a normal open face position led to cell death within 24 hours. In order to investigate the maturation and differentiation of the cultured nerve cells, we looked for synapse formation by staining the synaptic vesicle protein synaptophysin (p38). It could be immunostained after three days in vitro (DIV) only in the neuronal perikarya, in perikarya and axons after six DIV, and in varicosities and contact points between axon terminals and adjacent axons or perikarya after 10-12 DIV. It appears that this simple culture method, which (i) yields highly enriched (> 95%) neuronal cultures with more than 85% cells surviving after five days in vitro, (ii) the absence of non-neuronal cells and (iii) the good maturation/differentiation of the cells, may be useful for the study of the neurochemical, physiological or regulatory mechanisms involved in nerve cell development.