Cryopreservation of zebrafish larvae and embryos for biomedical research

项目摘要 斑马鱼是最广泛使用的研究模型系统之一，因为它们易于繁殖，操纵， 形象正是这种巨大的效用导致了数以万计的转基因、报告基因和突变株系的产生 对我们理解生物学、表征疾病和发现新疗法都有帮助， 仅举几个例子。尽管斑马鱼的饲养成本相对较低，但无限期地维持大量活鱼的库存， 与设施空间，人员和维护成本有关，这也有可能失去不可替代的研究线， 灾难性的事件和基因改造。在此能力，一个安全可靠的冷冻保存方法， 斑马鱼品系将为研究界提供一种储存宝贵资源的手段。因此，该项目 旨在开发一种适合不同储存条件的斑马鱼胚胎和幼体冷冻保存方案， 通过广泛的传播努力，利用易于实施的方案培训外部实验室。 虽然该领域在建立一系列鱼类精子库方面相对成功，但卵子/胚胎 尽管低温保存具有明显的优点，但仍遇到了严峻的挑战。冷冻保存精子需要 与冷冻保存胚胎相比，冷冻保存胚胎可以更快， 解冻后直接使用。然而，两种主要的低温保存方法-缓慢冷却和玻璃化-每一种都有 仍然无法克服的严重限制。缓慢冷却使用低浓度的冷冻保护剂 （CPA）还必须与可能损害细胞的大量冰形成竞争。或者，无冰方法，如 玻璃化需要高浓度的有毒CPA和高冷却速率，这限制了可扩展性。相反，我们提出了一个 相对未开发的方法称为中断冷却。 中断冷却使用低浓度的CPA，因为样品缓慢冷却到零度以下的高温（~-20 °C） 在快速冷却到深低温之前，冰的形成有限，然后将剩余的冰转化为低温 解冻的部分变成玻璃状的状态。这种灵活的方案既可以在高温下进行短期保存步骤， 零下的温度和长期保存解决方案的斑马鱼银行，是兼容的干冰， 运输和液氮的可靠内部存储。这将通过应用第一原则来实现， 冷冻保存设计的存储解决方案和中断冷却协议，将保护复杂的系统， 不良功能后果（具体目标1）。这些努力将通过应用从树蛙中吸取的教训得到加强， 性质，以提高解冻后斑马鱼的存活率（具体目标2）。最后，在具体目标3中，我们将执行广泛的 与非研究人员一起开展传播工作，并为更广泛的社区创建公开可用的培训工具。

PROJECT SUMMARY Zebrafish are one of the most broadly used research model systems because they are easy to breed, manipulate, and image. It is this immense utility that has led to the creation of tens of thousands of transgenic, reporter, and mutant lines that have been instrumental in our understanding of biology, characterizing disease, and discovering new therapeutics, to name only a few. Despite zebrafish being relatively cheap to house, maintaining large stocks of live fish indefinitely is associated with facility space, personnel, and maintenance costs that also risks the loss of irreplaceable research lines due to catastrophic events and genetic modifications. In this capacity, a safe and reliable method for cryopreservation of zebrafish lines would provide a means to bank precious resources for the research community. Consequently, this project aims to develop a zebrafish embryo and larvae cryopreservation protocol that is compatible with different storage temperatures and to train external labs with an easy to implement protocol through extensive dissemination efforts. While the field has been relatively successful in establishing sperm banks for a range of fish, egg/embryo cryopreservation has been met with severe challenges, despite the clear advantages. Cryopreservation of sperm requires at least 6 months to generate an adult strain, in contrast to cryopreservation of embryos that would enable more rapid and direct use upon thawing. However, the two main cryopreservation approaches – slow cooling and vitrification – each have severe limitations that have remained insurmountable. Slow cooling uses low concentrations of cryoprotectant agents (CPAs) yet must compete with extensive ice formation that can damage cells. Alternatively, ice-free methods, such as vitrification, require high concentration of toxic CPAs and high cooling rates that limit scalability. Instead, we propose a relatively unexplored method termed interrupted cooling. Interrupted cooling uses low concentrations of CPAs as samples are slowly cooled to high subzero temperatures (~-20°C) with limited ice formation, before a rapid cooling protocol to deep cryogenic temperatures that converts the remaining unfrozen fraction into a vitreous-like state. This flexible protocol enables both a short-term preservation step at high subzero temperatures and a long-term preservation solution for zebrafish banking that is compatible with dry ice for shipment and liquid nitrogen for reliable in-house storage. This will be achieved by applying first principles in cryopreservation to design a storage solution and interrupted cooling protocol that will protect complex systems without adverse functional consequences (Specific Aim 1). These efforts will be enhanced by applying lessons from wood frogs in nature to improve zebrafish survival after thawing (Specific Aim 2). Finally, in Specific Aim 3, we will perform extensive dissemination efforts with non-study staff and create publicly available training tools for the broader community.