Natural deep eutectic systems for nature‐inspired cryopreservation of cells

Abstract

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.

Natural Cryoprotective and Cytoprotective Agents in Cryopreservation: A Focus on Melatonin

“…Saccharides, such as fructose, galactose and glucose, are found in plants, fishes and insects exposed to ultralow temperatures. They can limit intracellular dehydration in the process of freezing, thus preserving the cellular membrane structure and its functions [ 30 ]. Trehalose, a natural nonreducing disaccharide, has shown good performance in cryopreservation, but due to its poor membrane permeability, it should be delivered into the cells through different strategies such as microinjection, pore formation or internal trehalose synthesis using genetic engineering techniques.…”

“…NADES have been recently explored as potential CPAs for the preservation of different types of cells, including lactobacillus [ 62 ], mouse fibroblast cells [ 63 ], mesenchymal stem cells [ 64 ], and Jurkat cells, [ 30 ], as well as in the development of DMSO-free protocols of cryopreservation of NK and T cells, that remains a challenge for the development of autologous and allogeneic cell therapy products of these lineages, such as CAR-T and CAR-NK cells (recently reviewed in [ 27 ]). In this respect, in Jurkat cells, an immortalized cell line used as a T lymphocyte model, a multicomponent osmolyte solution composed of the naturally occurring metabolites trehalose, glycerol, and isoleucine provided 84% post-thaw recovery that was comparable to the results obtained using 10% DMSO [ 65 ].…”

“…Natural CPAs have become a popular subject among researchers that have attempted to find an alternative to synthetic CPAs [ 28 , 29 , 30 ]. However, much less is known about their mechanism of action and efficacy in cryopreservation.…”

Differentiation of Human iPS Cells Into Sensory Neurons Exhibits Developmental Stage-Specific Cryopreservation Challenges

“…This result supports the transition from DMSO to non-DMSO in order to enable scale-up of the fill-and-finish process in a manufacturing setting, while adhering to a high batch consistency. Compared with DMSO whose cytotoxicity arises from dissolution, epigenetic and genomic alteration, these non-DMSO CPAs, typically with larger molecular weight than DMSO, can cause pre-freeze cell loss predominantly due to osmotic stress upon CPA introduction ( Best, 2015 ; Hornberger et al, 2021 ; Matsumura et al, 2021 ). Compared with D5 crest and D7 SN, which exhibited no significant cell loss over the course of prefreeze exposure to the non-DMSO CPA solution ( p > 0.05), D14 mSN experienced minor (10%) yet significant ( p < 0.05) decrease in viable cell count during the first 30 min of this process, suggesting that sensitivity to the osmotic stress varied by cell stage with the most mature cells being most sensitive.…”

A mixture of innate cryoprotectants is key for freeze tolerance and cryopreservation of a drosophilid fly larva

“…These mixtures have been proposed to serve different physiological functions, including survival of organisms in extreme drought and cold [17][18][19] and have also been proposed as new agents for cell cryopreservation [20]. We believe that there may be major practical consequences of exploring the complex composition of insect CP mixtures, as they could aid in the development of applicable cryogenic techniques.…”