Effects of astrocytes on neuronal attachment and survival shown in a serum-free co-culture system.

In order to study the neurosupportive effects of glial cells, we optimized a glial-neuron non-contact co-culture method. Astrocyte conditioned medium (ACM) and an astrocyte feeder layer were used to promote neuronal attachment and neuronal survival respectively. Neuron-enriched cultures were prepared from cortices of E-18 day rat embryos. Instead of plating cells in serum-supplemented medium, as an indispensable first-step procedure for many serum-free culture protocols, we found that coating the coverslips briefly with ACM was sufficient for the healthy attachment and neurite outgrowth of the dissociated neurons in serum-free medium. A high survival rate of the low density (4x10(4) cells/cm(2)) neuronal cultures was achieved by co-culturing primary neurons with an astrocyte feeder layer. This non-contact co-culture method could be easily implemented with ordinary culture dishes. Our serum-free chemically defined medium was MEM supplemented with insulin, transferrin, selenium and pyruvate. In this serum-free medium, glial cells did not proliferate and a neuron-enriched population was obtained without the need for mitotic inhibitors. Our experimental results reveal a critical role for astrocytes in neuronal attachment and growth. This method can be used to study glial-neuron interactions as well as culturing low-density population of pure neurons.