Department of Animal Science and Technology, Chung-Ang University, Ansung, Gyeonggi-Do, Korea.
PLoS One. 2013;8(1):e54889. doi: 10.1371/journal.pone.0054889. Epub 2013 Jan 22.
Development of techniques to isolate, culture, and transplant human spermatogonial stem cells (SSCs) has the future potential to treat male infertility. To maximize the efficiency of these techniques, methods for SSC cryopreservation need to be developed to bank SSCs for extended periods of time. Although, it has been demonstrated that SSCs can reinitiate spermatogenesis after freezing, optimal cryopreservation protocols that maximize SSC proliferative capacity post-thaw have not been identified. The objective of this study was to develop an efficient cryopreservation technique for preservation of SSCs. To identify efficient cryopreservation methods for long-term preservation of SSCs, isolated testis cells enriched for SSCs were placed in medium containing dimethyl sulfoxide (DMSO) or DMSO and trehalose (50 mM, 100 mM, or 200 mM), and frozen in liquid nitrogen for 1 week, 1 month, or 3 months. Freezing in 50 mM trehalose resulted in significantly higher cell viability compared to DMSO at all thawing times and a higher proliferation rate compared to DMSO for the 1 week freezing period. Freezing in 200 mM trehalose did not result in increased cell viability; however, proliferation activity was significantly higher and percentage of apoptotic cells was significantly lower compared to DMSO after freezing for 1 and 3 months. To confirm the functionality of SSCs frozen in 200 mM trehalose, SSC transplantation was performed. Donor SSCs formed spermatogenic colonies and sperm capable of generating normal progeny. Collectively, these results indicate that freezing in DMSO with 200 mM trehalose serves as an efficient method for the cryopreservation of SSCs.
开发分离、培养和移植人类精原干细胞（SSC）的技术具有治疗男性不育症的未来潜力。为了最大限度地提高这些技术的效率，需要开发 SSC 冷冻保存方法，以便将 SSC 储存一段时间。虽然已经证明 SSC 可以在冷冻后重新开始精子发生，但尚未确定最佳的冷冻保存方案，以最大限度地提高解冻后的 SSC 增殖能力。本研究的目的是开发一种有效的 SSC 冷冻保存技术。为了确定用于长期保存 SSC 的有效冷冻保存方法，分离的富含 SSC 的睾丸细胞被置于含有二甲亚砜（DMSO）或 DMSO 和海藻糖（50mM、100mM 或 200mM）的培养基中，并在液氮中冷冻 1 周、1 个月或 3 个月。与 DMSO 相比，在所有解冻时间点，50mM 海藻糖中的冷冻导致细胞活力显著提高，与 DMSO 相比，1 周冷冻期的增殖率更高。在 200mM 海藻糖中冷冻不会导致细胞活力增加；然而，与 DMSO 相比，冷冻 1 个月和 3 个月后，增殖活性显著更高，凋亡细胞的百分比显著更低。为了确认在 200mM 海藻糖中冷冻的 SSC 的功能，进行了 SSC 移植。供体 SSC 形成了精子发生集落，并且能够产生正常后代的精子。总的来说，这些结果表明，在含有 200mM 海藻糖的 DMSO 中冷冻是 SSC 冷冻保存的有效方法。
