The Effect of Cryoprotectants Concentration on Ice Crystal Propagation Velocity

Background: Two physicochemical effects occur during vitrification: nucleation and crystallization. Nucleation is a statistical occurrence by its nature. Thus, the more water molecules that are present the higher are the chances for nucleation to occur. Crystallization is a first-order transition where a water molecule is incorporated into ice crystal. Intracellular viscosity, which is the combination of water, salts, and cryoprotectants (CPs), affects both the nucleation and crystal growth rates. Ice velocity is inversely correlated with the viscosity and directly proportional to the function of the system's supercooling. However, little is known about the speed of ice crystal propagation in vitrification solutions containing different concentrations of CPs. Methods: This article describes the ice crystal propagation velocity while referring to vitrification. Ice crystal propagation velocity was measured in solutions containing different CP (dimethyl sulfoxide [DMSO], propylene glycol [PG], ethylene glycol [EG], and glycerol) concentrations at a supercooled temperature. The different CPs solutions were inserted into 0.25 mL straws and placed in different temperatures of an alcohol bath (T_c) at supercooling temperatures of -8°C to -10°C. Results: We found that ice crystal propagation is inversely correlated to CP concentrations. Interestingly, PG showed, with statistically significant results, lower ice crystal growth velocities up to concentrations of 30% (v/v), compared with DMSO, and EG at the same concentrations. The combination of EG with PG showed better results (0.25 mm/s) than EG with DMSO (0.39 mm/s) in terms of decreasing the ice crystal growth velocity. When the concentration was increased to 40% (v/v), EG showed the lowest ice crystal propagation velocity (0.09 mm/s), although not significantly different than PG and glycerol but significantly lower than DMSO (0.13 mm/s). Conclusion: These results suggest that current vitrification solutions are not optimized. Based on our results, we suggest that combining PG with EG has advantages over the combination of DMSO and EG, which might promote successful cell and tissue vitrification.