A Novel Method of Obtaining Cell Membrane Permeability Parameters Using Non-Ideal Thermodynamic Assumptions

  • Author / Creator
    Gabler Pizarro, Laura Adeline
  • Cryopreservation is the storage of biological matter at subzero temperatures to preserve it. As one can imagine, this comes with many challenges, including exposure to high solute concentrations as pure water in the extracellular solution freezes, cell dehydration as the intracellular water leaves the cells when the extracellular solution becomes hypertonic, intracellular ice formation, and more. These effects can be mitigated by optimizing the cooling rate, which depends on the cells’ permeability to water, and adding cryoprotective agents (CPAs) to the solutions. CPAs can be non-permeating or permeating, and for the latter, the permeability of the cells to CPAs also becomes an important factor. Mathematical modelling of the cryopreservation process is a useful tool to investigate all the different variables that effect the results of this process. To successfully design a cryopreservation protocol, the changing cell volume during cryopreservation can be modelled by obtaining cell-specific parameters that impact the cell volume, namely 𝐿𝑝 which is the permeability of the cell membrane to water, 𝑃𝑠 which is the permeability of the cell membrane to CPA, and the osmotically inactive fraction, 𝑏, which is the intracellular volume fraction that cannot leave the cells. These parameters have been found previously for different cell types under ideal and dilute assumptions, but biological solutions at subzero temperatures are far from ideal and dilute, especially when CPAs are included. The osmotic virial equation can be used to model the changing cell volume under non-ideal assumptions, and the intracellular environment is described using the grouped solute, which consists of all impermeant intracellular solutes grouped together. Therefore, two additional cell-specific parameters are required to model the cell volume during cryopreservation under non-ideal assumptions, which are the second and third osmotic virial coefficients of this grouped solute, 𝐡𝑔𝑔 and 𝐢𝑔𝑔𝑔.
    In this work, a novel fitting method is presented where kinetic cell volume data with 5x phosphate buffered saline (PBS) solution is used to fit for πΏπ‘βˆ— and π‘βˆ— (the asterisks indicating that these properties are obtained with non-ideal assumptions), and kinetic cell volume data with 3 molal dimethyl sulfoxide (DMSO) is used to fit for 𝐡𝑔𝑔, 𝐢𝑔𝑔𝑔, and π‘ƒπ‘ βˆ—. Because the fitting for πΏπ‘βˆ— and π‘βˆ— requires the parameters 𝐡𝑔𝑔 and 𝐢𝑔𝑔𝑔, the fitting process is done iteratively between both types of data, starting with 𝐡𝑔𝑔 = 𝐢𝑔𝑔𝑔 = 0. The iterative process converges to the final five cell-specific parameters. This was done for human umbilical vein endothelial cells (HUVECs) and H9C2 rat cardiomyocytes at room temperature. Additionally, the temperature dependence of πΏπ‘βˆ— and π‘ƒπ‘ βˆ— are obtained by using the parameters found from the new fitting method at room temperature to fit kinetic cell volume data with 5x PBS at 4 Β°C to obtain 𝑏4πΆβˆ— and πΈπ‘ŽπΏπ‘ which is the activation energy of πΏπ‘βˆ—, and to fit kinetic cell volume data with 3 molal DMSO at 4 Β°C to obtain πΈπ‘Žπ‘ƒπ‘  which is the activation energy of π‘ƒπ‘ βˆ—. This novel fitting method can be used to efficiently determine the five cell-specific fitting parameters, and the temperature dependence of the permeability parameters, required to model the changing cell volume during cryopreservation, an asset that will greatly impact the design of cryopreservation protocols.

  • Subjects / Keywords
  • Graduation date
    Fall 2021
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • 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.