Optimization of vitrification methods for Drosophila embryos

果蝇胚胎玻璃化冷冻方法的优化

• 批准号: 10163222
• 负责人: Rebecca Sandlin
• 金额: $ 21万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10163222
• 关键词: Address; Adult; Air; Alcohols; Alkanes; Animal Model; Automation; Biology; Chemistry; Complex; Cryopreservation; Cryopreserved Cell; Cryoprotective Agents; Crystallization; Development; Developmental Biology; Devices; Dimethyl Sulfoxide; Dose; Drosophila genus; Embryo; Engineering; Ensure; Excision; Exhibits; Food; Freezing; Generations; Genetic Drift; Glass; Goals; Gold; Home; Ice; Immersion; Industrialization; Kinetics; Laboratories; Laboratory Research; Liquid substance; Literature; Maintenance; Methods; Microfluidic Microchips; Microfluidics; Mutate; Mutation; Nature; Outcome; Paper; Permeability; Procedures; Propylene Glycols; Protocols documentation; Publishing; Recovery; Reporting; Research; Research Personnel; Resources; Review Literature; Rewarming; Risk; Series; Solid; Specimen; System; Techniques; Technology; Temperature; Terpenes; Toxic effect; Water; Work; base; cryobiology; cryogenics; crystallinity; design; experience; experimental study; improved; innovation; limonene; multidisciplinary; mutant; novel; process optimization; response; success; tool; uptake

PROJECT SUMMARY Drosophila research labs routinely maintain hundreds to thousands of unique strains, where each new generation must be transferred to fresh food every 4 to 6 weeks. Not only is this practice labor and resource intensive, but there is a risk that valuable strains will be lost or acquire additional mutations. This limitation would be overcome by the development of Drosophila cryopreservation methods. Thus, the overall goal of this proposal is to develop a simple, robust method to cryopreserve Drosophila embryos. Based on literature review and our expertise in cryobiology and Drosophila biology, we anticipate that vitrification is the optimal approach to cryopreservation. Vitrification is an `ice-free' method of cryopreservation where cells are loaded with high concentrations of cryoprotective agents (CPAs, e.g. dimethyl sulfoxide, propylene glycol, etc.) and rapidly cooled through the glass transition. The result is formation of an amorphous glassy state as opposed to crystalline ice. Nearly thirty years ago, a vitrification protocol for wild-type Drosophila was reported. However, this protocol is quite complex and results in a loss of nearly 70% of embryos upon development into adults. However, this breakthrough method represents an excellent starting point for us here, where we will take a comprehensive approach to both simplify and optimize the vitrification procedure by incorporating concepts and technologies from industrial chemistry, engineering and cryobiology. In Aim 1, we will simplify the embryo permeabilization procedure and then automate the liquid handling steps of the protocol by incorporating microfluidics. Automation will reduce user-error and decrease labor demands. In Aim 2, we will optimize the vitrification procedure by systematically evaluating a series of CPAs and CPA cocktail solutions. This information will then be used to increase the intraembryonic concentration of CPAs, thereby reducing the risk of ice crystallization upon cooling or rewarming. We will finally merge results from both Aims to validate this simplified and optimized vitrification procedure, where our goal is to achieve ≥60% survival of thawed embryos to adults. To better ensure the successful completion the work described here, we have assembled a multidisciplinary team including cryobiologists, engineers and Drosophila biology experts. The successful completion of this exploratory proposal will result in the development of a simplified and robust vitrification method for Drosophila embryos that can be quickly implemented into laboratories and resource centers.

项目摘要 果蝇研究实验室通常维持数百至数千种独特的菌株 必须每4至6周将一代转移到新鲜食品中。这个实践实验室和资源不仅 密集型，但有可能丢失或获得其他突变的风险。这个限制会 果蝇冷冻保存方法的发展要克服。这是该提议的总体目标 是为冷冻果蝇胚胎开发一种简单，坚固的方法。基于文献评论和我们的 冷冻生物学和果蝇生物学方面的专业知识，我们预计玻璃化是最佳方法 冷冻保存。玻璃化是一种“无冰”的冷冻保存方法，其中细胞充满了高 冷冻保护剂的浓度（CPA，例如二甲基亚氧化二甲基亚氧化物，丙二醇等），并迅速冷却 通过玻璃过渡。结果是形成了无定形的玻璃状态，而不是结晶冰。 近三十年前，据报道了野生型果蝇的玻璃化方案。但是，此协议是 非常复杂，导致在成人发育后丧失了近70％的胚胎。但是，这个 突破方法对我们来说代表了一个极好的起点，我们将在其中进行全面 通过合并概念和技术，可以简化和优化玻璃化程序 来自工业化学，工程和冷冻生物学。在AIM 1中，我们将简化胚胎通透性 程序，然后通过编码微流体来自动化协议的液体处理步骤。自动化 将减少用户纠正并减少劳动力需求。在AIM 2中，我们将通过 系统地评估一系列CPA和CPA鸡尾酒解决方案。然后将使用此信息 增加CPA的胚内浓度，从而降低冷却后冰结晶的风险 或重新加热。我们最终将合并两个目的的结果，以验证这种简化和优化的玻璃化 程序，我们的目标是实现对成年人融化的胚胎≥60％的存活率。更好地确保 成功完成此处描述的工作，我们组建了一个多学科团队 冷冻生物学家，工程师和果蝇生物学专家。该探索性建议的成功完成 将为果蝇胚胎的简化且可靠的玻璃化方法开发 迅速实施到实验室和资源中心。