Optimization of cryoprotectant loading into murine and human oocytes.
Optimization of cryoprotectant loading into murine and human oocytes.
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DOI：
10.1016/j.cryobiol.2013.11.002
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发表时间：
2014-02
期刊：
影响因子：
2.7
通讯作者：
Eroglu, All
中科院分区：
文献类型：
--
作者：
Karlsson, Jens O. M.;Szurek, Edyta A.;Higgins, Adam Z.;Lee, Sang R.;Eroglu, All
关键词：
将冷冻保护剂加载到卵母细胞中是冷冻保存过程的重要步骤，其中细胞暴露于潜在的破坏性渗透应力和化学毒性。因此，我们研究了使用基于物理的数学优化来指导小鼠和人卵母细胞冷冻保护剂加载方法的设计。我们首先检查了在23°C下将1.5 M二甲基亚砜（Me2SO）加载到小鼠卵母细胞中。传统的一步加载导致受精率（34%）和胚胎发育率（60%）显着低于未经处理的对照组（分别为95%和94%）。相比之下，数学优化的两步法产生了更高的受精率（85%）和发育率（87%）。为了检查卵母细胞损伤的原因，我们进行了实验以分离细胞收缩和Me2SO暴露时间的影响，揭示收缩和Me2SO暴露都不会单独损害受精和发育率。因此，在一步Me2SO添加过程中的损害似乎是由于Me2SO毒性和渗透胁迫的影响之间的相互作用。我们还研究了在30°C下Me2SO加载到小鼠卵母细胞中。在此温度下，与数学优化的两步加载（86%）和未处理的对照（96%）相比，一步加载（8%）后的受精率再次降低。此外，我们的计算机算法产生了一个有效的策略，减少Me2SO暴露时间，使用低渗稀释剂的冷冻保护剂解决方案。利用这种技术，1.5 M Me2SO仅在2.5分钟内成功加载，具有92%的受精率。基于这些有希望的结果，我们提出了新的方法来加载冷冻保护剂到人类卵母细胞，设计使用我们的数学优化方法。
Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethylsulfoxide (Me2SO) into mouse oocytes at 23°C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me2SO exposure time, revealing that neither shrinkage nor Me2SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me2SO addition appears to result from interactions between the effects of Me2SO toxicity and osmotic stress. We also investigated Me2SO loading into mouse oocytes at 30°C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me2SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me2SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach.
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