Yamashita N, Saito H
Pharmaceutical Research Center, Meiji Seika Kaisha, LTD., Tokyo, Japan.
Nihon Shinkei Seishin Yakurigaku Zasshi. 1994 Aug;14(4):227-34.
It has been difficult to put mature neurons of mammalian central nervous system (CNS) in vitro. Only a few reports have dealt with culture methods so far. They employed non-neuronal cell layers to make postnatal neurons attach better to a culture dish. We established a novel method for culturing mature functional hypothalamic neurons derived from 21 postnatal day rat brain without any help of non-neuronal cell layers. Nerve cells with a few processes could be isolated from sliced hypothalamic tissues by means of enzymatic and mechanical treatments. A new cell-collecting method was developed for these vulnerable cells by allowing the dissociated cell suspension to stand in a vertically held, wide-tipped syringe so that the cells were concentrated near the bottom liquid surface, from which they could be easily dropped into the medium, leaving most of the small debris in the syringe. Astrocyte conditioned medium prolonged neuronal survival, concentration dependently, for up to 28 days in vitro. This culture system may make it possible to study functions of single hypothalamic neurons.
在体外培养哺乳动物中枢神经系统（CNS）的成熟神经元一直很困难。到目前为止，只有少数报告涉及培养方法。他们采用非神经元细胞层以使出生后的神经元更好地附着在培养皿上。我们建立了一种新方法，可在不借助非神经元细胞层的情况下，培养源自出生后21天大鼠大脑的成熟功能性下丘脑神经元。通过酶促和机械处理，可以从下丘脑切片组织中分离出具有少量突起的神经细胞。针对这些脆弱的细胞，开发了一种新的细胞收集方法，即将解离的细胞悬液置于垂直握持的宽头注射器中静置，使细胞集中在底部液体表面附近，这样它们就可以很容易地滴入培养基中，而大部分小碎片则留在注射器中。星形胶质细胞条件培养基可浓度依赖性地延长神经元在体外的存活时间，长达28天。这种培养系统可能使研究单个下丘脑神经元的功能成为可能。
