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

由于过度捕捞和全球气候变化，世界各地的水生物种正在经历迅速减少，建立长期储存鱼类遗传资源的新技术已变得至关重要。该项目将研究生殖干细胞生物学的一个创新领域，以探索虹鱼(Oncorhynchus MykISS)精原干细胞玻璃化冷冻的最佳条件。玻璃化包括超快速冷却，导致细胞瞬间凝固而不结晶。玻璃化方案的开发是可取的，因为玻璃化速度很快，该过程通过消除细胞内的冰形成而避免了对细胞的损伤，而是创建了玻璃样(玻璃体)状态，从而导致更高的细胞存活率。这项研究将在日本东京的东京海洋科学技术大学与著名的鱼类生殖干细胞生物学专家吉崎五郎博士合作并在其监督下进行。这项拟议的研究将为干细胞冷冻生物学的基本方面提供深入的见解，并可能导致一种廉价、经济的方法来保存鱼类遗传资源，以保护濒危鱼类和水产养殖目的。该项目的拟议实验将使用GFP-VASA+转基因虹鱼来1)确定玻璃化过程对精原干细胞存活的影响，2)研究玻璃化冷冻精原干细胞的功能。干细胞的存活将通过使用流式细胞术进行量化，这将根据荧光模式将细胞群体区分为可存活或不可存活。生殖细胞移植试验将被用来确定存活的玻璃化干细胞是否保留了在受体体内定居和增殖的功能潜力。这项研究将提高对低温保存和玻璃化冷冻对干细胞功能的复杂影响的理解，同时提供必要的信息，成功地实施干细胞研究，作为一种新的人工生殖技术方法，以最大限度地提高遗传多样性，以保护受威胁的水生物种。该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).