权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Utilization of the high pressure freezing as a gamete cryopreservation procedure for mammalian bio-resource maintenances

利用高压冷冻作为哺乳动物生物资源维护的配子冷冻保存程序

基本信息

批准号：
19500364
负责人：
KOSHIMOTO Chihiro
金额：
$ 2.91万
依托单位：
University of Miyazaki
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2007
资助国家：
日本
起止时间：
2007 至 2009
项目状态：
已结题

项目摘要

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.

为了维持哺乳动物生物资源集落，玻璃化冷冻是一种简单可靠的冷冻保存方法，但由于冷冻液的非生理性渗透压和化学毒性，有限类型的细胞可以在不丧失活力的情况下冷冻。另一方面，为了制备电子显微镜观察用的样品，研制了高压冷冻机，实现了生物标本的玻璃体固定。通过使用这种为微观样品制备而开发的工程，我们正在设计一种新的冷冻保存技术，其中不需要冷冻保护剂。用该装置对小鼠受精卵进行冷冻，并与不同的冷冻保护剂进行冷冻，优选出毒性最小的冷冻溶液。冷冻代入后，按常规程序包埋、切片、染色，透射电镜观察细胞形态。而在低温保护剂浓度为零或更低的情况下冷冻的受精卵在高压冷冻后可能由于压力冲击而破裂，但当可渗透低温保护剂浓度高于1.5M时，高压冷冻受精卵保持细胞内微结构完整，不会破裂，也不会形成大冰晶。我们首次证明了高压法玻璃化的大细胞在不形成冰晶的情况下保持了完整的结构。这表明这项新技术可以发展成为一种通用的低温保存方案，使用极低的冷冻保护剂。