Dissociation, fractionation and culture of chick embryo sympathetic ganglionic cells

Summary

Sympathetic lumbar chains from 7 to 15 day old chick embryos were dissociated with consistently greater neuronal and non-neuronal cell yields when the source tissue was collected in Ca⁺⁺, Mg⁺⁺⁻ free solutions rather than standard balanced salt solutions or culture media. The 11-day embryonic material was used for further studies on the dissociation procedure and for a quantitative analysis of nerve growth factor-supported, dispersed cell cultures derived from it. Neuronal and non-neuronal elements attached to collagen-coated surfaces with a different time course, providing a way to fractionate the sympathetic dissociate into a neuron-free population of rapidly growing cells and an over 90% pure neuronal population with overall yields of about 10000 neurons per ganglion. From unfractionated harvests, about 25% of the seeded neurons attached to the culture dish: of these, 60% developed processes within the first day, survived for several days and underwent complete reaggregation into a number of neuronal masses interconnected by fibre tracts and overlying the growing non-neuronal population. In contrast, the purified neuronal fraction, while yielding initially the same number of attached neurons, led to cultures containing fewer fibre-bearing neurons, with none of them surviving beyond 48 h. The inability of the purified neurons to survive in culture was shown to be a direct consequence of the absence of non-neuronal cells. Seeding purified neurons on increasing numbers of pre-attached non-neuronal cells led to a progressive increase of fibre-bearing, long-term surviving neurons and a progressive decline in their tendency to coalesce into neuronal reaggregates.