This work aimed to evaluate the effect of two distinct cryopreservation procedures - conventional slow-freezing and vitrification, on survivability and mesenchymal marker expression stability of rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs). Cells at passage 2 were slowly frozen, using 10% of dimethylsulfoxide, or vitrified, using 40% of ethylene glycol, 0.5 M sucrose and 18% Ficoll 70. After three months storage in liquid nitrogen, viability, chromosomal stability, ultrastructure, surface and intracellular marker expression and differentiation potential of cells were evaluated immediately post-thawing/warming and after additional culture for 48-72 h. Our results showed decreased (P ≤ 0.05) viability of cells post-thawing/warming. However, after additional culture, the viability was similar to those in fresh counterparts in both cryopreserved groups. Increase (P ≤ 0.05) in the population doubling time of vitrified cells was observed, while doubling time of slow-frozen cells remained similar to non-cryopreserved cells. No changes in karyotype (chromosomal numbers) were observed in frozen/vitrified AF-MSCs, and histological staining confirmed similar differentiation potential of fresh and frozen/vitrified cells. Analysis of mesenchymal marker expression by qPCR showed that both cryopreservation approaches significantly affected expression of CD73 and CD90 surface markers. These changes were not detected using flow cytometry. In summary, the conventional slow-freezing and vitrification are reliable and effective approaches for the cryopreservation of rabbit AF-MSCs. Nevertheless, our study confirmed affected expression of some mesenchymal markers following cryopreservation.

中文翻译：

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缓慢冷冻或玻璃化后兔羊水来源的间充质干细胞的存活性。

这项工作旨在评估两种不同的冷冻保存程序（常规的慢速冷冻和玻璃化）对兔羊水来源的间充质干细胞（rAF-MSC）的存活率和间充质标记表达稳定性的影响。使用10％的二甲亚砜缓慢冷冻第2代细胞，或使用40％的乙二醇，0.5 M蔗糖和18％Ficoll 70进行玻璃化。在液氮中保存三个月后，其存活力，染色体稳定性，超微结构，表面和表面融化/加热后以及额外培养48-72小时后，立即评估细胞内标记物的表达和分化潜能。我们的结果显示，融化/变温后细胞活力降低（P≤0.05）。但是，经过额外的培养，在两个冷冻保存组中，其生存力都与新鲜的相似。观察到玻璃化细胞的群体倍增时间增加（P≤0.05），而慢速冷冻细胞的倍增时间与非冷冻保存的细胞相似。在冷冻/玻璃化的AF-MSCs中未观察到核型（染色体数）的变化，并且组织学染色证实了新鲜和冷冻/玻璃化的细胞具有相似的分化潜能。通过qPCR分析间充质标志物的表达表明，两种冷冻保存方法均会显着影响CD73和CD90表面标志物的表达。使用流式细胞仪未检测到这些变化。总之，常规的慢速冷冻和玻璃化是兔AF-MSC冷冻保存的可靠而有效的方法。不过，