Gelatin-methacryloyl hydrogel as an osmotic barrier to protect mouse testicular tissue from cryo-damage induced by high-concentration cryoprotectants.

RESEARCH QUESTION: Can a novel hydrogel-encapsulated cryopreservation system reduce cryoinjury while maintaining the viability and functionality of testicular tissue, and support subsequent in-vitro culture for fertility preservation?

DESIGN

Various pre-treatment methods that combined cryoprotectants (CPA) with gelatin methacryloyl (GelMA) hydrogels to enhance the rapid freezing of encapsulated testicular tissue were investigated. Key parameters, including CPA concentrations and compositions, were evaluated alongside different cryocarriers to establish an effective freezing system. Fresh and frozen-warmed testicular tissues were cultured in vitro for 1week, comparing the effects of two hydrogel systems: agarose and alginate methacryloyl (AlgMA), based on organ culture method.

RESULTS

Encapsulating testicular tissue with GelMA hydrogel, followed by the application of a 30% penetrating CPA, was identified as the optimal pre-treatment strategy. The combination of 15% dimethyl sulfoxide and 15% ethylene glycol significantly enhanced mitochondrial activity compared with 30% concentrations of either CPA alone (both P < 0.05). Among the cryocarriers tested, cryovials demonstrated superior preservation of cellular viability, mitochondrial function and antioxidant capacity. After in-vitro culture, AlgMA hydrogels further preserved basement membrane morphology and germ cell survival in frozen-warmed testicular tissues at levels comparable to fresh tissue, outperforming the traditional agarose hydrogels system in minimizing nuclear condensation, preventing cell loss and preserving tubular structure.

CONCLUSIONS

GelMA hydrogel functions as an osmotic barrier, regulating CPA diffusion rates to prevent apoptosis, oxidative stress and other cryoinjuries typically caused by direct exposure to high CPA concentrations. These findings advance the field of fertility preservation, showcasing the potential of novel materials and technologies to improve male fertility restoration outcomes.