Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, USA.
Theriogenology. 2011 Jul 15;76(2):280-9. doi: 10.1016/j.theriogenology.2011.02.004. Epub 2011 Mar 31.
As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN(2)). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a "closed" system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease.
随着猪模型在生物医学研究中的重要性不断增加，开发低成本、高通量的系统来冷冻保存猪种质资源以维护这些模型变得至关重要。然而，猪胚胎对低温极其敏感，成功的冷冻保存通常仅限于使用开放系统中的玻璃化，使胚胎直接与液氮（LN(2)）接触。这会增加病原体传播的风险。因此，“封闭”系统中猪胚胎的冷冻保存非常重要。在这项研究中，使用体外生产（IVP）的猪胚胎来研究封闭系统中冷冻保存的胚胎的冷冻存活率和发育潜力。使用第 4 天的胚胎研究了将细胞内脂质完全从卵裂球中分离出来的最佳离心力。通过在离心后立即或发育为囊胚后对去脂胚胎进行玻璃化来研究去脂胚胎的冷冻存活率。在这项研究中，以 13,000 g 的速度离心 30 分钟足以完全去除胚胎中的脂质；此外，这些胚胎能够以与仅离心 12 分钟相当的速率在冷冻保存后存活。当去脂胚胎在囊胚阶段进行玻璃化时，使用 OPS 和 0.25 mL 吸管玻璃化的胚胎在存活率方面没有差异。用每种方法冷冻的一些胚胎发育到足月。这些实验表明，可以在去除细胞内脂质后将猪胚胎在封闭系统中冷冻保存。这对于保存人类健康和疾病的猪模型具有重要意义。
