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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，因为它们与人类高度相似。为了充分利用这一优势进行中枢神经系统（CNS）的体外研究，需要建立灵长类神经元的原代培养。本研究首次建立了胎鼠脑细胞冻存及原代培养的最佳方法。成功的方案如下。小脑组织块分离自18日龄胎儿在培养基中补充10%DMSO首先冷冻？80℃下缓慢储存，然后储存在液氮中。冻存脑组织的活细胞回收率为新鲜脑组织的20-30%。免疫细胞化学结果显示，冻存对脑细胞无明显影响。接下来，将该方案稍作修改后用于灵长类动物（食蟹猴）脑细胞的冷冻保存和原代培养。食蟹猴胚胎冻存后进行原代培养的最佳胎龄为80日龄左右。从冻存的灵长类动物胎脑组织的80日龄胎儿的活细胞的回收率相当于从新鲜组织的80%以上。新鲜和冷冻保存的灵长类动物脑组织神经元的形态特征几乎没有区别。通过添加阿糖胞苷，建立了灵长类神经元的选择性培养。从冷冻保存的灵长类脑组织中培养的神经元自发形成网络，并且随机选择的培养神经元在没有任何刺激的情况下显示[Ca ~（2+）D_1] Ca ~（2+）D_2在Ca ~（2+）D_2中的同步振荡。因此，灵长类神经元即使在冷冻保存后也能在培养物中形成功能性突触。结论：灵长类动物脑神经元的原代培养是研究中枢神经系统生物医学的有效方法。