Shen Jing, Ye Na, Zhang Yuqi, Liu Chenxi, Li Weijie, Zhou Xinli
Institute of Biothermal Science & Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China.
Shanghai Co-Innovation Center for Energy Therapy of Tumors, Shanghai, 200093, China.
J Assist Reprod Genet. 2025 Sep 21. doi: 10.1007/s10815-025-03673-5.
Vitrification (ice-free) is considered more effective than slow freezing for oocyte cryopreservation. However, high concentrations of toxic cryoprotective agents (CPAs) are typically required, followed by multi-step washing to remove CPAs, risking osmotic shock and impairing oocytes development. This study aimed to establish a hydrogel encapsulation strategy using a microfluidic system to improve vitrification outcomes.
Three different sizes of oocyte-loaded sodium alginate hydrogel microspheres (OHMs) (~ 262 µm, ~ 193 µm, and ~ 156 µm) were prepared using an optimized three-channel microfluidic system. The effects of particle size, vitrification solutions (VS1, VS2, VS3, and VS4), CPA loading durations (4, 8, and 12 min), and warming/dilution procedures (one-step, two-step, and three-step) on oocyte vitrification were systematically evaluated.
OHMs with a particle size of 262 µm, loaded with VS3 (containing 10% DMSO) for 8 min and subjected to the two-step warming and dilution procedure, achieved excellent post-thaw survival (91.98%), cleavage (75.84%), and blastocyst rates (23.86%). The survival rate was comparable to that of the Cryotop group vitrified with 15% DMSO (91.81%), while the cleavage and blastocyst rates were significantly higher than those of the Cryotop group (67.42% and 20.03%, respectively). Besides, OHMs resulted in minimal oocyte volume changes upon CPA loading (minimum normalized volume of 0.8739) compared to the Cryotop multi-step method (0.4396).
Compared with the Cryotop carrier method, the microfluidic hydrogel microcapsule technique reduces CPA concentration, shortens loading time, and minimizes osmotic injury. This approach provides a promising strategy for fertility preservation and may be extended to other biological samples.
玻璃化（无冰）被认为在卵母细胞冷冻保存方面比慢速冷冻更有效。然而，通常需要高浓度的有毒冷冻保护剂（CPA），随后进行多步洗涤以去除CPA，这有导致渗透休克和损害卵母细胞发育的风险。本研究旨在建立一种使用微流控系统的水凝胶包封策略，以改善玻璃化效果。
使用优化的三通道微流控系统制备三种不同大小的负载卵母细胞的海藻酸钠水凝胶微球（OHM）（约262μm、约193μm和约156μm）。系统评估了粒径、玻璃化溶液（VS1、VS2、VS3和VS4）、CPA加载持续时间（4、8和12分钟）以及复温/稀释程序（一步、两步和三步）对卵母细胞玻璃化的影响。
粒径为262μm的OHM，加载VS3（含10%DMSO）8分钟，并采用两步复温和稀释程序，解冻后存活率（91.98%）、卵裂率（75.84%）和囊胚率（23.86%）优异。存活率与使用15%DMSO玻璃化的Cryotop组（91.81%）相当，而卵裂率和囊胚率显著高于Cryotop组（分别为67.42%和20.03%）。此外，与Cryotop多步法相比，OHM在加载CPA时导致卵母细胞体积变化最小（最小归一化体积为0.8739）（0.4396）。
与Cryotop载体法相比，微流控水凝胶微胶囊技术降低了CPA浓度，缩短了加载时间，并将渗透损伤降至最低。这种方法为生育力保存提供了一种有前景的策略，并且可能扩展到其他生物样本。
