Brewer Gregory J, Torricelli John R
Department of Neurology, Southern Illinois University School of Medicine, Springfield, Illinois 62794-9626, USA.
Nat Protoc. 2007;2(6):1490-8. doi: 10.1038/nprot.2007.207.
Here we present a protocol for extraction and culture of neurons from adult rat or mouse CNS. The method proscribes an optimized protease digestion of slices, control of osmolarity and pH outside the incubator with Hibernate and density gradient separation of neurons from debris. This protocol produces yields of millions of cortical, hippocampal neurons or neurosphere progenitors from each brain. The entire process of neuron isolation and culture takes less than 4 h. With suitable growth factors, adult neuron regeneration of axons and dendrites in culture proceeds over 1-3 weeks to allow controlled studies in pharmacology, electrophysiology, development, regeneration and neurotoxicology. Adult neurospheres can be collected in 1 week as a source of neuroprogenitors ethically preferred over embryonic or fetal sources. This protocol emphasizes two differences between neuron differentiation and neurosphere proliferation: adhesion dependence and the differentiating power of retinyl acetate.
在此，我们展示了一种从成年大鼠或小鼠中枢神经系统提取和培养神经元的方案。该方法规定了对切片进行优化的蛋白酶消化、在培养箱外使用Hibernate控制渗透压和pH值以及从碎片中进行神经元的密度梯度分离。此方案可从每个大脑中获得数百万个皮质、海马神经元或神经球祖细胞。神经元分离和培养的整个过程耗时不到4小时。在合适的生长因子作用下，培养的成年神经元轴突和树突的再生在1至3周内进行，以便在药理学、电生理学、发育、再生和神经毒理学方面进行可控研究。成年神经球可在1周内收集，作为神经祖细胞的来源，在伦理上比胚胎或胎儿来源更受青睐。该方案强调了神经元分化和神经球增殖之间的两个差异：黏附依赖性和视黄酸的分化能力。
