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Purpose <p>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.</p> Methods <p>Three different sizes of oocyte-loaded sodium alginate hydrogel microspheres (OHMs) (~ 262&#xa0;µm, ~ 193&#xa0;µm, and ~ 156&#xa0;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&#xa0;min), and warming/dilution procedures (one-step, two-step, and three-step) on oocyte vitrification were systematically evaluated.</p> Results <p>OHMs with a particle size of 262&#xa0;µm, loaded with VS3 (containing 10% DMSO) for 8&#xa0;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).</p> Conclusions <p>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.</p> Graphical Abstract <p></p>

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Optimization of mouse oocyte vitrification through microencapsulation in sodium alginate hydrogel

  • 期刊论文

  • 发表日期：

    2025年9月21日

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  • Jing Shen,
  • Na Ye,
  • Yuqi Zhang,
  • Chenxi Liu,
  • Weijie Li,
  • Xinli Zhou

摘要

Purpose

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.

Methods

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.

Results

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).

Conclusions

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.

Graphical Abstract