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

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.