Primary culture of postnatal rat hypothalamic neurons in astrocyte-conditioned medium

Mature functional hypothalamic neurons of male and female rats (21-day postnatal) were successfully cultured without attachment to non-neuronal cells in serum-free astrocyte-conditioned medium (ACM). A novel cell-collecting method was designed 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 lower liquid surface, from which position they could be easily dropped into the medium, leaving most of the small debris in the syringe. This method made it possible to study statistically the survival of cultured neurons. It was impossible to collect many viable cells by the commonly used dissociating technique for fetal rat brain. However, neuron-like cells with a few processes could be isolated from sliced hypothalamic tissues by means of enzymatic and mechanical treatments. The original processes disappeared within 1-2 days and some new processes were generated after 2-3 days in vitro; the cells survived for 28 days in vitro. The cells were identified as neurons by the immunostaining method for microtubule-associated protein 2 (MAP2) and neurofilament (NF). Such neurons were obtained from every site of hypothalamic tissue sampled. These phenomena were not observed in chemically-defined medium (CDM), CDM supplemented with basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) or nerve growth factor (NGF).