[Experimental techniques for developing new drugs acting on dementia (2)--Primary culture of mature and functional...

It has been difficult to put mature neurons of mammalian central nervous system (CNS) in vitro. Only a few reports have dealt with culture methods so far. They employed non-neuronal cell layers to make postnatal neurons attach better to a culture dish. We established a novel method for culturing mature functional hypothalamic neurons derived from 21 postnatal day rat brain without any help of non-neuronal cell layers. Nerve cells with a few processes could be isolated from sliced hypothalamic tissues by means of enzymatic and mechanical treatments. A new cell-collecting method was developed for these vulnerable cells by allowing the dissociated cell suspension to stand in a vertically held, wide-tipped syringe so that the cells were concentrated near the bottom liquid surface, from which they could be easily dropped into the medium, leaving most of the small debris in the syringe. Astrocyte conditioned medium prolonged neuronal survival, concentration dependently, for up to 28 days in vitro. This culture system may make it possible to study functions of single hypothalamic neurons.