EAPSI: Ultra-Rapid Freezing to Maintain Stem Cell Developmental Potential in Rainbow Trout

由于过度开发和全球气候变化，世界各地的水生物种正在迅速减少，因此建立新的鱼类遗传资源长期储存技术变得至关重要。本项目将研究生殖干细胞生物学的一个创新领域，以研究虹鳟精原干细胞玻璃化的最佳条件。玻璃化包括超高速冷却，导致细胞瞬间凝固而不结晶。由于玻璃化是快速的，并且通过消除细胞内冰形成的过程避免了对细胞的创伤，而是产生了玻璃样（玻璃体）状态，从而导致更高的细胞存活率，因此玻璃化方案的发展是可取的。这项研究将在日本东京的东京海洋科学技术大学进行，并在著名的鱼类生殖干细胞生物学专家吉崎五郎博士的指导下进行。该研究将为干细胞低温生物学的基本方面提供见解，并可能导致一种廉价、经济的方法用于保护濒危鱼类和水产养殖目的的鱼类遗传资源的实际应用。本项目拟进行的实验将使用Gfp-vasa+转基因虹鳟鱼，1)确定玻璃化过程对精原干细胞存活的影响，2)研究玻璃化后精原干细胞的功能。干细胞存活将通过流式细胞术进行量化，流式细胞术将根据荧光模式将细胞群分为有活力或无活力。生殖细胞移植试验将用于确定活的玻璃化干细胞是否保留其在受体中定植和增殖的功能潜力。本研究将提高对冷冻保存和玻璃化对干细胞功能的复杂影响的理解，同时为成功实施干细胞研究作为一种新的人工生殖技术方法提供必要的信息，以最大限度地提高濒危水生物种的遗传多样性。NSF EAPSI奖是与日本科学促进会（JSPS）合作资助的。

The establishment of novel technologies for the long-term storage of fish genetic resources has become critical as aquatic species around the world are experiencing rapid declines due to overexploitation and global climate change. This project will examine an innovative area of reproductive stem cell biology to investigate optimal conditions for vitrification of spermatogonial stem cells from Rainbow trout (Oncorhynchus mykiss). Vitrification involves ultra-rapid cooling that results in instant solidification of the cell without crystallization. Development of a vitrification protocol is desirable since vitrification is fast and the process avoids trauma to cells by eliminating intracellular ice formation and instead creates a glass-like (vitreous) state, resulting in higher cell survival. This study will be conducted at the Tokyo University of Marine Sciences and Technology in Tokyo, Japan in collaboration with and under the supervision of Dr. Goro Yoshizaki, a renowned expert in fish reproductive stem cell biology. The proposed study will provide insight into fundamental aspects of stem cell cryobiology and could lead to real-world application of an inexpensive, economical method for preserving fish genetic resources for the conservation of endangered fish and aquaculture purposes.The proposed experiments for this project will use Gfp-vasa+ transgenic rainbow trout to 1) determine the effects of the vitrification process on spermatogonial stem cell survival and 2) investigate the functionality of vitrified spermatogonial stem cells. Stem cell survival will be quantified by using flow cytometry, which will separate cell populations into viable or non-viable based on fluorescence patterns. A germ cell transplantation assay will be utilized to determine if the viable vitrified stem cells retain their functionality potential to colonize and proliferate in recipients. This research will improve understanding of the complex effects of cryopreservation and vitrification on stem cell function while providing the information necessary to successfully implement stem cell research as a novel artificial reproductive technology method to maximize genetic diversity for the conservation of threatened aquatic species. This NSF EAPSI award is funded in collaboration with the Japan Society for the Promotion of Science (JSPS).