FTIR Analysis of Molecular Changes Associated with Warming Injury in Cryopreserved Leukocytes

In this article, we explored the effects of cooling rate, dimethyl sulfoxide (DMSO) concentration, and thawing protocol on the post-thaw viability of frozen human white blood cells (WBCs). Different cooling rates (1, 2, 5, 10, 20, and 50 °C/min) at two DMSO concentrations (5 and 10% v/v) were tested as the samples were cooled to -120 °C. Frozen samples were thawed following either a fast (100 °C/min) or slow (2 °C/min) warming protocol applied in either a single stage or in two stages interrupted by a 6 min hold at -40, -50, -60, -70, or -80 °C. The highest post-thaw viability was obtained when WBCs were cooled at 2 °C/min in a 5% DMSO solution and warmed at the fastest rate (100 °C/min) without any interruption. Post-thaw viability decreased when the warming rate was reduced or when rapid warming was interrupted by a hold at a temperature below -60 °C. To elucidate the mechanisms of warming injury in addition to the biological response, several key interfacial and molecular phenomena require greater understanding; thus, we used Fourier transform infrared (FTIR) spectroscopy to investigate the roles of molecular structure and conformation in damage to cryopreserved WBCs during warming. During warming, FTIR spectra revealed the accumulation of cellular protein and lipid membrane damage below -60 °C if the samples were thawed slowly at 2 °C/min. The results presented here suggest that irreversible alterations of biomolecular structure are correlated with cell injury during warming; these deleterious effects appeared to be caused by one or more low-temperature kinetic processes, consistent with eutectic formation/melting and/or devitrification in the intracellular milieu.