Lin Haoyang, Liu Chenxi, Cao Yukun, Zhou Xinli
Institute of Biothermal Science & Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China; Shanghai Collaborative Innovation Center for Tumor Energy Therapy, Shanghai, 200093, China; Shanghai Technical Service Platform for Cryopreservation of Biological Resources, Shanghai, 200093, China.
Institute of Biothermal Science & Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China.
Cryobiology. 2025 Sep;120:105275. doi: 10.1016/j.cryobiol.2025.105275. Epub 2025 Jun 9.
Oocyte/embryo vitrification is one of the basic techniques employed in assisted reproduction and fertility preservation. However, the current process typically requires manual operation by experienced embryologists, which is both time-consuming and exhibits inconsistent outcomes. To resolve this issue, we herein developed an automated vitrification device for oocytes/embryos. The device consists of a microfluidic mixing unit, a microgrid capillary, and a mechanical sliding unit. The microfluidic mixing unit was adopted to determine continuous changes in cryoprotective agent (CPA) concentration, reducing osmotic damage during CPA loading/removal; and the microgrid capillary was used to load/remove the CPA and vitrify the oocytes/embryos in the same carrier so as to reduce cellular loss due to cell transfer. The medical absorbent dressing was placed under the carrier, and the excess liquid was absorbed to minimize the remaining CPA solution after CPA loading. Eight different loading/removal curves were developed for CPA loading and removal protocols, and we conducted oocyte vitrification with automated vitrification equipment. Our results revealed that a quadratic function-based, loading-removal curve achieved the highest oocyte survival rate within an 8-min loading and removal duration. In addition, there was no significant difference in oocyte survival rates between the automated vitrification device and the Cryotop multi-step equilibration method (90.33 % and 94.33 %, respectively); nor did the two methods differ in terms of survival or hatching rates of 8-cell embryos. The current system automates and standardizes the oocyte/embryo vitrification process while achieving survival and developmental potential.
卵母细胞/胚胎玻璃化是辅助生殖和生育力保存中采用的基本技术之一。然而，目前的过程通常需要经验丰富的胚胎学家手动操作，这既耗时，结果又不一致。为了解决这个问题，我们在此开发了一种用于卵母细胞/胚胎的自动化玻璃化装置。该装置由微流混合单元、微网格毛细管和机械滑动单元组成。采用微流混合单元来确定冷冻保护剂（CPA）浓度的连续变化，减少CPA加载/去除过程中的渗透损伤；微网格毛细管用于在同一载体中加载/去除CPA并玻璃化卵母细胞/胚胎，以减少细胞转移导致的细胞损失。将医用吸水敷料置于载体下方，吸收多余液体，以尽量减少CPA加载后剩余的CPA溶液。针对CPA加载和去除方案制定了8种不同的加载/去除曲线，并使用自动化玻璃化设备进行了卵母细胞玻璃化。我们的结果表明，基于二次函数的加载-去除曲线在8分钟的加载和去除持续时间内实现了最高的卵母细胞存活率。此外，自动化玻璃化装置与Cryotop多步平衡法之间的卵母细胞存活率没有显著差异（分别为90.33%和94.33%）；两种方法在8细胞胚胎的存活率或孵化率方面也没有差异。当前系统使卵母细胞/胚胎玻璃化过程自动化并标准化，同时实现了存活率和发育潜力。
