Optimization of vitrification methods for Drosophila embryos

项目总结 果蝇研究实验室经常维护数百到数千个独特的品系，其中每个新的 每隔4到6周，必须将一代人转移到新鲜食物中。这种做法不仅是劳力和资源 密集，但有价值的菌株将丢失或获得更多突变的风险。这一限制将 被果蝇冷冻保存方法的发展所克服。因此，这项提案的总体目标是 是开发一种简单、可靠的方法来冷冻保存果蝇胚胎。基于文献回顾和我们的 在低温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治疗 超低温保存。玻璃化冷冻是一种无冰的冷冻保存方法，在这种方法中，细胞中含有高水平的 低温保护剂(CPA，如二甲基亚砜、丙二醇等)的浓度并迅速冷却 通过玻璃过渡。其结果是形成了无定形的玻璃态，而不是结晶冰。 近30年前，报道了一项针对野生果蝇的玻璃化冷冻方案。然而，该协议是 相当复杂，在发育成成体后会损失近70%的胚胎。不过，这个 突破性的方法对我们来说是一个很好的起点，我们将在这里采取全面的 通过整合概念和技术来简化和优化玻璃化过程的方法 来自工业化学、工程学和低温生物学。在目标1中，我们将简化胚胎渗透 程序，然后通过结合微流体使协议的液体处理步骤自动化。自动化 将减少用户错误，减少人力需求。在目标2中，我们将通过以下方式优化玻璃化程序 系统评估一系列注册会计师和注册会计师鸡尾酒解决方案。然后这些信息将被用于 增加CPA的胚胎内浓度，从而降低冷却时冰结晶的风险 或者重新升温。我们最终将合并这两个目标的结果，以验证这种简化和优化的玻璃化 我们的目标是实现≥60%的成年解冻胚胎存活率。为了更好地确保 成功地完成了这里描述的工作，我们已经组建了一个多学科的团队，包括 低温生物学家、工程师和果蝇生物学专家。这一探索性提案的圆满完成 将导致开发一种简化和强大的果蝇胚胎玻璃化冷冻方法，这种方法可以 迅速落实到实验室和资源中心。

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.