Microencapsulation of oocytes for low-CPA (cryoprotectant) vitrification

描述（申请人提供）：卵母细胞冷冻保存对推进辅助生殖医学、维护动物资源和牲畜管理具有重要意义。然而，目前常用的卵母细胞冷冻保存方法，无论是慢速冷冻还是传统的玻璃化冷冻，都存在固有的缺陷。例如，缓慢冻结（冻结：液态水转变为冰晶）的方法与不可避免的细胞损伤有关，因为冰的形成和缓慢冻结诱导的细胞脱水。传统的玻璃化（玻璃化：将液态水转变为无定形、玻璃态而不是冰晶）方法所需的CPA（冷冻保护剂）浓度异常高（4 - 7 M），即使在短短几分钟的暴露时间内，也会对活细胞造成显著的代谢和渗透损伤。据推测，正是这些固有的缺陷导致了迄今为止卵母细胞冷冻保存的惨淡结果。R01提案中提出的研究目标是通过将细胞微包在海藻酸盐微胶囊中，以低cpa（低且无毒的冷冻保护剂量，= 1.5 M）浓度玻璃化来开发细胞低温保存的新策略。提出的低cpa玻璃化方法结合了目前常用的慢速冷冻和传统玻璃化技术的所有优点，同时避免了它们的所有缺点。本项目将使用自然繁殖（远交种）的Peromyscus卵母细胞作为生物学模型，以便从所提出的研究中获得的结果可以更易于转移，以实现包括人类在内的其他自然繁殖哺乳动物卵母细胞的低cpa玻璃化。此外，鉴于胚胎冷冻保存在一般情况下是成功的，本项目将以Peromyscus胚胎作为基准生物学模型来测试新方法。相信本研究和新型的低cpa玻璃化方法将对辅助生殖医学、动物资源维护和牲畜管理等卵母细胞冷冻保存领域产生重大影响。

DESCRIPTION (provided by applicant): Oocyte cryopreservation is of great importance to the advancement of assisted reproductive medicine, maintenance of animal resources, and livestock management. However, the commonly used methods today for oocyte cryopreservation by either slow-freezing or conventional vitrification have inherent drawbacks. For example, the slow-freezing (freezing: the transition of liquid water into ice crystal) approach is associated with inevitable cell injury due to ice formation and slow- freezing induced cell dehydration. The unusually high CPA (cryoprotectant) concentration (4 - 7 M) required by the conventional vitrification (vitrification: the transition of liquid water into an amorphous, glassy state rather than ice crystal) method can result in significant metabolic and osmotic injury in living cells even in a short exposure time of only a few minutes. Presumably, it is these inherent drawbacks that are responsible for the dismal outcome of oocyte cryopreservation to date. The goal of the proposed research outlined in this R01 proposal is to develop new strategies for cell cryopreservation by microencapsulating the cells in alginate microcapsule to vitrify at a low-CPA (low and non- toxic amount of cryoprotectants, = 1.5 M) concentration. The proposed low-CPA vitrification approach combines all the advantages of the commonly used slow-freezing and conventional vitrification techniques today while avoiding all their shortcomings. Oocytes of the naturally bred (outbred) Peromyscus will be used as the biological model in this project so that the results obtained from the proposed studies can be more transferable to achieve low-CPA vitrification of oocytes of other naturally bred mammals including humans. In addition, Peromyscus embryos will be used as the benchmark biological model in this project to test the new approach in view of the fact that embryo cryopreservation has been successful in general. It is believed that the proposed research and the novel low-CPA vitrification approach will have a significant impact on the field of oocyte cryopreservation for assisted reproductive medicine, maintenance of animal resources, and livestock management. PUBLIC HEALTH RELEVANCE: Oocyte cryopreservation is of great importance to the advancement of assisted reproductive medicine. We propose to develop a novel technology to achieve much improved performance for oocyte cryopreservation. This research will have a significant impact on the preservation of future fertility of women who may lose gonadal function because of exposure to environmental/occupational hazards or aggressive medical treatments such as extirpative surgery, radiation, and chemotherapy.