Long-term Cryopreservation of Human and other Mammalian Cells at -80 °C for 8 Years.

Cell medicine Pub Date : 2018-05-29 eCollection Date: 2018-01-01 DOI:10.1177/2155179017733148
Yoshitaka Miyamoto, Masashi Ikeuchi, Hirofumi Noguchi, Shuji Hayashi
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引用次数: 18

Abstract

Freezing is recognized as the most effective method of maintaining a stable supply of various cell types for long-term storage. However, cells might be damaged by environmental changes during the freezing process. There are various factors that influence the function of cells cultured after cryopreservation and thawing. These factors include cryopreservation solutions, biomaterials, freezing methods, and the freezing and preservation temperatures. There is also a risk of infection with mycoplasma in liquid nitrogen phase. Therefore, it is necessary to consider more useful and safe methods for freezing and storing various cells. In this study, we investigated the effects of temperature during long-term storage (8 years at -80 °C and in liquid nitrogen phase) on the quality of various cells (human hepatocellular carcinoma cells, bovine carotid artery normal endothelial cells, mouse fibroblast cells 3T3, and mouse embryo fibroblast cells STO). We examined the cell viability of cryopreserved human hepatocellular carcinoma cells at -80 °C using culture medium containing 10% DMSO, Cell Banker 1, and Cell Banker 2 as cryopreservation solutions. Among these solutions, Cell Banker 1 showed the highest efficiency. The viability of human hepatocellular carcinoma and bovine carotid artery normal endothelial cells in the Cell Banker 1 stored at -80 °C was over 90%, which was the same as that in liquid nitrogen phase. The cells stored at -80 °C had a morphology similar to that of the cells stored at liquid nitrogen phase. The proliferation of cells stored at -80 °C and in liquid nitrogen phase was not significantly different. Furthermore, none of the cells were infected with mycoplasma. There was no marked difference in the albumin secretion between the human hepatocellular carcinoma cells stored at -80 °C and those in liquid nitrogen phase. The short tandem repeats of the human hepatocellular carcinoma cells stored at -80 °C were identical to those stored in liquid nitrogen phase. In this report, various cells stored long-term at -80 °C were able to be used effectively after long-term storage. These findings can be applied to drug discovery, cell medicine, and cell therapy.

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人类和其他哺乳动物细胞-80°C长期冷冻保存8年。
冷冻被认为是维持各种细胞类型长期储存稳定供应的最有效方法。然而,在冷冻过程中,细胞可能会受到环境变化的破坏。影响细胞冷冻和解冻后功能的因素有很多。这些因素包括冷冻保存溶液、生物材料、冷冻方法以及冷冻和保存温度。在液氮期也有感染支原体的危险。因此,有必要考虑更有效和安全的方法来冷冻和储存各种细胞。在这项研究中,我们研究了长期储存(-80°C液氮期8年)温度对多种细胞(人肝癌细胞、牛颈动脉正常内皮细胞、小鼠成纤维细胞3T3和小鼠胚胎成纤维细胞STO)质量的影响。我们使用含有10% DMSO、cell Banker 1和cell Banker 2的培养基作为冷冻保存溶液,在-80°C下检测冷冻保存的人肝癌细胞的细胞活力。在这些溶液中,Cell Banker 1的效率最高。人肝细胞癌和牛颈动脉正常内皮细胞在-80℃的Cell Banker 1中保存的存活率均在90%以上,与液氮相相同。-80°C保存的细胞形态与液氮保存的细胞相似。-80℃和液氮条件下的细胞增殖无显著差异。此外,没有细胞感染支原体。-80℃保存的人肝癌细胞与液氮保存的人肝癌细胞的白蛋白分泌量无明显差异。在-80℃保存的人肝癌细胞的短串联重复序列与液氮保存的相同。在本报告中,在-80°C长期储存的各种细胞在长期储存后能够有效地使用。这些发现可以应用于药物发现、细胞医学和细胞治疗。
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Cell medicine
Cell medicine MEDICINE, RESEARCH & EXPERIMENTAL-
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