A SERUM-FREE, PYRUVATE-FREE MEDIUM THAT SUPPORTS NEONATAL NEURAL GLIAL CULTURES

Tissue culture media with serum generally cause excessive astrocyte pr oliferation in neonatal brain cultures, and often fail to support neon atal neurons, Published serum-free media for brain cultures contain so dium pyruvate, which interferes with lactate dehydrogenase (LDH) assay s for cell death, We wanted to use neonatal neural-glial cultures in L DH assays while avoiding astrocyte proliferation, so we developed a se rum-free medium without sodium pyruvate, Our initial medium was based on that of Romijn et al, (J Neurosci Methods 23:75-83, 1988), testing selected additives, Cell survival in 8-10-day-old cultures was measure d using [4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MT T), N-acetylcysteine, citrate, superoxide dismutase, ascorbate, supple mental amino acids, and high levels of transferrin improved survival, The optimized medium supported neonatal brain cells in reaggregates or in monolayers of 400 cells/mm(2) for several weeks with large, health y-appearing neurons and very little astrocyte proliferation, Neurons s tained strongly for the neuronal marker class III beta-tubulin and the synapse marker synaptophysin. Electron microscopy of reaggregate cult ures demonstrated abundant neurons with synapses in a dense neuropil, This medium will be useful for various in vitro applications, especial ly those using LDH assays or requiring the use of neonatal cells. (C) 1995 Wiley-Liss, Inc.