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A serum‐free, pyruvate‐free medium that supports neonatal neural/glial cultures
Author(s) -
Martin F. C.,
Wiley C. A.
Publication year - 1995
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.490410212
Subject(s) - sodium pyruvate , astrocyte , lactate dehydrogenase , glial fibrillary acidic protein , chemically defined medium , synaptophysin , cell culture , biology , neural cell , neuropil , in vitro , microbiology and biotechnology , biochemistry , andrology , cell , immunology , endocrinology , medicine , immunohistochemistry , central nervous system , enzyme , genetics
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, healthyappearing neurons and very little astrocyte proliferation. Neurons stained strongly for the neuronal marker class III b̃‐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. © 1995 Wiley‐Liss, Inc.