Cryopreservation of HeLa Cells at a High Hydrostatic Pressure of 1.0–1.5 kbar

Abstract

The idea of using a higher pressure to reduce damage due to freezing has been thoroughly theoretically examined, while there is little experimental evidence on cryopreservation of living systems at a higher hydrostatic pressure. A study was made to assess the viability of HeLa cells at a pressure of 1.0–2.0 kbar, the toxic effects of five classical cryoprotectants under these conditions, and the viability of HeLa cells during slow (conventional) cryopreservation at a pressure of 1.0 or 1.5 kbar. High resistance to higher hydrostatic pressure was experimentally demonstrated for HeLa cells; i.e., 100% cell survival was observed after cell exposure to a pressure of 1.0 kbar for 60 min. The toxic effect of the cryoprotectants increased under pressure in the following order: glycerol < ethylene glycol < 1,2-propanediol = DMSO < DMSO + formamide. Glycerol and ethylene glycol were the least toxic and additionally exerted substantial baroprotective effects. A high cell survival rate of 83 ± 16% was achieved with 10% glycerol used for cryopreservation at a pressure of 1.0 kbar according to fluorescence staining data, but did not exceed the survival rates observed in control normobaric experiments. Freezing was carried out via slow conventional cryopreservation. Different results might be obtained by using vitrification to freeze biological material at a higher pressure.