Freeze Filtration: Description and Evaluation of a Method for Obtaining Samples of Liquid Phase From Frozen Solutions

Abstract

The objective of this study was to develop a method for sampling the liquid phase of frozen solutions (i.e. the remaining liquid between ice crystals and other solids) for subsequent chemical analysis in order to determine the extent of ice formation and other physical-chemical effects of freezing upon solutions. Such information could enhance the development of cryoprotective agents (CP As). which would allow the frozen long term preservation of organs for transplantation. The traditional thermometric/ calorimetric methods for the measurement of ice formation are susceptible to bias by composition change of the liquid phase caused by secondary solid phase formations (i.e. solids besides ice). They are not able to determine the nature Df such composition changes, and some changes, like that of the pH, will go unnoticed. The new method uses subatmospheric pressure to aspirate liquid phase from a cooled sample holder into a collection vial. "Freeze Filtration" has been tested on four well-established cryoprotective agents comparing measurements in this study with literature values on these chemicals. Remarkable accuracy and repeatability have been achieved. Deviations from published values depend on the cryoprotectant used, which suggests a CPA-dependent methodical difference that may be rooted in bias within the literature values. Cells and solute precipitates can be isolated along with liquid phase, and the freeze-elevated concentrations of Na+, K+, Ca2+, and H+ of a single sample have been measured. Freeze Filtration differs from the older methods by providing a simple mode of operation, low cost, the ability to determine composition changes, and the ability to serve as a physical-chemical reference during actual cryosurvival studies. It is directly demonstrated that freezing at velocities and sample sizes applicable to organ freezing can result in substantial pH changes in the liquid phase and the precipitation of solutes. The data also indicate that 1,3-butanediol is not a good cryoprotectant.