National University Medical Institutes, Yong Loo Lin School of Medicine, Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore.
Fertil Steril. 2011 Jun;95(7):2399-403. doi: 10.1016/j.fertnstert.2011.03.035. Epub 2011 Apr 9.
To investigate the efficacy of vitrification, rapid freezing, and slow freezing in preserving testicular tissue for subsequent isolation of spermatogonial stem cells.
Experimental study.
University-based laboratory.
Immature mouse testicular tissue.
INTERVENTION(S): The tunica of the testis was manipulated before cryopreservation. The tunica was either breached with a fine needle or completely removed, or the testis was sectioned longitudinally into halves.
MAIN OUTCOME MEASURE(S): Cell viability by Trypan blue exclusion test and flow cytometry analysis of live-dead cytotoxicity test, measurement of hormonal production, enrichment of spermatogonial stem cells with use of magnetic-activated cell sorting technology.
RESULT(S): Samples with tunica minimally penetrated with a needle point gave the highest cell viability after freezing and thawing. Vitrification protocol with use of an ethylene glycol-sucrose-based vitrification solution (40% vol/vol ethylene glycol-0.6 mol/L sucrose) was able to maintain postwarming cell viability and functions similar to those of noncryopreserved controls and significantly better than both conventional slow and rapid freezing protocols. Primitive spermatogonial stem cells were enriched successfully from vitrified tissue via magnetic-activated cell sorting.
CONCLUSION(S): Vitrification of testicular tissue is a time- and cost-efficient strategy to preserve spermatogonial stem cells for potential transplantation procedure.
研究玻璃化、快速冷冻和慢速冷冻在保存睾丸组织以随后分离精原干细胞方面的疗效。
实验研究。
以大学为基础的实验室。
未成熟的小鼠睾丸组织。
在冷冻保存前对睾丸的被膜进行操作。被膜要么用细针刺破，要么完全切除，要么将睾丸纵向切成两半。
通过台盼蓝排斥试验和活/死细胞毒性试验的流式细胞术分析评估细胞活力，测量激素产生情况，利用磁性激活细胞分选技术富集精原干细胞。
用针尖最小程度穿透被膜的样本在冷冻和解冻后具有最高的细胞活力。使用基于乙二醇-蔗糖的玻璃化溶液（40%体积/体积乙二醇-0.6 mol/L 蔗糖）的玻璃化方案能够维持解冻后细胞活力和功能，与未经冷冻保存的对照相比显著更好，并且明显优于传统的慢速和快速冷冻方案。通过磁性激活细胞分选，可以从玻璃化组织中成功富集原始精原干细胞。
玻璃化睾丸组织是一种保存精原干细胞以备将来移植程序的高效省时策略。
