26. Freezing under pressure: A novel method for cryopreservation

Abstract

The ability to preserve cells, tissues, and organs with minimal damage for an extended period of time is essential for advancements in medicine and research. Current methods for cryopreservation are based on using high concentrations (up to 60% v/v) of cryoprotectants which are toxic to living cells. Moreover, complex tissues and organs have limited permeability to cryoprotectants and require a prolonged and often damaging perfusion process, which results in increased toxicity and unequal distribution of cryoprotectants. The effect of pressure on ice crystal formation and growth and on vitrification of water appears to be similar to the effect of cryoprotectants. Pressure decreases the freezing point of water and aqueous solutions, increases viscosity of water at the freezing point. It appears that high hydrostatic pressure also induces vitrification of water molecules and inhibits ice crystal growth. Unlike cryoprotectants, hydrostatic pressure can be rapidly and equally distributed throughout the entire volume of an organ, creating unique conditions for cryopreservation, which cannot be realized at ambient pressure. The effect of pressure and a low concentration of dimethyl sulfoxide (Me2SO) or glycerol, on hemolysis of human red blood cells after freezing and thawing were investigated. Pressure was applied during cooling and freezing the red blood cells and a minimum in hemolysis was reached at approximately 120 MPa. Either 5% v/v Me2SO or 8% v/v glycerol concentration in combination with 120 MPa pressure was sufficient to obtain 8% or less hemolysis of red blood cells after cooling at a 35 °C/min or a 160 °C/min rate. The preliminary results suggest that the method may help to solve the cryoprotectant toxicity problem. Since cryoprotectants in high concentrations are harmful to tissues and organs, the development of the method for freezing under pressure with a reduced cryoprotectant concentration may be another step towards successful cryopreservation and recovery of viable organs. More research is needed to optimize the method and determine if it is clinically applicable.