SERUM-FREE B27/NEUROBASAL MEDIUM SUPPORTS DIFFERENTIATED GROWTH OF NEURONS FROM THE STRIATUM, SUBSTANTIA-NIGRA, SEPTUM, CEREBRAL-CORTEX, CEREBELLUM, AND DENTATE GYRUS
SERUM-FREE B27/NEUROBASAL MEDIUM SUPPORTS DIFFERENTIATED GROWTH OF NEURONS FROM THE STRIATUM, SUBSTANTIA-NIGRA, SEPTUM, CEREBRAL-CORTEX, CEREBELLUM, AND DENTATE GYRUS
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DOI：
10.1002/jnr.490420510
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发表时间：
1995-12-01
影响因子：
4.2
通讯作者：
BREWER, GJ
中科院分区：
文献类型：
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作者：
BREWER, GJ
解决了有关神经元细胞培养的两个基本问题。一种专为海马神经元最佳生长而开发的无血清培养基能否支持大脑其他区域神经元的生长？文化中是否维持了区域特定的分化状态？为了回答这些问题，我们从其他六个大鼠大脑区域中分离出神经元，将它们置于 B27/Neurobasal(TM) 限定培养基中培养，并在培养 4 天后分析它们的形态和生长对细胞密度的依赖性。通过神经丝 200 抗体的免疫染色证实了神经元的身份。在几乎没有神经胶质细胞的情况下，来自每个大脑区域的神经元在培养物中保持了独特的形态。通过用木瓜蛋白酶消化从胚胎第 18 天大脑皮层分离的细胞显示出与海马神经元相同的高存活率，例如，以 160/mm(2) 铺板的细胞存活率为 70%。在这个年龄和密度下，隔膜神经元的存活率略低，为 45%。出生后第四天大脑中齿状颗粒神经元的存活率为 30-40%，明显较低，并且相对独立于铺板密度。这表明齿状颗粒神经元不依赖营养因子或接触。将从出生后第 7、8 或 9 天的大脑中分离的小脑颗粒神经元在 B27/Neurobasal 中的生长与在 BME/10% 血清中的生长进行比较。 4 天时在无血清培养基中的活力比在血清中的活力好得多，不需要将 KCl 升高至 25 mM，并且在没有神经胶质细胞大量生长的情况下发生。小脑颗粒神经元以 1,280 个细胞/mm(2) 铺板，维持培养三周，存活细胞密度为原始细胞密度的 17%。 4天后，从胚胎第18天黑质分离的细胞存活率为50%（浓度为160个细胞/mm(2)），与纹状体相似，但略低于海马神经元存活率。通过与酪氨酸羟化酶抗体的免疫反应性判断，黑质神经元的多巴胺能表型在培养物中维持超过2周。在此期间，当它们从体细胞中生长出来时，在这些过程中发现了免疫反应性。总之，这些研究表明 B27/Neurobasal 将成为维持许多大脑区域神经元分化生长的有用介质。讨论了不同神经元的通用生长培养基的潜在应用。 (C) 1995 Wiley-Liss, Inc.
Two 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/Neurobasal(TM) 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/mm(2). 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 KCl elevated to 25 mM, and occurred without substantial growth of glia. Cerebellar granule neurons plated at 1,280 cells/mm(2) 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/mm(2) 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. (C) 1995 Wiley-Liss, Inc.