Recent advances in the fundamental theories in cryobiology using thermodynamic principles have created new opportunities for innovative methodologies in cryobiology. This thesis tested the hypothesis that calculated indicators of the two-factor hypothesis of cryoinjury, depending on cellular osmotic properties, will describe outcomes of cryobiological experiments. In addition, this thesis demonstrated that knowledge gained from improved descriptions of cellular osmotic parameters allows better understanding of cryoinjury and cryoprotection. The main objective of this thesis was to develop approaches using simulations that can be applied to development of cryopreservation procedures for cell types of interest for therapies. In order for this approach to be successful, a method to more accurately describe the osmotic solution properties of the cell (i.e. osmolality as a function of molality for the cytoplasm) was developed. Also, in-depth examination into the correlation between predictions of the two types of cryoinjury and measured post-thaw biological outcomes was required. The work presented in this thesis has shown that simulations, based on cell-specific osmotic characteristics, and coupled with interrupted cooling procedures can be used to determine conditions that minimize the two identified damaging factors in cryopreservation. Based on results from this research, both intracellular supercooling and osmolality, as indicators of intracellular ice formation and solution effects injury, respectively, should be calculated when attempting to compare simulations with biological experimentation. This thesis has also shown a novel method of obtaining the solution properties (i.e. osmolality as a function of molality) of the cytoplasm of living cells using equilibrium cell volume measurements. Using these newly calculated parameters, this research also demonstrated the magnitude of error introduced by making dilute solution assumptions of the solution properties in cellular responses to low temperatures, including simulations of interrupted freezing procedures. Overall, the research work presented in this thesis has extended the approach to cryopreservation to include the properties of the cell and the physical conditions of the freezing environment, which was only possible through the linkage between biological experimentation and simulations.

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