| Project/Area Number |
10680775
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Laboratory animal science
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| Research Institution | The University of Tokyo |
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Principal Investigator |
KAWAMURA Seiji Graduate School of Agriculture and Life Sciences, Associate Professor, 大学院・農学生命科学研究科, 助教授 (10161366)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIKAWA Yasuhiro Graduate School of Agriculture and Life Sciences, Professor, 大学院・農学生命科学研究科, 教授 (80109975)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1998: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Cynomolgus monkey / cerebral cortex / primary culture / neuron / cryopreservation / synapse / fetus / 霊長類 / 脳 / ラット |
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| Research Abstract |
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.
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