24. Diffusion limited ice crystallisation + a comment on liquidus tracking

Abstract

Diffusion limited ice crystallisation. During conventional cryopreservation many of the changes which occur in a CPA solution following ice nucleation are not linear with temperature. Parameters such as the ice fraction, concentration of ionic species, osmolality, pH, viscosity and gas solubility, all vary in a non-linear manner with temperature. In addition, the biophysical characteristics of cells which determine the response to freezing, for example the cellular permeability to water, also change in a nonlinear manner with temperature. We have measured the viscosity of the residual unfrozen solution that cells are exposed to during conventional freezing, and the effect of high viscosities encountered on the diffusion of water at a constant temperature during freezing and during cooling at different linear rates has been estimated. At rates of cooling faster than 100 °C min−1 the diffusion distance during freezing was calculated to be less than 15 μ m. Validation of the diffusion calculations were confirmed by examination of the ultrastructure of the freeze concentrated matrix in samples prepared at a range of cooling rates. At a critical rate of cooling, water diffusion becomes limited by the high viscosity and the composition of the freeze concentrated matrix deviates from that of the equilibrium phase diagram, the effects on cell viability of this non equilibrium state are demonstrated. Liquidus tracking. In 1965 Farrant proposed the “liquidus tracking” method of preservation in which cryoprotectant is added progressively during cooling so that samples remain on or above the liquidus line of the equilibrium phase diagram. Low temperatures protect against cryoprotectant toxicity at high concentrations. This method has also been termed the equilibrium approach to vitrification. However due to practical issues associated with high viscosities and mixing at low temperatures the method was largely abandoned. Recently there has been revived interest in the technique as applied to the preservation of cartilage and encapsulated hepatocytes and also in ultrastructural studies where the method is known as progressive lowering of temperature (PLT). Planer products have modified their controlled rate freezer to deliver the process for cartilage. Asymptote are developing equipment for this process which will allow preservation of clinical biopsies with native immunology which can be immunolabelled and from which viable cells may be isolated for adoptive cell therapies and personalised medicine.