This study examined the post-thaw recovery of Jurkat cells cryopreserved in three combinations of five osmolytes including trehalose, sucrose, glycerol, mannitol, and creatine. Cellular response was characterized using low-temperature Raman spectroscopy, and variation of post-thaw recovery was analyzed using statistical modeling. Combinations of osmolytes displayed distinct trends of post-thaw recovery, and a nonlinear relationship between compositions and post-thaw recovery was observed, suggesting interactions not only between different solutes but also between solutes and cells. The post-thaw recovery for optimized cryoprotectants in different combinations of osmolytes at a cooling rate of 1°C/min was comparable to that measured with 10% dimethyl sulfoxide. Statistical modeling was used to understand the importance of individual osmolytes as well as interactions between osmolytes on post-thaw recovery. Both higher concentrations of glycerol and certain interactions between sugars and glycerol were found to typically increase the post-thaw recovery. Raman images showed the influence of osmolytes and combinations of osmolytes on ice crystal shape, which reflected the interactions between osmolytes and water. Differences in the composition also influenced the presence or absence of intracellular ice formation, which could also be detected by Raman. These studies help us understand the modes of action for cryoprotective agents in these osmolyte solutions.
Bibliographical noteFunding Information:
This study was funded by the National Institutes of Health under R01EB023880. Parts of this study were carried out in the Characterization Facility, University of Minnesota, which received partial support from NSF through the MRSEC program.
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- DMSO-free cryoprotectant
- Raman spectroscopy