Optimization of mouse oocyte vitrification through microencapsulation in sodium alginate hydrogel.

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.