OPTIMIZED SURVIVAL OF HIPPOCAMPAL-NEURONS IN B27-SUPPLEMENTED NEUROBASAL(TM), A NEW SERUM-FREE MEDIUM COMBINATION

We have systematically optimized the concentrations of 20 components o f a previously published serum-free medium (Brewer and Cotman, Brain R es 494: 65-74, 1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum-free medium supplement, B27, prod uced neuron survival above 60%, independent of plating density above 1 60 plated cells/mm2. For isolated cells (<100 cells/mm2), survival at 4 days was still above 45%, but could be rescued to the 60% level at 4 0 cells/mm2 by simply applying a coverslip on top of the cells. This s uggests a need for additional trophic factors. High survival was achie ved with osmolarity lower than found in Dulbecco's Modified Eagle's Me dium (DMEM), and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrous sulphate found in DME/F12. Neurob asal(TM) is a new medium that incorporates these modifications to DMEM . In B27/Neurobasal, glial growth is reduced to less than 0.5% of the nearly pure neuronal population, as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase. Excellent long-term viability is achieved after 4 weeks in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% v iability for cells plated at 160/mm2. Since the medium also supports t he growth of neurons from embryonic rat striatum, substantia nigra, se ptum, and cortex, and neonatal dentate gyrus and cerebellum (Brewer, i n preparation), support for other neuron types is likely. B27/Neurobas al should be useful for in vitro studies of neuronal toxicology, pharm acology, electrophysiology, gene expression, development, and effects of growth factors and hormones. (C) 1993 Wiley-Liss, Inc.