Cryopreservation of zebrafish larvae and embryos for biomedical research

项目概要 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、操作和 图像。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 这对于我们理解生物学、描述疾病和发现新疗法发挥了重要作用 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还是有必要的。 与设施空间、人员和维护成本相关，这也可能导致由于不可替代的研究而损失的风险 灾难性事件和基因改造。在此能力下，一种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方法。因此，本项目 旨在开发兼容不同存储的斑马鱼胚胎和幼虫冷冻保存方案 温度并通过广泛的传播工作来培训外部实验室易于实施的协议。 虽然该领域在为一系列鱼类、卵子/胚胎建立精子库方面相对成功 尽管冷冻保存具有明显的优势，但仍遇到了严峻的挑战。精子冷冻保存需要 至少需要 6 个月的时间才能产生成年品系，而冷冻保存胚胎则可以更快速、更快速地产生 解冻后直接使用。然而，两种主要的冷冻保存方法——缓慢冷却和玻璃化——各有其缺点。 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) 但必须与可能损害细胞的大量冰形成竞争。或者，也可以采用无冰方法，例如 玻璃化过程需要高浓度的有毒 CPA 和高冷却速率，从而限制了可扩展性。相反，我们建议 相对未经探索的方法称为间断冷却。 间断冷却使用低浓度的 CPA，将样品缓慢冷却至零下高温 (~-20°C) 有限的冰形成，然后快速冷却至深低温，将剩余的 未冻结的部分变成玻璃状状态。这种灵活的协议可以在高温度下实现短期保存步骤 零下温度和与干冰兼容的斑马鱼库长期保存解决方案 运输和液氮用于可靠的内部存储。这将通过应用第一原则来实现 冷冻保存来设计存储解决方案和间断冷却协议，以保护复杂的系统，而无需 不良功能后果（具体目标 1）。通过应用林蛙的经验教训，这些努力将得到加强 自然以提高斑马鱼解冻后的存活率（具体目标 2）。最后，在具体目标 3 中，我们将进行广泛的 与非研究人员进行传播工作，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.