Membrane hydration correlates to cellular biophysics during freezing in mammalian cells

Abstract

Cell survival during freezing applications in biomedicine is highly correlated to the temperature history and its dependent cellular biophysical events of dehydration and intracellular ice formation (IIF). Although cell membranes are known to play a significant role in cell injury, a clear correlation between the membrane state and the surrounding intracellular and extracellular water is still lacking. We previously showed that lipid hydration in LNCaP tumor cells is related to cellular dehydration. The goal of this study is to build upon this work by correlating both the phase state of the membrane and the surrounding water to cellular biophysical events in three different mammalian cell types: human prostate tumor cells (LNCaP), human dermal fibroblasts (HDF), and porcine smooth muscle cells (SMC) using Fourier Transform Infrared spectroscopy (FTIR). Variable cooling rates were achieved by controlling the degree of supercooling prior to ice nucleation (-3 degrees C and -10 degrees C) while the sample was cooled at a set rate of 2 degrees C/min. Membranes displayed a highly cooperative phase transition under dehydrating conditions (i.e. NT=-3 degrees C), which was not observed under IIF conditions (NT=-10 degrees C). Spectral analysis showed a consistently greater amount of ice formation during dehydrating vs. IIF conditions in all cell types. This is hypothesized to be due to the extreme loss of membrane hydration in dehydrating cells that is manifested as excess water available for phase change. Interestingly, changes in residual membrane conformational disorder correlate strongly with cellular volumetric decreases as assessed by cryomicroscopy. A strong correlation was also found between the activation energies for freezing induced lyotropic membrane phase change determined using FTIR and the water transport measured by cryomicroscopy. Reduced lipid hydration under dehydration freezing conditions is suggested as one of the likely causes of what has been termed as "solution effects" injury in cryobiology.

GMP Cryopreservation of Large Volumes of Cells for Regenerative Medicine: Active Control of the Freezing Process

“…In particular, in samples with controlled ice nucleation the calculated diffusion distance within the freeze concentrated matrix is significantly larger than that following spontaneous ice nucleation. 13 Effects on cell dehydration of controlled and spontaneous ice nucleation have previously been confirmed using Fourier transform infrared spectroscopy of mammalian cell lines 23,24 and horse spermatozoa 25 and it has been shown that cell dehydration is required to avoid lethal intracellular ice formation.…”

Thermodynamic and structural behaviour of equimolar POPC/CnE4 (n=8, 12, 16) mixtures by sorption gravimetry, 2H NMR spectroscopy and X-ray diffraction

“…the ability of different organisms to survive even complete dehydration. This gives many potential applications, such as tailored cryosurgery in medicine (Balasubramanian et al, 2009), but also in biopreservation, i.e. the storage of biological material (Aksan et al, 2009;Franca et al, 2007), even in the scope of space projects (Ricci et al, 2005;Rothschild and Mancinelli, 2001).…”

Comparative atomic-scale hydration of the ceramide and phosphocholine headgroup in solution and bilayer environments

“…10 One particularly fascinating aspect of lipid behavior is the effect that hydration has on lipid function. [11][12][13] For instance, in the stratum corneum, it is important to regulate hydration such that the skin can act as an effective barrier. [14][15][16][17] The importance of hydration is highlighted by the fact that when cells become dehydrated, they modify their membrane composition to retain suitable fluidity.…”