Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.
Institute of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.
Sci Rep. 2017 Aug 15;7(1):8206. doi: 10.1038/s41598-017-08586-7.
Cultured epidermal cell sheets (CES) containing undifferentiated cells are useful for treating skin burns and have potential for regenerative treatment of other types of epithelial injuries. The undifferentiated phenotype is therefore important for success in both applications. This study aimed to optimize a method for one-week storage of CES for their widespread distribution and use in regenerative medicine. The effect of storage temperatures 4 °C, 8 °C, 12 °C, 16 °C, and 24 °C on CES was evaluated. Analyses included assessment of viability, mitochondrial reactive oxygen species (ROS), membrane damage, mitochondrial DNA (mtDNA) integrity, morphology, phenotype and cytokine secretion into storage buffer. Lowest cell viability was seen at 4 °C. Compared to non-stored cells, ABCG2 expression increased between temperatures 8-16 °C. At 24 °C, reduced ABCG2 expression coincided with increased mitochondrial ROS, as well as increased differentiation, cell death and mtDNA damage. P63, C/EBPδ, CK10 and involucrin fluorescence combined with morphology observations supported retention of undifferentiated cell phenotype at 12 °C, transition to differentiation at 16 °C, and increased differentiation at 24 °C. Several cytokines relevant to healing were upregulated during storage. Importantly, cells stored at 12 °C showed similar viability and undifferentiated phenotype as the non-stored control suggesting that this temperature may be ideal for storage of CES.
培养的表皮细胞片（CES）含有未分化的细胞，可用于治疗皮肤烧伤，并有可能用于治疗其他类型的上皮损伤的再生治疗。因此，未分化表型对于这两种应用的成功都很重要。本研究旨在优化 CES 一周储存的方法，以广泛分布并用于再生医学。评估了储存温度 4°C、8°C、12°C、16°C 和 24°C 对 CES 的影响。分析包括对活力、线粒体活性氧（ROS）、膜损伤、线粒体 DNA（mtDNA）完整性、形态、表型和细胞因子分泌到储存缓冲液中的评估。在 4°C 下观察到最低的细胞活力。与未储存的细胞相比，在 8-16°C 之间，ABCG2 表达增加。在 24°C 下，ABCG2 表达减少与线粒体 ROS 增加、分化增加、细胞死亡和 mtDNA 损伤同时发生。P63、C/EBPδ、CK10 和 involucrin 荧光与形态观察相结合，支持 12°C 时保留未分化的细胞表型，16°C 时向分化过渡，24°C 时分化增加。在储存过程中，几种与愈合相关的细胞因子上调。重要的是，储存在 12°C 的细胞显示出与未储存的对照相似的活力和未分化表型，这表明该温度可能是 CES 储存的理想温度。
