Cryopreservation of zebrafish larvae and embryos for biomedical research

用于生物医学研究的斑马鱼幼虫和胚胎的冷冻保存

• 批准号: 10626493
• 负责人: Shannon Noella Tessier
• 金额: $ 64.59万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10626493
• 关键词: Adopted; Adult; Animals; Biological Models; Biological Products; Biology; Biomedical Research; Breeding; Communicable Diseases; Communities; Complex; Cryopreservation; Crystallization; Development; Disease; Dry Ice; Educational process of instructing; Embryo; Engineering; Ensure; Environmental Risk Factor; Event; Failure; Fishes; Freezing; Generations; Genes; Genetic; Genome; Glass; Health; Human; Human Resources; Ice; Image; Injectable; Interruption; Laboratories; Larva; Length; Liquid substance; Long-Term Effects; Maintenance; Mediating; Methods; Modeling; Modification; Molecular; Names; Nature; Nitrogen; Organism; Outcome; Oxygen; Procedures; Protocols documentation; Rana; Recovery; Reporter; Research; Research Personnel; Resources; Risk; Running; Sampling; Specimen; Sperm Banks; Standard Preparations; Survival Rate; System; Temperature; Testing; Time; Training; Transgenic Organisms; Trehalose; Validation; Vial device; Visit; Water; Wood material; Work; Zebrafish; animation; cell injury; cost; cryogenics; design; egg; embryo cryopreservation; experimental study; extracellular; flexibility; high throughput screening; imaging study; improved; improved outcome; inhibitor; model organism; mutant; novel therapeutics; preservation; prevent; reproductive; sperm cryopreservation; stress tolerance; success; tool; user-friendly

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.

项目总结 斑马鱼是应用最广泛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、报道和突变株系的创建 在我们对生物学的理解、疾病的特征和发现新的治疗方法方面都起到了重要作用 仅举几个例子。尽管斑马鱼的价格相对便宜，但无限期地保持大量活鱼的库存是 与设施空间、人员和维护成本相关，还有可能损失不可替代的研究线路 到灾难性事件和基因改造。在这种能力下，一种安全可靠的超低温保存方法 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，这个项目 旨在开发一种兼容不同保存方式的斑马鱼胚胎和仔鱼冷冻保存方案 并通过广泛的传播努力，培训外部实验室，使其具有易于实施的协议。 虽然该领域在为一系列鱼类、卵子/胚胎建立精子库方面取得了相对成功的成就 尽管冷冻保存有明显的优势，但它面临着严峻的挑战。精子的冷冻保存需要在 与胚胎冷冻保存相比，至少6个月才能产生成体菌株，这将使更快速和 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--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广泛结冰竞争。或者，无冰方法，如 玻璃化，需要高浓度有毒的CPA和限制可伸缩性的高冷却速度。相反，我们提出了一个 相对未被探索的方法被称为间断冷却。 间歇冷却使用低浓度的CPA，将样品缓慢冷却到零度以下的高温(~-20°C) 在冰形成有限的情况下，在快速冷却到深低温之前，将剩余的 解冻后的部分变成玻璃体状。这一灵活的协议实现了短期保存步骤 零度以下的温度和斑马鱼库的长期保存解决方案，与干冰兼容 装运和液氮，以实现可靠的内部存储。这将通过将第一原则应用于 低温保存以设计存储解决方案和中断冷却协议，从而保护复杂的系统，而无需 不利的功能后果(具体目标1)。这些努力将通过应用林蛙的经验教训来加强 自然改善斑马鱼解冻后的存活率(具体目标2)。最后，在具体目标3中，我们将广泛开展 与非学习人员一起开展宣传工作，并为更广泛的社区创建可公开使用的培训工具。