Effect of cell attachment and molecular events associated with freezing biophysics and the "two factor" injury hypothesis.

Abstract

Freezing of biological systems is generally intended to maximize cell survival (cryopreservation) or injury (cryosurgery) depending on the application. The biophysical contribution to cellular freeze injury is generally described by the “two factor” injury hypothesis. Slow freezing is associated with “solution effects” injury while rapid freezing is linked to the lethality of intracellular ice formation (IIF). The “two factor” hypothesis has been actively investigated for cell suspensions. While cell suspensions provide a fundamental understanding of cellular biophysics and injury, more complex systems (featuring cell attachment) are needed to potentially link to tissues. The effect of cell attachment in the context of the “two factor” hypothesis has not been extensively investigated especially for water transport biophysics. In addition, the “two factor” injury hypothesis implicates changes to cellular macromolecules (e.g. lipids and proteins) as potential freezing injury mechanisms. Currently very little is known about the molecular events associated with the biophysics of freezing cells. The specific aims (SA) of the dissertation are listed below, and it addresses some of the limitations identified. SA 1: Study the effect of cell attachment on the “two factor” hypothesis during freezing SA2: Investigate the molecular events (lipids and proteins) associated with the “two factor” biophysics of freezing The effect of cell attachment (SA 1) was studied using two mammalian cell types – human dermal fibroblasts (HDF), and porcine smooth muscle cells (SMC). The cellular systems that were evaluated include suspensions, monolayer (cell-cell interactions), and tissue equivalents (cell-cell and cell-ECM interactions). Cell based biophysical models were then used to compare the predicted biophysics as a function of the attachment state. The molecular events associated with the “two factor” biophysics (SA 2) were studied using three different mammalian cell types – HDF, SMC, and human LNCaP prostate tumor cells. Changes to membrane lipids and proteins during controlled freezing were evaluated using Fourier Transform Infra-Red spectroscopy (FTIR). The molecular events were then linked to cellular freezing biophysics assessed using cryomicroscope. The important findings of this dissertation are included below: 1. Cell attachment affects the “two factor” biophysics of freezing. Experimental data shows that IIF is enhanced for cells in the attached state as compared to suspensions. In addition, the results suggest that water transport is enhanced for cells in the attached state as compared to suspensions. However, the impact of increased water transport on cell survival for attached cells is unclear. 2. The study of molecular events shows that slow freezing affects membrane phase transition (liquid crystalline to gel phase), whereas rapid freezing is observed to maintain the high conformational disorder of the membranes. 3. Molecular events (i.e. membrane phase transition) measured using FTIR are linked to cellular biophysics measured using cryomicroscope. 4. The results show a link between cell and lipid membrane dehydration events. It is suggested that membranes can only tolerate dehydration to a certain extent. This connection is suggested as a potential link to a molecular mechanism of cell injury due to “solution effects”.