Department of Animal Science and Technology, Chung-Ang University, Anseong, Republic of Korea.
Department of Food Science and Technology, Chung-Ang University, Anseong, Republic of Korea.
Biopreserv Biobank. 2020 Jun;18(3):213-221. doi: 10.1089/bio.2019.0116. Epub 2020 Mar 27.
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 (, , , and ) 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.
精原干细胞 (SSCs) 的冷冻保存对于保存有价值的家畜和临床应用至关重要。尽管优化冷冻保护剂的平衡已成为提高冷冻保存效率的有前途的方法，但在 SSCs 的冷冻保存中尚未考虑标准的平衡方案。本研究旨在建立标准的平衡方案，以提高富含 SSCs 的鼠类生殖细胞的冷冻保存效率。在时间和温度依赖性平衡后，用 10%二甲基亚砜 (DMSO) 和 200mM海藻糖冷冻保存生殖细胞。为了研究不同平衡条件下的冷冻保存效率，在解冻后评估了存活和增殖率，然后分析了细胞毒性和细胞内海藻糖定量。使用免疫荧光和实时定量聚合酶链反应 (RT-qPCR) 分别测定蛋白质 (PLZF、GFRα1、VASA 和 c-Kit) 和基因 (,,, 和 ) 的表达。在室温 (RT; 163.7%±24.6%) 或 4°C (269.0%±18.2%) 平衡 20 分钟后，增殖率显著增加。与单独使用 10%DMSO 相比，在 10%DMSO 中添加 200mM 海藻糖可降低细胞毒性。此外，在平衡后观察到细胞内海藻糖。免疫荧光和 RT-qPCR 数据显示，经过平衡后，富含 SSCs 的鼠类生殖细胞在冷冻保存后保留了自我更新能力。最有效的方案是在 RT 或 4°C 下用 10%DMSO 和 200mM 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。这种改进的方法将有助于开发标准化的冷冻方案，用于富含 SSCs 的鼠类生殖细胞，并扩展其在各个领域的应用。
