Establishment of Primary Culture of Primate Brain Neurons.

灵长类脑神经元原代培养物的建立。

基本信息

批准号：
10680775
负责人：
KAWAMURA Seiji
金额：
$ 2.18万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680775/
关键词：
Cynomolgus monkey cerebral cortex primary culture neuron cryopreservation synapse fetus 霊長類脳ラット

项目摘要

Non-human primates are useful in biomedical studies because of their high similarity to humans. In order to make the most of this advantage in in vitro studies on central nervous system (CNS), primary culture of primate neurons should be established. In this study, the optimal procedure for the successful cryopreservation and primary culture of brain cells of fetal rats was first established. The successful protocol was as follows. Small cerebral tissue pieces isolated from 18-day-old fetuses in culture medium supplemented with 10% DMSO were first frozen to ?80℃ at slow rate, and then stored in liquid nitrogen. The recovery of viable cells from cryopreserved cerebral tissues was 20-30% of those from fresh tissues. By immunocytochemistry, no influence of cryopreservation on brain cells was observed. Next, this protocol was employed for cryopreservation and primary culture of brain cells of primate (Cynomolgus monkey) with slight modification. The optimal fetal age of cynomolgus monkey for appropriated primary culture after cryopreservation was around 80-day-old. The recovery of viable cells from the cryopreserved primate fetal cerebral tissues of the 80-day-old fetus corresponded to more than 80% of that from the fresh tissues. Morphological characteristics of neurons from the fresh and cryopreserved primate cerebral tissues were almost indistinguishable. By addition of cytosine arabinoside, selective culture of primate neurons was established. Cultured neurons from the cryopreserved primate cerebral tissues spontaneously formed networks, and randomly selected neurons in culture showed synchronous oscillations of [CaィイD12+ィエD1]ィイD2inィエD2 without any stimulation. Thus, primate neurons made functional synapses in culture even after cryopreservation. We concluded that primary culture of primate cerebral neuron was useful and effective method for biomedical study on CNS.

非人类灵长类动物在生物医学研究中很有用，因为它们与人类具有很高的相似性。为了充分利用对中枢神经系统（CNS）的体外研究的优势，应建立原发性神经元的原发性培养。在这项研究中，首先建立了成功的冷冻保存和原发性培养的最佳程序。成功的协议如下。首先以较慢的速度将其从18天大的胎儿中分离出的培养基中的18天胎儿片段被冷冻至80°℃，然后存储在液氮中。从冷冻保存的脑组织中恢复活细胞是新鲜组织中的细胞的20-30％。通过免疫细胞化学，未观察到冷冻保存对脑细胞的影响。接下来，该方案用于略微修饰的灵长类动物（cynomolgus猴子）的冷冻保存和原发性培养。冷冻保存后，用于分配原发性培养的cynomolgus猴子的最佳胎儿年龄约为80天。从80天大的胎儿的冷冻灵长类动物胎儿脑组织中恢复活细胞的对应于新鲜组织中的80％以上。来自新鲜和冷冻的灵长类动物脑组织的神经元的形态特征几乎是无法区分的。通过添加胞嘧啶阿拉伯糖苷，建立了灵长类动物神经元的选择性培养。在培养中赞助，赞助和随机选择的神经元的培养神经元在培养中，显示了[CAII D12+IE D1] II D2INI D2的同步振荡，而没有任何刺激。这，灵长类动物神经元即使在冷冻保存之后也可以在培养中产生功能突触。我们得出的结论是，灵长类动物脑神经元的原发性培养是对CNS生物医学研究的有效方法。