Serum-free culture of rat post-natal and fetal brainstem neurons

Serum-free medium is essential for cell culture studies in which complete c ontrol of the environment is required. Primary culture of post-natal brains tem neurons in defined medium has not been described in the literature, and successful culture of primary brainstem neurons is typically restricted to embryonic ages E14-E18. This study describes a method for culture of fetal and post-natal brainstem neurons using a serum-free culture medium. The cu lture system is based on Neurobasal(TM) medium supplemented with antioxidan t-rich B27. Media and supplements are commercially available products from Life Technologies. Neuron survival was optimized by replacing glutamine wit h GlutaMaxI, by matching osmolality with neuronal age, and by using Hiberna te(TM) medium to increase neuron survival during tissue dissociation. Fetal E14, E16, E20, and post-natal P3 and P6 cultures were examined after 4, 7, and 9 days in culture. Neuron and glial cells present in the cultures were identified using immunocytochemistry with antibodies raised against microt ubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP ), respectively. Fetal E14 cultures had more bipolar neurons than multipola r neurons compared with developmentally older P6 cultures. Early fetal cult ures had a higher percentage of neurons than late fetal and early post-nata l cultures. Neuron survival was similar between 4 and 9 days in culture for all age groups tested. This is the first reliable, defined culture medium that supports brainstem neurons from late fetal and early post-natal stages of the rat for up to 6 days post-partum. (C) 2000 Elsevier Science B.V. Al l rights reserved.