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.
Bibliographical noteFunding Information:
European Research Council, Grant/Award Number: ERC‐2016‐CoG 725034; National Institutes of Health, Grant/Award Numbers: R01EB023880, R25HL128372; National Science Foundation, Grant/Award Number: CON‐75851; Project 00074041 Funding information
This work was supported by the National Institutes of Health (R01EB023880, R25HL128372), National Science Foundation Graduate Research Fellowship (CON‐75851, Project 00074041) and ERC‐2016‐CoG 725034 (ERC Consolidator Grant Des.solve). The confocal light microscopy in this work was carried out using a Zeiss Scope with the assistance of Guillermo Marques at the University Imaging Centers, University of Minnesota. The flow cytometry in this work was carried out at the University of Minnesota Flow Cytometry Resource. The Raman spectroscopy in this work was carried out in the Characterization Facility, University of Minnesota, which receives partial support from the NSF through the MRSEC program.
- Raman spectroscopy
- amino acids
- differential scanning calorimetry
- natural deep eutectic solvents