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; Shanghai Technical Service Platform for Cryopreservation of Biological Resources, Shanghai 200093, China.
Department of Andrology, the Center for Men's Health, Urologic Medical Center, Shanghai Key Laboratory of Reproductive Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China.
Colloids Surf B Biointerfaces. 2024 Oct;242:114096. doi: 10.1016/j.colsurfb.2024.114096. Epub 2024 Jul 14.
Cryopreserved testicular tissue offers a promising method to restore fertility in male infertility patients. Current protocols rely on high concentrations of penetrating cryoprotectants (pCPAs), such as dimethyl sulfoxide (DMSO), which necessitating complex washing procedures and posing risks of toxicity. Hydrogel encapsulation presents a non-toxic alternative for cellular cryopreservation. This study investigates the effects of various types, concentrations, and thicknesses of hydrogel encapsulation on the cryopreservation of mouse testicular tissue. Testicular tissues loaded with varying concentrations of DMSO were encapsulated in alginate or gelatin-methacryloyl (GelMA) hydrogels. We evaluated hydrogels as potential CPAs to reduce pCPA concentrations and determine optimal combinations for cryopreservation. Post-cryopreservation, tissues were cultured using organ culture methods to assess spermatogenesis progression. Cryomicroscopy and differential scanning calorimetry (DSC) were used to examine ice crystal formation, melting enthalpy, and non-freezing water content in different hydrogels during cooling. Results indicate that 3 % alginate or 5 % GelMA hydrogel with thin encapsulation optimally preserves mouse testicular tissue. Using 20 % DMSO in 5 % GelMA thin encapsulation showed comparable apoptosis rates, improved morphology, higher mitochondrial activity, and enhanced antioxidant capacity compared to conventional 30 % DMSO without encapsulation. This suggests that hydrogel encapsulation reduces pCPA concentration by 10 %, thereby mitigating toxic damage. Hydrogel encapsulation can reduce basement membrane shrinkage of testicular tissue during cryopreservation. Moreover, frozen tissues remained viable with preserved germ cells after being cultured for one week on alginate methacryloyl (AlgMA) hydrogel using the gas-liquid interphase method. Cryomicroscopy and DSC studies confirmed the hydrogel's ability to inhibit ice crystal growth. In conclusion, this study introduces novel strategies for male fertility preservation and advances cryopreservation technology for clinical applications in assisted reproduction.
冷冻保存的睾丸组织为男性不育患者恢复生育能力提供了一种很有前途的方法。目前的方案依赖于高浓度的渗透保护剂（pCPA），如二甲基亚砜（DMSO），这需要复杂的洗涤程序，并存在毒性风险。水凝胶包封为细胞冷冻保存提供了一种无毒的替代方法。本研究调查了各种类型、浓度和厚度的水凝胶包封对小鼠睾丸组织冷冻保存的影响。用不同浓度 DMSO 加载的睾丸组织被包裹在藻酸盐或明胶甲基丙烯酰（GelMA）水凝胶中。我们评估了水凝胶作为潜在的 CPAs 来降低 pCPA 浓度，并确定冷冻保存的最佳组合。冷冻保存后，使用器官培养方法培养组织，以评估精子发生进展。冷冻显微镜和差示扫描量热法（DSC）用于检查不同水凝胶在冷却过程中冰晶的形成、熔融焓和非冻结水含量。结果表明，3%的藻酸盐或 5%的 GelMA 薄水凝胶包封能最佳地保存小鼠睾丸组织。在薄封装的 5% GelMA 中使用 20% DMSO 与不封装的传统 30% DMSO 相比，显示出相似的凋亡率、改善的形态、更高的线粒体活性和增强的抗氧化能力。这表明水凝胶包封可将 pCPA 浓度降低 10%，从而减轻毒性损伤。水凝胶包封可以减少睾丸组织冷冻保存过程中基膜的收缩。此外，在使用气-液界面法在 AlgMA 水凝胶上培养一周后，冷冻组织保持存活并保留有生殖细胞。冷冻显微镜和 DSC 研究证实了水凝胶抑制冰晶生长的能力。总之，本研究为男性生育力保存提供了新策略，并推进了冷冻保存技术在辅助生殖中的临床应用。
