Enabling the cryopreservation of embryos from aquatic species using ice recrystallization inhibitors

在大多数人类和动物系统中，精子冷冻保存是明确的，并且有成功的结局方案。然而，除了人类和少数几个农业上重要的物种外，胚胎冷冻不存在。对海洋胚胎和幼虫的冷冻保存几乎没有成功，只是在过去的一年里，一些水生生物的这种情况才有所改变。在过去的三十年里，胚胎冷冻保存技术的发展使家畜育种和人类生殖技术发生了革命性的变化。我们将研究两个水生胚胎或幼虫系统，包括珊瑚和鱼类。世界各地的珊瑚礁都受到威胁，增加对珊瑚礁低温生物学的了解和应用将使珊瑚礁的保护工作受益匪浅。我们相信，我们最近在新型冷冻保存技术方面的工作可以帮助改变这两个系统中的冷冻保存过程，这两个系统目前冷冻保存成功率较低。 冰晶形成和生长造成的物理和化学损伤是低温保存过程中活力下降和功能受损的最重要原因。在胚胎冷冻保存中，较低的表面积/体积比和较低的膜对水和低温保护剂的渗透性使得很难在胚胎中积累足够高的冷冻保护剂以避免细胞内/间质结冰。因此，迫切需要新的方法。近年来，小分子冰重结晶抑制物(Iris)的大型化学库已被开发并应用于哺乳动物细胞的冷冻保存。冰重结晶是在冻结和融化过程中发生的过程，在此过程中，大冰晶以牺牲小冰晶为代价长大，是细胞破坏的主要原因，降低了融化后的生存能力和效力。我们推测，使用新型小分子冰重结晶抑制剂控制冰的形成将减少冷冻损伤，并提高水生物种胚胎的解冻后存活率和细胞功能。 我们的多学科团队设定了四个互补的目标，以提高我们对IRI作为冷冻保护剂的有效性及其对水生物种胚胎的有益影响的理解。来自已建立的新鲜和海水模型系统的胚胎[即。斑马鱼(Danio Rerio)和珊瑚(Fungia Skaaria)将被评估。我们将评估使用冰重结晶抑制剂控制冰的冷冻保护效果，这是一种实用的海洋和淡水水生物种冷冻保存的新方法。其次，我们将发展胚胎低温生物学特性的知识，这将有助于进一步优化胚胎冷冻保存方案，识别潜在的冷冻损伤机制，并帮助将其翻译为用于人类健康研究和保护的努力。

In most human and animal systems, sperm cryopreservation is well defined and has successful outcome protocols. However, embryo cryopreservation does not exist except for humans and few agriculturally important species. Little cryopreservation success has been observed with marine embryos and larvae and only within the past year has this been changing for some aquatic organisms. The development of embryo cryopreservation technologies revolutionized breeding of domestic animals as well as human reproductive technologies over the past three decades. We will examine two aquatic embryo or larval systems, including coral and fish. Coral reefs are threatened around the world and their conservation would benefit enormously from increased understanding and application of their cryobiology. We believe that our recent work with novel cryopreservation technologies could help transform these cryopreservation processes in these two systems that are currently stalled with low cryopreservation success. The most significant cause of decreased viability and impaired function during cryopreservation is physical and chemical damage resulting from ice crystal formation and growth. In the context of embryo cryopreservation, a low surface area / volume ratio and a low membrane permeability to water and cryoprotectant makes it difficult to accumulate high enough concentrations of cryoprotectant into the embryo to avoid intracellular / interstitial ice formation. Consequently, new approaches are urgently required. Recently, large chemical libraries of small molecule ice recrystallization inhibitors (iRis) have been developed and used in the cryopreservation of mammalian cells. Ice recrystallization is the process that occurs during freezing and thawing where by large ice crystals grow larger at the expense of smaller ones and is a major cause of cellular damage that reduces postthaw viability and potency. We hypothesize that controlling ice formation using novel small molecule ice recrystallization inhibitors will reduce cryoinjury and enhance postthaw viability and cell function of embryos from aquatic species. Our multidisciplinary team has set four complementary aims to improve our understanding of the effectiveness of IRI as cryoprotectants and their beneficial impact on embryos from aquatic species. Embryos from established fresh and saltwater model systems [ie. zebrafish (Danio rerio) and coral (Fungia scutaria) will be evaluated. We will evaluate the cryoprotective effectiveness of using ice recrystallization inhibitors to control ice as a novel approach to the practical cryopreservation of marine and freshwater aquatic species. Secondarily we will develop knowledge on cryobiological properties of embryos which will help inform further optimization of embryo cryopreservation protocols, identify potential mechanism of cryoinjury and aid in the translation of this for use in human health research and conservation efforts.