Cryopreservation in trehalose preserves functional capacity of murine spermatogonial stem cells.
Cryopreservation in trehalose preserves functional capacity of murine spermatogonial stem cells.
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DOI：
10.1371/journal.pone.0054889
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Ryu BY
中科院分区：
文献类型：
--
作者：
Lee YA;Kim YH;Kim BJ;Kim BG;Kim KJ;Auh JH;Schmidt JA;Ryu BY
人精原干细胞的分离、培养和移植技术的发展具有治疗男性不育症的潜力。为了最大限度地提高这些技术的效率，需要开发SSC冷冻保存的方法，以延长SSC的储存时间。尽管已经证明SSC在冷冻后可以重新启动精子发生，但尚未确定解冻后最大化SSC增殖能力的最佳冷冻保存方案。本研究的目的是发展一种有效的冷冻保存技术，用于保存精原干细胞。为了鉴定用于长期保存SSC的有效冷冻保存方法，将富集SSC的分离的睾丸细胞置于含有二甲基亚砜（DMSO）或DMSO和海藻糖（50 mM、100 mM或200 mM）的培养基中，并在液氮中冷冻1周、1个月或3个月。在50 mM海藻糖中冷冻导致在所有解冻时间与DMSO相比细胞活力显著更高，并且在1周冷冻期内与DMSO相比增殖率更高。在200 mM海藻糖中冷冻不会导致细胞活力增加;然而，冷冻1个月和3个月后，与DMSO相比，增殖活性显著更高，凋亡细胞百分比显著更低。为了确认在200 mM海藻糖中冷冻的SSC的功能性，进行SSC移植。供体精原干细胞形成生精集落和精子，能够产生正常后代。总之，这些结果表明，在含有200 mM海藻糖的DMSO中冷冻是一种有效的冷冻保存SSC的方法。
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.
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