微流控芯片用于卵母细胞冷冻保存的实验研究

Experimental Study of Microfluidic Chip for Cryopreservation of Oocytes

低温保存对卵母细胞造成渗透损伤、毒性损伤和冰晶损伤,使得细胞冻后质量难以提高.本文首次提出将微流控法添加-去除保护剂分别与三种冷冻载体(OPS、QC及Cryotop)搭配使用,对猪卵母细胞进行冷冻保存,并与传统冷冻法进行比较;然后,首次选用透明陶瓷和玻璃制作集成一体化芯片,对猪卵母细胞进行冷冻保存,以冷冻保存后的细胞存活率和发育率为判断依据,筛选出较好的方案;最后,对冻后卵母细胞的早期凋亡情况、胞内活性氧水平和线粒体膜电位水平进行分析.结果表明,微流控添加-去除保护剂组卵母细胞冻后存活率以及卵裂率都显著高于传统冷冻组,可以有效降低卵母细胞的早期凋亡率和胞内活性氧水平,减小线粒体损伤,提高细胞的冻后质量.透明陶瓷一体化芯片保存卵母细胞得到的存活率和卵裂率与传统OPS冷冻的保存结果无显著差异.微流控芯片技术为卵母细胞的低温保存提供新的思路,有较好的应用前景.

Cryopreservation will cause osmotic damage, toxic damage and ice crystal damage to oocytes, thus it is difficult to improve the quality of the frozen oocytes. In this paper, microfluidic and stepwise cryoprotectant（CPA）loading-unloading protocols were combined with three kinds of cryopreservation devices（OPS, QC and Cryotop）to cryopreserve the porcine oocytes for the first time. Moreover, PDMS-transparent ceramic and PDMS-glass integration microfluidic chips were employed for oocytes cryopreservation for the first time, the survival rate and development rate of cryopreserved oocytes were used to evaluate optimal cryopreservation protocol. Finally, early apoptosis rate, intracellular reactive oxygen species（ROS） and mitochondrial membrane potential of oocytes were analyzed. The results showed that the survival rate and cleavage rate of frozen oocytes by microfluidic loading-unloading of CPAs were significantly higher than traditional loading-unloading group. Microfluidic method could reduce the early apoptosis rate, intracellular ROS and mitochondrial damage, improve the quality of frozen oocytes. The survival rate and cleavage rate of oocytes cryopreserved by PDMS-transparent ceramic integrated chip were not significantly different from traditional OPS method. Microfluidic chip technology provides a new idea for oocytes cryopreservation and has good application prospects.