微流控芯片加载低温保护剂过程中卵母细胞的损伤评估

The Injury Evaluation During Cryoprotectant Loading With Microfluidic Chip

卵母细胞的低温保存为辅助生殖技术和胚胎工程技术提供了更大的发展空间,而低温保存需要添加高浓度保护剂,会对细胞造成渗透损伤及毒性损伤.与分步法添加固定浓度的保护剂不同,微流控法能够实现保护剂浓度的连续性变化,关于微流控法连续性添加保护剂时卵母细胞的损伤评估还未见报道.本文首先采用数值模拟的方法,模拟细胞在不同加载时间、不同加载线型(线性、S型、凹型)、不同凹型加载(低凹型、高凹型)中细胞的渗透行为,计算各方案中细胞的传统的损伤评估参数:体积变化极值(ΔV)、积累性渗透损伤值(AOD)及毒性损伤值(J).在此基础上,藉由信息熵理论首次提出了综合损伤评估参数s,并通过猪卵母细胞微流控加载后孤雌激活实验的结果验证评估效果.结果表明,对于不同的加载方案,传统的损伤评估参数结果之间出现分歧,无法得到统一的结论.通过分析囊胚率同综合损伤评估参数s值的关系,发现二者呈负相关关系,且相关系数很高,说明综合损伤评估参数s能够较好地对细胞损伤进行评估,为细胞损伤评估开辟了新思路.

With the help of the oocyte cryoproservation, the assisted reproductive technology and the embryo engineering technology have tremendous development potential. However, the high concentration of cryoprotectants(CPAs) in cryopreservation will cause osmotic damage and toxic damage to oocytes. Unlike adding a fixed concentration of CPA in step-by-step method, the microfluidic method can achieve continuous CPA concentration change. The oocytes damage evaluation in CPA continuous loading by microfluidic method has not been reported. In this paper, the osmotic behavior of M Ⅱ porcine oocytes in different loading time, different loading line types and different concave loading types were simulated. Three traditional parameters ΔV(maximum volume change), AOD(accumulative osmotic damage) and J(accumulative chemical damage) were used to evaluate the damage of oocyte. A comprehensive parameter s, which based on the traditional parameters and the information entropy theory, was proposed to evaluate the damage. Oocytes loading experiments were conducted to verify the effectiveness of s. The results showed differences from the results of traditional damage assessment parameters, which cannot come to the unified conclusion. By analyzing the relationship between blastocyst rate and s, we found that there is a highly negative correlation between s and blastocysts rate. That indicates that the comprehensive damage assessment parameter s can be used to assess cell damage and breaks a new path for cell damage evaluation.