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.
Wang
X F
XF
Brain Research Centre, University of British Columbia and Vancouver Hospital and Health Sciences Centre, 2550 Willow Street, Vancouver, BC, Canada. xfwang@ecc.ubc.ca
Cynader
M S
MS
D000818:Animals; D001253:Astrocytes / Q000502:physiology*; D002448:Cell Adhesion; D002450:Cell Communication; D002470:Cell Survival; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000196:embryology; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D051381:Rats
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free