微流控法去除低温保护剂对卵母细胞发育的影响

Effect of cryoprotectant removal by microfluidic chip on developmental capacity of oocytes

为了减小低温保护剂去除过程对卵母细胞造成的渗透损伤和毒性损伤,本文利用微流控芯片对猪二次减数分裂中期(MⅡ-stage)卵母细胞低温保护剂的去除方案进行了优化研究。首先分析了微流控去除方法中去除时间、去除液成分及浓度对卵母细胞存活率及体外发育情况的影响,然后将微流控去除方法与传统的一步法、两步法进行了比较。研究结果表明,微流控法中去除总时间为8 min时,卵母细胞存活率(95.99%±4.64%)及桑椹胚率(74.17%±1.18%)与新鲜细胞(98.53%±2.94%;78.22%±1.34%)相比,差异无统计学意义;1 mol/L蔗糖去除液最有利于卵母细胞低温保护剂去除后的存活及体外发育;微流控法去除低温保护剂后,卵母细胞的存活率、体外发育情况等,均好于传统去除方法。本文研究结果提示,以微流控法去除低温保护剂可减小对卵母细胞的损伤,从而可能进一步提高卵母细胞的低温保存效果。

In order to reduce osmotic damage and chemical toxicity of cryoprotectants （CPA） to oocytes during unloading process, the microfluidic chip was used to remove CPA from porcine M II oocytes in this study. Firstly, the effects of unloading time, composition and concentration of diluting solutions of microfluidic method on survival rate and developmental capacity of oocytes were studied, then microfluidic method was compared with traditional one-step and two-step CPA unloading protocols. The results showed that when the total time is 8 minutes, the survival rate and morula rate of oocytes treated with microfluidic method could achieve 95.99% ±4.64% and 74.17% ±1.18%, respectively, which were not significantly different from fresh control group （98.53% ±2.94%; 78.22% ±1.34%）. In addition, I mol/L sucrose diluting solutions were more beneficial than other solutions, and it was also showed that microfluidic method achieved better survival, cleavage rate of oocytes than traditional methods. Microfluidic CPA removal protocol can reduce the damage to oocytes during unloading process, and may further improve the cryopreservation effect of oocytes.