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.
Martin
F C
FC
Department of Pathology, University of California, La Jolla, USA.
Wiley
C A
CA
D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D046508:Culture Techniques / Q000379:methods*; D007150:Immunohistochemistry; D007770:L-Lactate Dehydrogenase / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D009457:Neuroglia / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D011773:Pyruvates / Q000494:pharmacology; D019289:Pyruvic Acid
D000975:Antioxidants; D011773:Pyruvates; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase