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In a typical cryopreservation protocol, the system to be preserved is first equilibrated with chemicals known as cryoprotective agents (CPAs). CPAs have been shown to alleviate cell damage from either the solute effects or the formation of intracellular ice during the subsequent freezing process. Thus, an extensive body of literature reporting the effects of CPAs on cellular systems has been accumulated over the last 50 years; detailing largely experimental interactions between cell systems and chemicals. Recent advances in computational methodology now offer an additional dimension in our ability to understand the molecular interactions between cell membranes, idealized as lipid bilayers and CPAs at atomistic scales. Computer simulations provide unique capabilities for analyzing biomembrane properties from atomistic perspective with a degree of detail that is hard to reach by other techniques. The excellent agreement with the experiment obtained in various molecular dynamics (MD) studies [1] on simple model membranes has raised the confidence in applying the molecular dynamics simulations to even more complex systems. Dimethylsulfoxide (DMSO) is one of the most widely used solvents in cell biology and cryopreservation. During a typical cryopreservation protocol the DMSO composition of aqueous buffers inside and outside of the cell is known to differ considerably. To model and understand the structural changes in cell membranes in such a situation we performed MD simulations of an idealized lipid bilayer membrane which separates two aqueous reservoirs with and without DMSO. Zwitterionic dimyritoylphosphatidylcholine (DMPC) lipid bilayers was chosen as the model membrane.

In the present study, we report the effects of cooling ejaculated and epididymal bovine sperm from the same animals with and without a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal bovine sperm cell suspensions were obtained at a cooling rate of 20 °C/min under two different conditions: i) in the absence of cryoprotective agents, CPAs; and ii) in the presence of 0.7 M glycerol. Using previously published values, the bovine sperm cell was modeled as a cylinder of length 39.8 μm and a radius of 0.4 μm with an osmotically inactive cell volume, Vb, of 0.61Vo, where Vo is the isotonic cell volume. The subzero water transport response is analyzed to determine the variables governing the rate of water loss during cooling of bovine spermatozoa, i.e. the membrane permeability parameters (reference membrane permeability, Lpg and activation energy, ELp). The predicted best-fit permeability parameters ranged from, Lpg = 0.021 to 0.038 μm/min-atm and ELp = 27.8 to 41.1 kcal/mol. The subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal bovine spermatozoa under corresponding cooling conditions. If this observation is found to be more generally valid for other mammalian species as well, then the sperm extracted from the testicles of an animal during post-mortem can also be optimally cryopreseved using procedures similar to those derived for ejaculated sperm.

We report in this work our observations from detailed atomistic investigations of the interactions between solutions containing 11.3 mol% of dimethylsulfoxide (DMSO) and three fully hydrated bilayers of lipid molecules, (BLMs). The BLMs considered were: dipalmitoyl-phosphatidylcholine (DPPC), palmitoyloleoyl-phosphatidylcholine (POPC) and dimyristoylphosphatidylcholine (DMPC); all in the fluid phase and under equilibrium conditions at 323K and 298K, respectively. All of our simulations were performed over 100 ns total simulation time and in the absence of any externally applied stress to the membranes. In all three systems investigated, in the presence of DMSO, we observed that small hydrophobic pores start to open across the membranes at various times during the simulations ranging from 10 ns for the DMPC, 45ns for POPC to 50ns for DPPC membrane. By carefully analyzing the membranes structures we concluded that hydrophobic pores open and close continuously during the simulation beyond the above mentioned time marks. Interestingly, as there is no external stress applied to the membranes these hydrophobic pores are presumably nucleated by thermal fluctuations. In all three systems we also observe that after some time after the first hydrophobic pore nucleation the pore starts to grow and transforms into a hydrophilic pore which continue to grow at an even higher rate. Our MD simulation studies of various BLMs indicate that the presence of DMSO may lower the pore edge line tension leading to pore nucleation and growth due to only thermal fluctuations.