中文摘要：

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.

英文摘要：

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） Me2 SO 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.