Freeze-injury at the plasma membrane level has been identified as being crucial for the survival of living matter. Since plasma membranes consist of several micro domains that make the structure rather complex, this study attempted to use simple model membranes to investigate the changes of phospholipid bilayers at sub-zero temperatures. Egg yolk L-α-phosphatidylcholine (EPC) and 1, 2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC) that mimic plasma membranes in their unique ways were used to prepare large unilamellar vesicles (LUV), which were the model membranes of this study.
At cooling rates of 0.5 and 10�C/min, LUV were freeze-concentrated in the unfrozen matrix as a result of the advancing extraliposomal ice front and the decreasing phase volume of the unfrozen matrix, both of which led to membrane lesion. At the slow cooling rate of 0.5�C/min, an additional freezing stress imposed by the osmotic gradient across the bilayers, due to the increase of solute concentration in the unfrozen matrix, promoted leakage of LUV.
The gel-liquid crystal phase transition temperature of phospholipids played an important role in determining if the LUV could withstand freezing stress when the LUV were held at a defined sub-zero temperature for a given period of holding time. EPC LUV were more leaky than DPPC LUV when they were held at the high sub-zero temperatures and their leakage increased with increasing holding time. The leakiness of EPC LUV could be related to the fluid and deformable nature of the EPC above its phase transition temperature. In contrast, DPPC LUV with a higher gel-liquid crystal phase transition temperature compared to EPC may become increasingly fragile at lower sub-zero temperatures, which led to the increase of leakage when the DPPC LUV were held at the lower sub-zero temperatures. These results indicated that the determination of the fatty acid profile of the plasma membranes was essential to aid in developing the most suitable holding temperature and time during the cryopreservation of biological specimens. Adding to the integrity of LUV that depended on the gel-liquid crystal phase transition temperature of phospholipids, intraliposomal ice formation also depended on the phase transition temperature of phospholipids. Intraliposomal ice formation was only observed for DPPC LUV but not for EPC LUV.
In addition to the extraliposomal ice formation, other physical changes such as the eutectic crystallization of sodium chloride (NaCl) and ice mixture on the stability of LUV were also investigated. The eutectic crystallization of NaCl/ice mixture was governed by the intra- and extraliposomal distribution of NaCl and was more likely to occur at the physiological NaCl concentrations compared to lower NaCl concentrations. The eutectic crystallization of NaCl/ice mixture further increased the leakage of LUV.
The understanding of the freezing behaviour and the mechanisms of freeze-injury of LUV allowed the use of the current model membranes for further investigations of the cryoprotective actions of cryoprotective agents (CPA). Partial phase diagrams of sugar-salt-water, dimethyl sulfoxide (DMSO)-salt-water and ethylene glycol (EG)-salt-water systems that resembled extraliposomal solute compositions were constructed and the phase volume of ice and unfrozen matrix was estimated from the freezing curves. Ice reduction was the major mechanism by which the non-permeable and permeable CPA protected the LUV from freeze-injury. Other cryoprotective mechanisms of the non-permeable and permeable CPA through the dilution and spacing out of the LUV in the unfrozen matrix as well as the suppression of the eutectic crystallization of NaCl/ice mixture were not ruled out. Non-permeable CPA were more effective in preventing leakage of DPPC than EPC LUV. Unlike the non-permeable CPA, permeable CPA were more effective for EPC than DPPC LUV that had been subjected to freezing and thawing processes. At room temperature, however, DMSO and EG were detrimental to the stability of DPPC LUV. The choice of CPA is strictly dependent on the type of phospholipids that varied in their acyl chain length and phase transition temperature.
In summary, this study provides insights of the freeze-injury of LUV and the cryoprotective mechanisms of the non-permeable and permeable CPA which are beneficial to the field of cryopreservation that often depends on empirical trial and error methods. By integrating a comprehensive molecular-based understanding, an optimal cryopreservation procedure could be designed.
|Siow, Lee Fong, n/a
|University of Otago. Department of Food Science
|Australiasian Digital Theses Program
|http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Lee Fong Siow
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