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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Mathematical modelling of cellular osmotic responses to low temperatures is being increasingly used to overcome obstacles in the successful cryopreservation of cells and tissues. Current cryobiological models often contain simplifying assumptions regarding the solution behaviour of the complicated, multisolute intra- and extra-cellular solutions. In order to obtain more accurate predictions of cryobiological outcomes, equations derived from thermodynamic principles that more accurately describe the biological solution behaviour could be used to greatly advance the design of novel cryopreservation protocols. The general hypothesis of this thesis is that the application of the multisolute osmotic virial equation, with mixing rules derived from thermodynamic first principles, to solutions of interest in cryobiology will result in more accurate predictions of the multisolute solution behaviour, which will lead to improved cryobiological modelling and increased understanding of cellular responses to cryopreservation. Specifically, this thesis demonstrates that the osmotic virial coefficients, obtained from single-solute solution data, can be used in the multisolute osmotic virial equation to accurately predict the multisolute solution behaviour, without the need to fit multisolute solution data. The form of the multisolute osmotic virial equation proposed in this thesis was used to predict the solution behaviour of a range of multisolute solutions of interest in cryobiology. The equation commonly used in cryobiology to describe cellular osmotic equilibrium is based on ideal, dilute solution assumptions. In this thesis, a non-ideal osmotic equilibrium equation was derived and, combined with the multisolute osmotic virial equation, used to more accurately predict the osmotic equilibrium of human erythrocytes. The improved equations proposed in this thesis were combined with experimental measurements of the incidence of intracellular ice formation in order to further the understanding of the role of several important cryobiological parameters on the formation of intracellular ice. This thesis work has significantly contributed to the field of cryobiology by substantially improving the accuracy of two key equations used in the modelling of cellular osmotic responses to cryopreservation. The combination of accurate mathematical modelling and results from experiments will allow increased understanding of cellular responses to cryopreservation, leading to the design of novel cryopreservation protocols. / Chemical Engineering and Medical Sciences

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