Optimization of vitrification methods for Drosophila embryos

果蝇胚胎玻璃化冷冻方法的优化

基本信息

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
项目类别：
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

项目摘要

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.

项目概要果蝇研究实验室通常维护数百到数千个独特的菌株，其中每个新菌株每 4 至 6 周必须将一代转移到新鲜食物中，这种做法不仅耗费人力和资源。密集，但存在有价值的菌株丢失或获得额外突变的风险。因此，该提案的总体目标是通过开发果蝇冷冻保存方法来克服。基于文献综述和我们的研究，开发一种简单、可靠的方法来冷冻保存果蝇胚胎。在冷冻生物学和果蝇生物学方面的专业知识，我们预计玻璃化冷冻是最佳方法玻璃化冷冻是一种“无冰”冷冻保存方法，其中细胞负载高浓度。冷冻保护剂（CPA，例如二甲亚砜、丙二醇等）浓度并快速冷却通过玻璃化转变，结果是形成非晶态玻璃态，而不是结晶冰。近三十年前，报道了野生型果蝇的玻璃化冷冻方案。然而，该方案是。相当复杂，并导致在发育成成人时损失近 70% 的胚胎。突破性方法对我们来说是一个极好的起点，我们将采取全面的方法通过结合概念和技术来简化和优化玻璃化程序的方法在目标 1 中，我们将简化胚胎透化。程序，然后通过结合微流体技术来自动化协议的液体处理步骤。将减少用户错误并减少劳动力需求在目标 2 中，我们将通过以下方式优化玻璃化程序。然后系统地评估一系列注册会计师和注册会计师鸡尾酒解决方案。增加 CPA 的胚胎内浓度，从而降低冷却时冰结晶的风险我们最终将合并两个目标的结果来验证这种简化和优化的玻璃化。程序，我们的目标是实现解冻胚胎成体存活率≥60%，以更好地确保。成功完成此处描述的工作，我们组建了一个多学科团队，包括冷冻生物学家、工程师和果蝇生物学专家成功完成了这一探索性提案。将导致开发出一种简化且稳健的果蝇胚胎玻璃化冷冻方法迅速应用于实验室和资源中心。