Natural deep eutectic systems for nature-inspired cryopreservation of cells

Natural deep eutectic systems (NADES) are emerging as potential cryoprotective agents (CPA) for cell preservation. In this investigation, we develop an optimized CPA formulation using trehalose-glycerol NADES (T:G) diluted in Normosol-R and supplemented with isoleucine. Differential scanning calorimetry (DSC) is used to define the thermophysical properties of NADES-based solutions, and Raman spectroscopy is used to characterize the effect of NADES on ice formation and hydrogen bonding. Jurkat cells are cryopreserved in each solution, and post-thaw cell recovery, apoptosis, and growth are quantified. Raman spectra and heat maps show that NADES suppresses both ice formation and dehydration of the nonfrozen region. Supplementing NADES with isoleucine does not affect the solution's thermophysical properties but significantly improves the cells' survival and proliferation post-thaw. The study indicates that thermophysical properties of CPA solutions alone cannot predict optimal cell survival, suggesting that stabilization of biological structures by CPAs may play a role in successful cryopreservation.