Research question

Can specimen types (cells versus tissues) and additive cryoprotectant agents contribute to efficient cryopreservation of primate spermatogonial stem cells (SSC)?

Design

Testicular tissues or cells from four prepubertal monkeys were used in this study. The freezing efficacy of testicular tissue was compared with cell suspensions using conventional freezing media (1.4 mol/l dimethyl sulfoxide [DMSO]) and the efficacy of cryoprotectant additives (1.4 mol/l DMSO combined with trehalose 200 mmol/l, hypotaurine 14 mmol/l, necrostatin-1 50 µmol/l or melatonin 100 µmol/l) was evaluated in testicular tissue freezing.

Results

The survival rate (46.0 ± 4.8% versus 33.7 ± 6.0%; P = 0.0286) and number of recovered cells (5.0 ± 1.5 × 10⁶ cells/g versus 0.7 ± 0.8 × 10⁶ cells/g; P = 0.0286) were significantly higher in frozen tissues than in frozen cell suspensions. After tissue freezing, a higher number of recovered PGP9.5⁺ cells were observed with 200 mmol/l trehalose treatment than in DMSO controls (2.4 ± 0.6 × 10⁶ cells/g versus 1.1 ± 0.3 × 10⁶ cells/g; P = 0.0164). Normal establishment of donor-derived colony was observed in SSC after tissue freezing with 200 mmol/l trehalose.

Conclusions

Testicular tissue freezing is more effective than single cell suspension freezing for higher recovery of undifferentiated spermatogonia. Moreover, it was verified that slow freezing using 200 mmol/l trehalose, 1.4 mol/l DMSO and 10% KnockOut™ Serum Replacement in Dulbecco's phosphate-buffered saline is an effective cryopreservation protocol for primate testicular tissue.

中文翻译：

---

用于保存雄性灵长类动物 (Macaca fascicularis) 青春期前生育能力的有效冷冻保存方案

研究问题

样本类型（细胞与组织）和添加剂冷冻保护剂是否有助于灵长类动物精原干细胞 (SSC) 的有效冷冻保存？

设计

本研究中使用了来自四只青春期前猴子的睾丸组织或细胞。将睾丸组织的冷冻功效与使用常规冷冻培养基（1.4 mol/l 二甲基亚砜 [DMSO]）的细胞悬液和冷冻保护添加剂（1.4 mol/l DMSO 与 200 mmol/l 海藻糖、亚牛磺酸 14 mmol/l l、necrostatin-1 50 µmol/l 或褪黑激素 100 µmol/l) 在睾丸组织冷冻中进行评估。

结果

存活率（46.0 ± 4.8% vs 33.7 ± 6.0%；P  = 0.0286）和回收细胞数（5.0 ± 1.5 × 10 ^{6 个}细胞/g vs 0.7 ± 0.8 × 10 ^{6 个}细胞/g；P  = 0.0286）显着在冷冻组织中比在冷冻细胞悬液中更高。组织冷冻后，用 200 mmol/l 海藻糖处理观察到更多数量的回收 PGP9.5 ⁺细胞比在 DMSO 对照中（2.4 ± 0.6 × 10 ^{6 个}细胞/g vs 1.1 ± 0.3 × 10 ^{6 个}细胞/g；P  = 0.0164）。在用 200 mmol/l 海藻糖冷冻组织后，在 SSC 中观察到供体来源集落的正常建立。

结论

睾丸组织冷冻比单细胞悬液冷冻更有效，可提高未分化精原细胞的恢复率。此外，经证实，在 Dulbecco 磷酸盐缓冲盐水中使用 200 mmol/l 海藻糖、1.4 mol/l DMSO 和 10% KnockOut™ 血清替代物进行缓慢冷冻是灵长类动物睾丸组织的有效冷冻保存方案。