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Obtaining Temperature Dependence of Cell Membrane Permeability Parameters Using Non-Ideal Thermodynamic Assumptions to Mathematically Model Real Cryopreservation Protocols

  • Author / Creator
    Yadegari, Faranak
  • Cryopreservation is the process of preserving biological matter such as cells, tissues, and organs, at sub-zero temperatures for long-term storage. Cells at low temperatures are susceptible to mechanical damage due to intracellular ice formation and osmotic injuries related to increasing concentration of solutes as the pure water solidifies. Cryobiological damage can be mitigated by controlling the cooling rate and using cryoprotective agents (CPAs). Cell injuries at low temperatures are governed by the transport of water and CPAs across the cell membrane leading to cell volume changes, known as the cell osmotic response. Mathematical modeling of the cell osmotic response to non-isotonic solutions at different temperatures is helpful for optimizing cryopreservation protocols. It has been shown that intra- and extracellular solutions at low temperatures are generally thermodynamically non-ideal. Thus, the changing cell volume under non-ideal thermodynamic assumptions can be modeled using the osmotic virial equation proposed by Elliott et al., and obtaining cell membrane permeabilities to water and CPA, LP^* and PS^, the osmotically inactive fraction of the cell, b^ (the asterisks express that these properties are obtained with non-ideal thermodynamic assumptions), and the second and third osmotic virial coefficients of the grouped intracellular solute Bgg, and Cggg. Grouped solute is in fact all the non-permeating intracellular solutes treated as a single solute. The temperature dependence of the cell membrane permeability parameters plays an important role in optimizing a cryopreservation protocol by determining the optimum cooling rate, and the steps for the CPA addition or removal.
    In this work, we present a new two-part fitting method to obtain the five cell-type-specific parameters at room temperature and 0 ºC and model the temperature dependence of the permeability parameters using the Arrhenius equation for five cell types, namely, human umbilical vein endothelial cells (HUVECs), H9c2 rat myoblasts, porcine corneal endothelial cells (PCECs), Jurkat T-lymphocyte cell line, and human cerebral microvascular endothelial cells (hCMECs/D3 cell line). Unlike the previous works in this area, the fitting method in this work is based on both equilibrium and kinetic cell volume data, enabling us to overcome the limitations of previous methods, expand our measurements to lower temperatures, and investigate the temperature dependence of the cell-type-specific properties. The data collected from equilibrium cell volume experiments are used to fit for b^, Bgg, and Cggg . Then, the three measured parameters and the data obtained from the kinetic cell volume experiments are used to fit for the two permeability parameters, L_p^ and Ps^*. We also investigated the possibility that the third osmotic virial coefficient, Cggg, being equal to zero would result in a better fit to the experimental data for different cell types at different temperatures, which has not been investigated in previous studies. Finally, the temperature dependence of Lp^* and Ps^* was modeled using Arrhenius equations and the activation energies related to each permeability parameter, E(aLp^* ) and E(aPs^* ) were found.
    In the final chapter of this thesis, we use the presented model and the calculated parameters for HUVECs as an example to investigate the impact of the non-ideal thermodynamic model on predicting the changing cell volume during the cryopreservation protocol to show the effectiveness of the proposed mathematical model in having a better understanding of the cell osmotic response and consequently, optimizing the cryopreservation protocol.

  • Subjects / Keywords
  • Graduation date
    Spring 2023
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/r3-m1mt-n193
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.