A Generalized Method for the Minimization of Cellular Osmotic Stresses and Strains During the Introduction and Removal of Permeable Cryoprotectants

The successful freeze preservation of mammalian cells and tissues usually requires the presence of high concentrations of cryoprotective agents (CPAs) such as glycerol, ethylene glycol, or dimethylsulfoxide. Unfortunately, the addition of these permeable agents to cells and tissues prior to freezing and their removal after thawing has been documented to be as damaging as the freeze-thaw process itself. This damaging process has been hypothesized to result from the drastic alterations in cell size caused by the osmotic stresses usually imposed upon cells during the introduction and removal of the cryoprotectants. Consequently, on the basis of a nonequilibrium thermodynamic model for the transport of water and a permeable CPA across cell membranes, a method has been developed to minimize these potentially lethal transient changes in cell size. This method involves the simultaneous variation of both the extracellular CPA and electrolyte or osmotic extender osmolalities in a balanced, prescribed manner so that both the cellular water content and the total intracellular ionic strength remain constant as the intracellular CPA osmolaity is either raised or lowered. The theoretical analysis indicates that many of the resulting protocols are practical from the clinical point of view.