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，并有成功的结果方案。然而，除了人类和少数农业上重要的物种外，胚胎冷冻保存并不存在。海洋胚胎和幼体的低温保存很少成功，仅在过去一年中，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彻底改变了家畜育种和人类生殖技术。我们将研究两种水生胚胎或幼虫系统，包括珊瑚和鱼类。珊瑚礁在世界各地都受到威胁，对珊瑚礁的保护将从增加对其低温生物学的理解和应用中受益匪浅。我们相信，我们最近的工作与新的低温保存技术可以帮助改变这些低温保存过程在这两个系统中，目前停滞不前的低温保存成功。在低温保存过程中，最重要的原因是冰晶形成和生长造成的物理和化学损伤。在胚胎低温保存的情况下，低的表面积/体积比和低的膜对水和冷冻保护剂的渗透性使得很难在胚胎中积累足够高浓度的冷冻保护剂来避免细胞内/间质结冰。因此，迫切需要新的办法。近年来，小分子冰重结晶抑制剂（iRis）的大型化学文库已被开发出来，并用于哺乳动物细胞的低温保存。冰重结晶是在冷冻和解冻过程中发生的一个过程，在这个过程中，大的冰晶会以牺牲小的冰晶为代价变大，这是导致细胞损伤的主要原因，会降低解冻后的生存能力和效力。我们假设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，提高水生物种胚胎的解冻后活力和细胞功能。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。来自已建立的淡水和咸水模型系统的胚胎。斑马鱼（Danio rerio）和珊瑚（Fungia scutaria）将进行评估。我们将评估使用冰重结晶抑制剂来控制冰的冷冻保护效果，作为海洋和淡水水生物种实际冷冻保存的新方法。其次，我们将发展关于胚胎低温生物学特性的知识，这将有助于进一步优化胚胎低温保存方案，确定低温损伤的潜在机制，并帮助将其翻译用于人类健康研究和保存工作。

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.