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Multiscale Transport and Osmotic Tolerance in Liver Cells and Tissues

Date

2023-08-18

Journal Title

Journal ISSN

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Type

Thesis

Degree Level

Doctoral

Abstract

Cryopreservation enables the storage of biological samples for later use while preserving all aspects of biological interest by cooling them to a temperature where chemical reactions are sufficiently slowed. However, there have been considerable challenges in preserving complex tissues and organs due to excessive ice formation, severe thermal stress, chilling, ischemic injury, and the osmotic stress caused by highly viscous cryoprotectants (CPA). To overcome these challenges, mathematical modeling approaches have proven effective in predicting cell and tissue responses to osmotic stress and developing an optimal method for loading and unloading CPA. Predicting optimal cryopreservation protocols requires an accurate estimation of cell volume, solute concentration, and water permeability parameters. A key bottleneck in this process is the requirement of careful measurement of these parameters from the cellular to the tissue scale and the difficulty of studying these in their native three-dimensional (3D) structures: little is known about the detailed responses of individual cells and nuclei in monolayers and tissues to anisosmotic media. Over the course of four projects, my study has mainly used two approaches to overcome these barriers. It focused on real-time monitoring of cellular morphometric parameters using modern four-dimensional imaging techniques and employed mathematical models for solute and water permeability estimation. In the first project, I characterized the osmotic behavior in HepG2 cells, which serve as a model for hepatocytes, and determined the mechanism of osmoregulation within these cells. I illustrate that HepG2 cells are non-ideal osmometers by showing the difference between the expected behavior of cells in anisosmotic environments and by making predictions about their volume regulation mechanisms. Second, I compared cell volume measurement techniques for adherent cell monolayers, which included using a calcein fluorescence quenching technique to investigate the volumetric responses of HepG2 monolayers. My follow-up study uses modern 3D imaging techniques to simultaneously measure real-time cell and nuclear volume changes in adherent cells in an aniosomotic medium, including during the addition and removal of CPA. My results demonstrate that both cells and nuclei regulate their volume in response to osmotic stress. Consequently, cells and nuclear permeability to water (Lp) and CPA (Ps) are inferred during perfusion with anisosmotic and CPA solutions for adherent cell monolayers. Thirdly, I show that osmotic damage is time dependent and that the flavonoid silymarin enhances resistance to osmotic stress and may improve cryosurvival in HepG2 cells. Finally, I extend the 3D imaging technique to track and quantify three dimensional changes in cell and nuclear morphology in response to anisosmotic medium. I then estimate the volume within complex liver tissue, specifically a precision-cut liver slice (PCLS). This method allows the quantification of the expansion and contraction of the whole PCLS during CPA equilibration, as well as the tracking of nuclei and cell volume. By demonstrating the nonideality of liver cells and the complex interplay between cytoplasm and nuclear volumes, we can inform biophysical models, which may have profound implications for our understanding of cell physiology and the mechanism of osmoregulation. Furthermore, the methods described in this study can be adapted to enhance cryopreservation strategies for adherent cells, other complex tissues, and organs. Altogether, this research contributes to the development of a new cryopreservation method for liver cells and tissues and will have a broad impact on the field of tissue transplantation and biomedical research.

Description

Keywords

cryopreservation, cryoprotectant, osmotic damage, liver cells, liver tissue slices, three-dimensional imaging, image analysis, silymarin, volume regulation

Citation

Degree

Doctor of Philosophy (Ph.D.)

Department

Biology

Program

Biology

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DOI

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