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定义的，并且具有成功的结果协议。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。在海洋胚胎和幼体中观察到的冷冻保存成功率很低，只是在过去的一年里，一些水生生物的情况才有所改变。胚胎冷冻技术的发展在过去的三十年里彻底改变了家畜的饲养以及人类的生殖技术。我们将研究两种水生胚胎或幼体系统，包括珊瑚和鱼类。珊瑚礁在世界各地都受到威胁，对珊瑚礁低温生物学的进一步了解和应用将使珊瑚礁的保护受益匪浅。我们相信，我们最近的工作与新的冷冻保存技术可以帮助改变这些冷冻保存过程中，这两个系统，目前停滞不前，低冷冻保存成功。低温保存过程中活力下降和功能受损的最重要原因是冰晶形成和生长造成的物理和化学损伤。在胚胎冷冻保存的背景下，低表面积/体积比和对水和冷冻保护剂的低膜渗透性使得难以将足够高浓度的冷冻保护剂累积到胚胎中以避免细胞内/间质冰形成。因此，迫切需要采取新的办法。最近，已经开发了小分子冰重结晶抑制剂（iRis）的大型化学文库，并将其用于哺乳动物细胞的冷冻保存。冰重结晶是在冷冻和解冻期间发生的过程，其中大冰晶以较小的冰晶为代价而变大，并且是降低解冻后活力和效力的细胞损伤的主要原因。我们假设，控制冰的形成，使用新的小分子冰重结晶抑制剂将减少冷冻损伤，提高解冻后的生存能力和细胞功能的胚胎从水生物种。我们的多学科团队已经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。胚胎从建立淡水和咸水模型系统[即。将对斑马鱼（Danio rerio）和珊瑚（Fungia scutaria）进行评价。我们将评估使用冰重结晶抑制剂来控制冰作为海洋和淡水水生物种的实际冷冻保存的新方法的冷冻保护效果。其次，我们将开发有关胚胎的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，确定冷冻损伤的潜在机制，并帮助将其用于人类健康研究和保护工作。

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.