Serum‐free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus

Journal of Neuroscience Research - Tập 42 Số 5 - Trang 674-683 - 1995
Gregory J. Brewer1
1Southern Illinois University School of Medicine, Springfield 62794-1220, USA.

Tóm tắt

AbstractTwo fundamental questions about neuron cell culture were addressed. Can one serum‐free medium that was developed for optimum growth of hippocampal neurons support the growth of neurons from other regions of the brain? Is the region specific state of differentiation maintained in culture? To answer these questions, we isolated neurons from six other rat brain regions, placed them in culture in B27/NeurobasalTM defined medium, and analyzed their morphology and growth dependence on cell density after 4 days in culture. Neuronal identity was confirmed by immunostaining with antibodies to neurofilament 200. Neurons from each brain region maintained distinctive morphologies in culture in the virtual absence of glia. Cells isolated from embryonic day 18 cerebral cortex by digestion with papain showed the same high survival as hippocampal neurons, e.g., 70% survival for cells plated at 160/mm2. At this age and density, neurons from the septum showed slightly lower survival, 45%. Survival of dentate granule neurons from postnatal day four brains was 30‐40%, significantly lower, and relatively independent of plating density. This suggests an absence of dependence on trophic factors or contact for dentate granule neurons. Growth of cerebellar granule neurons isolated from postnatal day 7, 8, or 9 brains in B27/Neurobasal was compared to growth in BME/10% serum. Viability in serum‐free medium at 4 days was much better than that in serum, did not require KCI elevated to 25 mM, and occurred without substantial growth of glia. Cerebellar granule neurons plated at 1,280 cells/mm2 were maintained in culture for three weeks with 17 of the original cell density surviving. Survival of cells isolated from embryonic day 18 substantia nigra was 50% at 160 cells/mm2 after 4 days, similar to that of striatum, but slightly less than hippocampal neuron survival. The dopaminergic phenotype of the substantia nigral neurons was maintained over 2 weeks in culture as judged by immunoreactivity with antibodies to tyrosine hydroxylase. During this time, immunoreactivity was found in the processes as they grew out from the soma. Together, these studies suggest that B27/Neurobasal will be a useful medium for maintaining the differentiated growth of neurons from many brain regions. Potential applications of a common growth medium for different neurons are discussed. © 1995 Wiley‐Liss, Inc.

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Tài liệu tham khảo

10.1016/0006-8993(87)90766-9

10.1016/0306-4522(88)90279-5

10.1002/cne.901870302

Bjorklund A, 1983, Fluorescence histochemistry of biogenic monoamines, Handbook of Chemistry and Neuroanatomy, 1, 50

10.1016/0165-3806(87)90024-1

10.1002/jnr.490330302

Brewer GJ, 1993, Optimized survival of hippocampal neurons in B27‐supplemented Neurobasal, a new serum‐free combination, J Neurosci Res, 35, 567, 10.1002/jnr.490350513

10.1016/0304-3940(89)90458-8

Cohen G, 1983, The pathobiology of Parkinson's disease: Biochemical aspects of dopamine neuron senescence, J Neural Transm, Suppl, 19, 89

10.1111/j.1471-4159.1972.tb01431.x

Fallon JH, 1985, The Rat Nervous System, 353

10.1083/jcb.110.4.1319

Ip NY, 1993, Cultured hippocampal neurons show responses to BDNF, NT‐3, and NT‐4, but not NGF, J Neurosci, 13, 3394, 10.1523/JNEUROSCI.13-08-03394.1993

10.1016/0306-4522(93)90535-N

10.1016/0306-4522(93)90112-S

10.1016/0012-1606(92)90284-N

Seress L, 1981, Structure of the granular layer of the rat dentate gyros: A light microscopic and Golgi study, J Anat, 133, 181

10.1016/0165-3806(90)90007-L

10.1002/cne.903300305

10.1002/cne.901990207

10.1002/cne.901990208

10.1016/0006-8993(76)90506-0

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Journal of Neuroscience Research

Tập 42 Số 5

674-683

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