Collaborative Research: Cryopreservation of Zebrafish Oocytes by an Interdisciplinary Approach

这项研究的总体目标是开发一种新的斑马鱼卵母细胞冷冻保存技术。斑马鱼是研究人类疾病以及胚胎发育、器官发育和衰老等其他研究领域的有价值的模式系统。斑马鱼卵母细胞或胚胎的成功冷冻保存是维持突变系和转基因系数量成倍增长的迫切需要。成功地储存斑马鱼配子或胚胎不仅可以显著降低成本，还可以避免与持续繁殖相关的感染和遗传漂移的风险。此外，开发一种成功的斑马鱼卵母细胞冷冻保存技术将是保护其他鱼类物种的主要踏脚石，其中许多物种正在迅速减少。目前，鱼类精子的冷冻保存虽然取得了有限的成功，但由于其大尺寸和多隔室结构、极低的膜透过率和低温保护剂以及对冷害和细胞内结冰的敏感性等几个主要障碍，鱼类胚胎仍然不能冷冻。为了绕过这些障碍，该项目建议使用多学科方法冷冻保存未受精的斑马鱼卵母细胞。这项建议预测，斑马鱼卵母细胞将比胚胎更容易冷冻保存，因为它们更小，单细胞结构要简单得多，对冷冻保护剂的渗透性和耐受性更高。智力优势：这个项目具有重要意义和创新，因为它提出通过结合工程学(例如，膜渗透性和细胞内冰形成的建模，CPA加载/移除和冷却曲线的数学和实验优化)和细胞/分子生物学方法(例如，膜运输的分子操作)来解决一个基本的科学问题(即，鱼类卵母细胞的冷冻保存)。拟议的机械方法是新颖的，灵感来自于在自然界中观察到的适应方案(例如，青蛙、迟滞、咸虾、细菌和酵母等生物的生存策略，它们能够适应包括冻结和干燥在内的极端条件)；一个关键的适应是细胞内和细胞外糖的积累。最近的研究表明，糖在哺乳动物细胞的冷冻保存和干燥过程中具有良好的作用，但必须克服膜对糖的通透性障碍才能使这一策略有效。初步研究表明，糖与低浓度常规穿透性冷冻保护剂的结合可以弥补前者的低渗透，同时降低后者的细胞毒性。因此，该项目的中心假设是，将细胞内和细胞外的糖与少量穿透性冷冻保护剂相结合，将保护斑马鱼卵母细胞免受与冷冻相关的压力。利用质量传输和非平衡相变的工程模型，结合分子生物学技术，这一建议将通过追求三个具体目标来检验中心假说：(1)检验认为斑马鱼卵母细胞的渗透限制可以通过包括生物学、物理化学和工程方法在内的多学科策略来克服的工作假说；(2)检验细胞内和细胞外的糖类与渗透性CPA的优化组合可以使斑马鱼卵母细胞成功超低温保存的工作假说；(3)检验认为为了最大限度地提高活性能，应该尽可能快地冷却斑马鱼卵母细胞，而不会造成有害的细胞内冰晶形成的工作假说。本文提出的跨学科方法有望克服与斑马鱼种质冷冻相关的障碍，并在成功冷冻斑马鱼卵母细胞方面取得重大进展。这项拟议的工作还有望在低温生物现象的生物热质传递建模和计算机辅助生物热过程设计方面取得进展。拟议研究的广泛影响将通过以下方式实现：(1)在当地高中开展教育推广活动，以斑马鱼为教学工具，激发学生对科学和技术的好奇心/兴趣；(2)为K-12和本科生开发教材；(3)为高中生创建暑期研究计划；(4)培训研究生和博士后研究员。拟议的外联活动预计将扩大和加强格鲁吉亚医学院与当地高中和社区之间正在进行的伙伴关系。拟议编写的教材和课程模块将加强维拉诺瓦大学及以后的生物工程课程。

The overall goal of the proposed research is to develop a novel cryopreservation technique for zebrafish oocytes. The zebrafish is a valuable model system for human diseases and other research areas such as embryogenesis, organ development, and aging. Successful cryopreservation of zebrafish oocytes or embryos is urgently needed to maintain exponentially growing numbers of mutant and transgenic lines. Successful banking of zebrafish gametes or embryos would not only significantly reduce costs, but also avoid risks of infection and genetic drift associated with continuous breeding. Furthermore, development of a successful cryopreservation technique for zebrafish oocytes would be a major stepping-stone towards conservation of other fish species, many of which are rapidly declining. Cryopreservation of fish sperm is currently practiced albeit with limited success, whereas fish embryos remain non-freezable due to several major obstacles such as their large size and multi-compartmental structure, extremely low membrane permeability to water and cryoprotective chemicals, and susceptibility to chilling injury and intracellular ice formation. To circumvent these obstacles, this project proposes to cryopreserve unfertilized zebrafish oocytes using a multidisciplinary approach. This proposal predicts that zebrafish oocytes will be more amenable to cryopreservation than embryos, based on their smaller size, much simpler single-cell structure, and higher permeability and tolerance to cryoprotectants.Intellectual merit: This project is significant and innovative because it proposes to tackle a fundamental scientific problem (i.e., cryopreservation of fish oocytes) by combining engineering (e.g., modeling membrane permeability and intracellular ice formation, mathematical and experimental optimization of CPA loading/removal and cooling profiles) and cell/molecular biology approaches (e.g., molecular manipulation of the membrane transport). The proposed mechanistic approach is novel and inspired by adaptation schemes observed in nature (e.g., survival strategies by organisms such as frogs, tardigrades, brine shrimp, bacteria, and yeast, which are able to adapt to extreme conditions including freezing and desiccation); a key adaptation is the accumulation of intra- and extracellular sugars. Recent studies have demonstrated beneficial effect of sugars during cryopreservation and desiccation of mammalian cells, whereas the membrane permeability barrier to sugars must be overcome for this strategy to be effective. Preliminary studies have demonstrated that the combination of sugars with low concentrations of conventional penetrating cryoprotectants can compensate for the low permeation of the former wile reducing the cytotoxicity of the latter. Thus, the central hypothesis of this project is that combination of intra- and extracellular sugars with small amounts of a penetrating cryoprotectant will protect zebrafish oocytes against freezing-associated stresses. Using engineering models of mass transport and non-equilibrium phase transformation, in combination with molecular biology techniques, this proposal will test the central hypothesis by pursuing three specific aims: (1) test the working hypothesis that osmotic limitations of zebrafish oocytes can be overcome by a multi-disciplinary strategy including biological, physico-chemical, and engineering approaches; (2) test working hypothesis that an optimized combination of intra- and extracellular sugars with a penetrating CPA permits successful cryopreservation of zebrafish oocytes; (3) test working hypothesis that to maximize viability, zebrafish oocytes should be cooled as rapidly as possible without causing formation of deleterious intracellular ice crystals. The interdisciplinary approach proposed here is expected to overcome the obstacles associated with cryopreservation of zebrafish germplasm, and lead to significant advances towards successful cryopreservation of zebrafish oocytes. The proposed work is also expected to lead to advances in the state-of-the-art of bioheat/mass-transfer modeling of cryobiological phenomena, and in computer-aided optimization of biothermal process design.Broader impacts of the proposed research will be attained by: (1) educational outreach activities in local high schools using zebrafish as a teaching tool to awaken curiosity/interest among the students in science and technology; (2) developing teaching materials for K-12 as well as undergraduate bioengineering students; (3) creating a summer research program for high school students; (4) training graduate students and postdoctoral fellows. The proposed outreach activities are expected to broaden and strengthen the ongoing partnership of the Medical College of Georgia with local high school and communities. The proposed development of educational materials and course modules will enhance the bioengineering curriculum at Villanova University and beyond.