Introduction: Human corneal stromal stem cells (CSSCs) have gained increasing attention in the treatment of corneal stromal scars. In view of this, the preparation and storage of CSSCs are critical to maintaining the regenerative potential of CSSCs. The goal of the study was to investigate the human serum (HS) concentration in the cryomedia that could best preserve CSSCs. Materials and Methods: Three different cryopreservation media, varying in HS concentration were evaluated in their ability to preserve the viability and phenotype of CSSCs: 2% HS (FS1), 4% HS (FS2), and 90% HS (FS3). After thawing, CSSCs morphology, recovery rate, cell proliferation, relative gene expression of CSSC markers (ABCG2, SOX2, NANOG, PAX6, and SIX3), and their anti-inflammatory response (level of TNFAIP6) were compared with those of unfrozen CSSCs (control). Results: Cryopreserved CSSCs had similar cell morphology as the control. Cell viability was significantly higher using FS2 (92.7 ± 1.3%) compared with FS1 (88 ± 0.8%, p = 0.018). Doubling times of CSSCs were maintained in all cryopreserved conditions, as in the control (p > 0.05), which were 0.9 ± 0.1 days and 1.8 ± 0.0 days at passages 3 and 4, then increased to 18.2 ± 1.9 days at passage 6 (p > 0.05). The expression level of stem cell/progenitor cell markers investigated was not affected by the cryopreservation with any of the three media. In addition, cryopreserved CSSCs have a similar expression level of TNFAIP6 after stimulation with proinflammatory cytokines as the control (p > 0.05). Conclusion: Our results indicated that all three cryopreservation media maintained CSSCs phenotype after undergoing one freezing/thawing cycle. Impact Statement Corneal stromal stem cells (CSSCs) offer an alternative for the treatment of corneal stromal scars. Cryopreservation of CSSCs is necessary as it enables feasibility of using CSSCs as a cell therapy candidate. The current study shows that media used to cryopreserve CSSCs could be optimized to maintain cell viability, phenotype, and potency of CSSCs after thawing.

中文翻译：

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冷冻保存培养基对人角膜基质干细胞保存的评价。

简介：人类角膜基质干细胞（CSSC）在角膜基质瘢痕的治疗中受到越来越多的关注。有鉴于此，CSSC的制备和储存对于保持CSSC的再生潜力至关重要。该研究的目的是研究冷冻培养基中人血清（HS）的浓度，该浓度可以最佳地保存CSSC。材料和方法：评估了三种不同的冷冻保存介质，它们的HS浓度各不相同，以保持CSSC的存活力和表型：2％HS（FS1），4％HS（FS2）和90％HS（FS3）。解冻后，将CSSC的形态，恢复率，细胞增殖，CSSC标记（ABCG2，SOX2，NANOG，PAX6和SIX3）的相对基因表达及其抗炎反应（TNFAIP6的水平）与未冻结的CSSC进行比较（控制）。结果：冷冻保存的CSSC具有与对照相似的细胞形态。与FS1（88±0.8％，p = 0.018）相比，使用FS2（92.7±1.3％）的细胞活力明显更高。在所有低温保存条件下，CSSC的倍增时间均保持不变（与对照组相比，p> 0.05），在第3和4代分别为0.9±0.1天和1.8±0.0天，然后在第6代增加至18.2±1.9天（p > 0.05）。所研究的干细胞/祖细胞标记物的表达水平不受三种培养基中任何一种的冷冻保存的影响。此外，冷冻保存的CSSC在用促炎细胞因子刺激后与对照相比具有相似的TNFAIP6表达水平（p> 0.05）。结论：我们的结果表明，所有三种冷冻保存培养基经过一个冷冻/融化循环后均保持CSSCs表型。影响力声明角膜基质干细胞（CSSC）为角膜基质瘢痕的治疗提供了另一种选择。CSSC的超低温保存是必要的，因为它使将CSSC用作细胞治疗候选物的可行性成为可能。当前的研究表明，用于冷冻保存CSSC的培养基可以进行优化，以在融化后保持CSSC的细胞活力，表型和效力。