Cryopreservation of spermatogonial stem cells (SSCs) is essential for preservation of valuable livestock and clinical applications. Although optimal equilibration of cryoprotectants has emerged as a promising approach to improve the cryopreservation efficiency, standard equilibration protocols have not yet been considered in cryopreservation of SSCs. This study aimed to establish a standard equilibration protocol to improve the cryopreservation efficiency of murine germ cells enriched for SSCs. After time- and temperature-dependent equilibration, the germ cells were cryopreserved with 10% dimethyl sulfoxide (DMSO) and 200 mM trehalose. To investigate cryopreservation efficiency at different equilibration conditions, the survival and proliferation rates were assessed after thawing, and then, cytotoxicity and intracellular trehalose quantification were analyzed. Protein (PLZF, GFRα1, VASA, and c-Kit) and gene (Bcl6b, Erm, Dazl, and Sycp1) expression was determined using immunofluorescence and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. The proliferation rate increased significantly following equilibration for 20 minutes at room temperature (RT; 163.7% ± 24.6%) or 4°C (269.0% ± 18.2%). Cytotoxicity was reduced in 10% DMSO with 200 mM trehalose compared with that of 10% DMSO alone. Also, intracellular trehalose was observed after equilibration. The immunofluorescence and RT-qPCR data revealed that the murine germ cells enriched for SSCs retained their self-renewal ability after cryopreservation following equilibration. The most effective protocol was equilibration with 10% DMSO and 200 mM trehalose for 20 minutes at RT or 4°C, which is due to synergistic effects of intracellular and extracellular trehalose. This improved methodology will contribute toward the development of a standardized freezing protocol for murine germ cells enriched for SSCs and thereby expand their application in various fields.

中文翻译：

---

平衡时间和温度对小鼠精原干细胞冷冻保存的影响。

精原干细胞（SSCs）的冷冻保存对于珍贵的家畜和临床应用至关重要。尽管最佳的冷冻保护剂平衡已成为提高冷冻保存效率的一种有前途的方法，但在SSC的冷冻保存中尚未考虑标准的平衡方案。这项研究旨在建立一个标准的平衡方案，以提高富含SSCs的鼠生殖细胞的冷冻保存效率。经过时间和温度依赖性平衡后，将生殖细胞用10％二甲基亚砜（DMSO）和200 mM海藻糖冷冻保存。为了研究在不同平衡条件下的冷冻保存效率，解冻后评估了存活率和增殖率，然后，分析了细胞毒性和细胞内海藻糖的定量。蛋白质（PLZF，GFRα1，VASA和c-Kit）和基因（Bcl6b，呃，DAZL和SYCP1分别使用免疫荧光和实时定量聚合酶链反应（RT-qPCR）确定表达。在室温（RT; 163.7％±24.6％）或4°C（269.0％±18.2％）平衡20分钟后，增殖速率显着增加。与单独的10％DMSO相比，含200 mM海藻糖的10％DMSO降低了细胞毒性。另外，平衡后观察到细胞内海藻糖。免疫荧光和RT-qPCR数据显示，富含SSC的鼠生殖细胞在平衡后冷冻保存后保留了其自我更新能力。最有效的方案是在室温或4°C下用10％DMSO和200 mM海藻糖平衡20分钟，这是由于细胞内和细胞外海藻糖的协同作用。