微流控线性加载低温保护剂减少猪MⅡ期卵母细胞的渗透损伤
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上海理工大学 生物热科学研究所,上海理工大学 生物热科学研究所,上海农业科学院 畜牧兽医研究所 动物遗传工程研究室,上海农业科学院 畜牧兽医研究所 动物遗传工程研究室,上海理工大学 生物热科学研究所,上海理工大学 生物热科学研究所,上海理工大学 生物热科学研究所

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国家自然科学基金(51376132, 50906057),上海市自然科学基金(13ZR1429200)和上海市国家重点实验室(1N15301101)资助项目


Linear Loading Cryoprotectants (CPAs) With Microfluidic Method Reduces Osmotic Damage to Porcine MⅡ-stage Oocytes
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Institute of Biothermal Technology, University of Shanghai for Science and Technology,Institute of Biothermal Technology, University of Shanghai for Science and Technology,Animal and Veterinary Rechear Institute, SAAS,Animal and Veterinary Rechear Institute, SAAS,Institute of Biothermal Technology, University of Shanghai for Science and Technology,Institute of Biothermal Technology, University of Shanghai for Science and Technology,Institute of Biothermal Technology, University of Shanghai for Science and Technology

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This work was supported by grants from The National Natural Science Foundation of China (51376132, 50906057), The Natural Science Foundation of Shanghai of China (13ZR1429200) and The National Key Laboratory of China (1N15301101)

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    摘要:

    在卵母细胞低温保存中,通常需要加载冷冻保护剂来抑制冰晶对细胞的损伤,但高浓度冷冻保护剂的加载会对细胞造成渗透损伤.为了减小细胞的渗透损伤,本文设计并制作了适合卵母细胞冷冻保护剂加载的微流体装置,研究了微流控线性加载30% (v/v)二甲基亚砜(Me2SO)低温保护剂时细胞内保护剂浓度变化、细胞体积变化,以及对细胞存活率与发育率的影响,并与传统的加载方法(一步法、分步法)做了比较.结果表明:微流控法能够实现卵母细胞冷冻保护剂的连续线性加载,避免了卵母细胞体积的骤变,显著减小了细胞的渗透损伤,提高了细胞的存活率.其中细胞的最小渗透体积减小为0.86V0,细胞的存活率达到92.8%,比一步法高33%,比两步法高16.3%,但与四步法之间无显著性差异.经孤雌激活后体外培养,细胞的卵裂率和囊胚率分别达到75.8%和27.4%,都显著高于一步法和分步法(P < 0.05).因此,微流控线性加载低温保护剂能够显著减小细胞的渗透损伤,为卵母细胞低温保存技术提供新思路.

    Abstract:

    Loading cryoprotectants (CPAs) into oocytes is critical to restrain ice formation during cryopreservation. However, high concentration of CPAs may cause osmotic and toxic damage to oocytes. In order to minimize the osmotic damage, a microfluidic device for loading CPAs into oocytes was designed and fabricated in this study. 30% (v/v) Me2SO was linear loaded into porcine MⅡ-stage oocyte with the microfluidic device, the intracellular CPA concentration, the cell volumetric changes and the effect on the survival rate and developmental rate of oocytes were investigated and compared with the traditional methods including the one-step method and the step-wise methods. The results showed that the microfluidic method can realize continuous CPA loading to oocytes, reduce the osmotic shock to cells and enhance the cell viability. The lowest penetration volume of oocyte reached 0.86V0. The cell viability was 92.8% with the microfluidic method, ~33% higher than one-step method, ~16.3% higher than two-step method, and no significant difference with four-step method. After parthenogenetic activation and cultured in vitro, the cleavage rate and the blastocyst rate of oocytes were 75.8% and 27.4%, which were significantly higher than the one-step method and the step-wise methods (P < 0.05). In conclusion, linear loading CPAs with the microfluidic method can significantly alleviate the osmotic damage to oocytes, which may provide a new path for oocyte cryopreservation.

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杨云,周新丽,戴建军,张德福,邵文琪,衣星越,陶乐仁.微流控线性加载低温保护剂减少猪MⅡ期卵母细胞的渗透损伤[J].生物化学与生物物理进展,2016,43(6):616-623

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  • 收稿日期:2016-01-13
  • 最后修改日期:2016-03-07
  • 接受日期:2016-03-16
  • 在线发布日期: 2016-06-21
  • 出版日期: 2016-09-20