The Role of Natural Deep Eutectic Solvents - ACS Publications

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Cite This: ACS Sustainable Chem. Eng. 2017, 5, 9542-9553

How Do Animals Survive Extreme Temperature Amplitudes? The Role of Natural Deep Eutectic Solvents Ana Gertrudes,†,‡ Rita Craveiro,§ Zahara Eltayari,§ Rui L. Reis,†,‡ Alexandre Paiva,§ and Ana Rita C. Duarte*,†,‡ †

3B’s Research Group - Biomaterials, Biodegradable and Biomimetic, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Avepark, 4805-017 Barco, Guimarães, Portugal ‡ ICVS/3B’s PT Government Associated Laboratory, Braga, Guimarães, Portugal § LAQV-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal

ABSTRACT: Recent findings have reported the reason why some living beings are able to withstand the huge thermal amplitudes between winter and summer in their natural habitats. They are able to produce metabolites decreasing deeply the crystallization temperature of water, avoiding cell disrupture due to the presence of ice crystals and overcoming osmotic effects. In vitro, the possibility to cool living cells and tissues to cryogenic temperatures in the absence of ice can be achieved through a vitrification process. Vitrification has been suggested as an alternative approach to cryopreservation and could hereafter follow an interesting biomimetic perspective. The metabolites produced by these animals are mostly sugars, organic acids, choline derivatives, or urea. When combined at a particular composition, these compounds form a new liquid phase which has been defined as Natural Deep Eutectic Solvents (NADES). In this review, we relate the findings of different areas of knowledge from evolutive biology, cryobiology, and thermodynamics and give a perspective to the potential of NADES in the development of new cryoprotective agents. KEYWORDS: Deep eutectic solvents, Cryopreservation, Vitrification, Cryoprotective agents, Glycerol

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INTRODUCTION

The ability of living beings to adapt to extreme environments has intrigued and fascinated many scientists from different fields of research. Likewise, the ability to survive extreme temperature amplitudes throughout the year is also remarkable. The understanding of the capacity of living beings to adapt to these conditions may reveal important clues for the development of efficient methods for organ cryopreservation. Nature has provided solutions to allow living beings to inhabit locations with extreme temperature differences, and these living beings have adapted themselves to overcome potential chilling injury in tissues. In this paper, we review different animal species which have been studied and their mechanisms of survival. Figure 1 illustrates the geographic distribution of the animal species which have been studied and are here reviewed. © 2017 American Chemical Society

MECHANISMS TO SURVIVE EXTREME TEMPERATURE AMPLITUDES

Considering, for example, Alaska, the temperature differences between winter and summer may reach more than 60 °C.1 Cold temperature triggers chilling injury of living systems, mainly by ice nucleation and osmotic stress. The decrease in temperature promotes a new equilibrium where available water in the liquid state tends to form ice crystals.2,3 These can disrupt biological structures, namely cell membranes, organelles, and extracellular matrix in tissues and organs.4 For this reason, the size and localization of ice crystals formed during cryopreservation has shown to be a determinant for survival rates.3 Furthermore, the Received: May 30, 2017 Revised: August 7, 2017 Published: September 20, 2017 9542

DOI: 10.1021/acssuschemeng.7b01707 ACS Sustainable Chem. Eng. 2017, 5, 9542−9553

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ACS Sustainable Chemistry & Engineering

Figure 1. Geographical distribution of the animal species reviewed.

water occurs in the tissues. Differential scanning calorimetry (DSC) was used to identify the glass transition temperature (Tg) of beetle larvae sealed in an aluminum pan, cooled from 10 to −150 °C, and then warmed applying different rates.21 Interesting to notice is the fact that two glass transition temperatures were identified, suggesting two different compartments, one richer in water than the other. In a vitrification process, ice nucleation is inhibited,5,22 and this detail is the reason why vitrification is considered an “ice-free” cryopreservation technique. In the case of earthworms, DSC analysis has shown no evidence of ice formation up to −60 °C.23 In the winter season, earthworm specimens can inhabit soils with low subzero temperatures,24 where glycogen catabolism and amino acid content has been shown to occur is response to body fluid freezing.25,26 A two-step approach to survive lethal cold environments seems to be used by these animals: First, a slow absorption of sugar to prepare the tissues for a significant osmolality increment occurs, and then, in a second step, the polyols are promptly produced when the cold hits.27 Polyols found in cold-resistant living beings comprehend, for example, sorbitol, mannitol, erythritol, threitol, arabinitol, and ribitol.6,12,16 A similar dual strategy has been observed in other insects, such as rice water weevil, coldig moth, and many others.16,17,19,28 This overwinter tactic seems to work as a progressive creation of a system comprising sugars which are mixed with polyols. Moreover, the observation of amphibians led to the discovery of another two-step approach for cold resistance in wood frogs. For a long time, various specimens of frogs were known to survive subzero conditions. Glucose was the first metabolite to be detected in the cold-resistance strategy of frogs,29,30 and its synthesis regulation was found to be modified in frogs that can tolerate freezing.31,32 For example, higher amounts of membrane glucose transporters are expressed in cold-tolerant frogs than in intolerant specimens.33 Most recently, urea was also found to be part of the cold resistance strategy of terrestrial frogs. Urea is a well-known nitrogenous waste that can denature proteins and be highly toxic. However, urea action in the biological systems can be modified by particular solvent conditions.34,35 Marine beings were found to accumulate not only urea but also betaine, trimethylamine oxide (TMAO), and sarcosine in stressful biochemical situations.27 It has been proven that the addition of urea and betaine in specific quantities allows mammalian cells

reduction of available liquid water increases the concentration of the solutes in the cells, inducing osmotic stress.4,5 The strategy that living organisms have found to survive extreme temperature amplitudes relies on the orchestrated production of different biological metabolites. For example, in the case of the stonefly from Alaska (Nemoura arctica), glycerol has been found to increase by 3 orders of magnitude in cold acclimation, leading to a survival rate of 85% when they are subjected to −15 °C for 2.5 weeks.6 A group of exceptional animals, with a size of microns, is tardigrades. Tardigrades, can survive in extreme conditions, hot and cold temperatures, high saline concentrations, and ionic radiation.7,8 These resistant small invertebrates support a dehydrated state in cold conditions for long periods and later can be revived by rehydration. In the 1990s, Westh and Ramlov studied the variation of trehalose in tardigrades when subjected to dehydration. Trehalose quantity increases near 23 fold during dehydration, and the level is reduced when hydration is initiated.9,10 Later, they related the trehalose level and the desiccant state with increased freeze tolerance.11 Not only are the metabolites accumulated by the organisms crucial but also is the amount of water that remains in the system during freezing. In 1995, Somme and Meier reported the survival of hydrated tardigrades after almost 600 days at −20 °C and dehydrated tardigrades for 3040 days at the same temperature.7 Furthermore, trehalose was found to be accumulated in several other animals with cold resistance, namely, beetles, cabage root fly, gall fly, stonefly, and damaging pests such as codling moth and rice weevil6,12−19 Beetles from central Europe (Ips typographus) endure winter temperatures of −20 °C, and it has been confirmed that seasonal production of trehalose, other sugars, and polyols is part of a strategy for cold survival. During autumn, accumulation of trehalose and glucose was observed, and then, when conditions undergo a threshold point (humidity and temperature), a massive production of polyols was initiated.12 In spring, the identified cryoprotective metabolites were practically cleared from the tissues.20 Also, it has been noticed that available free water in the organism follows an inverse pattern of lowering during cold to be again increased in the spring.20,21 Sformo and co-workers not only demonstrated that Alaska beetle larvae can survive below subzero temperatures but also that vitrification of 9543


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Figure 2. Schematic representation of dual step strategy for overwinter survival in cold-tolerant insects and frogs.

Table 1. Metabolites Found Responsible for Overwinter Survival of Animals Living in Environments with Extreme Temperature Amplitudes Animal

Component 1

Component 2

Component 3

Component 4

ref

Tardigrades Beetles Beetles Cabbage root fly Common fruit fly Stonefly Codling moth

Trehalose Trehalose Glycerol Trehalose Trehalose Trehalose Trehalose

− Polyols (sorbitol and mannitol) − − Amino acids (glycine, lysine, and tryptophan) Proline Polyols (manitol and sorbitol)

Glycerol Trehalose Glucose

− − − − − − Amino acids (alanine) − − −

9, 11 12 21 13 14 6 16

Gall fly Rice weevil Earthworm

− Glucose − − Glucose Glycerol Other sugars (frutose and glucose) Polyols (sorbitol) Polyols (sorbitol and mannitol) Amino acids (proline, glutamine, and alanine)

15, 18 19 26

Earthworm Frog Frog

Glucose Urea Urea

− − −

14 31, 36 38

Glucose Glucose

Trehalose − − − − Amino acids (aspartic acid, alanine, glutamatic acid, leucine, isoleucine, valine, aspargine, serine)

to survive in high saline media conditions.34 Moreover, recent studies show that unique solvent systems comprising urea can even lead to protein stabilization.35 The wood frog Rana sylvatica initiates a process of urea accumulation when drought occurs in its habitat, normally in autumn and early winter. Costanzo and Lee works registered a 25-fold higher urea content in outdoor individuals collected in November compared to the ones collected in April.36 When the temperature drops to near −20 °C, an increase in glucose production is triggered.36,37 Very similar to insects, the two-tep strategy starts with urea accumulation, and then, a cold freeze initiates the glucose increment, balancing osmolality of the tissues to avoid osmotic stress during water freezing. These natural cryoprotectants flow into the frog’s cells and serve to diffuse the concentration gradient between the interior and exterior of the cells as extracellular water freezes, preventing cell shrinkage. Comparative analysis of organs from frozen and unfrozen test frogs show not only that glucose was increased in the frozen subject but also that the water content in the tissue was reduced.36 Moreover, the levels of urea and glucose accumulated for the cold-resistance

strategy in frogs have been confirmed to be an adaption of a population to habitat characteristics. In 2013, Costanzo and coworkers demonstrated that wood frog populations from Alaska had higher levels of protective metabolites and had better survival rates in extreme conditions than the ones from Ohio.31 The freeze-tolerant terrestrial frogs survival approach also appears to work as a gradual construction of a system with urea mixed with sugars, in an analogous process to the insects strategy. Figure 2 presents a schematic representation of this dual step strategy for overwinter survival. In both systems, other metabolites remain unraveled, and recently, amino acids were discovered to be part of wood frogs preparation for winter cold.38 A summary of the different biological metabolites which are synthesized by the different organisms to ensure that life is possible below 0 °C is listed in Table 1. Besides the presented natural cryoprotectants, many other low weight molecules are produced during drought and cold conditions. For instance, the variation of free amino acids concentration in animals during the cold has been described in some animals. Proline content has slightly increased when 9544


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Figure 3. Schematic diagram of the processes involved in cryopreservation either by slow freezing or vitrification.

Table 2. Summary of Advantages and Disadvantages of Slow Freezing and Vitrification Processes Slow freezing

Vitrification

Advantages

Low concentration of CPA (1.5 M) Low risk of chemical damage Technically simpler

Disadvantages

Longer process Longer exposure time Freezing machine required Ice crystals formed Poor control on solute penetration

Shorter exposure time Faster process No machine required No ice crystals formed Control of solute penetration Low concentration of CPA (3−6 M) High risk of chemical damage More clinical expertise

comparing cold- and warm-acclimated stoneflies,6 while other amino acids, such as glutamine, alanine, lysine, leucine, and phenylalanine, have been reported in insects, earthworms, and amphibians associated with sugars and polyols as part of the cryoprotective strategy.14,17,26,38 This ability of some animals to vitrify or to reduce the crystallization temperature of water inside their cells for winter survival provides insights to answer unmet needs of cryopreservation for tissues and organs.

assisted reproductive clinics in the last decades. On the topic of human health, the advances of cryopreservation techniques helps to respond to hospital transplantations demand for tissues and organs. Even with all of the improvements in cryobiology through the last 60 years, techniques are still not satisfactory for tissue and organs.5 Cryopreservation methods are slow freezing and vitrification, and both aim to overcome ice damage and the presence of solutes in toxic concentrations, which can have a detrimental effect on cell viability during freezing and thawing steps.40,41 A schematic diagram of the processes involved in cryopreservation is presented in Figure 3. Slow freezing makes use of cryoprotectants agents (CPA) and slow cooling rates to delocalize water from the cells to the cryopreservation media, where ice will be formed instead inside the cells.40 Associated disadvantages are the demanding tight control of the cooling process to avoid chilling injury and the need of expensive equipment, and it is a time-consuming method that generally destroys the extracellular matrix making it useless for tissues and organs.5,40 In vitrification, a high concentration of cryoprotectant agents is added to the medium, thus avoiding the crystallization of water. Because ice formation is avoided, there is no need for a slow cooling process; therefore, vitrification of the living system is achieved by a fast cooling step below the glass transition temperature.42 Vitrification is defined as a change in the structure of the material between a rubber-like state and a glass-like state.

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CRYOPRESERVATION AND VITRIFICATION Cryopreservation is a method to store biological systems for a significant period of time and then recover them with no changes in the metabolism and little or no damage to the biological structure. Cryopreservation combines extreme cold temperatures to ensure months or even years of storing with cryoprotectant agents used to protect cells during freezing and thawing steps.4 Vast development was achieved in the 20th century regarding preservation of cells, tissues, and organs with relevant impact in different aspects of our society.39 Simple and robust methods for single cell cryopreservation made possible the generalization of in vitro cellular studies, with high contribution for biological and medical scientific developments worldwide. Commercially, the progresses in cryopreservation opened new market opportunities for specimens’ storage and applications: ovules, sperm, embryos, stem cells, seeds, and many others. Examples are the increasing number of cell banks and 9545


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Figure 4. Molecular structure of penetrating (MW < 100 Da) and nonpenetrating (MW 180−600 Da) cryopreservants.

This glass transition is a second-order transition that occurs at the glass transition temperature (Tg). In 1968, the success of the vitrification process to preserve biological activity and molecular stability was proven in human erythrocytes using a concentrated solution of glycerol.43,44 Cryopreservation by vitrification has been growing exponentially since the early 1980s, and in 1985, Rall and co-workers demonstrated for the first time the vitrification of mouse embryos.45 Today, it is mostly accepted that vitrification performs better than slow freezing as demonstrated in systematic reviews and meta-analysis of human embryo cryopreservation for reproductive medicine.41,46 Vitrification avoids chilling injury from ice crystals and does not require optimal cooling/warming rates due to the thermal behavior which presents no crystallization step.47 The vitrification process challenges lie mostly on sample size and potential toxic effect of the high concentration of cryoprotectants. Additionally, the osmotic effects of adding and removing the cryoprotective agents are still major drawbacks hindering the application of vitrification in the preservation of cells, tissues, and organs. Table 2 summarizes the advantages and disadvantages slow freezing versus vitrification. Cryoprotectants comprise a large range of molecules, such as sugars, polyalchools, amides, amino acids, polysaccharides, and even macromolecules, such as proteins.4,48 Cryoprotectants can be divided into two categories, the penetrating cryoprotectants which are generally low molecular weight (MW) agents (below 100 Da) and the nonpenetrating cryoprotectants, namely, sugars with a MW in the range of 180−600 Da (Figure 4). Other nonpenetrating cryoprotectants used have high molecular weight molecules, with MW > 1000 Da, such as ficoll, dextran, polyvinylpyrrolidone, polyethylene glycol, and polyvinyl alcohol49,50 The cryoprotectant agents’ mechanism of action involves various factors and is not fully understood. General features of cryoprotectant agents are its viscosity in high concentration and the ability to interact with water molecules by hydrogen bonding.4,5 Different agents and mixtures are available, but an explanation for the difference in efficiency is not yet recognized. Furthermore, currently used cryoprotectants may have inherent toxicity, limiting the concentrations in which they may be used.42

Glycerol is a polyol that can be found in organisms, and it is involved in fatty acids biochemistry.51 For decadesn it has been used as a cryoprotectant agent. Since the use of glycerol as a cryoprotectant agent in vitrification of spermatozoa by Polge and co-workers in the 1940s,52 this has been the standard for the preservation of cell lines, mammalian embryos, and more. Although glycerol is considered a low toxic molecule in shortterm exposure, it has been reported to damage some tissues,53,54 and the ability to preserve larger human tissues and organs has not yet been discovered. The introduction of sugars, for example, sucrose and trehalose, to cryoprotective solutions allow for the concentration reduction of other compounds, namely, 2,3-butanediol, to achieve an amorphous state. For example, if 6% w/v of sucrose is added, the concentration of 2,3-butanediol can be reduced about 6% w/v, which can be significant in terms of toxicity.4 Trehalose is a nonreducing disaccharide present in living systems from animals and plants to yeasts and bacteria. It functions as an energy source, osmoregulator, and structural element of bacterial membranes and is a stress-responsive metabolite during dehydration.55,56 Trehalose, such as other sugar molecules, can be maintained in a vitreous or glass state with a characteristic Tg. Crowe and his group developed diverse studies regarding the trehalose role in stabilization of biological samples during dehydration and freezing; however, the mechanism is not fully unraveled.57−59 The vitrification of intracellular water by the increased concentration of trehalose or other sugars is a valid hypothesis to explain the mechanism in dry and freezing environments.8,56,60,61 In 1991, Koster hypothesized that the presence of a particular combination of sugars in desiccation-tolerant seeds, which have a glass transition temperature above 0 °C, is the reason that these seeds are tolerant to desiccation. On the other hand, the sugars present on nontolerant seeds present a subzero glass transition temperature.61,62 Facts contributing to the ability to achieve a glass state are tissue dehydration and accumulation of sugar molecules that enhance the system’s viscosity. The presence of trehalose and glycerol mixtures are also known to have effects of the stabilization of enzymes as reported by Barreca et al.63 9546


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NATURAL DEEP EUTECTIC SOLVENTS A notoriously wide range of sugar, polyols, and amino acids has been proven to be part of a multicomponent system for the resistance to extreme temperature differences in the animal kingdom. This knowledge was the basis for cryoprotectant solutions development in the last century; however, this knowledge has not until recently been related with deep eutectic systems. Inspired in nature, Choi and co-workers have related the presence of naturally occurring molecules in plant cells with their cellular metabolism and physiology.64 These compounds, which are present in large amounts in cells, are proposed to form a third type of liquid, one separate from water and lipids, the so-called natural deep eutectic solvents (NADES).64 NADES are defined as a mixture of two or more solid or liquid components, such as sugars, amino acids, organic acids, or choline derivatives, which at a particular composition present a high melting point depression, compared to its individual constituents, becoming liquid at room temperature or near room temperature.65 Eutectic solvents have scarcely been reported in the literature until the beginning of the 21st century,66,67 and the most common are based on choline chloride, carboxylic acids, and other hydrogen-bond donors, for example, urea, succinic acid, and glycerol. Durand et al. has also recognized that deep eutectic solvents are not only present in plants but also in animals.68 The role of a third liquid phase in an organism’s biochemistry has not yet been fully explored; however, there are some reports in the literature that relate the presence of NADES to the ability to transport and dissolve poorly water-soluble molecules as well as to improve the activity and stability of enzymes. NADES play an important role in the biotransformation of these poorly water-soluble molecules, providing a substrate in which both solutes and enzymes can be solubilized.68 Several natural deep eutectic systems have been reported in the literature, and the number continues to increase as more information on NADES is unraveled. Certain combinations of the metabolites synthesized by extreme cold-tolerant animals (listed in Table 1) have demonstrated the ability to form NADES. Table 3 presents a summary of the systems reported in

components of the system results in a supermolecular structure.64,73,74 The more distinct characteristic of the NADES system is the reduced melting temperature compared to its individual components. This depression in the melting point is accepted to be a consequence of the charge of the hydrogen bond network formation.64,65,75 The molar ratio of the components is hence critical to have a single liquid NADES system. Depending of the molar ratio and temperature, the mixture can be above the eutectic point, and a single liquid phase composed solely by a supramolecular structure/complex is obtained A two-phase system composed of a liquid phase and a solid phase can also be obtained, or in some cases, a single liquid phase can be obtained but composed by the liquid supramolecular structure and by the solid solubilized in it. Naturally, in these cases, the properties of the mixture differs from that of a NADES. However, other properties are of great importance, namely, density, polarity, and viscosity. NADES density is higher than water, and it decreases when water is added to the system.74,76,77 In terms of polarity, NADES are, in general, polar systems, and their polarity is increased by inclusion of organic acids73,76 and by the presence of water. In fact, the water percentage can only be raised until a limit point, after which the hydrogen bonds between the molecules which constitute the NADES are disrupted and the characteristic properties of NADES are lost. For example, experiences with 1,2-propanediol and choline chloride in the molar ratio 1:1 have shown that above 50 wt % of water the structural supermolecular complex of the NADES system is completely disrupted.73 The high viscosity is another inherent characteristic of the NADES system, which is explained by the extensive hydrogen bond interactions between components. The type of hydrogen bond donor has been associated with viscosity.74 This property was also found to be modified by the percentage of water in the system; i.e., with increasing water content, the viscosity decreases.73,74 Below the critical limit, water in NADES is part of the system and cannot be evaporated.64 Accumulation of NADES can ensure that a minimum quantity of water molecules is retained in the living being. Overall, NADES are systems with no or low water content, and the water percentage extensively alters its properties (density, polarity, and viscosity); therefore; the water content of the systems is a critical parameter which should be rigorously controlled. The thermal behavior of NADES systems have been studied in the past few years. Besides melting, also the thermal decomposition and the glass transition temperature have been studied for NADES. Craveiro and co-workers have shown that NADES are glass formers.76 The thermal properties of NADES have been described for different systems, and at least one glass transition temperature has been detected. However, few studies couple differential scanning calorimetry (DSC) with polarized optical microscopy (POM). In the study reported by Craveiro, it is possible to observe the glassy state of NADES for the system glucose:tartaric acid in a molar ratio of 1:1. In order to show the influence of NADES in the vitrification process of water and its impact in the melting and crystallization phenomena, this work shows preliminary experimental data on studies carried out by adding different amounts of NADES. In order to perform these studies, NADES composed of proline and glucose was prepared in a molar ratio of 5:3, respectively, since these metabolites are often found in animals that live in extreme cold environments (Table 3) and the ability of these compounds to form a NADES has been previously reported.66

Table 3. NADES Described in Literature Comprising Only Metabolites Found in Extreme Cold-Tolerant Animals Component 1

Component 2

Component 3

Ratio

ref

proline proline proline serine glutamic salt glucose urea urea urea

sorbitol glucose glucose glucose glucose frutose sorbitol glucose glucose

− − − − − − NH4Cl CaCl2 CaCl2

1:1 5:3 1:1 5:4 1:1 1:1 2:7:1 4:5:1 3:6:1

69 66, 69 68, 69 69 69 64 70, 71 71 72

the literature where the constituents are metabolites found in extreme cold-tolerant animals. As can be noticed, not many of the systems reported in Table 1 have been described. The peculiar and distinct feature of NADES is the fact that its properties are not the summation of each component characteristics; NADES possesses distinguished features from its pure components. Its chemistry is based in the complexation of a hydrogen bond acceptor and a hydrogen bond donor. The hydrogen bond network formed between the two or more 9547


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Figure 5. Thermogram obtained at 10 °C min−1 for pure proline, pure glucose, and NADES pro:glc in a 5:3 molar ratio. Several heating and cooling cycles are presented.

Figure 6. Thermogram obtained at 10 °C min−1 for pure water and different mixtures of water and NADES pro:glc in a 5:3 molar ratio, pro:glc (5:3): 20, 50, and 80 wt %. DSC thermograms were collected between −90 and 30 °C. At least three heating and cooling cycles are presented. 9548


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ACS Sustainable Chemistry & Engineering Differential scanning calorimetry (DSC) experiments were carried out for DES composed of proline and glucose with a molar ratio of 5:3, respectively, pro:glc (5:3). This NADES is composed of metabolites found in animals that live in extreme cold environments (Table 2), and its DSC is also presented in Figure 5. NADES pro:glc (5:3) is liquid at room temperature. The only thermal event presented in the DSC thermogram between −90 and 40 °C is glass transition, with a Tg located at −26 °C (Figure 5), and it is very distinct from that of its pure compounds, showing no crystallization or melting events. Figure 6 shows the thermograms of pure water and of water mixed with different amounts of pro:glc (5:3), which shows that DES has a clear effect on the thermal behavior of water. Pure water (upper left) shows a typical water crystallization peak at ca. −14.9 °C, which has a distinctive form due to the self-heating of the sample78 and a melting point at −4.3 °C. As 20 wt % of pro:glc (5:3) is added to water (upper right), a decrease in the crystallization and melting temperature is observed, −7.1 and −15.1 °C, respectively. When 50 wt % of pro:glc (5:3) is added (lower left), a crystallization event is observed during the cooling cycle; nevertheless, it occurs at a significantly lower temperature when compared with crystallization of pure water since crystallization temperature occurs at −35.9 °C followed by the respective melting at −20.2 °C. When higher amounts of pro:glc (5:3) are added to water, more specifically 80 wt % (lower right), no thermal events are observed at the studied temperature range, except for glass transition; thus, no crystallization is observed at temperatures above −50 °C. At these conditions, the interactions established between water and DES are strong enough to maintain water liquid even at such extreme low temperatures. Additionally it is crucial to study the biocompatibility of NADES toward living beings. The toxicity and biocompatibility of different NADES have been reported in the literature mostly for different choline chloride-based systems.65,79,80 The results presented demonstrate that NADES can be biocompatible and nontoxic and therefore could be used as new cryoprotective agents. The understanding of the physicochemical and thermal properties of NADES, coupled with the knowledge of a thorough study of their biological performance, may open new perspectives in vitrification processes of cells and tissues and ultimately organs.81

cryopreservation by vitrification of complex structures, such as tissues and organs, without compromising their function.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ana Rita C. Duarte: 0000-0003-0800-0112 Notes

The authors declare no competing financial interest. Biographies

Ana Gertrudes was born in Coimbra in 1986. She is graduated in Biochemistry by University of Coimbra, and completed her MSc in Biochemistry in 2011 by the same university. In 2011, she performed an internship as researcher in drug discovery for the biopharmaceutical Anacor, USA. After it and until 2015, Ana Gertrudes was part of the development team of the Portuguese biotech Stemmatters, for injectable medical devices and combined ATMPs for arthritis. Currently, she performs duties as quality assurance manager in the clinical research organization Blueclinical. Her experience combines knowledge from GMP and GCP working environments, polysaccharides extraction and characterization, primary and immortalized cells manipulation (suspension, 2D and 3D cultures) and characterization. Since her work with combined ATMPs, she became interest in cell preservations techniques that could avoid frozen as solution for stable shelf products with higher viabilities.

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CONCLUSIONS Cellular water is considered the key of life; however, different biological metabolites are synthesized by different organisms to ensure that life is possible below the ambient water freezing point many times using dehydration strategies. The overview of those dehydration strategies under extreme cold conditions has shown an unexpected increase in metabolites, such as sugars, polyols, and amino acids, with increased osmolality and a significant reduction of available free water, concomitant to a body fluid viscosity increment. Moreover, the survival of animals and vitrification of water in the organisms with this cellular biochemistry has been proven, revealing that the systems composed of those metabolites are virtually water-free but can still support life. The possibility of the metabolites to form NADES is an explanation for the overwinter natural strategy of survival of some animals. Nature observations were many times used as a starting point for successful improvements in a diverse scientific landscape. A better understanding of cryoprotectant agents from extreme cold-tolerant animals and an innovative developmental approach based on NADES holds a new path for

Rita Craveiro was born in 1983 in Covilhã. She graduated in chemistry in 2008 from the Universidade de Coimbra and received her MSc in 2010 from the Universidade de Aveiro. She obtained her PhD in Sustainable Chemistry in 2015 from Universidade Nova de Lisboa, in alternative processes for biobased polymer processing. She is currently a Post-Doc at the Faculdade de Ciências e Tecnologia of the Universidade Nova de 9549


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and the editor-in-chief of the Journal of Tissue Engineering and Regenerative Medicine (Wiley-Blackwell). Rui L. Reis education background includes: (i) a graduation in Metal. Eng., U. Porto, Portugal, 1990, (ii) a Master degree by research on Mater. Sci. and Eng. − Biomaterials−obtained in a joint program of the six major technical Universities in Portugal, awarded by U. Porto, Portugal, 1994 (iii) a PhD on Polymer Eng. − Biomaterials & Tissue Engineering, U. Minho, Portugal, degree that was prepared in co-operation with Brunel University, London, UK, 1999, (iv) a Doctor of Science (D.Sc.) degree on Biomedical Engineering - Biomaterials & Tissue Engineering, by U. Minho, Portugal, 2007. He is coauthor of 1047 ISI listed publications (855 full papers in scientific journals), with 23850 citations and an hindex of 76, around 250 book chapters, 35 patents and 7 books. He is PI of projects totalizing around 45 MEuros, including the very prestigious European Research Council (ERC) Advanced Grant. He was awarded several major national and international scientific and innovation awards, including both the Jean Leray and George Winter Awards from the European Society for Biomaterials (ESB), the Clemson Award (Soc. For Biomaterials, USA) and the TERMIS-EU awards, both for contributions to the Literature and the UNESCO Life Sciences International Award, among many others. He was also awarded a honoris causa degree by the University of Granada, Spain and is a member of the National Academy of Engineering (USA). Rui L. Reis has been involved in biomaterials research since 1990. His main area of research is the development of biomaterials from natural origin polymers (starch, chitin, chitosan, casein, soy, algae based materials, silk fibroin, gellan gum, carragenan, hyaluronic acid, xanthan, marine collagen, etc.) that in many cases his group originally proposed for a range of biomedical applications, including bone replacement and fixation, drug delivery carriers, partially degradable bone cements and tissue engineering scaffolding. Lately the research of his group has been increasingly focused on tissue engineering, regenerative medicine, stem cells and drug delivery applications. His research group works with bone marrow, adipose-derived, umbilical cord (blood and matrix), amniotic origin (fluid and membrane) and embryonic stem cells.

Lisboa and associated laboratory LAQV@Requimte, and is involved in various projects and in the cosupervision of two master thesis works. Her main research interests are focused on the area of green chemistry and in the development, characterization and application of deep eutectic solvents in areas that range from polymer processing, biomass processing to biocatalysis and enantiomeric separations. She also works in areas such as supercritical fluids and their applications with eutectic solvents. Currently, she is the author and coauthor of several scientific papers, mainly in the area of eutectic solvents, polymer processing and characterization.

Zahara Eltayari is a 20 year old student from the Faculdade de Ciências e Tecnologia of the Universidade Nova de Lisboa. She is currently studying Chemical and Biochemical Engineering and anything she thinks that might be an opportunity to grow up as a student and as a future professional she likes to participate. She already did three summer internships and attended to several Scientific Conferences. One of the internships she did was in 2016 at the LAQV@Requimte group where she joined the research team to start developing the project about the study of the cryopreservation methods using Natural Deep Eutetic Solvents in the water vitrification process. Zahara is going now to start her master degree and her biggest goal is to specialize in areas such as the Biochemical Engineering, Bioenergetics and Biology. She is also looking forward to do her master thesis somewhere abroad in order to enrich her knowledge and understand a bit more what kind of research projects are being made around the world and what areas she would like to work in the future, as an engineer or as a researcher.

Alexandre Paiva was born in Lisbon, Portugal in 1978. He graduated in Chemical Engineering from the Faculdade de Ciências e Tecnologia of the Universidade Nova de Lisboa in 2001 and completed his PhD in Chemical Engineering−thermodynamics and Biocatalysis from the Technische Universität Hamburg-Harburg in Germany, 2008.From 2010 to 2012 he was an invited professor at the Instituto Piaget were he lectured Introduction to Chemical Engineering and Transport Phenomenon I and II. Since 2008 he is also an assistant lecturer in Biocatalysis. From 2008 to 2016 he was a Postdoc researcher at REQUIMTE, a nonprofit scientific organization, working on the “Biodiesel production from green sources using supercritical technology” project. Since 2016 he is a senior researcher at LAQV@ REQUIMTE. Alexandre Paiva has more than 30 papers in international

Rui L. Reis, PhD, DSc, Hon. Causa MD, FBSE, FTERM is 50 years. He is the Vice-Rector for R&D of University of Minho (UMinho), Portugal. He is the Director of the 3B’s Research Group and of the ICVS/3B́ s PT Government Associate Laboratory of UMinho. He is also the CEO of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine (TERM). He is the World President of TERMIS (Tissue Engineering & Regenerative Medicine International Society) 9550


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(3) Wesley-Smith, J.; Berjak, P.; Pammenter, N. W.; Walters, C. Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: An ultrastructural study of factors affecting cell and ice structures. Ann. Bot. 2014, 113 (4), 695− 709. (4) Fuller, B. J. Cryoprotectants: The essential antifreezes to protect life in the frozen state. Cryo-Letters 2004, 25 (6), 375−388. (5) Taylor, M. J.; Song, Y. C.; Brockbank, K. G. M. Vitrification in Tissue Preservation: New Developments. In Life in the Frozen State; Fuller, B. J., Lane, N., Benson, E. E., Eds.; CRC Press, 2004; Chapter 22. (6) Walters, K. R.; Sformo, T.; Barnes, B. M.; Duman, J. G. Freeze tolerance in an arctic Alaska stonefly. J. Exp. Biol. 2009, 212 (2), 305− 312. (7) Sømme, L.; Meier, T. Cold tolerance in Tardigrada from Dronning Maud Land, Antarctica. Polar Biol. 1995, 15 (3), 221−224. (8) Wright, J. C. Cryptobiosis 300 Years on from van Leuwenhoek: What Have We Learned about Tardigrades? Zool. Anz. 2001, 240 (3−4), 563−582. (9) Westh, P.; Ramlov, H. Trehalose accumulation in the tardigrade Adorybiotus coronifer during anhydrobiosis. J. Exp. Zool. 1991, 258, 303−311. (10) Hengherr, S.; Heyer, A. G.; Köhler, H. R.; Schill, R. O. Trehalose and anhydrobiosis in tardigrades - Evidence for divergence in responses to dehydration. FEBS J. 2008, 275 (2), 281−288. (11) Ramlo̷ v, H.; Westh, P. Survival of the cryptobiotic eutardigrade Adorybiotus coronifer during cooling to −196 °C: Effect of cooling rate, trehalose level, and short-term acclimation. Cryobiology 1992, 29 (1), 125−130. (12) Košt ál , V.; Doleža l, P.; Rozsypal, J.; Moravcová, M.; Zahradníčková, H.; Šimek, P. Physiological and biochemical analysis of overwintering and cold tolerance in two Central European populations of the spruce bark beetle, Ips typographus. J. Insect Physiol. 2011, 57 (8), 1136−1146. (13) Koštál, V.; Šimek, P. Dynamics of cold hardiness, supercooling and cryoprotectants in diapausing and non-diapausing pupae of the cabbage root fly, Delia radicum L. J. Insect Physiol. 1995, 41 (7), 627− 634. (14) Overgaard, J.; Malmendal, A.; Sørensen, J. G.; Bundy, J. G.; Loeschcke, V.; Nielsen, N. C.; Holmstrup, M. Metabolomic profiling of rapid cold hardening and cold shock in Drosophila melanogaster. J. Insect Physiol. 2007, 53 (12), 1218−1232. (15) Lee, R.; Dommel, R.; Joplin, K.; Denlinger, D. Cryobiology of the freeze-tolerant gall fly Eurosta solidaginis: overwintering energetics and heat shock proteins. Clim. Res. 1995, 5, 61−67. (16) Rozsypal, J.; Koštál, V.; Zahradníčková, H.; Šimek, P. Overwintering Strategy and Mechanisms of Cold Tolerance in the Codling Moth (Cydia pomonella). PLoS One 2013, 8 (4), e61745. (17) Lalouette, L.; Koštál, V.; Colinet, H.; Gagneul, D.; Renault, D. Cold exposure and associated metabolic changes in adult tropical beetles exposed to fluctuating thermal regimes. FEBS J. 2007, 274 (7), 1759− 1767. (18) Morrissey, R. E.; Baust, J. G. The ontogeny of cold tolerance in the gall fly, Eurosta solidagensis. J. Insect Physiol. 1976, 22 (3), 431−437. (19) Lee, K. Y.; Chang, Y. D.; Kim, Y. G. Trehalose, a Major Sugar Cryoprotectant of the Overwintering Rice Water Weevil, Lissorhoptrus oryzophilus (Coleoptera: Curculionidae). J. Asia-Pac. Entomol. 2002, 5 (1), 35−41. (20) Koštál, V.; Zahradníčková, H.; Šimek, P.; Zelený, J. Multiple component system of sugars and polyols in the overwintering spruce bark beetle, Ips typographus. J. Insect Physiol. 2007, 53 (6), 580−586. (21) Sformo, T.; Walters, K.; Jeannet, K.; Wowk, B.; Fahy, G. M.; Barnes, B. M.; Duman, J. G. Deep supercooling, vitrification and limited survival to −100 °C in the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae). J. Exp. Biol. 2010, 213, 502−509. (22) Luyet, B., Gehenio, M. P.Life and Death at Low Temperatures; Biodynamica, 1940. (23) Holmstrup, M.; Bayley, M.; Ramløv, H. Supercool or dehydrate? An experimental analysis of overwintering strategies in small permeable

scientific peer-reviewed journals. His main areas of expertise are sub-, supercritical fluids, biocatalysis, deep eutectic solvents and the application of these solvents in the valorization of agro-industrial byproducts. He is/was involved in 1 Marie Curie project, 4 scientific projects financed by the Portuguese Foundation for Science and Technology, being the principal investigator in 2 of those projects. He is/was also involved in 4 projects in collaboration with Portuguese and international companies. He is the main supervisor of 4 PhDs. and the cosupervisor of 2 more. He is/was the supervisor of more than 20 Master degree thesis.

Ana Rita C. Duarte was born in Lisbon in 1978. Currently research assistant at the 3B’s research group at Universidade do Minho, She graduated in Chemical Engineering by Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa in 2002 and completed her PhD on Exploring supercritical fluid technology for the preparation of controlled drug delivery systems in 2006 by the same University. In 2006/2007 she was a researcher at Techniche Universiteit Delft, The Netherlands. From 2007 she was a postdoc at the group where now she currently working, the 3B’s research group. The International Society for Advancement of Supercritical Fluids granted her thesis the Best Thesis Award in 2007. At the moment, she has 79 papers listed in web of knowledge with a total of 1617 citations, and an h-index of 25. Her main research interests are the use of green technologies for the development of biomaterials. In particular, the use of water, ionic liquids, supercritical fluids together with the exploration of natural deep eutectic solvents for natural-based polymer processing are her main scientific interests.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support of FCT through the project Des.zyme - Biocatalytic separation of enantiomers using Natural Deep Eutectic Solvents (PTDC/ BBB-EBB/1676/2014). The funding received from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement numbers REGPOT-CT2012-316331POLARIS, as well as from European Regional Development Fund (ERDF) under the project “Novel smart and biomimetic materials for innovative regenerative medicine approaches” RL1ABMR-NORTE-01-0124-FEDER-000016), cofinanced by North Portugal Regional Operational Programme (ON.2, O Novo Norte), under the National Strategic Reference Framework (NSRF), are also appreciatively acknowledged.

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REFERENCES

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