A serum-free, pyruvate-free medium that supports neonatal neural/glial cultures

Tissue culture media with serum generally cause excessive astrocyte proliferation in neonatal brain cultures, and often fail to support neonatal neurons. Published serum-free media for brain cultures contain sodium pyruvate, which interferes with lactate dehydrogenase (LDH) assays for cell death. We wanted to use neonatal neural-glial cultures in LDH assays while avoiding astrocyte proliferation, so we developed a serum-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 measured using 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). N-acetylcysteine, citrate, superoxide dismutase, ascorbate, supplemental amino acids, and high levels of transferrin improved survival. The optimized medium supported neonatal brain cells in reaggregates or in monolayers of 400 cells/mm2 for several weeks with large, healthy-appearing neurons and very little astrocyte proliferation. Neurons stained strongly for the neuronal marker class III beta-tubulin and the synapse marker synaptophysin. Electron microscopy of reaggregate cultures demonstrated abundant neurons with synapses in a dense neuropil. This medium will be useful for various in vitro applications, especially those using LDH assays or requiring the use of neonatal cells.