Membrane hydraulic permeability changes during cooling of mammalian cells

Abstract

In order to predict optimal cooling rates for cryopreservation of cells, the cell-specific membrane hydraulic permeability and corresponding activation energy for water transport need to be experimentally determined. These parameters should preferably be determined at subzero temperatures in the presence of ice. There is, however, a lack of methods to study membrane properties of cells in the presence of ice. We have used Fourier transform infrared spectroscopy to study freezing-induced membrane dehydration of mouse embryonic fibroblast (3T3) cells and derived the subzero membrane hydraulic permeability and the activation energy for water transport from these data. Coulter counter measurements were used to determine the suprazero membrane hydraulic permeability parameters from cellular volume changes of cells exposed to osmotic stress. The activation energy for water transport in the ice phase is about three fold greater compared to that at suprazero temperatures. The membrane hydraulic permeability at 0 °C that was extrapolated from suprazero measurements is about five fold greater compared to that extrapolated from subzero measurements. This difference is likely due to a freezing-induced dehydration of the bound water around the phospholipid head groups. Using Fourier transform infrared spectroscopy, two distinct water transport processes, that of free and membrane bound water, can be identified during freezing with distinct activation energies. Dimethylsulfoxide, a widely used cryoprotective agent, did not prevent freezing-induced membrane dehydration but decreased the activation energy for water transport.

The effect of solution nonideality on modeling transmembrane water transport and diffusion-limited intracellular ice formation during cryopreservation

“…In addition, the effect of CPA on E Lp is still in debate in the literature. Akhoondi et al 33 and Smith et al 34 reported that the presence of DMSO dramatically decreases E Lp . Devireddy et al 35 also found the presence of glycerol greatly reduces E Lp while McCaa et al 36 identified that human monocytes have a higher E Lp in the presence of DMSO.…”

Osmotic Stress and Membrane Phase Changes During Freezing of Stallion Sperm: Mode of Action of Cryoprotective Agents1

“…Methods to derive membrane hydraulic permeability parameters from freezing-induced membrane phase behavior data determined by FTIR are described in detail elsewhere [21,22]. It is assumed that the freezing-induced shift in mCH 2 is proportional to the reduction in cellular volume that occurs due to water transport out of the cell in response to cooling.…”

“…Freezing induces even larger changes in the membrane phase state, with membranes rearranging in a tightly packed gel phase due to the removal of the bound water surrounding the phospholipid head groups [20,21]. The latter increases the activation energy for water transport through membranes at subzero temperatures [22]. Permeating cryoprotectants such as GLY and dimethyl sulfoxide (DMSO) increase membrane permeability to water, allowing cells to continue to dehydrate further at low subzero temperatures and avoid intracellular ice formation [23].…”

Membrane Stability during Biopreservation of Blood Cells

“…Recent studies have demonstrated how cryoprotective agents such as glycerol or dimethyl sulphoxide (DMSO) affect sub-zero membrane phase behavior and membrane hydraulic permeability parameters of mammalian cells [24,38]. Interestingly, freezing-induced membrane phase changes are not prevented in the presence of cryoprotective additives (CPA) but they occur more gradual over a wider temperature regime.…”

“…The reason why disaccharides are used instead of monosaccharides in freeze-drying protocols is related to the difference regarding the glass transition temperature. The glass transition temperature of a formulation based on monosaccharides is lower water [24]. These effects of CPAs on membrane hydraulic permeability parameters have some important practical applications as they result in a decrease of the optimal cooling rate for cryopreservation to technically more easily achievable rates.…”