Development of supercooling preservation method of adherent cultured human cells

Cryopreservation of mammalian cells is an important technology; however, freezing damage due to osmotic pressure differences and ice crystal formation is inevitable. In addition, cryopreserved cells cannot be used immediately after thawing in many cases. Therefore, in this study, we developed a method for supercooling and preserving adherent cells using a precision temperature-controlled CO2 incubator. The effects of the cooling rate from 37 to -4°C, the warming rate from -4 to 37°C and a preservation solution on cell viability after storage were examined. Human hepatocarcinoma-derived cell line HepG2 cells, preserved with HypoThermosol FRS at -4°C with a cooling rate of -0.028°C/min (24 h from 37°C to -4°C) and warming to 37°C at a rate of +1.0°C/min (40 min from -4 to 37°C), displayed high cell viability after 14 days of preservation. The superiority of supercooling preservation at -4°C was demonstrated by comparing the obtained results with that of refrigerated preservation at +4°C. Cells preserved for 14 days under optimal conditions showed no cell shape abnormalities and may be used for experiments immediately after thawing. The optimized supercooling preservation method determined in this study is suitable for the temporary preservation of adherent cultured cells.