| Project/Area Number |
19500364
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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 | University of Miyazaki |
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Principal Investigator |
KOSHIMOTO Chihiro University of Miyazaki, フロンティア科学実験総合センター, 教授 (70295210)
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| Co-Investigator(Kenkyū-buntansha) |
KASAI Magosaburou 高知大学, 教育研究部自然科学系, 教授 (60152617)
EDASHIGE Keisuke 高知大学, 教育研究部自然科学系, 教授 (30175228)
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| Project Period (FY) |
2007 – 2009
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| Project Status |
Completed (Fiscal Year 2009)
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| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2009: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2008: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
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| Keywords | 凍結保存 / 実験動物 / バイオリソース / 生殖細胞凍結保存 / 高圧凍結法 / 胚 / 精子 |
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| Research Abstract |
In order to maintain mammalian bio-resource colony, vitrification is an easy and reliable cryopreservation option, but limited types of cell can be frozen without a loss of viability because of unphysiological osmolality and chemical toxicity of freezing solution. On the other hand, high pressure freezing machine has been developed for the sample preparation for electron microscopic observation, by which a vitreous fixation of biological specimen is achieved. By using this engineering that were developed for microscopic sample preparation, we are devising a new cryopreservation technique where cryoprotectant is not required. Mouse zygote was frozen by using this apparatus with various cryoprotectant agents to optimize freezing solution with minimal toxicity. Freezing substitution was implemented and the samples embedded, sliced and stained under the regular procedures and observed by transmission electron microscope (TEM) to evaluate cell eumorphism. While many zygotes frozen with zero or lower concentration of cryoprotectant were fractured after high pressure freezing probably caused from pressure impact, However, when the concentration of permeable cryoprotectant higher than 1.5M, high pressure frozen zygotes maintain intact intracellular microstructures without fracturing or large ice crystal formation. We have firstly demonstrated that vitrified larger cell by high pressure method maintains an intact structures without forming ice crystal. This suggests that this new technique could be developed as universal cryopreservation protocol with very low cryoprotectant agents.
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