Effect of high DMSO concentration on albumin during freezing and vitrification†
IR spectroscopy was used to probe the composition of the freeze concentrated liquid (FCL) during near equilibrium freezing of albumin–dimethyl sulfoxide (DMSO) solutions down to −70 °C. While cooling down to −32 °C, preferential entrapment of DMSO within ice occurred, which was attributed to formation of DMSO–water complexes. These complexes subsequently collapsed on cooling below −32 °C with albumin being preferentially entrapped in ice. Heterogeneity induced in the FCL as a result, was more pronounced in solutions containing lower solute concentrations and thus larger volume fractions of ice. Crystallization of DMSO was not observed on cooling below its eutectic temperature and the FCL vitrified instead at −58 °C, as confirmed by differential scanning calorimetry and low temperature X-ray crystallography. The increase in viscosity of the FCL upon glass transition prevented further compositional changes. Isothermal measurements confirmed that preferential exclusion of either species from ice occurred solely during advance of the ice interfaces and was hence governed by its relative diffusivity. Adverse effects of high DMSO concentrations on the protein were observed between −35 °C and −55 °C and devitrification during thawing proved to be even more detrimental in vitrified samples. Thawing rate had a significant effect on the extent of structural unfolding of the protein in vitrified samples, but not on the ones which were simply frozen. These results will be relevant to vitrification of solutions containing proteins, cells and small tissue systems as such as monocytes, oocytes, ova, embryos, zygotes, vein rings/segments and pancreatic islets.