![]() ![]() In clinical practice, glycerol is the standard CPA for RBCs, but its toxicity and complex deglycerolization limit its application. ![]() Additionally, a combination of proline and trehalose has been reported for the cryopreservation of red blood cells (RBCs). When used alone in GLC-82, HeLa and MCF-10 cells, frozen in the presence of betaine, the post-thawing viability was significantly higher. ![]() On the other hand, betaine showed, by DSC, the ability to supresses water crystallization. In all cases the mixture showed better results than Me 2SO or trehalose alone. Many authors reported the mixture of trehalose with Me 2SO to cryopreserve different cells lines, such as stem cells, primary hepatocytes, and HepG2 cells, ,, ,, , ]. For the past few years, many reports have been published using these metabolites in combination with Me 2SO to improve post-thawing cell viability. Some components of NADES, such as trehalose, glucose, sorbitol, and proline, are known to be produced by animals and plants that live in extreme cold environments and are involved in many metabolic pathways during winter. The small additions of water can tailor their intrinsic viscosity, as well as, decrease their toxicity. The components are mixed at a particular molar ratio, presenting a high melting point depression, compared to its individual constituents, therefore becoming liquid at room temperature or near room temperature. These systems can be formed by natural primary metabolites, such as sugars, amino acids, organic acids, or choline derivative, and therefore called natural deep eutectic systems (NADES). However, less toxic alternatives would allow to preserve more cell and tissue types for biomedical research.ĭeep eutectic systems (DES) have gained attention in the last years due to their sustainable properties such as high biodegradability, cost-effectiveness, and low toxicity. Me 2SO remains a solvent of choice in biomedical research, despite the physiological interference and cytotoxicity. However, it is known that it is toxic for cells, because it penetrates the cell membrane immediately after contact, causing cellular damage by altering mitochondrial membrane potential and increasing reactive oxygen species, and must be removed as soon as the cells are thawed. The use of dimethyl sulfoxide (Me 2SO) as CPA remains the standard protocol used in cryopreservation of mammalian cells in common laboratories. If the heating process is too slow, small ice crystals will recrystalize into bigger ones which will cause cell death. The use of a CPA can also play an important role during the thawing step because they can also avoid the recrystallization process. Many studies have been reported for the cryopreservation of mammalian cells but cellular function can be highly affected by environmental changes during the freezing process, such as cryopreservation solutions, biomaterials, freezing methods, and freezing/preservation temperatures.Ĭryoprotection usually involves the treatment of cells with a cryoprotectant agent (CPA), which will cause osmotic dehydration of cells, thus preventing the formation of intracellular ice crystals that would cause the bursting of cells. However, the cryopreservation of mammalian cells with high post-thawing cell survival remains a major challenge. The preservation of biological material in a stable state is fundamental for biological/medical sciences. In overall, the results demonstrate the high potential of NADES to be used in cryobiology as alternative CPAs. Additionally, we have shown that NADES can act as CPA when cells are frozen at −20 ☌. Moreover, the results presented herein showed that NADES do not need to be removed from the freezing media after thawing the cells, which is a great advantage of these materials. For Hacat cell line a significant improvement on post-thawing recovery was observed. After freeze/thawing cycle, it was possible to observe that for L929 cells, NADES presented similar behaviour to Me 2SO. To test NADES as CPAs, two cell lines were used, L929 and HacaT cells. Moreover, this cell line was highly tolerant to 10% (w/v) of NADES when compared to Me 2SO. All systems showed very little cytoxicity towards L929 cells at concentrations high as 1–2 M. Several combinations between natural primary metabolites that have been identified in animals that live in extreme cold climates were prepared. This work aimed at evaluating the potential of using natural deep eutectic systems (NADES) as cryoprotectant agents (CPAs). ![]()
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