High entropy solutions, methods for producing the same, and devices and methods for preserving biological matter

Abstract

The present disclosure provides high entropy solutions which do not freeze at subzero centigrade temperatures and methods for producing the high entropy solutions. Furthermore, the present disclosure provides devices and methods for preserving biological matter by cooling and / or storage at subzero centigrade temperatures with high entropy solutions without ice formation.

Description

Attorney Reference: BCHR-001WO HIGH ENTROPY SOLUTIONS, METHODS FOR PRODUCING THE SAME, AND DEVICES AND METHODS FOR PRESERVING BIOLOGICAL MATTER WITH HIGH ENTROPY SOLUTIONS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 670,528, filed July 12, 2024, the disclosure of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present disclosure relates to high entropy solutions which do not freeze at subzero centigrade temperatures and methods for producing high entropy solutions. Furthermore, the present disclosure relates to devices and methods for preserving biological matter by cooling and / or storage at subzero centigrade temperatures with high entropy solutions without ice formation. BACKGROUND

[0003] Preservation of biological matter such as molecules, cells, complex organs or organisms, tissues, or foods is essential to current medical and research applications, and to the food and pharmaceutical industry. Life processes are temperature-dependent chemical reactions, and the time of preservation of biological matter can be extended by preserving the matter at increasingly low temperatures. Conventional sub-normothermic preservation across a wide range of biological matter is performed at or around 4 ºC. Although preservation at even lower temperatures could further extend the period of preservation, this further extension is often hindered by the formation of ice at subzero centigrade temperatures, which yields chemical and mechanical effects that prove detrimental to the biological matter.

[0004] As such, methods and devices for the cryopreservation of a biological matter generally seek, where possible, to avoid or reduce the nucleation and growth of ice in theAttorney Reference: BCHR-001WO sample. In systems wherein it is concluded that ice growth cannot be avoided, methods and devices generally seek to minimize said growth, and / or localize it to the areas of the sample wherein it will cause the least damage. As such, the avoidance of damage due to ice growth is one of the central challenges of cryopreservation, and methods and devices by which to achieve this avoidance are sought.

[0005] Many works teach methods by which to achieve this avoidance, either by achieving principally “ice-free” conditions at sub-0 °C temperatures or by achieving an amount or mode of ice growth deemed favorable. See Eva Puschmann et al., “Liquidus Tracking: Large scale preservation of encapsulated 3-D cell cultures using a vitrification machine”, Cryobiology 2017 June; U.S. Patent No. 9723831B2; and U.S. Patent Publication No. US20220071196A1. Prior art can be subdivided into three general classes, based on the thermodynamic approach employed to avoid or reduce the growth of ice: 1) cooling the sample to temperatures mildly (at most approximately -20°C) beneath the equilibrium melting point of the solution, at which the liquid in the system may remain thermodynamically metastable and ice-free in the supercooled state (generally termed “supercooling”, “sub-cooling”, or similar); 2) cooling the sample to temperatures beneath the glass transition temperature of the solution, at which, depending on the thermal rates employed, the liquid in the system is stabilized in an ice-free solid glassy or “vitreous” state (generally termed “vitrification” or similar), and 3) cooling the samples to any temperature beneath the equilibrium melting point of the solution, at which ice is allowed or driven to form (generally termed “freezing”, “partial freezing”, or similar).

[0006] Prior works teaching methods of the first class, generally termed supercooling or similar, include the following. Usta and colleagues (U.S. Publication No. 2021 / 0039057A1) teach a method by which to supercool an aqueous sample (i.e. cool it to or maintain it in a thermodynamically metastable liquid state at temperatures beneath the equilibrium melting point of the sample) that entails applying to it an immiscible phase which separates the surface of the aqueous sample from air. They teach that this method may be applied to the preservation of biological samples at temperatures between 0°C and -20°C. Wei (U.S. Publication No. 2023 / 0292741A1) teaches a method by which to supercool a biological sample by placing it in contact with peptoid polymers or salts thereof. They teach that this method may be applied in a similar temperature range to that indicated by Usta, from 0°C to about -20°C. Fujikawa and colleagues (U.S. Patent No. 7,837,895B2) teach a method by which to supercool a biological sample that entails preparing it in an aqueous solution that contains flavonoid glycosides. TheyAttorney Reference: BCHR-001WO claim a similar temperature range of application, between 0°C and -15°C. As such, these prior works provide a unique means by which to avoid ice formation in the supercooled liquid state (use of an immiscible phase to stabilize supercooling; use of peptoid molecules that stabilize supercooling; use of flavonoid glycosides), and indicate that supercooling may be achieved at temperatures down to approximately -15 to -20 °C. This temperature range is generally consistent with the state of knowledge pertaining to supercooling of aqueous systems for preservation of biological matter, which indicates that in general, systems of practical volume (> microLiter sizes) can be supercooled for preservation-relevant periods (hours or greater) to temperatures not more than about ten degrees below the melting point of ice. Furthermore, there is extensive prior art pertaining to more complicated pieces of mechanical or thermal machinery that may be used to facilitate supercooling of aqueous samples, both for biological preservation (U.S. Patent Publication No. 2022 / 0325937A1) and for storage or treatment of food and beverages (U.S. Patent Publication No. 2007 / 0163275A1, No. 2019 / 0142037A1, and No. 2015 / 0323237A1).

[0007] Prior methods of the second class, broadly termed “vitrification”, are extremely numerous, but are unified by the definitional requirement of vitrification that the biological sample be cooled to (and stored at) a temperature beneath its glass transition temperature. Vitrification is defined as the process of a liquid undergoing the glass transition and thereby becoming a glass, typically defined as an amorphous solid of viscosity 1013poise or higher. This process is desirable because in the solid glass state, the practical possibility of ice crystallization is generally considered to be entirely eliminated. The general definition of vitrification as requiring cooling to and / or storage at beneath the glass transition temperature of the aqueous or biological system involved is so well accepted that it is rarely, in the modern era, explicitly commented upon or claimed in detail within the patent literature. The glass transition temperatures of different aqueous solutions of relevance to biology (i.e., solutions that are not generally acutely toxic) may vary extremely (for existence, ultra-high concentration sugar solutions can have glass transition temperatures above 0°C, while common commercial solutions of interest to medical cryopreservation have glass transition temperatures on the order of -120°C), but the defining requirement of vitrification as taking place beneath the glass transition temperature remains, regardless of what that temperature may be.

[0008] Vitrification of large systems (such as tissues and organs) is often hindered by an acute dependence on cooling and warming rate. Given the general relative instability of theAttorney Reference: BCHR-001WO liquid state at temperatures increasingly far from the equilibrium melting point and yet above the glass transition temperature, in order to avoid ice crystallization during cooling to or heating from the glass transition temperature, fast cooling or warming rates are required. Due to asymmetries in the thermodynamic driving forces at play, the warming rates required to avoid ice formation upon warming from the vitrified state are generally an order of magnitude larger than those required for cooling en route to the vitrified state. Given these rate-dependencies, many works of prior art teach methods by which to rapidly cool a biological sample to (or typically past) the glass transition temperature, or by which to rapidly warm a biological sample from said temperature upon thawing.

[0009] Furthermore, this characteristic rate dependence of vitrification is a function of system chemistry and / or concentration. Many aqueous chemical systems, at sufficient concentrations, will vitrify at easily attainable cooling and warming rates. However, these concentrations are generally toxic to living biological material of interest (such as human tissues and organs, marine organisms, cell therapies, etc). Thus, solutions of relevance to preservation of living biological matter generally have an absolute maximum concentration of 8-10 Molar, or approximately 35 mol%, and at these concentrations, the instability of the liquid at temperatures higher than the glass transition necessitate relatively fast cooling (and especially warming rates). Examples of prior patent art seeking to achieve vitrification preservation of biological samples by affecting cooling and warming rates include U.S. Patent No. 9,538,745 B2, U.S. Patent Publication No. 2016 / 0102286A1, U.S. Patent Publication No. 2022 / 0412635A1, U.S. Patent Publication No. 2018 / 0094232A1, U.S. Patent No. 9,557,090 B2, and International Publication No. WO 2018 / 064976 A1. In this prior art, and in the broader scientific literature, reported vitrification solutions include up to at most 5-6 principal non-water components contributing to their thermodynamic stability against ice formation, with principal component here defined as any non-water component with a molar concentration in solution not less than one tenth that of the most abundant non-water component in solution.

[0010] The confluence of chemistries and cooling / warming rates required to achieve vitrification also introduce a unique and potentially disastrous mode of failure: cracking. Like any glass, the glasses formed by vitrified aqueous liquids are extremely brittle. When coupled with the extremely low thermal conductivity of concentrated aqueous system and the high thermal rates required, the risk of cracking induced by thermal stresses becomes acute, introducing a mode of damage characteristic of vitrification, but not of supercooling.Attorney Reference: BCHR-001WO

[0011] Based on prior art in the domain of ice-free low-temperature preservation of biological samples, the following conclusions may be reached: ice-free preservation by supercooling is generally limited (by the stability of the metastable supercooled state) to -15 °C to -20 °C; ice-free preservation by vitrification requires cooling biological samples to and storing samples at temperatures beneath the glass transition temperature of the preservation solution, and typically requires certain cooling and warming rates to avoid ice formation at temperatures warmer than the glass transition temperature, which are increasingly difficult to achieve in increasingly large biological systems given the low thermal diffusivity of water and aqueous solutions. Based on the available published literature, the general range of glass transition temperatures exhibited by preservation solutions of relevance to biological systems (i.e. of minimal toxicity) is approximately -110 °C to -140 °C, and that the range over which supercooling techniques operate is approximately 0 °C to -20 °C.

[0012] The temperature range in between the ranges accessible to supercooling and vitrification, i.e. the range from approximately -21°C to approximately -110°C, is generally not accessible to ice-free techniques. Thus, to achieve cryopreservation of a biological sample in this range, ice formation must generally be accepted, per the aforementioned third class of techniques. Prior art teaching means of cryopreservation in this temperature range whilst tolerating ice formation are taught for example by U.S. Patent No. 8,037,696 B2 or Canadian Patent No. 2753291C.

[0013] In practice, this third class of techniques can be tolerated by cellular systems (given their relative lack of structural or complexity), but they cannot generally be applied to good effect in complex tissues or organs, which will be irreversibly damaged by extensive interior ice formation. Ice formation can be tolerated to a limited degree by organs at higher temperatures, if the solution chemistry is chosen so as to limit total ice growth (International Publication No. WO2018 / 232110A1). However, at these higher temperatures, supercooling could also be applied to avoid ice entirely, limiting their utility. It should also be noted that the preservation periods afforded by these higher temperatures are limited to the order of days-to- weeks, whereas at temperatures around or beneath -80 °C or -100 °C, preservation durations are extended to the order of months-to-years.

[0014] In light of all of the above, it is desirable to achieve ice-free cryopreservation of a biological sample at temperatures beneath those currently accessible to known supercooling techniques whilst avoiding the complications of known vitrification techniques (acute thermalAttorney Reference: BCHR-001WO rate dependences, difficulty in scaling to larger volumes because of these dependences, acute risk of cracking, etc.). SUMMARY

[0015] The inventors discovered high entropy solutions which do not freeze when cooled to and warmed from a temperature between 0 °C and about -200 °C and methods for producing the high entropy solutions. The present disclosure further provides methods and devices or apparatuses enabling ice-free preservation of a biological matter with high entropy solutions at all temperatures less than 0°C, and in particular at temperatures below the range currently accessible to supercooling techniques disclosed in prior art. This discovery is paradigm-shifting because of the potential to enable cryopreservation of arbitrarily large biological matters at a temperature between 0 °C and about -100 °C while reducing or eliminating the risk of ice formation. Embodiments of the invention satisfy this desire.

[0016] Aspects of the present disclosure include high entropy solutions which do not freeze when cooled to and warmed from a temperature between 0 °C and about -200 °C. More specifically, the high entropy solutions of the present disclosure comprise water and two or more non-water chemical components with different physical or thermodynamic properties. In some embodiments, an entropy of mixing of the high entropy solution is equal to or greater than 7 J / (mol K). In some embodiments, the high entropy solution is comprised of ten or more principal components, attaining a sufficient entropy to suppress ice formation based on the number of components alone.

[0017] Aspects of the present disclosure further include methods for producing high entropy solutions which do not freeze when cooled to and warmed from a temperature between 0 °C and about -200 °C. More specifically, the methods for producing high entropy solutions comprise a) selecting two or more non-water chemical components with different physical or thermodynamic properties and water; b) determining each amount of the non-water chemical components and amount of the water to increase an entropy of mixing; and c) mixing the non- water chemical components and the water with the determined amounts to produce the high entropy solution.Attorney Reference: BCHR-001WO

[0018] In some embodiments, the number of non-water chemical components is between 2 and 100. In some embodiments, the entropy of mixing increases with the number of the non- water chemical components.

[0019] In some embodiments, the non-water chemical components are non-water carbon-based components with molar volumes between about 40 mL / mol and about 400 mL / mol or between about 40 mL / mol and about 150 mL / mol.

[0020] In some embodiments, molar volumes of non-water chemical components are different from one another, and molar volumes of non-water chemical components are different from molar volume of the water. In other embodiments, mol fractions of the non-water chemical components are different from one another, and mol fractions of the non-water chemical components are different from mol fraction of the water.

[0021] In some embodiments, an entropy of mixing increases when differences in molar volumes of the non-water chemical components increase. In other embodiments, an entropy of mixing increases when differences in mol fractions of the non-water chemical components increase. In some embodiments, the mol fractions of the non-water chemical components ascend in magnitude with ascending molar volumes of the non-water chemical components.

[0022] In some embodiments, mol fraction of the water is between 0.65 and 0.99. In some embodiments, mol fraction of the water is constant while mol fractions of the non-water chemical components vary to increase the entropy of mixing.

[0023] In some embodiments, the number of the non-water chemical components is equal to or greater than 3, and mol fraction of at least one of the non-water chemical components are constant and mol fractions of at least two of the non-water chemical components vary to increase the entropy of mixing.

[0024] In some embodiments, the entropy of mixing in the liquid phase may be calculated by entropy of mixing equation:

[0025] ∆^^ = −^^^^ ln ^^ + ∑^^ ^^ ln^^^^^

[0026] wherein ^^and ^^are respectively mol and volume fractions of water; ^^and ^^are respectively mol and volume fractions of each of n non-water chemicaln is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^is entropy of mixing. The entropy of mixing equation for calculating the entropy of mixing in the liquid phase is not limited to the equation set forth above.Attorney Reference: BCHR-001WO

[0027] In some embodiments, the mol fractions and the volume fractions of the non- water chemical components, the mol fraction and the volume fraction of the water, and the number of the non-water chemical components are determined to increase the entropy of mixing. In other embodiments, the mol fractions and the volume fractions of the non-water chemical components and the mol fraction and the volume fraction of the water are necessary to find or approach a local or global extremum in the entropy of mixing equation.

[0028] In some embodiments, optimization of the entropy of mixing equation is performed by using a computational optimization method. In certain embodiments, the computational optimization method includes a brute force optimization method, a binary search optimization method, a convex optimization method, a stochastic optimization method, or a particle swarm optimization method.

[0029] In some embodiments, the number of non-water chemical components is equal to or greater than 3 and the high entropy solution comprises a) water with molar volume about 18 mL / mol; b) first group comprising one or more non-water chemical components in molar volume range between about 40 mL / mol and about 75 mL / mol; c) second group comprising non-water chemical components in molar volume range between about 75 mL / mol and about 100 mL / mol; and d) third group comprising one or more non-water chemical components in molar volume range between about 100 mL / mol and about 150 mL / mol. In some embodiments, the molar volumes of the non-water chemical components in the first group are different from one another. In other embodiments, the molar volumes of the non-water chemical components in the second group are different from one another. In still other embodiments, the molar volumes of the non-water chemical components in the third group are different from one another. In some embodiments, the non-water chemical components in the first group are different from one another. In other embodiments, the non-water chemical components in the second group are different from one another. In still other embodiments, the non-water chemical components in the third group are different from one another.

[0030] In some embodiments, the water is replaced by a saline solution, a hypothermic organ preservation solution, or a base solution containing electrolytes.

[0031] Various non-water chemical components (NWCCs) can be used in the high entropy solutions. Such components can also be used as the principal non-water chemical components (PNWCCs) of high entropy solutions. In some embodiments, any of the non-water chemical components of the present disclosure is selected from glycols, sugar alcohols,Attorney Reference: BCHR-001WO methylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers. In other embodiments, any of the non-water chemical components of the present disclosure is selected from ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di-ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol, threitol, ethanol, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose, and trehalose.

[0032] In some embodiments, the components used as NWCCs or PNWCCs are selected from the group consisting of 1,2,4-butanetriol, 1,2,6-hexanetriol, 1,2-butanediol, 1,2-hexanediol, 1,2-pentanediol, 1,2-propanediol, 1,3-butanediol, 1,3-diaminopropane, 1,3-dihydroxyacetone, 1,3-dimethlurea, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1-2- hydroxyethyl-2-pyrrolidinone, 1-amino-2-propanol, 1-methyl-pyrrolidinone, 1-propanol, 2,3- butanediol, 2,5-dimethyl-2,5-hexanediol, 2-hydroxymethyl-1,3-propanediol, 2- isopropoxyethanol, 2-Methoxyethanol, 2-methyl-1,3-propanediol, 2-methyl-2,4-pentanediol, 2- propanol, 2-pyrrolidone, 3-amino-1-propanol, 3-methoxy-1,2-propanediol, 3-methoxy-1- butanol, 3-methoxy-1-propanol, 3-methyl-1,3-butanediol, Acetamide, Acetic acid, Acetone, Allantoin, Aspartame, Betaine, Citric acid, Diacetone alcohol, Diacetyl, Di-ethylene-glycol, Diethylene-glycol-diethyl-ether, Diethylene-glycol-dimethyl-ether, Diethylene-glycol- monoethyl-ether, Diethylene-glycol-monomethyl-ether, Diglycerol, Dimethyl isosorbide, Dimethyl sulfone, Dimethyl sulfoxide, Dipropylene-glycol-dimethyl-ether, Dipropylene-glycol- monomethyl-ether, Erythritol, Ethanol, Ethanolamine, Ethylene diamine, Ethylene glycol, Ethylene-glycol-diethyl-ether, Ethylene-glycol-dimethyl-ether, Ethylene-glycol-monoethyl- ether, Ethylene-glycol-monopropyl-ether, Formamide, Formic acid, Fructose, Galatose, Glucose, Glycerol, Glycerol formal, Glycine, Hydroxy-acetone, Isoascorbic acid, Isosorbide, Lactic acid, L-Arginine, Malic acid, Maltitol, Maltose, Mannitol, Meglumine, Methanol, Methyl-(S)-(-)-lactate, N,N-dimethylacetamide, N,N-dimethylformamide, N,N- dimethylpropionamide, N-Acetylethanolamine, N-acetylglycine, N-Formylmorpholine, Nicotinamide, N-methyl-2-pyrrolidone, N-methylacetamide, N-Methyldiethanolamine, N- methylformamide, Oxalic acid, PEG-200, PEG-300, PEG-400, Proline, Propionamide, Propylene-glycol-monomethyl-ether, Propylene-glycol-monopropyl-ether, Pyridine, Raffinose, Sorbitol, Sucrose, Sulfolane, Tartaric acid, Taurine, Tert-butanol, Tetraethylene glycol, Tetraethylene-glycol-dimethyl-ether, Tetrahydro-4-pyranol, Tetrahydrofurfuryl alcohol,Attorney Reference: BCHR-001WO Trehalose, Triethanolamine, Tri-ethylene-glycol, Triethylene-glycol-dimethyl-ether, Triethylene-glycol-monobutyl-ether, Triethylene-glycol-monoethyl-ether, Triethylene-glycol- monomethyl-ether, Triglycerol, Trimethylamine n-oxide, Trimethylolpropane, Tripropylene- glycol-monomethyl-ether, TRIS, Urea, and Xylitol.

[0033] In certain embodiments, the high entropy solution of the present disclosure comprises a) water at a mol fraction between 0.65 and 0.99; b) hexylene glycol at a mol fraction greater than all other non-water chemical components; c) sorbitol at a mol fraction less than the mol fraction of the hexylene glycol but greater than all other non-water chemical components; d) glucose at a mol fraction less than the mol fractions of the sorbitol and the hexylene glycol but greater than all other non-water chemical components; e) xylitol at a mol fraction less than the mol fractions of the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; f) butylene glycol at a mol fraction less than the mol fractions of the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non- water chemical components; g) propylene glycol at a mol fraction less than the mol fractions of the butylene glycol, the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; h) glycerol at a mol fraction less than the mol fractions of the propylene glycol, the butylene glycol, the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; i) ethanol at a mol fraction less than the mol fractions of the glycerol, the butylene glycol, the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; and j) ethylene glycol at a mol fraction less than all other non-water chemical components, wherein an entropy of mixing equal to or greater than 7 J / (mol K) as calculated using the entropy of mixing equation that accepts both mol fractions and volume fractions as arguments.

[0034] In some embodiments, the high entropy solution of the present disclosure further comprises trehalose, sucrose, polyethylene glycol, polyvinyl alcohol, or another polymer with molar volume greater than 150 mL / mol in an amount less than 50% by mass. In other embodiments, the high entropy solution of the present disclosure further comprises a surfactant in an amount less than 50% by mass.

[0035] In some embodiments, the non-water chemical components are biocompatible. In other embodiments, the non-water chemical components are non-toxic.Attorney Reference: BCHR-001WO

[0036] Aspects of the present disclosure include devices for preserving biological matter without freezing at sub-0°C temperatures. More specifically, the device of the present disclosure comprises a) the high entropy solution of the present disclosure, b) a biological matter in contact with the high entropy solution; and c) a cooling system configured to cool the biological matter and high entropy solution to below 0 °C, thereby preserving the biological matter without ice formation at a temperature between 0 °C and about -200 °C.

[0037] Aspects of the present disclosure further include methods for preserving a biological matter without freezing at sub-0°C temperatures. More specifically, the methods for preserving a biological matter comprise a) placing the biological matter in contact with the high entropy solution of the present disclosure; b) cooling the biological matter and the high entropy solution to a temperature lower than 0 °C; and c) storing the biological matter without ice formation at a temperature between 0 °C and about -200 °C.

[0038] In some embodiments, the biological matter is loaded with the high entropy solution by a perfusion process, a diffusion process, a submersion process, or a convection process. In certain embodiments, the perfusion process is powered by gravity, application of pressure to a transfusion bag, a pump, or a machine perfusion device.

[0039] In some embodiments, the biological matter is loaded with the high entropy solution at hypothermic temperatures between about 20 °C and about -20 °C.

[0040] In some embodiments, the biological matter is loaded with the high entropy solution and submerged in the high entropy solution.

[0041] In some embodiments, the high entropy solution and the biological matter are placed within an isochoric chamber under isochoric conditions. In certain embodiments, the isochoric chamber is hermetically sealed.

[0042] In some embodiments, the cooling system of the device uses dry ice to cool the biological matter and the high entropy solution to below 0 °C. In other embodiments, the cooling system is a freezer or a refrigerator to cool and / or store the biological matter and the high entropy solution. In other embodiments, the cooling system of the device uses liquid nitrogen to cool the biological matter and the high entropy solution to below 0 °C.

[0043] In some embodiments, the cooling system controls the rate of cooling of the biological matter and the high entropy solution, wherein the rate of cooling is between about 0.01°C / min and about 10 °C / min.Attorney Reference: BCHR-001WO

[0044] In some embodiments, the biological matter and the high entropy solution are cooled to a temperature above the glass transition temperature of the high entropy solution without ice formation. In other embodiments, the biological matter and the high entropy solution are cooled to a temperature equal to or beneath the glass transition temperature of the high entropy solution without ice formation.

[0045] In some embodiments, the biological matter and the high entropy solution are first cooled to about -80 °C or -100 °C and then stored at a temperature between about -80 °C or -100 °C and about -200 °C.

[0046] In some embodiments, the biological matter is a human cell, a human tissue, a human organ, a whole human body, a non-human cell, a non-human tissue, a non-human organ, a whole non-human body, an organism, a biomedical product, an agricultural product, a food product, a fruit, or a beverage. BRIEF DESCRIPTION OF THE FIGURES

[0047] FIGS. 1A-1D show homogeneous ice nucleation temperature as a function of differing thermodynamic parameters. FIG. 1A depicts solute mol fraction; FIG. 1B depicts size-dependent (Flory) entropy of mixing, FIG. 1C depicts ideal entropy of mixing, and FIG. 1D depicts volume fraction of water.

[0048] FIG. 2 shows visual inspection of standard ice-free cryopreservation solutions after 1 hour at -80°C on dry ice.

[0049] FIG. 3 shows visual inspection of standard ice-free cryopreservation solutions after 1 hour at -196°C in liquid nitrogen. DETAILED DESCRIPTION

[0050] Aspects of the present disclosure provide high entropy solutions which do not freeze when cooled to and warmed from subzero centigrade temperatures and methods for producing high entropy solutions. Aspects of the present disclosure further provide devices and methods for preserving unfrozen biological matter by cooling and / or storage at subzero centigrade temperatures with high entropy solutions. More specifically, aspects of the present disclosure provide high entropy solutions, devices and methods for reducing or eliminating the risk of ice formation in biological matter by placing the biological matter in contact with theAttorney Reference: BCHR-001WO high entropy solution designed to have a high entropy of mixing, and thereby an enhanced capacity to avoid the growth of ice; cooling the biological sample to a sub-zero centigrade temperature in an ice-free state, including optionally a temperature above the glass transition temperature of the solution; and storing the biological matter at the same or a lower temperature in an ice-free state.

[0051] Cryopreservation is a method for preserving a biological tissue, such as a human kidney, by cooling it to a very low temperature that is below 0 °C. However, the cooling process can result in the transition of liquid water into a crystalline state, i.e. the formation of ice. Additionally, even if the liquid water does not crystallize during the cooling step, the sample is typically warmed to room temperature before use. For example, a human kidney can be collected from a donor, cooled, stored for a period of time, warmed to room temperature, and then implanted into a recipient. However, the liquid water can also crystallize while warming the biological tissue from the low temperature back to room temperature.

[0052] Traditional cryopreservation methods can involve contacting the kidney or other biological tissue with a cryopreservation aqueous solution. After being incorporated into the kidney, the cryopreservation solution helps inhibit the transition from liquid water in the kidney to crystalline ice during cooling and warming. For example, cooling the kidney can result in the transition of liquid water into an amorphous solid state (i.e. a glass state) instead of the transition from liquid water into a crystalline ice state. As used herein, the term “freezing” refers to a phase transition into a solid crystalline state. For example, the freezing can be the transition of liquid water into a solid crystalline ice state or the transition of water in an amorphous solid state into a solid crystalline ice state. In some cases, the ice has that phase of ice Ih.

[0053] Therefore, a cryopreservation solution can help inhibit ice formation by retaining the water in an amorphous solid state (i.e. a glass state) instead of allowing it to form a crystalline ice state.

[0054] However, many traditional cryopreservation solutions are unable to prevent ice formation during certain cooling or warming procedures. In some cases, the rate of cooling or warming (e.g. in °C / min) influences the chance that crystalline ice will form. Such ice formation can damage the biological tissue, e.g. a human kidney, potentially rendering it unusable. Exemplary cooling rates and warming rates are 1 °C / min, 5 °C / min, and 10 °C / min.Attorney Reference: BCHR-001WO

[0055] The presently described high entropy solutions can be more effective than traditional cryopreservation solutions at preventing ice formation during the steps of cooling to and warming from temperatures below 0 °C. In some cases, the present high entropy solutions can completely prevent ice formation during such steps, even while descending to temperatures around -200 °C.

[0056] It has been found that the entropy of mixing (i.e. ∆Sm) influences the chance that ice will form during the cooling and warming procedures. The entropy of mixing refers to the change in entropy when mixing the water and other components with one another to form the high entropy solution. By optimizing the entropy of mixing, the ability to inhibit ice formation can also be optimized.

[0057] It was found that the entropy of mixing can be improved by increasing the total number of components in the solution. For example, an original solution might include water at a mol fraction of 0.8 and a first chemical component at a mol fraction of 0.2. However, a new solution might keep the mol fraction of water at 0.8 while splitting the remaining mol fraction between four different chemical components. In other words, each of the chemical components has a mol fraction of 0.05. Therefore, even though the total mol fraction of chemical components remained constant at 0.2, the new solution can have a better entropy of mixing than the original solution. Hence, the new solution can have an better ability to resist ice formation than the original solution.

[0058] It was also found that the entropy of mixing can be improved by using chemical components with a wide variety of molar volumes. Molar volume is the molar mass of a compound divided by its density. For example, glucose has a molar mass of 180.06 g / mol, a density of 1.56 g / ml, and a molar volume of 115.42 ml / mol. For example, an original solution might include two chemical components with similar molecules, e.g. glucose with a molar volume of 115.42 ml / mol and fructose with a molar volume of 112.60 ml / mol. However, a new solution might use glucose (115.42 ml / mol) and sucrose (215.28 ml / mol). Whereas the original solution had components with very similar molar volumes (115.42 and 112.60 ml / mol), the new solution had components with very different molar volumes (115.42 and 215.28 ml / mol). Accordingly, the new solution would have an improved entropy of mixing and a better resistance to ice formation than the original solution. In some cases, the difference in molar volumes can be quantified with statistical measurements such as weighted standard deviation or weighted coefficient of variation (CV). As used herein, the CV of a data set is the weightedAttorney Reference: BCHR-001WO sample standard deviation divided by the weighted mean. The mean and the sample standard deviation must be weighted based on the relative amounts of each chemical component. For instance, a high entropy solution could have 0.8 mol fraction water, 0.15 mol fraction glucose (mv = 115), and 0.05 mol fraction sucrose (mv = 215). The weighted average molar volume of the non-water components will be 140 ml / mol due to the equation (75% * 115) + (25% * 215). The 75% value was obtained because the glucose mol fraction of 0.015 is 75% of the total mol fraction of 0.020 (0.015 + 0.005). The weighted sample standard deviation of the two components is 50 ml / mol. Hence, the CV of the non-water components is 0.36 due to the equation 50 / 140.

[0059] It was found that the entropy of mixing is improved when using a higher average molar volume. For example, it was found that high entropy solutions with average molar volumes of about 30 ml / mol tended to have better entropies of mixing than solutions with molar volumes of about 25 ml / mol.

[0060] It was also determined that the variables influencing the entropy of mixing could be rationally controlled in a manner that provided optimal entropies of mixing.

[0061] The entropy of mixing of an aqueous solution a small amount of chemical components can be around 6 J / (mol K). However, the present high entropy solutions can have significantly higher entropies of 7 J / (mol K) or more, such as 8 or more, 9 or more, 10 or more, 11 J / (mol K) or more, 12 or more, or 13 J / (mol K) or more.

[0062] In some cases, the freezing process can be influenced by the pressure being exerted on the solution. For example, atmospheric pressure on the surface of the Earth is about 101 kPa in many cases. Thus, in some cases the behaviors described herein for the high entropy solutions are behaviors with a pressure of 101 kPa. 1. Definitions

[0063] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0064] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, betweenAttorney Reference: BCHR-001WO the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0066] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antisense oligonucleotide” includes a plurality of such oligonucleotides and reference to “the nucleic acid modification” includes reference to one or more nucleic acid modifications and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0067] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0068] As used herein, the term “Interfacial free energy” refers to a critical quantity governing the behavior in processes such as crystal nucleation and growth.

[0069] As used herein, the term “high entropy solution” refers to a solution designed to have high entropy of mixing and high stability against ice formation. The high entropy solution of the present disclosure does not freeze at subzero centigrade temperatures. As such, the highAttorney Reference: BCHR-001WO entropy solution may be used for preserving unfrozen biological matter at subzero centigrade temperature without ice formation.

[0070] As used herein, the term “mixing of entropy” refers to the change in the entropy, an extensive thermodynamic quantity, when two or more different chemical substances or components are mixed. In thermodynamics, the entropy of mixing is the increase in the total entropy when several initially separate systems of different composition, each in a thermodynamic state of internal equilibrium, are mixed without chemical reaction by the thermodynamic operation of removal of impermeable partition(s) between them, followed by a time for establishment of a new thermodynamic state of internal equilibrium in the new unpartitioned closed system.

[0071] As used herein, the term “a non-water chemical component” (NWCC) refers to a non-water carbon-based component with molar volumes between about 40 mL / mol about 400 mL / mol or a non-water organic chemical component with molar volumes between about 40 mL / mol about 400 mL / mol. For example, the non-water chemical component of the present disclosure is selected from glycols, sugar alcohols, methylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers. In certain embodiments, the non-water chemical component of the present disclosure is, but not limited to, ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di-ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol, threitol, ethanol, lactitol, isomalt, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose, or trehalose.

[0072] In some cases, the “principal non-water chemical components” (PNWCCs) can have the same properties as the NWCCs. Hence, in some cases, each component that can be used as a NWCC can also be used as an PNWCC.

[0073] A “non-principal non-water component” (N-PNWCCs) is another component in the high entropy solution that is not water and not a PNWCC. In some cases, the concentration in mol fraction of each N-PNWCC is relatively low, such as 0.01 or less, 0.005 or less, 0.001 or less, or 0.0001 or less. In some cases, the sum of the mol fractions of all N-PNWCCs is relatively low, such as 0.1 or less, 0.05 or less, 0.01 or less, or 0.001 or less.

[0074] In some cases, the N-PNWCC is a purposefully added compound that has a purpose separate from changing the freezing properties of the solution. For example, the N- PNWCC can be a salt or other electrolyte, a biologically active compound (e.g. a drug, aAttorney Reference: BCHR-001WO vasodilator, an anti-cogulant), or a polymer for providing oncotic support or membrane perfusion. In some cases, the N-PNWCC is simply a trace contaminant in the high entropy solution and not an component that was intentionally added to the solution. In some cases, the high entropy solution includes dust as an N-PNWCC. For example, dead skin cells from human workers in a laboratory can create microscopic dust that floats through the air and lands into the high entropy solution.

[0075] As used herein, the term “glycol” refers to any of a class of organic compounds belonging to the alcohol family, in the molecule of a glycol, two hydroxyl (―OH) groups are attached to different carbon atoms. Examples of a glycol include, but not limited to, ethylene glycol (CH₂OH₂, also known as 1,2-ethanediol), ethylene glycol, propylene glycol (C₃H₈O₂, also known as propane-1,2-diol), butylene glycol, hexylene glycol, di-ethylene glycol, polyethylene glycol, propanediol, and glycerol.

[0076] As used herein, the term, “a sugar alcohol”, also known as a polyol, refers to ingredients used as sweeteners and bulking agents. Examples of sugar alcohols include, but not limited to, mannitol, sorbitol, xylitol, erythritol, lactitol, isomalt, maltitol and hydrogenated starch hydrolysates (HSH).

[0077] As used herein, the term “a methylated organic compound” refers to a compound with the addition of a methyl group on a substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom.

[0078] As used herein, the term “a sugar” refers to sweet-tasting, soluble carbohydrates. Simple sugars, also called monosaccharides, include glucose, fructose, and galactose. Compound sugars, also called disaccharides or double sugars, are molecules made of two bonded monosaccharides; common examples are sucrose (glucose + fructose), lactose (glucose + galactose), and maltose (two molecules of glucose). Longer chains of monosaccharides (>2) are not regarded as sugars and are called oligosaccharides or polysaccharides.

[0079] As used herein, the term “a polyol” refers to a compound (as sorbitol or pentaerythritol) containing usually several alcoholic hydroxyl groups.

[0080] As used herein, the term “an alcohol” refers to any of a class of organic compounds characterized by one or more hydroxyl (―OH) groups attached to a carbon atom of an alkyl group (hydrocarbon chain). Alcohol may be considered as organic derivatives of water (H2O) in which one of the hydrogen atoms has been replaced by an alkyl group, typicallyAttorney Reference: BCHR-001WO represented by R in organic structures. For example, alcohol includes, not limited to, ethanol, propanol, methyl alcohol, isopropyl alcohol, alcohol, methanol, ethyl alcohol, allyl alcohol, 1- butanol, methanol, grain alcohol.

[0081] As used herein, the term “an amide”, “an organic amide” and “a carboxamide”, used interchangeably herein, refer to a functional group containing a carbonyl group linked to a nitrogen atom or any compound containing the amide functional group. As such, an amide is a compound with the general formula R−C(=O)−NR′R″, where R, R', and R″ represent any group, typically organyl groups or hydrogen atoms. The amide group is called a peptide bond when it is part of the main chain of a protein, and an isopeptide bond when it occurs in a side chain, as in asparagine and glutamine. It can be viewed as a derivative of a carboxylic acid (R−C(=O)−OH) with the hydroxyl group (−OH) replaced by an amine group (−NR′R″); or, equivalently, an acyl (alkanoyl) group (R−C(=O)−) joined to an amine group. For example, common examples of amides are formamide (H−C(=O)−NH2), acetamide (H3C−C(=O)−NH2), benzamide (C6H5−C(=O)−NH2), and dimethylformamide (H−C(=O)−N(−CH3)2). Simple examples of primary amides are ethanamide (CH3CONH2), and propanamide (C2H5CONH2). Some uncommon examples of amides are N-chloroacetamide (H3C−C(=O)−NH−Cl) and chloroformamide (Cl−C(=O)−NH2).

[0082] As used herein, the term “an amine” refers to derivatives of ammonia (NH3), wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group (these may respectively be called alkylamines and arylamines; amines in which both types of substituents are attached to one nitrogen atom may be called alkylarylamines). Examples of amines are, but not limited to, methylamine, amino acid, propylamine, allylamine, biogenic amine, trimethylamine, and aniline.

[0083] As used herein, the term “an amino acid” refers to an organic compound that contains amino and carboxylic groups. Amino acids can be classified according to the locations of the core structural functional groups (alpha- (α-), beta- (β-), gamma- (γ-) amino acids, etc.), other categories relate to polarity, ionization, and side chain group type (aliphatic, acyclic, aromatic, polar, etc.). For example, amino acids are, but not limited to, alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0084] As used herein, the term “an organic polymer” refers to a macromolecule in which the primary backbone of the molecular chain is composed, at least partially, of carbonAttorney Reference: BCHR-001WO atoms. Organic polymers have carbon covalent bonds in their chain. Polymer backbones consisting of carbon along with other atoms – such as oxygen, nitrogen, and others – are more common than polymers with only carbon comprising the backbone. Examples of organic polymers are polysaccharide, polypeptides (proteins), polyester, nylon, polycarbonate, low density polyethylene (LDPE), high density polyethylene (HDPE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), nylon, Teflon, and thermoplastic polyurethane (TPU).

[0085] As used herein, the term “University of Wisconsin solution” and “UW solution” refers to a hyperkalemic, hyperosmolar solution that prevents cell swelling, maintains stable transmembrane electrical gradients upon reperfusion, by preventing efflux of intracellular potassium during storage, and contains a variety of oxygen free radical scavengers. The University of Wisconsin solution is well known preservation solution in the art and contains lactobionate, raffinose, and hydroxyethyl starch as osmotic agents and other components including glutathione, adenosine, and the free-radical scavenger allopurinol. Examples of the University of Wisconsin solution include, but not limited to, SPS-1®.

[0086] As used herein, the term “Celsior®solution” refers to an organ preservation solution. The Celsior®solution was developed for heart preservation not only as a storage medium but also as a perfusion fluid during initial donor-heart arrest, poststorage graft reimplantation, and early reperfusion. Celsior formulation prevents cell swelling (by mannitol and lactobionate), oxygen-derived free radical injury (by reduced glutathione, histidine, and mannitol), and contracture by enhancement of energy production (glutamate) and limitation of calcium overload (high magnesium content, slight degree of acidosis).

[0087] As used herein, the term “Custodiol®HTK solution” and “Histidine-Tryptophan- Ketoglutarate solution”, interchangeably used, refers to a high-flow, low-potassium preservation solution used for organ transplantation. Custodiol®HTK solution is for perfusion and flushing of donor kidneys, liver, pancreas and heart prior to removal from the donor or immediately after removal from the donor. The solution is left in the organ vasculature during hypothermic storage and transportation (not for continuous perfusion) to the recipient. HTK solution is based on the principle of inactivating organ function by withdrawal of extracellular sodium and calcium, together with intensive buffering of the extracellular space by means of histidine / histidine hydrochloride, so as to prolong the period during which the organs will tolerate interruption ofAttorney Reference: BCHR-001WO oxygenated blood. The composition of HTK is similar to that of intracellular fluid. All of the components of HTK occur naturally in the body. The osmolarity of HTK is 310 mOsm / L.

[0088] As used herein, the term “Del Nidos cardioplegia solution (DNS)” refers to a solution for improving myocardial protection for both pediatric and adult patients undergoing cardiac surgery, which is well known in the art. DNS was originally developed for pediatric cardiac surgery as a single-dose formula. Although DNS has been studied in pediatric patients, its effectiveness in adults is still under investigation. DNS contains a base solution of Plasma- Lyte A (Baxter Healthcare Corp, Deerfield, Ill), which has an electrolyte composition similar to extracellular fluid. In the original formulation, the concentrations of electrolytes before the addition of cardioplegic additives are 140 mEq / L sodium, 5 mEq / L potassium, 3 mEq / L magnesium, 98 mEq / L chloride, 27 mEq / L acetate, and 23 mEq / L gluconate, but this has been modified by many clinicians. The formulation of both original and modified versions serves as the crystalloid component, which is mixed with blood at a ratio of 4 parts crystalloid to 1 part fully oxygenated patient whole blood.

[0089] As used herein, the term “LM5 solution” refers to a hypertonic carrier solution. The LM solution contains glucose, mannitol, alpha-lactose monohydrate, potassium chloride, potassium phosphate dibasic trihydrate, gluthathione, adenine HCl, sodium bicarbonate. Carrier solution consists of solution ingredients that are not explicit cryoprotectants. The role of the carrier solution is to provide basic support for cells at temperatures near freezing. It contains salts, osmotic agents, pH buffers, and sometimes nutritive ingredients or apoptosis inhibitors. The ingredients are usually present at near isotonic concentration (300 milliosmoles) so that cells neither shrink nor swell when held in carrier solution. Carrier solution is sometimes called “base perfusate.” The carrier solution typically used with M22 cryoprotectant solution is called LM5.

[0090] As used herein, the term “B2 solution” and “B2 preservation solution”, interchangeably used, refers to a preservation solution used for optimizing preservation within kidney and other organ perfusion models, which is well known in the art.

[0091] As used herein, the term “Phosphate buffered saline solution” and “PBS solution, interchangeably used, refers to one of the most commonly used buffers in different biological research applications for maintaining the pH of a solution and comprising a combination of water and salts. This solution consists of sodium chloride, sodium phosphate, and depending onAttorney Reference: BCHR-001WO the intended application, it can also include potassium chloride and potassium phosphate. It is a non-toxic solution used in many laboratories.

[0092] As used herein, the term “saline solution” refers to a mixture of salt and water. Normal or isotonic saline solutions contain 0.9 percent sodium chloride (salt), a concentration similar to human tears. Hypotonic saline solutions contain half as much salt, or 0.45 percent sodium chloride.

[0093] As used herein, the term “filtered seawater” refers to seawater from the ocean that has been filtered by any standard means of filtration.

[0094] As used herein, the term “cryopreservation” refers to a process that preserves organelles, cells, tissues, or any other biological constructs by cooling the samples to very low temperatures. Cryopreservation process is the use of very low temperatures to preserve structurally intact living cells and tissues for a long period of time.

[0095] As used herein, the term “optimize” refers to finding optimal values for the specific parameters of a given system to fulfill all design requirements. As used herein, the term “optimization” refers to the process of finding optimal values for the specific parameters of a given system to fulfill all design requirements.

[0096] As used herein, the term “brute force optimization method” and “brute force method”, interchangeably used, refers to general problem-solving technique and algorithmic paradigm that consists of systematically checking all possible candidates for whether or not each candidate satisfies the problem's statement. As such, the brute force optimization methods try to calculate all possible solutions and decide afterwards which one is the best. These methods are feasible only for small problems (in terms of the dimensionality of the phase space), since the number of possible states of the system increases exponentially with the number of dimensions. In the case of continuous predictor variables, the number of states is infinite. Despite these drawbacks, brute force methods do have a few benefits: the implementation of brute force algorithms is rather simple, and in the case of discrete systems, all possible states are checked. As a consequence, brute force optimization methods are often seen as reference methods for calculating the number of states, or the number of calculations necessary to find the optimum with a probability of 100%. Hence, it can be used for the estimation of the effort to solve a problem.

[0097] As used herein, the term “binary search optimization method” and “binary search algorithm”, interchangeably used, refers to search algorithm used to find the position of a targetAttorney Reference: BCHR-001WO value within a sorted array. It works by repeatedly dividing the search interval in half until the target value is found or the interval is empty. The search interval is halved by comparing the target element with the middle value of the search space.

[0098] As used herein, the term “convex optimization method” and “convex optimization”, interchangeably used, refers to a subfield of mathematical optimization that studies the problem of minimizing convex functions over convex sets (or, equivalently, maximizing concave functions over convex sets). Many classes of convex optimization problems admit polynomial-time algorithms, whereas mathematical optimization is in general NP-hard.

[0099] As used herein, the term “stochastic optimization method”, “stochastic optimization” and “stochastic optimization algorithm”, interchangeably used, refers to a collection of methods for minimizing or maximizing an objective function when randomness is present. As such, stochastic optimization algorithms provide an alternative approach that permits less optimal local decisions to be made within the search procedure that may increase the probability of the procedure locating the global optima of the objective function.

[0100] As used herein, the term “a particle swarm optimization method” and “particle swarm optimization (PSO)”, interchangeably used, refers to a computational method that optimizes a problem by iteratively trying to improve a candidate solution with regard to a given measure of quality. Particle swarm optimization is the best used to find the maximum or minimum of a function defined on a multidimensional vector space. Assume we have a function^^^^ that produces a real value from a vector parameter ^ (such as coordinate ^^, ^^ in a plane)and ^ can take on virtually any value in the space (for example, ^^^^ is the altitude and we can find one for any point on the plane), then we can apply PSO. The PSO algorithm will return the parameter ^ it found that produces the minimum ^^^^.

[0101] The terms “biological matter” and “biological sample” are interchangeably used herein.

[0102] The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to an individual organism, e.g., a mammal, including, but not limited to, murines, simians, humans, non-human primates, ungulates, felines, canines, bovines, ovines, mammalian farm animals, mammalian sport animals, and mammalian pets. In some cases, an “individual” is a human.Attorney Reference: BCHR-001WO

[0103] The use of the terms “a,” “an,” and “the,” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0104] The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments of the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the embodiments of the disclosure.

[0105] As used herein, the term “about” used in connection with an amount indicates that the amount can vary by 10% of the stated amount. For example, “about 100” means an amount of from 90-110. Where about is used in the context of a range, the “about” used in reference to the lower amount of the range means that the lower amount includes an amount that is 10% lower than the lower amount of the range, and “about” used in reference to the higher amount of the range means that the higher amount includes an amount 10% higher than the higher amount of the range. For example, from about 100 to about 1000 means that the range extends from 90 to 1100.

[0106] The term “and / or” as used herein a phrase such as “A and / or B” is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used herein a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).Attorney Reference: BCHR-001WO

[0107] It is understood that aspects and embodiments of the present disclosure described herein include “comprising,” “consisting,” and “consisting essentially of” aspects and embodiments.

[0108] It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of this disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to this disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0109] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. 2. Entropy manipulation (1) Overview

[0110] The invention of the present disclosure is based upon manipulation of the entropy of a solution by manipulation of its composition, and in particular the manipulation of the entropy of mixing. Increasing the entropy of an aqueous system increases the entropy difference between the liquid phase and ice, which in turn increases the interfacial free energy. The interfacial free energy dominates the nucleation barrier for ice in the solution, or the energetic barrier to the formation of ice. At sufficiently high liquid entropies, the interfacial free energy and the resultant nucleation barrier become sufficiently high that ice will not form on time scales relevant to cryopreservation (days to years). As such, by designing a preservation solution to have sufficiently high entropy, ice-free cryopreservation can be achieved at a temperature between 0 °C and about 0 K, including those above the glass transition temperature and beneath the range currently accessible to supercooling (approximately -20 °C beneath the equilibriumAttorney Reference: BCHR-001WO melting point). The present disclosure provides methods by which to increase the entropy of an aqueous solution via manipulation of its composition, methods by which to apply these solutions to the cryopreservation of biological matters, specific compositions of interest, and related devices.

[0111] In order to make clear the process of entropy manipulation, the relevant thermodynamic principles at play are outlined herein. The classical nucleation barrier, which ultimately dictates the homogeneous nucleation temperature in all but the most viscous systems,is given by ^^∗~ ^^∆^^, wherein ∆^ is the difference in Gibbs free energy between the solid andliquid the thermodynamic driving force for nucleation and ^ is theinterfacial free energy. In other words, the likelihood of ice formation from an aqueous liquid isinversely proportional to the nucleation barrier ^^∗~ ^^∆^^. Here, ∆^ is the square of the freeenergy difference between the solid and ^ is the interfacial free energy.

[0112] The interfacial free energy is proportional to the entropy difference between thetwo phases, the liquid and ice, as defined by Turnbull and others: ^ = !"∆^. Herein T is thetemperature, ∆^ is the entropy difference between the liquid and solid phases ∆^ = ^#^$%^& −^^'(, and c is a factor assumed constant with respect to temperature, which includes the molar surface area of the solid phase and the effect of finite thickness on the interface. In certain embodiments, c is typically taken as ~0.5. Herein, ∆^ is in actuality a function of temperature ∆^^"^, as are all extensive thermodynamic variables.

[0113] For aqueous solutions / non-pure systems, ∆^ is replaced by the chemical potential difference between ice and the liquid water in solution ∆µ because ice Ih does not incorporate solutes. However, these driving forces will be of the same general energetic scale and will thus not affect analyses herein.

[0114] For a pure material, ∆^ = ∆* − "∆^, where ∆* is the enthalpy differencebetween phases, and the nucleation barrier can thus be written as ^^∗~ ^+∆,^^Critically, itcan be easily shown that for water, ∆* is of the same energy1012 / 456^.The denominator may thus be taken to scale as ^"∆^^ , leaving ^^∗~ ^+∆,^^^+∆,^^ ~"∆^, multipliedby a constant of order 10-21for water-ice, which renders the final units of joules.Attorney Reference: BCHR-001WO

[0115] Incorporating this barrier into the nucleation rate equation 2 ~ 78^9 :&^∗;+ < andassuming that the kinetic pre-factor A can be taken as approximately rate as a function of temperature is 2^"^ ~ 78^9^∆^^. The cancellation ofterm from 2 implies that the temperature dependence of the nucleation rate is driven principally by the temperature dependence of ∆^^"^itself. The nucleation rate 2 has units ice nuclei per second per m3of aqueous sample, and the likelihood of nucleation occurring at all on experimentally relevant time scales is proportional to J.

[0116] The nucleation barrier ^^∗, the nucleation rate J, and the likelihood of ice nucleation are thus dominated by the entropy difference between the solid and liquid phases. As such, if this entropy difference can be engineered to increase, the probability of ice nucleation will accordingly decrease, and at sufficiently high entropy differences, the likelihood of ice nucleation will approach zero. Given that ice forms a chemically pure crystal, i.e. has virtually no solubility with most solutes, the entropy for the ice phase is not a function of composition. Thus, to increase the entropy difference between the liquid and ice phases, the magnitude of the entropy of the liquid phase (which will always be larger than that of the ice phase) must be increased. (2) Size dependent entropy estimation

[0117] The present disclosure provides a general method by which to increase the magnitude of the entropy of the liquid phase by manipulating the magnitude of the entropy of mixing. The entropy of mixing is one component of the total liquid entropy, and the component that dominates in solutions of interest to biological preservation. The entropy of mixing for anygeneric aqueous organic solution can be calculated as ∆^^ = −^^∑^^=> ^^ln ^^^^^ , wherein R isthe ideal gas constant, x is the mol fraction of each of nsolution (such that∑^^=> ^^ = 1), and v is the volume fraction of each of n components in solution (such that∑^^=> ^^ = 1). The mol fractions and volume fractions of each of the n components in solutionare related by the molar volumes mv of each constituent, such that ^ ?@ ^A@^ = ^A .@

[0118] For an aqueous solution, the entropy of mixing may also be written as ∆^^=−^^^^ ln ^^ + ∑^^ ^^ ln ^^^^^, wherein ^^ and ^^ are the mol and volume fractions of water,which is takenand ^^and ^^are the mol and volume fractions of each of non-water solutes, taken as the 2nd throughth components. Herein, n is an integer. This formulation estimates the entropy of mixing for a wide array of aqueous organic solutionsAttorney Reference: BCHR-001WO and provides several salient insights according to which a method to systematically enhance the entropy is devised.

[0119] In some embodiments, the magnitude of the function S appears to increase monotonically with the number of components n, for component numbers n from 2 to at least 100. In other embodiments, the minimum value of the magnitude of S for a given number of components n is reached when the molar volumes and mol fractions of each component are equal to one another, implying that increasing the differences in molar volume between the constituent components will increase the magnitude of the entropy of mixing. In other embodiments, increasing the average molar volume of the non-water components, which for a given mol fraction of water is equivalent to increasing the average molar volume of the entire solution (water included), increases the entropy of mixing. In other embodiments, for a given number of components in solution n, and for a finite set of components with an according finite set of molar volumes, the entropy of mixing equation S is convex and possesses a global extremum, which may be obtained via any chosen optimization process, including but not limited to a brute force optimization process, a stochastic optimization process, or a convex optimization process.

[0120] Based on these insights, the present disclosure provides methods for designing the composition of a solution with high or optimized entropy, and methods for cryopreserving a biological matter using the composition of a solution with high or optimized entropy. According to the basic requirements, the terms “solutions with optimized entropy,” “solutions with increased entropy,” “high entropy solutions,” and “high entropy aqueous solutions,” used interchangeably herein, refers to high entropy solutions with sufficient entropies of mixing to suppress ice formation upon cooling and warming to a temperature between 0 and about -200 °C. 3. High Entropy Solutions

[0121] Aspects of the present disclosure include a high entropy solution which does not freeze upon cooling to and warming from subzero centigrade temperatures. Aspects of the present disclosure provide a high entropy solution for preserving an unfrozen biological matter at subzero centigrade temperatures without ice formation. As used herein, “unfrozen biological matter” refers to biological matter where 1% or less by mass of the biological matter is ice. Aspects of the present disclosure provide a high entropy solution for reducing or eliminating the risk of ice formation in biological matter by placing the biological matter in contact with theAttorney Reference: BCHR-001WO high entropy solution designed to have a high entropy of mixing. As such, the high entropy solution of the present disclosure has high stability against the ice formation. Therefore, the high entropy solution of the present disclosure enhances capacity to avoid the growth of ice; cooling the biological matter to a sub-zero centigrade temperature in an ice-free state, including optionally a temperature above or below the glass transition temperature of the solution; and storing the biological matter at the same or a lower temperature in an ice-free state. As used herein, an “ice-free state” refers to a composition where about 1% or less by mass of the composition is ice. (1) Overview

[0122] In some cases, the high entropy solutions can be described as having non-water chemical components (NWCCs). In some cases, the high entropy solutions can be described as having principal non-water chemical components (PNWCCs)

[0123] In some cases, the high entropy solution comprises: a. water; b. two or more non-water chemical components with different physical or thermodynamic properties; and c. an entropy of mixing equal to or greater than 7 J / (mol K).

[0124] In some cases, the high entropy solution comprises: a. water; b. two or more non-water chemical components with different physical or thermodynamic properties; and c. an entropy of mixing equal to or greater than 7 J / (mol K), wherein there is at least one temperature between 0 °C and -200 °C wherein the solution is not actively freezing, such as at a pressure of 101 kPa, such as when being cooled at a rate of 10 °C / min.

[0125] In some cases, the high entropy solution comprises: a. water; b. two or more non-water chemical components with different physical or thermodynamic properties; and c. an entropy of mixing equal to or greater than 7 J / (mol K),Attorney Reference: BCHR-001WO wherein the high entropy solution does not freeze at a temperature between 0 °C and about -200 °C, such as at a pressure of 101 kPa, such as when being cooled at a rate of 10 °C / min.

[0126] In some cases, the high entropy solution comprises: a. water; b. two or more non-water chemical components with different physical or thermodynamic properties; and wherein there is at least one temperature between 0 °C and -200 °C wherein the solution is not actively freezing, such as at a pressure of 101 kPa, such as when being cooled at a rate of 10 °C / min.

[0127] In some cases, the high entropy solution comprises: a. water; and b. at least 10 principal non-water chemical components (PNWCCs), wherein the mol fraction of the most abundant PNWCC is not more than 10 times the mol fraction of the least abundant PNWCC.

[0128] In some cases, the high entropy solution comprises: a. water; and b. at least 10 principal non-water chemical components (PNWCCs), wherein the mol fraction of the most abundant PNWCC is not more than 10 times the mol fraction of the least abundant PNWCC, and wherein there is at least one temperature between 0 °C and -200 °C wherein the solution is not actively freezing, such as at a pressure of 101 kPa, such as when being cooled at a rate of 10 °C / min.

[0129] In some cases, the high entropy solution has a freezing point ranging from -1 °C to -273 °C, such as from -1 °C to -200 °C, such as from -1 °C to -150 °C, from -1 °C to -100 °C, from -1 °C to -80 °C, or from -1 °C to -50 °C. In some cases, the high entropy solution has freezing point ranging from -20 °C to -180 °C, such as from -20 °C to -150 °C, or from -50 °C to -150 °C. In some cases, such freezing point is due to cooling or warming at a rate of 0.1 °C / min, 1 °C / min, or 10 °C / min.Attorney Reference: BCHR-001WO

[0130] In some cases, the high entropy solution does not have a measurable freezing point, as it reaches its glass transition temperature before freezing can occur, even at very slow cooling rates (such as 0.1 °C per minute). In some cases, the high entropy solution does not have a measurable freezing point between -1 °C and -273 °C when cooled from 0 °C to -273 °C at a cooling rate of 0.1 °C per minute and a pressure of 101 kPa.

[0131] As discussed above, in some cases the solution includes 2 or more non-water chemical components (NWCCs). In some cases, the mol fraction of water in those solutions is 0.9 or less, such as 0.8 or less, or 0.7 or less. In some cases, the mol fraction of water ranges from 0.6 to 0.9, such as from 0.7 to 0.8. “Mol fraction” is used interchangeably with “mole fraction”. Mol fraction can be expressed as a decimal number (e.g. 0.8) or a percentage (e.g. 80%). In some cases, the sum of the mol fractions of the 2 or more NWCCs is 0.1 or more, such as 0.2 or more, or 0.3 or more. In some cases, the sum of the mol fraction of the 2 or more NWCCs ranges from 0.1 to 0.4, such as from 0.15 to 0.3. For example, if the NWCCs include ethanol at 0.05 mol fraction, glycerol at 0.05 mol fraction, and ethylene glycol at 0.05 mol fraction, then the sum of the mol fractions of the NWCCs is 0.15. In some cases, each NWCC has a mol fraction ranging from 0.01 to 0.10. In some cases, there are 3 or more NWCCs, such as 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more. In some cases, the average molar volume of the high entropy solution is greater than the molar volume of water, i.e. 18 ml / mol. In some cases, the average molar volume is greater than 20 ml / mol, such as greater than 25 ml / mol, greater than 30 ml / mol, greater than 40 ml / mol, greater than 50 ml / mol, greater than 60 ml / mol, or greater than 70 ml / mol. In some cases, the average molar volume ranges from 18 ml / mol to 50 ml / mol. In some cases, the entropy of mixing is at least 7 J / (mol-K), such as at least 9 J / (mol-K), such as at least 11 J / (mol-K).

[0132] As discussed above, in some cases the solution includes at least 10 principal non- water chemical components (PNWCCs). In some cases, the mol fraction of water in those solutions is 0.9 or less, such as 0.8 or less, or 0.7 or less. In some cases, the mol fraction of water ranges from 0.6 to 0.9, such as from 0.7 to 0.8. In some cases, the sum of the mol fractions of the 10 or more PNWCCs is 0.1 or more, such as 0.2 or more, or 0.3 or more. In some cases, the sum of the mol fraction of the 2 or more PNWCCs ranges from 0.1 to 0.4, such as from 0.15 to 0.3. For example, if the PNWCCs include ethanol at 0.05 mol fraction, glycerol at 0.05 mol fraction, and ethylene glycol at 0.05 mol fraction, then the sum of the mol fractions of the PNWCCs is 0.15. In some cases, each PNWCCs has a mol fraction ranging from 0.01 toAttorney Reference: BCHR-001WO 0.10. In some cases, the average molar volume of the high entropy solution is greater than the molar volume of water, i.e. 18 ml / mol. In some cases, the average molar volume is greater than 20 ml / mol, such as greater than 25 ml / mol, greater than 30 ml / mol, greater than 40 ml / mol, greater than 50 ml / mol, greater than 60 ml / mol, or greater than 70 ml / mol. In some cases, the average molar volume ranges from 18 ml / mol to 50 ml / mol. In some cases, the entropy of mixing is at least 7 J / (mol-K), such as at least 9 J / (mol-K), such as at least 11 J / (mol-K), such as at least 13 J / (mol-K).. (2) Aspects of the high entropy solutions

[0133] In some aspects, a high entropy solution comprises preserving an unfrozen biological matter at subzero centigrade temperatures without ice formation. More specifically, the high entropy solution of the present disclosure comprises water and two or more non-water chemical components with different physical or thermodynamic properties. In some embodiments, the physical properties of non-water chemical components include, but are not limited to, color, solubility, electrical or thermal conductivity, volume, odor, texture, density, melting point, and boiling point. As such, in certain embodiments, the high entropy solution of the present disclosure comprises water and two or more non-water chemical components with different color, solubility, electrical or thermal conductivity, volume, odor, texture, density, melting point, and boiling point. In some embodiments, the thermodynamic property refers to a physical quantity that is measurable, and whose value characterizes a system in thermal equilibrium. For example, different thermodynamic properties include, but are not limited to, variables like internal energy, enthalpy, entropy, Helmholtz free energy, or Gibbs free energy. As such, in certain embodiments, the high entropy solution of the present disclosure comprises water and two or more non-water chemical components with different internal energy, enthalpy, entropy, Helmholtz free energy, or Gibbs free energy.

[0134] In some embodiments, an entropy of mixing of the high entropy solution is equal to or greater than 7 J / (mol K). In other embodiments, the entropy of mixing in the high entropy solution is equal to or greater than 7 J / (mol K) and less than 30 J / (mol K). In certain embodiments, the entropy of mixing in the high entropy solution is equal to or greater than 7 J / (mol K), equal to or greater than 8 J / (mol K), equal to or greater than 9 J / (mol K), equal to or greater than 10 J / (mol K), equal to or greater than 11 J / (mol K), equal to or greater than 12 J / (mol K), equal to or greater than 13 J / (mol K), equal to or greater than 14 J / (mol K), equal to or greater than 15 J / (mol K), equal to or greater than 16 J / (mol K), equal to or greater than 17Attorney Reference: BCHR-001WO J / (mol K), equal to or greater than 18 J / (mol K), equal to or greater than 19 J / (mol K), equal to or greater than 20 J / (mol K), equal to or greater than 21 J / (mol K), equal to or greater than 22 J / (mol K), equal to or greater than 23 J / (mol K), equal to or greater than 24 J / (mol K), equal to or greater than 25 J / (mol K), equal to or greater than 26 J / (mol K), equal to or greater than 27 J / (mol K), equal to or greater than 28 J / (mol K), or equal to or greater than 29 J / (mol K).

[0135] In some embodiments, the number of non-water chemical components with different physical or thermodynamic properties is between 2 and 150. In other embodiments, the number of non-water chemical components with different physical or thermodynamic properties is between 2 and 100. In still other embodiments, the number of non-water chemical components is with different physical or thermodynamic properties between 3 and 100. In further embodiments, the number of non-water chemical components with different physical or thermodynamic properties is between 4 and 100. For example, the number of non-water chemical component with different physical or thermodynamic properties is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,110, 120, 130, 140, or 150.

[0136] In some embodiments, the entropy of mixing increases with the number of the non-water chemical components.

[0137] In some embodiments, the non-water chemical components are non-water carbon-based components with molar volumes between about 40 mL / mol about 400 mL / mol. The terms “non-water chemical components,” “non-water carbon-based components” and “non- water organic chemical components” are interchangeably used herein. In some embodiments, the non-water chemical components are non-water carbon-based components with molar volumes between about 40 mL / mol and 300 mL / mol, between about 40 mL / mol and about 250, between about 40 and about 200 mL / mol, or between about 40 mL / mol and about 150 mL / mol. In certain embodiments, the non-water chemical components are non-water carbon-based components with molar volume about 40 mL / mol, about 45 mL / mol, about 50 mL / mol, about 55 mL / mol, about 60 mL / mol, about 65 mL / mol, about 70 mL / mol, about 75 mL / mol, about 80 mL / mol, about 85 mL / mol, about 90 mL / mol, about 95 mL / mol, about 100 mL / mol, about 105 mL / mol, about 110 mL / mol, about 115 mL / mol, about 120 mL / mol, about 125 mL / mol, about 130 mL / mol, about 135 mL / mol, about 140 mL / mol, about 145 mL / mol, about 150 mL / mol,Attorney Reference: BCHR-001WO about 155 mL / mol, about 160 mL / mol, about 165 mL / mol, about 170 mL / mol, about 175 mL / mol, about 180 mL / mol, about 185 mL / mol, about 190 mL / mol, about 195 mL / mol, about 200 mL / mol, about 250 mL / mol, about 300 mL / mol, about 350 mL / mol or about 400 mL / mol. Herein, the non-water carbon-based components refer to non-water organic chemical components comprising carbon molecules.

[0138] In some embodiments, molar volumes of the non-water chemical components are different from one another, and molar volumes of the non-water chemical components are different from molar volume of the water.

[0139] In some embodiments, mol fractions of the non-water chemical components are different from one another, and mol fractions of the non-water chemical components are different from mol fraction of the water. (mol fraction of water + mol fractions of total other non-water chemical components = 1).

[0140] In some embodiments, an entropy of mixing increases when differences in molar volumes of the non-water chemical components increase. In other embodiments, an entropy of mixing increases when differences in mol fractions of the non-water chemical components increase. In some embodiments, the mol fractions of the non-water chemical components ascend in magnitude with ascending molar volumes of the non-water chemical components.

[0141] In some embodiments, mol fraction of the water is between 0.65 and 0.99 (equivalently 65 to 99 mol %). In some embodiments, mol fraction of the water is constant while mol fractions of the non-water chemical components vary to increase the entropy of mixing.

[0142] In some embodiments, when the number of the non-water chemical components is equal to or greater than 3, mol fraction of at least one of the non-water chemical components may be constant and mol fractions of at least two of the non-water chemical components may vary to increase the entropy of mixing. In other embodiments, when the number of the non- water chemical components is equal to or greater than 4, mol fraction of at least one of the non- water chemical components may be constant and mol fractions of at least three of the non-water chemical components may vary to increase the entropy of mixing. In still other embodiments, when the number of the non-water chemical components is equal to or greater than n, mol fraction of at least one of the non-water chemical components may be constant and mol fractions of at least n-1 of the non-water chemical components may vary to increase the entropy of mixing, wherein n is between 3 and 150 or between 3 to 100.Attorney Reference: BCHR-001WO

[0143] In some embodiments, the entropy of mixing in the liquid phase may be calculated by entropy of mixing equation:

[0144] ∆^^ = −^^^^ ln ^^ + ∑^^ ^^ ln^^^^^

[0145] wherein ^^and ^^are respectively mol and volume fractions of water; ^^and ^^are respectively mol and volume fractions of each of n non-water chemical components; n is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^is entropy of mixing. The entropy of mixing equation for calculating the entropy in the liquid phase is not limited to the equation set forthabove.

[0146] In some embodiments, the mol fractions and the volume fractions of the non- water chemical components, the mol fraction and the volume fraction of the water, and the number of the non-water chemical components are determined to increase the entropy of mixing. In other embodiments, the mol fractions and the volume fractions of the non-water chemical components and the mol fraction and the volume fraction of the water are necessary to find or approach a local or global extremum in the entropy of mixing equation.

[0147] In certain embodiments, the high entropy solution of the present disclosure comprises: a) water at a mol fraction between 0.65 and 0.99; b) first non-water chemical component at a mol fraction and a molar volume greater than all other non-water chemical components; c) second non-water chemical component at a mol fraction and a molar volume less than the mol fraction and the molar volume of the first non-water chemical component but greater than all other non-water chemical components; d) third non-water chemical component at a mol fraction and a molar volume less than the mol fractions and the molar volumes of the first non-water chemical component and the second non-water chemical component but greater than all other non-water chemical components, wherein an entropy of mixing is equal to or greater than 7 J / (mol K) as calculated using entropy of mixing equation that accepts both mol fractions and volume fractions as arguments.

[0148] In certain embodiments, the high entropy solution further comprises e) fourth non-water chemical component at a mol fraction and a molar volume less than the mol fractions and the molar volumes of the first non-water chemical component, the second non-water chemical component and the third non-water chemical component, but greater than all other non-water chemical components, wherein an entropy of mixing is equal to or greater than 7Attorney Reference: BCHR-001WO J / (mol K) as calculated using entropy of mixing equation that accepts both mol fractions and volume fractions as arguments.

[0149] In certain embodiments, the high entropy solution further comprises f) fifth non- water chemical component at a mol fraction and a molar volume less than the mol fractions and the molar volumes of the first non-water chemical component, the second non-water chemical component, the third non-water chemical component and the fourth non-water chemical component, but greater than all other non-water chemical components, wherein an entropy of mixing is equal to or greater than 7 J / (mol K) as calculated using entropy of mixing equation that accepts both mol fractions and volume fractions as arguments.

[0150] In certain embodiments, the high entropy solution further comprises g) sixth non- water chemical component at a mol fraction and a molar volume less than the mol fractions and the molar volumes of the first non-water chemical component, the second non-water chemical component, the third non-water chemical component, the fourth non-water chemical component and the fifth non-water chemical component, but greater than all other non-water chemical components, wherein an entropy of mixing is equal to or greater than 7 J / (mol K) as calculated using entropy of mixing equation that accepts both mol fractions and volume fractions as arguments.

[0151] In certain embodiments, the high entropy solution further comprises h) seventh non-water chemical component at a mol fraction and a molar volume less than the mol fractions and the molar volumes of the first non-water chemical component, the second non-water chemical component, the third non-water chemical component, the fourth non-water chemical component, the fifth non-water chemical component and the sixth non-water chemical component, but greater than all other non-water chemical components, wherein an entropy of mixing is equal to or greater than 7 J / (mol K) as calculated using entropy of mixing equation that accepts both mol fractions and volume fractions as arguments.

[0152] In certain embodiments, the high entropy solution of the present disclosure further comprises, but is not limited to, consecutively one or more of tenth to one-hundredth non-water chemical components, wherein each non-water chemical component has a mol fraction and a molar volume less than the mol fractions and the molar volumes of all lower numbered non-water chemical components, but greater than all other non-water chemical components. The mol fractions of the non-water chemical components ascend in magnitude with ascending molar volumes of the non-water chemical components.Attorney Reference: BCHR-001WO

[0153] In some embodiments, the number of non-water chemical components is equal to or greater than 3 and the high entropy solution comprises a) water with molar volume about 18 mL / mol; b) first group comprising one or more non-water chemical components in molar volume range between about 40 mL / mol and about 75 mL / mol; c) second group comprising non-water chemical components in molar volume range between about 75 mL / mol and about 100 mL / mol; and d) third group comprising one or more non-water chemical components in molar volume range between about 100 mL / mol and about 150 mL / mol.

[0154] In some embodiments, the molar volumes of the non-water chemical components in the first group are different from one another. In other embodiments, the molar volumes of the non-water chemical components in the second group are different from one another. In still other embodiments, the molar volumes of the non-water chemical components in the third group are different from one another. In some embodiments, the non-water chemical components in the first group are different from one another. In other embodiments, the non-water chemical components in the second group are different from one another. In still other embodiments, the non-water chemical components in the third group are different from one another.

[0155] In some embodiments, any of the non-water chemical components of the present disclosure is selected from glycols, sugar alcohols, methylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers. In other embodiments, any one of the non-water chemical components of the present disclosure is selected from ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di-ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol, threitol, ethanol, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose, and trehalose. In certain embodiments, any one of the non-water chemical components of the high entropy solution are selected from hexylene glycol, sorbitol, glucose, xylitol, butylene glycol, propylene glycol, glycerol, ethylene glycol, and ethanol.

[0156] In some embodiments, the components used as NWCCs or PNWCCs are selected from the group consisting of 1,2,4-butanetriol, 1,2,6-hexanetriol, 1,2-butanediol, 1,2-hexanediol, 1,2-pentanediol, 1,2-propanediol, 1,3-butanediol, 1,3-diaminopropane, 1,3-dihydroxyacetone, 1,3-dimethlurea, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1-2- hydroxyethyl-2-pyrrolidinone, 1-amino-2-propanol, 1-methyl-pyrrolidinone, 1-propanol, 2,3- butanediol, 2,5-dimethyl-2,5-hexanediol, 2-hydroxymethyl-1,3-propanediol, 2-Attorney Reference: BCHR-001WO isopropoxyethanol, 2-Methoxyethanol, 2-methyl-1,3-propanediol, 2-methyl-2,4-pentanediol, 2- propanol, 2-pyrrolidone, 3-amino-1-propanol, 3-methoxy-1,2-propanediol, 3-methoxy-1- butanol, 3-methoxy-1-propanol, 3-methyl-1,3-butanediol, Acetamide, Acetic acid, Acetone, Allantoin, Aspartame, Betaine, Citric acid, Diacetone alcohol, Diacetyl, Di-ethylene-glycol, Diethylene-glycol-diethyl-ether, Diethylene-glycol-dimethyl-ether, Diethylene-glycol- monoethyl-ether, Diethylene-glycol-monomethyl-ether, Diglycerol, Dimethyl isosorbide, Dimethyl sulfone, Dimethyl sulfoxide, Dipropylene-glycol-dimethyl-ether, Dipropylene-glycol- monomethyl-ether, Erythritol, Ethanol, Ethanolamine, Ethylene diamine, Ethylene glycol, Ethylene-glycol-diethyl-ether, Ethylene-glycol-dimethyl-ether, Ethylene-glycol-monoethyl- ether, Ethylene-glycol-monopropyl-ether, Formamide, Formic acid, Fructose, Galatose, Glucose, Glycerol, Glycerol formal, Glycine, Hydroxy-acetone, Isoascorbic acid, Isosorbide, Lactic acid, L-Arginine, Malic acid, Maltitol, Maltose, Mannitol, Meglumine, Methanol, Methyl-(S)-(-)-lactate, N,N-dimethylacetamide, N,N-dimethylformamide, N,N- dimethylpropionamide, N-Acetylethanolamine, N-acetylglycine, N-Formylmorpholine, Nicotinamide, N-methyl-2-pyrrolidone, N-methylacetamide, N-Methyldiethanolamine, N- methylformamide, Oxalic acid, PEG-200, PEG-300, PEG-400, Proline, Propionamide, Propylene-glycol-monomethyl-ether, Propylene-glycol-monopropyl-ether, Pyridine, Raffinose, Sorbitol, Sucrose, Sulfolane, Tartaric acid, Taurine, Tert-butanol, Tetraethylene glycol, Tetraethylene-glycol-dimethyl-ether, Tetrahydro-4-pyranol, Tetrahydrofurfuryl alcohol, Trehalose, Triethanolamine, Tri-ethylene-glycol, Triethylene-glycol-dimethyl-ether, Triethylene-glycol-monobutyl-ether, Triethylene-glycol-monoethyl-ether, Triethylene-glycol- monomethyl-ether, Triglycerol, Trimethylamine n-oxide, Trimethylolpropane, Tripropylene- glycol-monomethyl-ether, TRIS, Urea, and Xylitol.

[0157] In some embodiments, the water is replaced by a saline solution, a hypothermic organ preservation solution, a carrier solution, or a base solution containing electrolytes. For example, the saline solution is selected from the group consisting of University of Wisconsin solution (UW solution), Celsior®solution, Custodiol®HTK solution, Del Nidos cardioplegia solution (DNS), LM5 solution, B2 solution, phosphate buffered saline solution (PBS solution), filtered seawater, and another saline solution. Examples of the University of Wisconsin solution include, but not limited to, SPS-1®. Examples of the saline solution normal or isotonic saline solutions containing 0.9 percent sodium chloride (salt) and hypotonic saline solutions containing half as much salt, or 0.45 percent sodium chloride.Attorney Reference: BCHR-001WO

[0158] In certain embodiments, the high entropy solution of the present disclosure comprises a) water at a mol fraction between about 0.65 and about 0.99; b) hexylene glycol at a mol fraction greater than all other non-water chemical components; c) sorbitol at a mol fraction less than the mol fraction of the hexylene glycol but greater than all other non-water chemical components; d) glucose at a mol fraction less than the mol fractions of the sorbitol and the hexylene glycol but greater than all other non-water chemical components; e) xylitol at a mol fraction less than the mol fractions of the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; f) butylene glycol at a mol fraction less than the mol fractions of the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; g) propylene glycol at a mol fraction less than the mol fractions of the butylene glycol, the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; h) glycerol at a mol fraction less than the mol fractions of the propylene glycol, the butylene glycol, the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; i) ethanol at a mol fraction less than the mol fractions of the glycerol, the butylene glycol, the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; and j) ethylene glycol at a mol fraction less than all other non- water chemical components, wherein an entropy of mixing equal to or greater than 7 J / (mol K) as calculated using the entropy of mixing equation that accepts both mol fractions and volume fractions as arguments.

[0159] In some embodiments, the high entropy solution of the present disclosure further comprises trehalose, sucrose, polyethylene glycol, polyvinyl alcohol, or another polymer with molar volume greater than 150 mL / mol in an amount less than 50% by mass. In other embodiments, the high entropy solution of the present disclosure further comprises a surfactant in an amount less than 50% by mass.

[0160] In some embodiments, the non-water chemical components are biocompatible. In other embodiments, the non-water chemical components are non-toxic.

[0161] In some embodiments, the high entropy solution of the present disclosure does not freeze at a temperature between 0 °C and about -273 °C (around 0 K) or between 0 °C and about -200 °C. In certain embodiments, the high entropy solution of the present disclosure does not freeze at a temperature between 0 °C and about -200 °C, between 0 °C and about -80 °C,Attorney Reference: BCHR-001WO between about -80 °C and about -100 °C, between about -100 °C and about -150 °C, between about -150 °C and about -200 °C, or between about -200 °C and about -273 °C. 4. Methods for Producing High Entropy Solutions

[0162] Aspects of the present disclosure include a method of producing a high entropy solution which does not freeze at subzero centigrade temperatures. Aspects of the present disclosure provide a method for producing a high entropy solution for preserving an unfrozen biological matter at subzero centigrade temperatures without ice formation. Aspects of the present disclosure provide method for producing a high entropy solution for reducing or eliminating the risk of ice formation in biological matter by placing the biological matter in contact with the high entropy solution designed to have a high entropy of mixing. The methods of the present disclosure entail manipulating the entropy of mixing of a solution to ensure that it has sufficient thermodynamic stability so as to avoid or reduce ice growth upon cooling and warming at desired cooling and warming rates. Therefore, the high entropy solution produced by the method of the present disclosure enhances capacity to avoid the growth of ice; cooling the biological matter to a sub-zero centigrade temperature in an ice-free state, including optionally a temperature above the glass transition temperature of the solution; and storing the biological matter at the same or a lower temperature in an ice-free state.

[0163] In some aspects, the present disclosure provides a method for producing a high entropy solution comprising: a) selecting two or more non-water chemical components with different physical or thermodynamic properties and water; b) determining each amount of the non-water chemical components and amount of the water to increase an entropy of mixing; and c) mixing the non-water chemical components and the water with the determined amounts to produce the high entropy solution, wherein the high entropy solution does not freeze at a temperature between 0 °C and about -200 °C.

[0164] In some embodiments, the entropy of mixing in the high entropy solution is equal to or greater than 7 J / (mol K). In other embodiments, the entropy of mixing in the high entropy solution is equal to or greater than 7 J / (mol K) and less than 30 J / (mol K). In certain embodiments, the entropy of mixing in the high entropy solution is equal to or greater than 7 J / (mol K), equal to or greater than 8 J / (mol K), equal to or greater than 9 J / (mol K), equal to or greater than 10 J / (mol K), equal to or greater than 11 J / (mol K), equal to or greater than 12 J / (mol K), equal to or greater than 13 J / (mol K), equal to or greater than 14 J / (mol K), equal toAttorney Reference: BCHR-001WO or greater than 15 J / (mol K), equal to or greater than 16 J / (mol K), equal to or greater than 17 J / (mol K), equal to or greater than 18 J / (mol K), equal to or greater than 19 J / (mol K), equal to or greater than 20 J / (mol K), equal to or greater than 21 J / (mol K), equal to or greater than 22 J / (mol K), equal to or greater than 23 J / (mol K), equal to or greater than 24 J / (mol K), equal to or greater than 25 J / (mol K), equal to or greater than 26 J / (mol K), equal to or greater than 27 J / (mol K), equal to or greater than 28 J / (mol K), or equal to or greater than 29 J / (mol K). (1) Step a)

[0165] In some embodiments, the a) comprising selecting a number of non-water chemical components with different physical or thermodynamic properties includes a-1) determining the number of non-water chemical components to produce the high entropy solution and then a-2) selecting specific non-water chemical components with different physical or thermodynamic properties and water.

[0166] In some embodiments, a number of non-water chemical components with different physical or thermodynamic properties are selected. In some embodiments, the physical properties of non-water chemical components include, but are not limited to, color, solubility, electrical or thermal conductivity, volume, odor, texture, density, melting point, and boiling point. As such, in certain embodiments, the high entropy solution of the present disclosure comprises water and two or more non-water chemical components with different color, solubility, electrical or thermal conductivity, volume, odor, texture, density, melting point, and boiling point. In some embodiments, the thermodynamic property refers to a physical quantity that is measurable, and whose value characterizes a system in thermal equilibrium. For example, different thermodynamic properties include, but are not limited to, variables like internal energy, enthalpy, entropy, Helmholtz free energy, or Gibbs free energy. As such, in certain embodiments, the high entropy solution of the present disclosure comprises water and two or more non-water chemical components with different internal energy, enthalpy, entropy, Helmholtz free energy, or Gibbs free energy.

[0167] In some embodiments, the number of non-water chemical components with different physical or thermodynamic properties is between 2 and 150. In other embodiments, the number of non-water chemical components with different physical or thermodynamic properties is between 2 and 100. In still other embodiments, the number of non-water chemical components is with different physical or thermodynamic properties between 3 and 100. In further embodiments, the number of non-water chemical components with different physical orAttorney Reference: BCHR-001WO thermodynamic properties is between 4 and 100. For example, the number of non-water chemical component with different physical or thermodynamic properties is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, or 150.

[0168] In some embodiments, the entropy of mixing increases with the number of the non-water chemical components.

[0169] In some embodiments, the non-water chemical components are non-water carbon-based components with molar volumes between about 40 mL / mol about 400 mL / mol. The terms “non-water chemical components,” “non-water carbon-based components” and “non- water organic chemical components” are interchangeably used herein. In some embodiments, the non-water chemical components are non-water carbon-based components with molar volumes between about 40 mL / mol and 300 mL / mol, between about 40 mL / mol and 250, between about 40 and 200 mL / mol, or between about 40 mL / mol and 150 mL / mol. In certain embodiments, the non-water chemical components are non-water carbon-based components with molar volume about 40 mL / mol, about 45 mL / mol, about 50 mL / mol, about 55 mL / mol, about 60 mL / mol, about 65 mL / mol, about 70 mL / mol, about 75 mL / mol, about 80 mL / mol, about 85 mL / mol, about 90 mL / mol, about 95 mL / mol, about 100 mL / mol, about 105 mL / mol, about 110 mL / mol, about 115 mL / mol, about 120 mL / mol, about 125 mL / mol, about 130 mL / mol, about 135 mL / mol, about 140 mL / mol, about 145 mL / mol, about 150 mL / mol, about 155 mL / mol, about 160 mL / mol, about 165 mL / mol, about 170 mL / mol, about 175 mL / mol, about 180 mL / mol, about 185 mL / mol, about 190 mL / mol, about 195 mL / mol, about 200 mL / mol, about 250 mL / mol, about 300 mL / mol, about 350 mL / mol or about 400 mL / mol. Herein, the non-water carbon-based components refer to non-water organic chemical components comprising carbon molecules.

[0170] In some embodiments, any of the non-water chemical components of the present disclosure is selected from glycols, sugar alcohols, methylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers. In other embodiments, any of the non-water chemical components of the present disclosure is selected from ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di-ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol,Attorney Reference: BCHR-001WO threitol, ethanol, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose, and trehalose.

[0171] In some embodiments, the non-water chemical components are biocompatible. In other embodiments, the non-water chemical components are non-toxic.

[0172] In some embodiments, the water is replaced by a saline solution, a hypothermic organ preservation solution, a carrier solution, or a base solution containing electrolytes. For example, the saline solution is selected from the group consisting of University of Wisconsin solution, Celsior®solution, Custodiol®HTK solution, Del Nidos solution, LM5 solution, B2 solution, phosphate buffered saline solution (PBS solution), filtered seawater, and another saline solution. Examples of the University of Wisconsin solution include, but not limited to, SPS-1®. Examples of the saline solution normal or isotonic saline solutions containing 0.9 percent sodium chloride (salt) and hypotonic saline solutions containing half as much salt, or 0.45 percent sodium chloride. (2) Step b) and c)

[0173] The method for producing a high entropy solution further comprises: b) determining each amount of the non-water chemical components and amount of the water to increase an entropy of mixing; and c) mixing the non-water chemical components and the water with the determined amounts to produce the high entropy solution, wherein the high entropy solution does not freeze at a temperature between 0 °C and about -200 °C. In some embodiments, the b) determining each amount of the non-water chemical components and amount of the water to increase an entropy of mixing comprises b-1) determining molar volumes of the non-water chemical components and water, and b-2) determining mol fractions of the non-water chemical components and water.

[0174] In other embodiments, this step is performed by optimizing an entropy of mixing equation so as to identify the amounts of two or more of the non-water chemical components necessary to find or approach a local or global extremum in the entropy of mixing. In certain embodiments, optimization value of the entropy of mixing is equal to or greater than 7 J / (mol K). In certain embodiments, optimization value of the entropy of mixing is equal to or greater than 7 J / (mol K) and less than 30 J / (mol K).

[0175] In some embodiments, molar volumes of the non-water chemical components are different from one another, and molar volumes of the non-water chemical components are different from molar volume of the water.Attorney Reference: BCHR-001WO

[0176] In some embodiments, mol fractions of the non-water chemical components are different from one another, and mol fractions of the non-water chemical components are different from mol fraction of the water. (mol fraction of water + mol fractions of total other non-water chemical components = 1).

[0177] In some embodiments, an entropy of mixing increases when differences in molar volumes of the non-water chemical components increase. In other embodiments, an entropy of mixing increases when differences in mol fractions of the non-water chemical components increase. In some embodiments, the mol fractions of the non-water chemical components ascend in magnitude with ascending molar volumes of the non-water chemical components.

[0178] In some embodiments, mol fraction of the water is between 0.65 and 0.99 (equivalently 65 to 99 mol %). In some embodiments, mol fraction of the water is constant while mol fractions of the non-water chemical components vary to increase the entropy of mixing.

[0179] In some embodiments, when equal to or greater than 3 of the non-water chemical components are selected, mol fraction of at least one of the non-water chemical components may be constant and mol fractions of at least two of the non-water chemical components may vary to increase the entropy of mixing. In other embodiments, when equal to or greater than 4 of the non-water chemical components are selected, mol fraction of at least one of the non-water chemical components may be constant and mol fractions of at least three of the non-water chemical components may vary to increase the entropy of mixing. In still other embodiments, when equal to or greater than n of the non-water chemical components are selected, mol fraction of at least one of the non-water chemical components may be constant and mol fractions of at least n-1 of the non-water chemical components may vary to increase the entropy of mixing, wherein n is between 3 and 150 or between 3 to 100.

[0180] In some embodiments, the entropy of mixing in the liquid phase may be calculated by entropy of mixing equation:

[0181] ∆^^ = −^^^^ ln ^^ + ∑^^ ^^ ln^^^^^mol and volume fractions of water; ^^and ^^are respectively mol and volume fractions of each of n non-water chemical components; n is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^is entropy of mixing. The entropy of mixing equation forAttorney Reference: BCHR-001WO calculating the entropy of mixing of the liquid phase is not limited to the equation set forth above.

[0183] In some embodiments, the mol fractions and the volume fractions of the non- water chemical components, the mol fraction and the volume fraction of the water, and the number of the non-water chemical components are determined to increase the entropy of mixing. In other embodiments, the mol fractions and the volume fractions of the non-water chemical components and the mol fraction and the volume fraction of the water are necessary to find or approach a local or global extremum in the entropy of mixing equation.

[0184] In some embodiments, i) a mol fraction of the water is determined to be between 0.65 and 0.99; ii) a mol fraction and a molar volume of first non-water chemical component is determined to be greater than all other non-water chemical components; iii) a mol fraction and a molar volume of second non-water chemical component is determined to be less than the mol fraction and the molar volume of the first non-water chemical component but greater than all other non-water chemical components; and iv) a mol fraction and a molar volume of third non- water chemical component is determined to be less than the mol fractions and the molar volumes of the first non-water chemical component and the second non-water chemical component but greater than all other non-water chemical components; v) a mol fraction and a molar volume of fourth non-water chemical component is determined to be less than the mol fractions and the molar volumes of the first non-water chemical component, the second non- water chemical component and the third non-water chemical component, but greater than all other non-water chemical components; vi) a mol fraction and a molar volume of fifth non-water chemical component is determined to be less than the mol fractions and the molar volumes of the first non-water chemical component, the second non-water chemical component, the third non-water chemical component and the fourth non-water chemical component, but greater than all other non-water chemical components; vii) a mol fraction and a molar volume of sixth non- water chemical component is determined to be less than the mol fractions and the molar volumes of the first non-water chemical component, the second non-water chemical component, the third non-water chemical component, the fourth non-water chemical component and the fifth non-water chemical component, but greater than all other non-water chemical components; and viii) a mol fraction and a molar volume of seventh non-water chemical component are determined to be less than the mol fractions and the molar volumes of the first non-water chemical component, the second non-water chemical component, the third non-water chemicalAttorney Reference: BCHR-001WO component, the fourth non-water chemical component, the fifth non-water chemical component and the sixth non-water chemical component, but greater than all other non-water chemical components, wherein an entropy of mixing is equal to or greater than 7 J / (mol K) as calculated using entropy of mixing equation that accepts both mol fractions and volume fractions as arguments.

[0185] In certain embodiments, selecting first to nth non-water chemical components and determining mol fractions and molar volumes of the non-water chemical components, wherein the mol fractions and the molar volumes of the water chemical components are determined to be less than the mol fractions and the molar volumes of all lower numbered non- water chemical components, but greater than all other non-water chemical components, wherein n is between 3 to 150 or between 3 to 100. The mol fractions of the non-water chemical components ascend in magnitude with ascending molar volumes of the non-water chemical components.

[0186] In some embodiments, when the number of non-water chemical components is selected to be equal to or greater than 3, i) molar volume of water is determined to be about 18 mL / mol; ii) one or more non-water chemical components in first group are determined to be in molar volume range between about 40 mL / mol and about 75 mL / mol; iii) one or more non- water chemical components in second group are determined to be in molar volume range between about 75 mL / mol and about 100 mL / mol; and iv) one or more non-water chemical components in third group are determined to be in molar volume range between about 100 mL / mol and about 150 mL / mol.

[0187] In some embodiments, the molar volumes of the non-water chemical components in the first group are different from one another. In other embodiments, the molar volumes of the non-water chemical components in the second group are different from one another. In still other embodiments, the molar volumes of the non-water chemical components in the third group are different from one another. In some embodiments, the non-water chemical components in the first group are different from one another. In other embodiments, the non-water chemical components in the second group are different from one another. In still other embodiments, the non-water chemical components in the third group are different from one another.

[0188] In some embodiments, the entropy of mixing in the liquid phase may be calculated by entropy of mixing equation:

[0189] ∆^^ = −^^^^ ln ^^ + ∑^^ ^^ ln^^^^^Attorney Reference: BCHR-001WO

[0190] wherein ^^and ^^are respectively mol and volume fractions of water; ^^and ^^are respectively mol and volume fractions of each of n non-water chemical components; n is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^is entropy of mixing. The entropy of mixing equation for calculating total liquid entropy is not limited to the equation set forth above.

[0191] In some embodiments, the mol fractions and the volume fractions of the non- water chemical components, the mol fraction and the volume fraction of the water, and the number of the non-water chemical components are determined to increase the entropy of mixing. In other embodiments, the mol fractions and the volume fractions of the non-water chemical components and the mol fraction and the volume fraction of the water are necessary to find or approach a local or global extremum in the entropy of mixing equation.

[0192] In some embodiments, optimization of the entropy of mixing equation is performed by using a computational optimization method. Herein, optimizing the entropy of mixing refers to finding optimal values for the specific parameters of an entropy of mixing equation system to fulfill all design requirements, wherein the optimal value of the entropy of mixing is equal to or greater than 7 J / (mol K) and wherein the specific parameters of an entropy of mixing equation are mol fraction and volume fraction of non-water chemical components and water.

[0193] In certain embodiments, the computational optimization method includes, but not limited to, a brute force optimization method, a binary search optimization method, a convex optimization method, a stochastic optimization method, or a particle swarm optimization method. The brute force optimization method, the binary search optimization method, the convex optimization method, the stochastic optimization method, or the particle swarm optimization method are well known optimization algorithms or optimization methods in the art.

[0194] In some aspects, the present disclosure provides a method for producing a high entropy solution which does not freeze at sub-0°C temperatures comprise of the following steps:

[0195] a) selecting a number of non-water chemical components to include in solution, wherein the number is between 4 and about 100;

[0196] b) selecting individual non-water chemical components with molar volumes significantly different from one another, and from water, such that the solution comprises:

[0197] i) water with molar volume about 18 mL / mol;Attorney Reference: BCHR-001WO

[0198] ii) at least one non-water chemical component with molar volume in the range between about 40 mL / mol and about 75 mL / mol;

[0199] iii) at least one non-water chemical component with molar volume in the range between about 75 mL / mol and about 100 mL / mol; and

[0200] iv) at least one non-water chemical component with molar volume in the range between about 100 mL / mol and about 150 mL / mol;

[0201] c) selecting a total mol fraction of water within the range wherein both entropic effects dominate and tolerable degrees of toxicity are plausible, taken as 0.65 to 0.99 (equivalently 65 to 99 mol %), wherein the mol fraction of water dictates the mol fraction of total other components, as mol fraction other components + mol fraction water = 1;

[0202] d) for the selected group of non-water chemical components, having an according group of molar volumes, and the selected mol fraction of water, minimizing theentropy equation ∆^^ = −^^^^ ln ^^ + ∑^^= ^^ ln ^^^^^, solving for the mol fractions ^^ ofeach non-water component; and

[0203] e) mixing the solution according to the calculated mol fractions, thereby yielding an aqueous solution of optimized entropy, which will optimally prevent ice formation.

[0204] In some embodiments of the methods above, the absolute value (or equivalently the magnitude) of the entropy of mixing for the solution calculated according to the entropyequation ∆^^ = −^^^^ ln ^^ + ∑^^= ^^ ln ^^^^^, will equal or exceed 7 J / mol K. Suchsolutions are high entropy solutions.

[0205] In some embodiments of the methods above, the water in solution, as evaluated exactly or approximately on a mass or volume basis from the calculated mol fraction, may be replaced with a physiological saline solution, clinical organ preservation solution, or other carrier solution.

[0206] In some embodiments, the high entropy solution produced by the method of the present disclosure does not freeze at a temperature between about 0 °C and about -273 °C (around 0 K) or between 0 °C and about -200 °C. In certain embodiments, the high entropy solution produced by the method of the present disclosure does not freeze at any temperature between 0 °C and about -200 °C, between 0 °C and about -80 °C, between about -80 °C and about -100 °C, between about -100 °C and about -150 °C, between about -150 °C and about - 200 °C, or between about -200 °C and about -273 °C.Attorney Reference: BCHR-001WO 5. Devices for Preserving Biological Matters

[0207] Aspects of the present disclosure provide a device for preserving a biological matter at subzero centigrade temperatures without ice formation. More particularly, the device of the present disclosure comprises the high entropy solution of the present disclosure; a biological matter in contact with the high entropy solution; and a cooling system configured to cool the biological matter and high entropy solution to below 0°C. Aspects of the present disclosure provide a device for reducing or eliminating the risk of ice formation in biological matter by placing the biological matter in contact with the high entropy solution designed to have a high entropy of mixing while preserving the biological matter at subzero centigrade temperature. Therefore, the device comprising high entropy solution of the present disclosure enhances preservation capacity to avoid the growth of ice; cooling the biological matter to a sub-zero centigrade temperature in an ice-free state, including optionally a temperature above the glass transition temperature of the solution; and storing the biological matter at the same or a lower temperature in an ice-free state.

[0208] High entropy solutions of the present disclosure are precisely described in the above sections (“3. High Entropy Solutions” and “4. Methods for Producing High Entropy Solutions”) and are incorporated into this section.

[0209] In some embodiments, the device of the present disclosure further comprises a chamber. In some embodiments, the high entropy solution and the biological matter are placed within the chamber. In certain embodiments, the chamber is rigid and volume constant container. In certain embodiments, the chamber is an air-free container. In certain embodiments, the chamber is an isochoric chamber. In certain embodiments, the isochoric chamber is hermetically sealed. In certain embodiments, the high entropy solution and the biological matter are placed within the isochoric chamber and cooled under isochoric conditions.

[0210] In some embodiments, the biological matter is loaded with the high entropy solution by a perfusion process. In certain embodiments, the perfusion process is powered by gravity. In certain embodiments, the perfusion process is powered by application of pressure to a transfusion bag. In certain embodiments, the perfusion process is powered by a pump. In certain embodiments, the perfusion process is powered by a machine perfusion device.

[0211] In certain embodiments, the biological matter is loaded with the high entropy solution by a diffusion process.Attorney Reference: BCHR-001WO

[0212] In certain embodiments, the biological matter is loaded with the high entropy solution by a submersion process. In other embodiments, the biological matter is loaded with the high entropy solution and submerged in the high entropy solution.

[0213] In certain embodiments, the biological matter is loaded with the high entropy solution by a convection process.

[0214] In some embodiments, the biological matter is loaded with the high entropy solution at hypothermic temperatures between about 35 °C and about -20 °C, between about 30 °C and about -20 °C, between about 25 °C and about -20 °C, or between about 20 °C and about -20 °C. In certain embodiments, the biological matter is loaded with the high entropy solution at hypothermic temperatures between about 20 °C and about -20 °C.

[0215] In some embodiments, the cooling system of the device uses dry ice to cool the biological matter and the high entropy solution to below 0 °C. For example, the cooling system uses dry ice to cool the biological matter and the high entropy solution to about -20 °C, about -30 °C, about -40 °C, about -50 °C ̧about -60 °C, about -70 °C¸ about -80 °C, about -90 °C, orabout -100 °C. As such, the biological matter and the high entropy solution may be cooled and / or stored by using dry ice.

[0216] In some embodiments, the cooling system of the device uses liquid nitrogen to cool the biological matter and the high entropy solution to below 0 °C. For example, the cooling system uses liquid nitrogen to cool the biological matter and the high entropy solution to about -20 °C, about -30 °C, about -40 °C, about -50 °C ̧about -60 °C, about -70 °C ̧about -80 °C,about -90 °C¸ about -100 °C, about -110 °C ̧about -120 °C, about -130 °C¸ about -140 °C,about -150 °C, about -160 °C, about -170 °C, about -180 °C, about -190 °C, or about -200 °C. As such, the biological matter and the high entropy solution may be cooled and / or stored by using liquid nitrogen.

[0217] In some embodiments, the cooling system of the device may be a freezer or a refrigerator to cool and / or store the biological matter and the high entropy solution. The freezer or the refrigerator enables to cool the biological matter and the high entropy solution to below0 °C, such as about -20 °C, about -30 °C, about -40 °C, about -50 °C ̧about -60 °C, about -70 °C ̧about -80 °C, about -90 °C ̧about -100 °C, about -110 °C¸ about -120 °C, about -130 °C ̧about -140 °C, about -150 °C, about -160 °C, about -170 °C, about -180 °C, about -190 °C, or about -200 °C.Attorney Reference: BCHR-001WO

[0218] In some embodiments, the cooling system of the device controls the rate of cooling of the biological matter and the high entropy solution. In certain embodiments, the rate of cooling is between about 0.01 °C / min and about 10 °C / min, between about 0.05 °C / min and about 10 °C / min, between about 0.05 °C / min and about 5 °C / min, between about 0.1 °C / min and about 10 °C / min, between about 0.1 °C / min and about 9 °C / min, between about 0.1 °C / min and about 8 °C / min, between about 0.1 °C / min and about 7 °C / min, between about 0.1 °C / min and about 6 °C / min, between about 0.1 °C / min and about 5 °C / min, between about 0.5 °C / min and about 10 °C / min, between about 0.5 °C / min and about 5 °C / min, between about 0.5 °C / min and about 4 °C / min, between about 0.5 °C / min and about 3 °C / min, between about 0.5 °C / min and about 2 °C / min, between about 0.5 °C / min and about 1 °C / min. In certain embodiments, the biological matter is cooled at a rate between about 0.1 °C / min and about 5 °C / min or between about 0.5 °C / min and about 1 °C / min.

[0219] In some embodiments, the biological matter and the high entropy solution are cooled to a temperature above the glass transition temperatures of the high entropy solution without ice formation. In other embodiments, the biological matter and the high entropy solution are cooled to a temperature equal to or beneath the glass transition temperature of the high entropy solution without the ice formation. The general range of glass transition temperatures exhibited by standard preservation solutions of relevance to biological systems (i.e. of minimal toxicity) is approximately -100 °C to -140 °C. The general range of glass transition temperatures exhibited by high entropy solutions of relevance to biological systems (i.e. of minimal toxicity) is approximately -65 °C to -100 °C.

[0220] In some embodiments, the biological matter and the high entropy solution are first cooled to a temperature between about -50 and about -90 °C, about -60 °C and about - 90 °C, about -70 °C and about -90 °C or about -80 °C and about -90 °C. In certain embodiments, the biological matter and the high entropy solution are first cooled to a temperature about -50 °C, about -55 °C, about -60 °C, about -65 °C, about -70 °C, about -71 °C, about -72 °C, about -73 °C, about -74 °C, about -75 °C, about -76 °C, about -77 °C, about - 78 °C, about -79 °C, about -80 °C, about -81 °C, about -82 °C, about -83 °C, about -84 °C, about -85 °C, about -90 °C.

[0221] In some embodiments, after the biological matter and the high entropy solution are first cooled to the above temperature, and then the biological matter and the high entropy solution are stored at a temperature between about -60 °C and about -200 °C, between about -Attorney Reference: BCHR-001WO 65 °C and about -200 °C, between about -70 °C and about -200 °C, between about -75 °C and about -200 °C, between about -80 °C and about -200 °C, between about -85 °C and about - 200 °C, between about -90 °C and about -200 °C, between about -95 °C and about -200 °C, or between about -100 °C and about -200 °C. In certain embodiments, the biological matter and the high entropy solution are stored at a temperature about -80 °C, about -85 °C, about -90 °C, about -95 °C, about -100 °C, about -105 °C, about -110 °C, about -115 °C, about -120 °C, about -125 °C, about -130 °C, about -135 °C, about -140 °C, about -145 °C, about -150 °C, about -155 °C, about -160 °C, about -165 °C, about -170 °C, about -175 °C ̧about -180 °C, about -185 °C, about -190 °C, about -195 °C, or about -200 °C.

[0222] In some embodiments, the biological matter is a human cell, a human tissue, a human organ, a whole human body. In other embodiments, the biological matter is a non-human cell, a non-human tissue, a non-human organ, a whole non-human body, an organism. In certain embodiments, the non-human cell is, but not limited to, a plant cell, an animal cell, or a microbial cell.

[0223] In certain embodiments, the human cell or non-human cell is, but not limited to, a blood cell, a neuron cell, a stem cell, a bone cell, a muscle cell, a sperm cell, a female egg cell, a fat cell, a nerve cell. In certain embodiments, the human cell or tissue or non-human cell or tissue is, but not limited to, obtained from a kidney, a liver, a heart, a lung, a brain, a spleen, a stomach, a pancreas, a urinary bladder, a gallbladder, bile duct, duodenum, colon, large intestine, small intestine, anus, muscles, arteries, a lymph node, a limb, or skin. In certain embodiments, the human organ or non-human organ is but not limited to, obtained from a kidney, a liver, a heart, a lung, a brain, a spleen, a stomach, a pancreas, a urinary bladder, a gallbladder, bile duct, duodenum, colon, large intestine, small intestine, anus, muscles, arteries, a lymph node, a limb.

[0224] In some embodiments, the whole human body is preserved at a temperature less than 0 °C to reduce or suspend metabolism. In other embodiments, the whole non-human body is preserved at a temperature less than 0 °C to reduce or suspend metabolism.

[0225] In some embodiments, the biological matter is a biomedical product. In some embodiments, the biomedical product is, but not limited to, artificial organs, drugs, and medicines. In certain embodiments, medical instruments can be sterilized by the method of the present disclosure during long term preservation.Attorney Reference: BCHR-001WO

[0226] In some embodiments, the biological matter is an agricultural product, a food product, a fruit, or a beverage. In certain embodiments, the fruit includes a whole fruit, a fresh- cut fruit, or fruit arils. In other embodiments, the fruit is, but not limited to, an apple, a pear, a pomegranate, an orange, a grapefruit, a mandarin, a lime, a lemon, a nectarine, an apricot, a peach, a plum, a banana, a mango, a strawberry, a raspberry, a blueberry, a kiwifruit, a passionfruit, watermelons, a melon, a honeydew melon, a cantaloupe, a tomato, or an avocado. In certain embodiments, the agricultural product includes, but is not limited to, vegetables, legumes, or grains. In other embodiments, the vegetables are, not limited to, lettuce, spinach, beet, cabbage, cauliflower, brussels sprout, broccoli, pumpkin, cucumber, zucchini, potato, sweet potato, yam, celery, asparagus, onion, garlic, shallot, or carrot. In some embodiments, the legumes are, but not limited to, tofu, soybeans, chickpea flour, lentil flour, soy flour, haricot beans, red kidney beans, chickpeas, lentils, green peas, green beans, butter beans, or snow peas.

[0227] In some embodiments, the beverage includes fruit juice, vegetable juice, or combination thereof. In other embodiments, the beverage includes milk. In certain embodiments, the fruit juice is, but not limited to, orange juice, apple juice, grape juice, tomato juice, pomegranate juice, or mixed fruit juice. In certain embodiments, the vegetable juice is, but not limited to, carrot juice, kale juice, spinach juice, or mixed vegetable juice. In some embodiments, the food product includes dairy product or milk product.

[0228] In some embodiments, liquid biological matter, such as fruit juice or vegetable juice, is collected in an impermeable but flexible container. For example, liquid biological matter is collected in a polyethylene sterile bag. The bag is then sealed with negligible headspace and placed inside the device chamber. 6. Methods for Preserving Biological Matters

[0229] Aspects of the present disclosure provide a method for preserving a biological matter at subzero centigrade temperatures without ice formation. More particularly, the method of the present disclosure comprises a) placing the biological matter in contact with the high entropy solution of the present disclosure; b) cooling the biological matter and the high entropy solution to a temperature lower than 0°C; and c) storing the biological matter without ice formation, or without significant ice formation, at a temperature between 0 °C and about -200 °C. As used herein, the term “significant ice formation” means that about 1% or less by mass is ice. Aspects of the present disclosure provide a method for reducing or eliminating the risk ofAttorney Reference: BCHR-001WO ice formation in biological matter by placing the biological matter in contact with the high entropy solution designed to have a high entropy of mixing while preserving the biological matter at subzero centigrade temperature. Therefore, the method for preserving a biological matter at subzero centigrade temperatures without ice formation comprising enhances preservation capacity to avoid the growth of ice; cooling the biological matter to a sub-zero centigrade temperature in an ice-free state, including optionally a temperature above the glass transition temperature of the solution; and storing the biological matter at the same or a lower temperature in an ice-free state.

[0230] High entropy solutions of the present disclosure are precisely described in the above sections (“3. High Entropy Solutions” and “4. Methods for Producing High Entropy Solutions”) and are incorporated into this section.

[0231] In some embodiments, the method of the present disclosure is designed to reduce or eliminate the risk of ice formation during sub-zero Centigrade preservation of a biological matter. As such, the method of the present disclosure enables to cryopreserve a biological matter without ice formation by using a high entropy solution of the present disclosure which is designed to have high entropy of mixing and not to freeze when cooled to and warmed from a temperature between 0 °C and about -200 °C. Furthermore, cryopreservation is achieved at temperatures warmer than the glass transition temperature of the solution, though cryopreservation at lower temperatures can also be achieved.

[0232] In some embodiments, the method comprises placing the biological matter and the high entropy solution of the present disclosure within a chamber. In certain embodiments, the chamber is rigid and constant volume container. In certain embodiments, the chamber is an air-free container. In certain embodiments, the chamber is an isochoric chamber. In certain embodiments, the isochoric chamber is hermetically sealed. In some embodiments, the method of the present disclosure comprises placing the biological matter and the high entropy solution of the present disclosure within the isochoric chamber and cooling them under isochoric conditions.

[0233] In some embodiments, the biological matter is loaded with the high entropy solution by a perfusion process. In certain embodiments, the perfusion process is powered by gravity. In certain embodiments, the perfusion process is powered by application of pressure toAttorney Reference: BCHR-001WO a transfusion bag. In certain embodiments, the perfusion process is powered by a pump. In certain embodiments, the perfusion process is powered by a machine perfusion device.

[0234] In certain embodiments, the biological matter is loaded with the high entropy solution by a diffusion process.

[0235] In certain embodiments, the biological matter is loaded with the high entropy solution by a submersion process. In other embodiments, the biological matter is loaded with the high entropy solution and submerged in the high entropy solution.

[0236] In certain embodiments, the biological matter is loaded with the high entropy solution by a convection process.

[0237] In some embodiments, the biological matter is loaded with the high entropy solution at hypothermic temperatures between about 35 °C and about -20 °C, between about 30 °C and about -20 °C, between about 25 °C and about -20 °C, or between about 20 °C and about -20 °C. In certain embodiments, the biological matter is loaded with the high entropy solution at hypothermic temperatures between about 20 °C and about -20 °C.

[0238] In some embodiments, the biological matter and the high entropy solution are cooled to a temperature above the glass transition temperatures of the high entropy solution without ice formation. In other embodiments, the biological matter and the high entropy solution are cooled to a temperature equal to or beneath the glass transition temperature of the high entropy solution without the ice formation. The general range of glass transition temperatures exhibited by preservation solutions of relevance to biological systems (i.e. of minimal toxicity) is approximately -100 °C to -140 °C.

[0239] In some embodiments, the biological matter and the high entropy solution are first cooled to a temperature between about -50 and about -90 °C, about -60 °C and about -90 °C, about -70 °C and about -90 °C or about -80 °C and about -90 °C. In certain embodiments, the biological matter and the high entropy solution are first cooled to a temperature about -50 °C, about -55 °C, about -60 °C, about -65 °C, about -70 °C, about -71 °C, about -72 °C, about - 73 °C, about -74 °C, about -75 °C, about -76 °C, about -77 °C, about -78 °C, about -79 °C, about -80 °C, about -81 °C, about -82 °C, about -83 °C, about -84 °C, about -85 °C, about -90 °C.

[0240] In some embodiments, after the biological matter and the high entropy solution are first cooled to the above temperature, and then the biological matter and the high entropy solution are stored at a temperature between about -60 °C and about -200 °C, between about -65Attorney Reference: BCHR-001WO °C and about -200 °C, between about -70 °C and about -200 °C, between about -75 °C and about -200 °C, between about -80 °C and about -200 °C, between about -85 °C and about -200 °C, between about -90 °C and about -200 °C, between about -95 °C and about -200 °C, or between about -100 °C and about -200 °C. In certain embodiments, the biological matter and the high entropy solution are stored at a temperature about -80 °C, about -85 °C, about -90 °C, about -95 °C, about -100 °C, about -105 °C, about -110 °C, about -115 °C, about -120 °C, about -125 °C, about -130 °C, about -135 °C, about -140 °C, about -145 °C, about -150 °C, about -155 °C, about -160 °C, about -165 °C, about -170 °C, about -175 °C ̧about -180 °C, about -185 °C, about -190 °C, about -195 °C, or about -200 °C.

[0241] In some embodiments, the biological matter is cooled at a rate between about 0.01 °C / min and about 10 °C / min, between about 0.05 °C / min and about 10 °C / min, between about 0.05 °C / min and about 5 °C / min, between about 0.1 °C / min and about 10 °C / min, between about 0.1 °C / min and about 9 °C / min, between about 0.1 °C / min and about 8 °C / min, between about 0.1 °C / min and about 7 °C / min, between about 0.1 °C / min and about 6 °C / min, between about 0.1 °C / min and about 5 °C / min, between about 0.5 °C / min and about 10 °C / min, between about 0.5 °C / min and about 5 °C / min, between about 0.5 °C / min and about 4 °C / min, between about 0.5 °C / min and about 3 °C / min, between about 0.5 °C / min and about 2 °C / min, between about 0.5 °C / min and about 1 °C / min. In certain embodiments, the biological matter is cooled at a rate between about 0.1 °C / min and about 5 °C / min or between about 0.5 °C / min and about 1 °C / min.

[0242] In some embodiments, the biological matter and the high entropy solution are cooled and / or stored by using dry ice. In other embodiments, the biological matter and the high entropy solution are cooled and / or stored in a refrigerator or a freezer.

[0243] In some embodiments, the biological matter is a human cell, a human tissue, a human organ, a whole human body. In other embodiments, the biological matter is a non-human cell, a non-human tissue, a non-human organ, a whole non-human body, an organism. In certain embodiments, the non-human cell is, but not limited to, a plant cell, an animal cell, or a microbial cell.

[0244] In certain embodiments, the human cell or non-human cell is, but not limited to, a blood cell, a neuron cell, a stem cell, a bone cell, a muscle cell, a sperm cell, a female egg cell, a fat cell, a nerve cell. In certain embodiments, the human cell or tissue or non-human cell or tissue is, but not limited to, obtained from a kidney, a liver, a heart, a lung, a brain, a spleen, aAttorney Reference: BCHR-001WO stomach, a pancreas, a urinary bladder, a gallbladder, bile duct, duodenum, colon, large intestine, small intestine, anus, muscles, arteries, a lymph node, a limb, or skin. In certain embodiments, the human organ or non-human organ is but not limited to, obtained from a kidney, a liver, a heart, a lung, a brain, a spleen, a stomach, a pancreas, a urinary bladder, a gallbladder, bile duct, duodenum, colon, large intestine, small intestine, anus, muscles, arteries, a lymph node, a limb.

[0245] In some embodiments, the whole human body is preserved at a temperature less than 0 °C to reduce or suspend metabolism. In other embodiments, the whole non-human body is preserved at a temperature less than 0 °C to reduce or suspend metabolism.

[0246] In some embodiments, the biological matter is a biomedical product. In some embodiments, the biomedical product is, but not limited to, artificial organs, drugs, and medicines. In certain embodiments, medical instruments can be sterilized by the method of the present disclosure during long term preservation.

[0247] In some embodiments, the biological matter is an agricultural product, a food product, a fruit, or a beverage. In certain embodiments, the fruit includes a whole fruit, a fresh- cut fruit, or fruit arils. In other embodiments, the fruit is, but not limited to, an apple, a pear, a pomegranate, an orange, a grapefruit, a mandarin, a lime, a lemon, a nectarine, an apricot, a peach, a plum, a banana, a mango, a strawberry, a raspberry, a blueberry, a kiwifruit, a passionfruit, watermelons, a melon, a honeydew melon, a cantaloupe, a tomato, or an avocado. In certain embodiments, the agricultural product includes, but is not limited to, vegetables, legumes, or grains. In other embodiments, the vegetables are, not limited to, lettuce, spinach, beet, cabbage, cauliflower, brussels sprout, broccoli, pumpkin, cucumber, zucchini, potato, sweet potato, yam, celery, asparagus, onion, garlic, shallot, or carrot. In some embodiments, the legumes are, but not limited to, tofu, soybeans, chickpea flour, lentil flour, soy flour, haricot beans, red kidney beans, chickpeas, lentils, green peas, green beans, butter beans, or snow peas.

[0248] In some embodiments, the beverage includes fruit juice, vegetable juice, or combination thereof. In other embodiments, the beverage includes milk. In certain embodiments, the fruit juice is, but not limited to, orange juice, apple juice, grape juice, tomato juice, pomegranate juice, or mixed fruit juice. In certain embodiments, the vegetable juice is, but not limited to, carrot juice, kale juice, spinach juice, or mixed vegetable juice. In some embodiments, the food product includes dairy product or milk product.Attorney Reference: BCHR-001WO

[0249] In some embodiments, liquid biological matter, such as fruit juice or vegetable juice, is collected in an impermeable but flexible container. For example, liquid biological matter is collected in a polyethylene sterile bag. The bag is then sealed with negligible headspace and placed inside the device chamber.

[0250] In certain embodiments, the methods of the present disclosure entail manipulating the entropy of mixing of a solution to ensure that it has sufficient thermodynamic stability so as to avoid or reduce ice growth upon cooling and warming at desired cooling and warming rates; loading a biological matter with this solution, either by internal perfusion, external diffusion, or both; and cooling the sample to a temperature colder than 0 °C. The biological matter may then optionally be stored for some period at the same temperature to which it was cooled, or it may be stored at a lower temperature. In other embodiments, it may also optionally be rewarmed, and the solution may be removed via perfusion, diffusion, or both, and the sample may then be evaluated, transplanted, or otherwise put to use in a living biological system. EXAMPLES OF NON-LIMITING ASPECTS OF THE DISCLOSURE

[0251] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

[0252] Aspect 1. A high entropy solution comprising: a. water; b. two or more non-water chemical components with different physical or thermodynamic properties; and c. an entropy of mixing equal to or greater than 7 J / (mol K), wherein the high entropy solution does not freeze at a temperature between 0 °C and about -200 °C.Attorney Reference: BCHR-001WO The high entropy solution of aspect 1, wherein the number of non-water chemical components is between 2 and 100. The high entropy solution of aspect 2 or 3, wherein the entropy of mixing increases with the number of the non-water chemical components. The high entropy solution of any one of aspects 1-3, wherein the non-water chemical components are non-water carbon-based components with molar volumes between about 40 mL / mol and about 400 mL / mol. The high entropy solution of aspect 4, wherein the non-water chemical components are non-water carbon-based components with molar volumes between about 40 mL / mol and about 150 mL / mol. The high entropy solution of any one of aspects 1-5, wherein molar volumes of non- water chemical components are different from one another, and molar volumes of non- water chemical components are different from molar volume of the water. The high entropy solution of any one of aspects 1-6, wherein the entropy of mixing increases when the average molar volume of the non-water chemical components increases. The high entropy solution of any one of aspects 1-7, wherein the average molar volume of the high entropy solution is greater than 18 ml / mol. The high entropy solution of aspect 8, wherein the average molar volume of the high entropy solution is greater than 30 ml / mol.Attorney Reference: BCHR-001WO The high entropy solution of any one of aspects 1-9, wherein the entropy of mixing of the high entropy solution is at least 11 J / (mol K). The high entropy solution of any one of aspects 1-10, wherein mol fractions of the non- water chemical components are different from one another, and mol fractions of the non- water chemical components are different from mol fraction of water. The high entropy solution of any one of aspects 1-11, wherein the mol fractions of the non-water chemical components ascend in magnitude with ascending molar volumes of the non-water chemical components. The high entropy solution of any one of aspects 1-12, wherein mol fraction of the water is between 0.65 and 0.99. The high entropy solution of any one of aspects 1-13, wherein mol fraction of the water is constant while mol fractions of the non-water chemical components vary to increase the entropy of mixing. The high entropy solution of any one of aspects 1-14, wherein the number of the non- water chemical components is equal to or greater than 3, and the mol fraction of at least one of the non-water chemical components is constant and mol fractions of at least two of the non-water chemical components vary to increase the entropy of mixing. The high entropy solution of any one of aspects 1-15, wherein the entropy of mixing in the liquid phase is calculated by entropy of mixing equation: ^Attorney Reference: BCHR-001WO wherein ^^and ^^are respectively mol and volume fractions of water; ^^and ^^are respectively mol and volume fractions of each of n non-water chemical components; n is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^is entropy of mixing. 17. The high entropy solution of any one of aspects 1-16, wherein the entropy of mixing in the liquid phase is calculated by ideal entropy of mixing equation: ^ =−^ ^^ ^^wherein ^^of n non-water chemical components; n is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^.^&(F#is ideal entropy of mixing. 18. The high entropy solution of aspect 17, wherein the mol fractions and the volume fractions of the non-water chemical components, the mol fraction and the volume fraction of the water, and the number of the non-water chemical components are determined to increase the entropy of mixing. 19. The high entropy solution of aspect 17 or 18, wherein the mol fractions and the volume fractions of the non-water chemical components and the mol fraction and the volume fraction of the water are necessary to find or approach a local or global extremum in the entropy of mixing equation. 20. The high entropy solution of any one of aspects 1-19, wherein the number of non-water chemical components is equal to or greater than 3 and the high entropy solution comprises:Attorney Reference: BCHR-001WO a. water with molar volume about 18 mL / mol; b. a first group comprising one or more non-water chemical components in molar volume range between about 40 mL / mol and about 75 mL / mol; c. a second group comprising one or more non-water chemical components in molar volume range between about 75 mL / mol and about 100 mL / mol; and d. a third group comprising one or more non-water chemical components in molar volume range between about 100 mL / mol and about 150 mL / mol. The high entropy solution of aspect 20, wherein the molar volumes of the non-water chemical components in the first group are different from one another, the molar volumes of the non-water chemical components in the second group are different from one another, and the molar volumes of the non-water chemical components in the third group are different from one another. The high entropy solution of any one of aspects 1-21, wherein the water is replaced by a saline solution, a hypothermic organ preservation solution, or a base solution containing electrolytes. The high entropy solution of aspect 22, wherein the saline solution is selected from the group consisting of University of Wisconsin solution, Celsior® solution, Custodiol® HTK solution, Del Nidos solution, LM5 solution, B2 solution, phosphate buffered saline solution (PBS solution), Belzer Machine Perfusion Solution (Belzer MPS), filtered seawater, Lactated Ringers solution, isotonic saline solution containing 0.9 percent sodium chloride (salt) and hypotonic saline solution containing 0.45 percent sodium chloride.Attorney Reference: BCHR-001WO The high entropy solution of any one of aspects 1-23, wherein any of the non-water chemical components is selected from glycols, sugar alcohols, methylated organic compounds, methoxylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers. The high entropy solution of aspect 24, wherein any of the non-water chemical components is selected from ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di-ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol, threitol, ethanol, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose, trehalose, 1,2,4-butanetriol, 1,2,6-hexanetriol, 1,2-butanediol, 1,2-hexanediol, 1,2-pentanediol, 1,2-propanediol, 1,3-butanediol, 1,3-diaminopropane, 1,3-dihydroxyacetone, 1,3-dimethlurea, 1,3-propanediol, 1,4-butanediol, 1,5- pentanediol, 1,6-hexanediol, 1-2-hydroxyethyl-2-pyrrolidinone, 1-amino-2-propanol, 1- methyl-pyrrolidinone, 1-propanol, 2,3-butanediol, 2,5-dimethyl-2,5-hexanediol, 2- hydroxymethyl-1,3-propanediol, 2-isopropoxyethanol, 2-Methoxyethanol, 2-methyl-1,3- propanediol, 2-methyl-2,4-pentanediol, 2-propanol, 2-pyrrolidone, 3-amino-1-propanol, 3-methoxy-1,2-propanediol, 3-methoxy-1-butanol, 3-methoxy-1-propanol, 3-methyl-1,3- butanediol, Acetamide, Acetic acid, Acetone, Allantoin, Aspartame, Betaine, Citric acid, Diacetone alcohol, Diacetyl, Di-ethylene-glycol, Diethylene-glycol-diethyl-ether, Diethylene-glycol-dimethyl-ether, Diethylene-glycol-monoethyl-ether, Diethylene- glycol-monomethyl-ether, Diglycerol, Dimethyl isosorbide, Dimethyl sulfone, Dimethyl sulfoxide, Dipropylene-glycol-dimethyl-ether, Dipropylene-glycol-monomethyl-ether, Erythritol, Ethanol, Ethanolamine, Ethylene diamine, Ethylene glycol, Ethylene-glycol- diethyl-ether, Ethylene-glycol-dimethyl-ether, Ethylene-glycol-monoethyl-ether, Ethylene-glycol-monopropyl-ether, Formamide, Formic acid, Fructose, Galatose, Glucose, Glycerol, Glycerol formal, Glycine, Hydroxy-acetone, Isoascorbic acid,Attorney Reference: BCHR-001WO Isosorbide, Lactic acid, L-Arginine, Malic acid, Maltitol, Maltose, Mannitol, Meglumine, Methanol, Methyl-(S)-(-)-lactate, N,N-dimethylacetamide, N,N- dimethylformamide, N,N-dimethylpropionamide, N-Acetylethanolamine, N- acetylglycine, N-Formylmorpholine, Nicotinamide, N-methyl-2-pyrrolidone, N- methylacetamide, N-Methyldiethanolamine, N-methylformamide, Oxalic acid, PEG- 200, PEG-300, PEG-400, Proline, Propionamide, Propylene-glycol-monomethyl-ether, Propylene-glycol-monopropyl-ether, Pyridine, Raffinose, Sorbitol, Sucrose, Sulfolane, Tartaric acid, Taurine, Tert-butanol, Tetraethylene glycol, Tetraethylene-glycol- dimethyl-ether, Tetrahydro-4-pyranol, Tetrahydrofurfuryl alcohol, Trehalose, Triethanolamine, Tri-ethylene-glycol, Triethylene-glycol-dimethyl-ether, Triethylene- glycol-monobutyl-ether, Triethylene-glycol-monoethyl-ether, Triethylene-glycol- monomethyl-ether, Triglycerol, Trimethylamine n-oxide, Trimethylolpropane, Tripropylene-glycol-monomethyl-ether, TRIS, and Urea. The high entropy solution of any one of aspects 1-25, comprising: a. water at a mol fraction between 0.65 and 0.99; b. hexylene glycol at a mol fraction greater than all other non-water chemical components; c. sorbitol at a mol fraction less than the mol fraction of the hexylene glycol but greater than all other non-water chemical components; d. glucose at a mol fraction less than the mol fractions of the sorbitol and the hexylene glycol but greater than all other non-water chemical components; e. xylitol at a mol fraction less than the mol fractions of the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components;Attorney Reference: BCHR-001WO f. butylene glycol at a mol fraction less than the mol fractions of the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non- water chemical components; g. propylene glycol at a mol fraction less than the mol fractions of the butylene glycol, the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; h. glycerol at a mol fraction less than the mol fractions of the propylene glycol, the butylene glycol, the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; i. ethanol at a mol fraction less than the mol fractions of the glycerol, the butylene glycol, the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; j. ethylene glycol at a mol fraction less than all other non-water chemical components; and k. an entropy of mixing equal to or greater than 7 J / (mol K) as calculated using the entropy of mixing equation that accepts both mol fractions and volume fractions as arguments. The high entropy solution of any one of aspects 1-26, wherein the high entropy solution further comprises trehalose, sucrose, polyethylene glycol, polyvinyl alcohol, or another polymer with molar volume greater than 150 mL / mol in an amount less than 50% by mass. The high entropy solution of any one of aspects 1-27, wherein the high entropy solution further comprises a surfactant in an amount less than 50% by mass.Attorney Reference: BCHR-001WO The high entropy solution of any one of aspects 1-28, wherein the non-water chemical components are biocompatible. The high entropy solution of any one of aspects 1-29, wherein the non-water chemical components are non-toxic. The high entropy solution of any one of aspects 1-30, further comprising a pH adjusting compound. The high entropy solution of any one of aspects 1-31, further comprising a vasodilation inducing compound. The high entropy solution of any one of aspects 1-32, further comprising a compound providing oncotic support to an organ. A method of producing a high entropy solution, the method comprising: a. selecting two or more non-water chemical components with different physical or thermodynamic properties and water; a b. determining each amount of the non-water chemical components and amount of the water to increase an entropy of mixing; and c. mixing the non-water chemical components and the water with the determined amounts to produce the high entropy solution, wherein the high entropy solution does not freeze at a temperature between 0 °C and about -200 °C. The method of any one of aspect 34, wherein the entropy of mixing in the high entropy solution is equal to or greater than 7 J / (mol K).Attorney Reference: BCHR-001WO The method of any one of aspects 34-35, wherein the number of the non-water chemical components is between 2 and 100. The method of any one of aspects 34-36, wherein the entropy of mixing increases with the number of the non-water chemical components. The method of any one of aspects 34-37, wherein the non-water chemical components are non-water carbon-based components with molar volumes between about 40 mL / mol and about 400 mL / mol. The method of aspect 38, wherein the non-water chemical components are non-water carbon-based components with molar volumes between about 40 mL / mol and about 150 mL / mol. The method of any one of aspects 34-39, wherein molar volumes of non-water chemical components are different from one another, and molar volumes of non-water chemical components are different from molar volume of the water. The method of any one of aspects 34-40, wherein mol fractions of the non-water chemical components are different from one another, and mol fractions of the non-water chemical components are different from mol fraction of water. The method of any one of aspects 34-41, wherein the mol fractions of the non-water chemical components ascend in magnitude with ascending molar volumes of the non- water chemical components.Attorney Reference: BCHR-001WO The method of any one of aspects 34-42, wherein mol fraction of the water is between 0.65 and 0.99. The method of any one of aspects 34-43, wherein mol fraction of the water is constant while mol fractions of the non-water chemical components vary to increase the entropy of mixing. The method of any one of aspects 34-44, wherein the number of the non-water chemical components is equal to or greater than 3, and mol fraction of at least one of the non- water chemical components are constant and mol fractions of at least two of the non- water chemical components vary to increase the entropy of mixing. The method of any one of aspects 34-45, wherein the number of non-water chemical components is equal to or greater than 3 and the high entropy solution comprises: a. water with molar volume about 18 mL / mol; b. a first group comprising one or more non-water chemical components in molar volume range between about 40 mL / mol and about 75 mL / mol; c. a second group comprising one or more non-water chemical components in molar volume range between about 75 mL / mol and about 100 mL / mol; and d. a third group comprising one or more non-water chemical components in molar volume range between about 100 mL / mol and about 150 mL / mol. The method of aspect 34-46, wherein the molar volumes of the non-water chemical components in the first group are different from one another; the molar volumes of the non-water chemical components in the second group are different from one another; and the molar volumes of the non-water chemical components in the third group are different from one another.Attorney Reference: BCHR-001WO The method of any one of aspects 34-47, wherein the water is replaced by a physiological saline solution, a hypothermic organ preservation solution, or a base solution containing electrolytes. The method of any one of aspects 34-48, wherein any of the non-water chemical components is selected from glycols, sugar alcohols, methylated organic compounds, methoxylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers. The method of aspect 49, wherein any of the non-water chemical components is selected from ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di-ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol, threitol, ethanol, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose, trehalose, of ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di- ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol, threitol, ethanol, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose, trehalose, 1,2,4-butanetriol, 1,2,6-hexanetriol, 1,2-butanediol, 1,2-hexanediol, 1,2-pentanediol, 1,2-propanediol, 1,3-butanediol, 1,3-diaminopropane, 1,3- dihydroxyacetone, 1,3-dimethlurea, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1-2-hydroxyethyl-2-pyrrolidinone, 1-amino-2-propanol, 1-methyl- pyrrolidinone, 1-propanol, 2,3-butanediol, 2,5-dimethyl-2,5-hexanediol, 2- hydroxymethyl-1,3-propanediol, 2-isopropoxyethanol, 2-Methoxyethanol, 2-methyl-1,3- propanediol, 2-methyl-2,4-pentanediol, 2-propanol, 2-pyrrolidone, 3-amino-1-propanol, 3-methoxy-1,2-propanediol, 3-methoxy-1-butanol, 3-methoxy-1-propanol, 3-methyl-1,3- butanediol, Acetamide, Acetic acid, Acetone, Allantoin, Aspartame, Betaine, Citric acid,Attorney Reference: BCHR-001WO Diacetone alcohol, Diacetyl, Di-ethylene-glycol, Diethylene-glycol-diethyl-ether, Diethylene-glycol-dimethyl-ether, Diethylene-glycol-monoethyl-ether, Diethylene- glycol-monomethyl-ether, Diglycerol, Dimethyl isosorbide, Dimethyl sulfone, Dimethyl sulfoxide, Dipropylene-glycol-dimethyl-ether, Dipropylene-glycol-monomethyl-ether, Erythritol, Ethanol, Ethanolamine, Ethylene diamine, Ethylene glycol, Ethylene-glycol- diethyl-ether, Ethylene-glycol-dimethyl-ether, Ethylene-glycol-monoethyl-ether, Ethylene-glycol-monopropyl-ether, Formamide, Formic acid, Fructose, Galatose, Glucose, Glycerol, Glycerol formal, Glycine, Hydroxy-acetone, Isoascorbic acid, Isosorbide, Lactic acid, L-Arginine, Malic acid, Maltitol, Maltose, Mannitol, Meglumine, Methanol, Methyl-(S)-(-)-lactate, N,N-dimethylacetamide, N,N- dimethylformamide, N,N-dimethylpropionamide, N-Acetylethanolamine, N- acetylglycine, N-Formylmorpholine, Nicotinamide, N-methyl-2-pyrrolidone, N- methylacetamide, N-Methyldiethanolamine, N-methylformamide, Oxalic acid, PEG- 200, PEG-300, PEG-400, Proline, Propionamide, Propylene-glycol-monomethyl-ether, Propylene-glycol-monopropyl-ether, Pyridine, Raffinose, Sorbitol, Sucrose, Sulfolane, Tartaric acid, Taurine, Tert-butanol, Tetraethylene glycol, Tetraethylene-glycol- dimethyl-ether, Tetrahydro-4-pyranol, Tetrahydrofurfuryl alcohol, Trehalose, Triethanolamine, Tri-ethylene-glycol, Triethylene-glycol-dimethyl-ether, Triethylene- glycol-monobutyl-ether, Triethylene-glycol-monoethyl-ether, Triethylene-glycol- monomethyl-ether, Triglycerol, Trimethylamine n-oxide, Trimethylolpropane, Tripropylene-glycol-monomethyl-ether, TRIS, and Urea. The method of any one of aspects 34-50, wherein the non-water chemical components are biocompatible or non-toxic. The method of any one of aspects 34-51, herein the entropy of mixing in the liquid phase is calculated by entropy of mixing equation:Attorney Reference: BCHR-001WO ^ ∆^^ = −^ C^^ ln ^^ + D ^^ln^^^^E wherein ^^and^^and ^^are respectively mol and volume fractions of each of n non-water chemicaln is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^is entropy of mixing. 53. The high entropy solution of any one of aspects 34-51, wherein the entropy of mixing in the liquid phase is calculated by ideal entropy of mixing equation: ^ −^wherein ^^of n non-water chemical components; n is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^.^&(F#is ideal entropy of mixing. 54. The method of aspect 34-53, wherein the mol fractions and the volume fractions of the non-water chemical components, the mol fraction and the volume fraction of the water, and the number of the non-water chemical components are determined to increase the entropy of mixing. 55. The method of any one of aspects 34-54, wherein the mol fractions and the volume fractions of the non-water chemical components and the mol fraction and the volume fraction of the water are necessary to find or approach a local or global extremum in the entropy of mixing equation.Attorney Reference: BCHR-001WO 56. The method of any one of aspects 34-55, wherein optimization of the entropy of mixing equation is performed by using a computational optimization method. 57. The method of aspect 56, wherein the computational optimization method includes a brute force optimization method, a binary search optimization method, a convex optimization method, a stochastic optimization method, or a particle swarm optimization method. 58. A high entropy solution with principal non-water chemical components (PNWCCs), the high entropy solution comprising: a. water; and b. at least 10 PNWCCs, wherein the mol fraction of the most abundant PNWCC is not more than 10 times the mol fraction of the least abundant PNWCC. 59. The high entropy solution of aspect 58, wherein the at least 10 PNWCCs is 10 to about 100 PNWCCs. 60. The high entropy solution of any one of aspects 58-59, further comprising at least 1 non- principal non-water component (N-PNWCCs). 61. The high entropy solution of aspect 60, wherein the at least 1 N-PNWCC is 1 to about 100 N-PNWCCs. 62. The high entropy solution of any one of aspects 60-61, wherein each amount of N- PNWCC is such that the most abundant N-PNWCC is less than one tenth the mol fraction of the most abundant PNWCC.Attorney Reference: BCHR-001WO The high entropy solution of any one of aspects 58-62, wherein the PNWCCs are non- water carbon-based components with molar volumes between about 40 mL / mol and about 400 mL / mol. The high entropy solution of any one of aspects 58-63, wherein the PNWCCs are non- water carbon-based components with molar volumes between about 40 mL / mol and about 150 mL / mol. The high entropy solution of any one of aspects 58-64, wherein the average molar volume of the high entropy solution is greater than 18 ml / mol. The high entropy solution of aspect 65, wherein the average molar volume of the high entropy solution is greater than 30 ml / mol. The high entropy solution of any one of aspects 58-66, wherein the entropy of mixing of the high entropy solution is at least 11 J / (mol K). The high entropy solution of any one of aspects 58-67, wherein molar volumes of PNWCCs are different from one another, and molar volumes of PNWCCs are different from molar volume of the water. The high entropy solution of any one of aspects 58-68, wherein the weighted sample standard deviation of the molar volumes of the PNWCCs ranges from 1 ml / mol to 100 ml / mol, such as 5 ml / mol to 50 ml / mol or 10 ml / mol to 25 ml / mol. The high entropy solution of any one of aspects 58-69, wherein the entropy of mixing increases when the average molar volume of the PNWCCs increase.Attorney Reference: BCHR-001WO 71. The high entropy solution of any one of aspects 58-70, wherein mol fractions of the PNWCCs are different from one another, and mol fractions of the PNWCCs are different from mol fraction of water. 72. The high entropy solution of any one of aspects 58-71, wherein the mol fractions of the PNWCCs ascend in magnitude with ascending molar volumes of the PNWCCs. 73. The high entropy solution of any one of aspects 58-72, wherein mol fraction of the water is between 0.65 and 0.99. 74. The high entropy solution of any one of aspects 58-73, wherein mol fraction of the water is constant while mol fractions of the PNWCCs vary to increase the entropy of mixing. 75. The high entropy solution of any one of aspects 58-74, wherein the entropy of mixing in the liquid phase is calculated by entropy of mixing equation: ^ wherein ^^and^^and ^^are respectively mol and volume fractions of each of n PNWCCs; n is the number of the PNWCCs and n is an integer equal to or greater than 10; R is the ideal gas constant; and ∆^^is entropy of mixing. 76. The high entropy solution of any one of aspects 58-74, wherein the entropy of mixing in the liquid phase is calculated by ideal entropy of mixing equation: ^Attorney Reference: BCHR-001WO wherein ^^is the mol fractions of water; ^^is the mol fractions of each of n PNWCCs; n is the number of the PNWCCs and n is an integer equal to or greater than 10; R is the ideal gas constant; and ∆^^.^&(F#is ideal entropy of mixing. 77. The high entropy solution of any one of aspects 58-76, wherein the mol fractions and the volume fractions of the PNWCCs, the mol fraction and the volume fraction of the water, and the number of the PNWCCs are determined to increase the entropy of mixing. 78. The high entropy solution of any one of aspects 58-77, wherein the mol fractions and the volume fractions of the PNWCCs and the mol fraction and the volume fraction of the water are necessary to find or approach a local or global extremum in the entropy of mixing equation. 79. The high entropy solution of any one of aspects 58-78, wherein the number of PNWCCs is equal to or greater than 10 and the high entropy solution comprises: a. water with molar volume about 18 mL / mol; b. a first group comprising one or more PNWCCs in molar volume range between about 40 mL / mol and about 75 mL / mol; c. a second group comprising one or more PNWCCs in molar volume range between about 75 mL / mol and about 100 mL / mol; and d. a third group comprising one or more PNWCCs in molar volume range between about 100 mL / mol and about 150 mL / mol. 80. The high entropy solution of any one of aspects 58-79, wherein the molar volumes of the PNWCCs in the first group are different from one another, the molar volumes of the PNWCCs in the second group are different from one another, and the molar volumes of the PNWCCs in the third group are different from one another.Attorney Reference: BCHR-001WO The high entropy solution of any one of aspects 58-80, wherein the water is replaced by a saline solution, a hypothermic organ preservation solution, or a base solution containing electrolytes. The high entropy solution of any one of aspects 58-81, wherein the saline solution is selected from the group consisting of University of Wisconsin solution, Celsior® solution, Custodiol® HTK solution, Del Nidos solution, LM5 solution, B2 solution, phosphate buffered saline solution (PBS solution), Belzer Machine Perfusion Solution (Belzer MPS), filtered seawater, Lactated Ringers solution, isotonic saline solution containing 0.9 percent sodium chloride (salt) and hypotonic saline solution containing 0.45 percent sodium chloride. The high entropy solution of any one of aspects 58-82, wherein any of the PNWCCs is selected from glycols, sugar alcohols, methylated organic compounds, methoxylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers. The high entropy solution of any one of aspects 58-83, wherein any of the PNWCCs is selected from ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di- ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol, threitol, ethanol, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose, trehalose, 1,2,4-butanetriol, 1,2,6-hexanetriol, 1,2-butanediol, 1,2-hexanediol, 1,2-pentanediol, 1,2-propanediol, 1,3-butanediol, 1,3-diaminopropane, 1,3- dihydroxyacetone, 1,3-dimethlurea, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1-2-hydroxyethyl-2-pyrrolidinone, 1-amino-2-propanol, 1-methyl- pyrrolidinone, 1-propanol, 2,3-butanediol, 2,5-dimethyl-2,5-hexanediol, 2-Attorney Reference: BCHR-001WO hydroxymethyl-1,3-propanediol, 2-isopropoxyethanol, 2-Methoxyethanol, 2-methyl-1,3- propanediol, 2-methyl-2,4-pentanediol, 2-propanol, 2-pyrrolidone, 3-amino-1-propanol, 3-methoxy-1,2-propanediol, 3-methoxy-1-butanol, 3-methoxy-1-propanol, 3-methyl-1,3- butanediol, Acetamide, Acetic acid, Acetone, Allantoin, Aspartame, Betaine, Citric acid, Diacetone alcohol, Diacetyl, Di-ethylene-glycol, Diethylene-glycol-diethyl-ether, Diethylene-glycol-dimethyl-ether, Diethylene-glycol-monoethyl-ether, Diethylene- glycol-monomethyl-ether, Diglycerol, Dimethyl isosorbide, Dimethyl sulfone, Dimethyl sulfoxide, Dipropylene-glycol-dimethyl-ether, Dipropylene-glycol-monomethyl-ether, Erythritol, Ethanol, Ethanolamine, Ethylene diamine, Ethylene glycol, Ethylene-glycol- diethyl-ether, Ethylene-glycol-dimethyl-ether, Ethylene-glycol-monoethyl-ether, Ethylene-glycol-monopropyl-ether, Formamide, Formic acid, Fructose, Galatose, Glucose, Glycerol, Glycerol formal, Glycine, Hydroxy-acetone, Isoascorbic acid, Isosorbide, Lactic acid, L-Arginine, Malic acid, Maltitol, Maltose, Mannitol, Meglumine, Methanol, Methyl-(S)-(-)-lactate, N,N-dimethylacetamide, N,N- dimethylformamide, N,N-dimethylpropionamide, N-Acetylethanolamine, N- acetylglycine, N-Formylmorpholine, Nicotinamide, N-methyl-2-pyrrolidone, N- methylacetamide, N-Methyldiethanolamine, N-methylformamide, Oxalic acid, PEG- 200, PEG-300, PEG-400, Proline, Propionamide, Propylene-glycol-monomethyl-ether, Propylene-glycol-monopropyl-ether, Pyridine, Raffinose, Sorbitol, Sucrose, Sulfolane, Tartaric acid, Taurine, Tert-butanol, Tetraethylene glycol, Tetraethylene-glycol- dimethyl-ether, Tetrahydro-4-pyranol, Tetrahydrofurfuryl alcohol, Trehalose, Triethanolamine, Tri-ethylene-glycol, Triethylene-glycol-dimethyl-ether, Triethylene- glycol-monobutyl-ether, Triethylene-glycol-monoethyl-ether, Triethylene-glycol- monomethyl-ether, Triglycerol, Trimethylamine n-oxide, Trimethylolpropane, Tripropylene-glycol-monomethyl-ether, TRIS, and Urea.Attorney Reference: BCHR-001WO The high entropy solution of any one of aspects 58-84, wherein the high entropy solution further comprises trehalose, sucrose, polyethylene glycol, polyvinyl alcohol, or another polymer with molar volume greater than 150 mL / mol in an amount less than 50% by mass. The high entropy solution of any one of aspects 58-85, wherein the high entropy solution further comprises a surfactant in an amount less than 50% by mass. The high entropy solution of any one of aspects 58-86, wherein the non-water chemical components are biocompatible, non-toxic, or a combination thereof. The high entropy solution of any one of aspects 58-87, further comprising a pH adjusting compound. The high entropy solution of any one of aspects 58-88, further comprising a vasodilation inducing compound. The high entropy solution of any one of aspects 58-89, further comprising a compound providing oncotic support to an organ. A method of producing a high entropy solution with principal non-water chemical components (PNWCCs), the method comprising: a) selecting at least 10 PNWCCs, optionally selecting 1 or more non-principal non-water components (N-PNWCCs), and water; b) determining each amount of PNWCCs such that the mol fraction of the most abundant PNWCCs is not more than 10 times the mol fraction of the least abundantAttorney Reference: BCHR-001WO PNWCCs, thereby resulting in a sufficient entropy of mixing to suppress ice formation based on number of PNWCCs; and c) if 1 or more N-PNWCCs were selected, determining each amount of N- PNWCCs such that the most abundant N-PNWCCs is less than one tenth the mol fraction of the most abundant PNWCC; and d) mixing the PNWCCs, the N-PNWCCs, and the water with the determined amounts to produce the high entropy solution. 92. A high entropy solution comprising: water; and 2 or more principal non-water chemical components (PNWCCs), wherein the high entropy solution has an entropy of mixing of 7 J / (mol K) or more, wherein the high entropy solution does not freeze when cooling from 0 °C to a lower temperature or when warming from the lower temperature to 0 °C, wherein the lower temperature is -80 °C, wherein the cooling and warming are both performed at a rate of 1 °C / min and a pressure of 101 kPa. 93. The high entropy solution of aspect 92, wherein the 2 or more PNWCCs is 4 or more or more PNWCCs, such as 6 or more, 8 or more, 10 or more, or 12 or more. 94. The high entropy solution of any one of aspects 92-93, wherein the total mol fraction of the PNWCCs is 0.1 or more, such as 0.2 or more, 0.3 or more, or 0.4 or more. 95. The high entropy solution of any one of aspects 92-94, wherein the mol fraction of each PNWCC ranges from 0.001 to 0.015. 96. The high entropy solution of any one of aspects 92-95, wherein at least 1 PNWCC is an organic compound with a molar volume ranging from about 40 ml / mol to about 400 ml / mol, such as 60 ml / mol to 300 ml / mol, such as at least 2 PNWCCs, at least 3 PNWCCs, at least 4 PNWCCs, at least 5 PNWCCs, at least 6 PNWCCs, or at least 7 PNWCCs. 97. The high entropy solution of any one of aspects 92-96, wherein at least 1 PNWCC is selected from the group consisting of glycols, sugar alcohols, methylated organic compounds,Attorney Reference: BCHR-001WO methoxylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers, such as at least 2 PNWCCs, at least 3 PNWCCs, at least 5 PNWCCs, or at least 7 PNWCCs. 98. The high entropy solution of any one of aspects 92-97, wherein at least 1 PNWCC is selected from the group consisting of ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di-ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol, threitol, ethanol, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose, trehalose, 1,2,4-butanetriol, 1,2,6-hexanetriol, 1,2-butanediol, 1,2-hexanediol, 1,2-pentanediol, 1,2-propanediol, 1,3-butanediol, 1,3-diaminopropane, 1,3-dihydroxyacetone, 1,3-dimethlurea, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1-2-hydroxyethyl-2- pyrrolidinone, 1-amino-2-propanol, 1-methyl-pyrrolidinone, 1-propanol, 2,3-butanediol, 2,5- dimethyl-2,5-hexanediol, 2-hydroxymethyl-1,3-propanediol, 2-isopropoxyethanol, 2- Methoxyethanol, 2-methyl-1,3-propanediol, 2-methyl-2,4-pentanediol, 2-propanol, 2- pyrrolidone, 3-amino-1-propanol, 3-methoxy-1,2-propanediol, 3-methoxy-1-butanol, 3- methoxy-1-propanol, 3-methyl-1,3-butanediol, Acetamide, Acetic acid, Acetone, Allantoin, Aspartame, Betaine, Citric acid, Diacetone alcohol, Diacetyl, Di-ethylene-glycol, Diethylene- glycol-diethyl-ether, Diethylene-glycol-dimethyl-ether, Diethylene-glycol-monoethyl-ether, Diethylene-glycol-monomethyl-ether, Diglycerol, Dimethyl isosorbide, Dimethyl sulfone, Dimethyl sulfoxide, Dipropylene-glycol-dimethyl-ether, Dipropylene-glycol-monomethyl-ether, Erythritol, Ethanol, Ethanolamine, Ethylene diamine, Ethylene glycol, Ethylene-glycol-diethyl- ether, Ethylene-glycol-dimethyl-ether, Ethylene-glycol-monoethyl-ether, Ethylene-glycol- monopropyl-ether, Formamide, Formic acid, Fructose, Galatose, Glucose, Glycerol, Glycerol formal, Glycine, Hydroxy-acetone, Isoascorbic acid, Isosorbide, Lactic acid, L-Arginine, Malic acid, Maltitol, Maltose, Mannitol, Meglumine, Methanol, Methyl-(S)-(-)-lactate, N,N- dimethylacetamide, N,N-dimethylformamide, N,N-dimethylpropionamide, N- Acetylethanolamine, N-acetylglycine, N-Formylmorpholine, Nicotinamide, N-methyl-2- pyrrolidone, N-methylacetamide, N-Methyldiethanolamine, N-methylformamide, Oxalic acid, PEG-200, PEG-300, PEG-400, Proline, Propionamide, Propylene-glycol-monomethyl-ether, Propylene-glycol-monopropyl-ether, Pyridine, Raffinose, Sorbitol, Sucrose, Sulfolane, Tartaric acid, Taurine, Tert-butanol, Tetraethylene glycol, Tetraethylene-glycol-dimethyl-ether,Attorney Reference: BCHR-001WO Tetrahydro-4-pyranol, Tetrahydrofurfuryl alcohol, Trehalose, Triethanolamine, Tri-ethylene- glycol, Triethylene-glycol-dimethyl-ether, Triethylene-glycol-monobutyl-ether, Triethylene- glycol-monoethyl-ether, Triethylene-glycol-monomethyl-ether, Triglycerol, Trimethylamine n- oxide, Trimethylolpropane, Tripropylene-glycol-monomethyl-ether, TRIS, and Urea, such as at least 2 PNWCCs, at least 3 PNWCCs, at least 4 PNWCCs, at least 5 PNWCCs, at least 6 PNWCCs, or at least 7 PNWCCs. 99. The high entropy solution of any one of aspects 92-98, wherein the weighted sample standard deviation of the molar volumes of the PNWCCs ranges from 1 ml / mol to 100 ml / mol, such as 5 ml / mol to 50 ml / mol or 10 ml / mol to 25 ml / mol. 100. The high entropy solution of any one of aspects 92-99, wherein the average molar volume of the high entropy solution is 25 ml / mol or more, such as 30 ml / mol or more, 35 ml / mol or more, 40 ml / mol or more, 45 ml / mol or more, 50 ml / mol or more, or 60 ml / mol or more. 101. The high entropy solution of any one of aspects 92-100, wherein the lower temperature is -100 °C, -120 °C, -140 °C, -160 °C, -180 °C, or -200 °C. 102. The high entropy solution of any one of aspects 92-101, wherein the high entropy solution has an entropy of mixing of 8 J / (mol K) or more, such as 9 or more, 10 or more, or 11 or more. 103. The high entropy solution of any one of aspects 92-102, wherein the entropy of mixing is entropy of mixing according to the equation: ^wherein ^^and ^^and ^^are respectively mol and volume fractions of each of n non-water chemicalis the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^is entropy of mixing.Attorney Reference: BCHR-001WO 104. The high entropy solution of any one of aspects 92-102, wherein the entropy of mixing is ideal entropy of mixing according to the equation: ^ =−^ C^^ ln ^^ + D ^^ln^^^^E wherein ^^of n non-water chemical components; n is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^.^&(F#is ideal entropy of mixing. 105. The high entropy solution of aspect 92, wherein: 106. The high entropy solution of aspect 105, wherein the high entropy solution has an entropy of mixing of 11 J / (mol K) or more according to the equation: ^ wherein ^^of n non-water chemical components; n is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^.^&(F#is ideal entropy of mixing. 107. A device for preserving a biological matter without freezing at sub-0°C temperatures, the device comprising: a. the high entropy solution of any one of the preceding aspects; b. a biological matter in contact with the high entropy solution; and c. a cooling system configured to cool the biological matter and high entropy solution to below 0 °C.Attorney Reference: BCHR-001WO. The device of aspect 107, wherein the high entropy solution and the biological matter are placed within a chamber. . The device of aspect 108, wherein the chamber is an isochoric chamber under isochoric conditions. . The device of any one of aspects 108-109, wherein the isochoric chamber is hermetically sealed. . The device of any one of aspects 107-110, wherein the biological matter is loaded with the high entropy solution by a perfusion process, a diffusion process, a submersion process, or a convection process. . The device of aspect 111, wherein the perfusion process is powered by gravity, application of pressure to a transfusion bag, a pump, or a machine perfusion device. . The device of any one of aspects 107-112, wherein the biological matter is loaded with the high entropy solution at hypothermic temperatures between about 20 °C and about -20 °C. . The device of any one of aspects 107-113, wherein the biological matter is loaded with the high entropy solution and submerged in the high entropy solution. . The device of any one of aspects 107-114, wherein the cooling system uses dry ice to cool the biological matter and the high entropy solution to below 0 °C.Attorney Reference: BCHR-001WO. The device of any one of aspects 107-115, wherein the cooling system uses liquid nitrogen to cool the biological matter and high entropy solution to below 0 °C. . The device of any one of aspects 107-116, wherein the cooling system is a freezer or a refrigerator to cool and / or store the biological matter and the high entropy solution. . The device of any one of aspects 107-117, wherein the cooling system controls the rate of cooling of the biological matter and the high entropy solution, wherein the rate of cooling is between about 0.01°C / min and about 10 °C / min. . The device of any one of aspects 107-118, wherein the biological matter and the high entropy solution are cooled to a temperature above the glass transition temperature of the high entropy solution without significant ice formation because 1% or less by mass of the high entropy solution is ice. . The device of any one of aspects 107-119, wherein the biological matter and the high entropy solution are cooled to a temperature equal to or beneath the glass transition temperature of the high entropy solution without significant ice formation because about 1% or less by mass of the high entropy solution is ice. . The device of any one of aspects 107-120, wherein the biological matter and the high entropy solution are first cooled to about -80 °C and then stored at a temperature between about -80 °C and about -200 °C. . The device of any one of aspects 107-121, wherein the biological matter is a human cell, a human tissue, a human organ, a whole human body, a non-human cell, aAttorney Reference: BCHR-001WO non-human tissue, a non-human organ, a whole non-human body, an organism, a biomedical product, an agricultural product, a food product, a fruit, or a beverage. . The device of aspect 122, wherein the human tissue or organ is a kidney, a liver, a heart, a lung, a brain, a limb, or skin. . The device of aspect 122, wherein the whole human body is preserved at a temperature less than 0 °C to reduce or suspend metabolism. . A method of preserving a biological matter without freezing at sub-0°C temperatures, the method comprising: a. placing the biological matter in contact with the high entropy solution of any of the preceding aspects, or the high entropy solution produced by a method of any one of the preceding aspects; b. cooling the biological matter and the high entropy solution to a temperature lower than 0°C; and c. storing the biological matter without ice formation at a temperature between 0 °C and about -200 °C. . The method of aspect 125, wherein the biological matter is loaded with the high entropy solution by a perfusion process, a diffusion process, a submersion process, or a convection process. . The method of aspect 126, wherein the perfusion process is powered by gravity, application of pressure to a transfusion bag, a pump, or a machine perfusion device.Attorney Reference: BCHR-001WO. The method of any one of aspects 125-127, wherein the biological matter is loaded with the high entropy solution at hypothermic temperatures between about 20 °C and about -20 °C. . The method of any one of aspects 125-128, wherein the biological matter is loaded with the high entropy solution and submerged in the high entropy solution. . The method of any one of aspects 125-129, wherein the biological matter and the high entropy solution are cooled to a temperature above the glass transition temperature of the high entropy solution without ice formation. . The method of any one of aspects 125-130, wherein the biological matter and the high entropy solution are cooled to a temperature equal to or beneath the glass transition temperature of the high entropy solution without ice formation. . The method of any one of aspects 125-131, wherein the biological matter and the high entropy solution are cooled to about -80 °C. . The method of any one of aspects 125-132, wherein the biological matter and high entropy solution are first cooled to about -80 °C, then optionally transported at about -80 °C, then stored at a temperature between about -130 °C and about -200 °C. . The method of any one of aspects 125-133, wherein the biological matter is cooled at a rate between about 0.01°C / min and about 10 °C / min. . The method of any one of aspects 125-134, wherein the biological matter and the high entropy solution are cooled by using dry ice.Attorney Reference: BCHR-001WO 136. The method of any one of aspects 125-135, where the biological matter and the high entropy solution are transported or stored on dry ice. 137. The method of any one of aspects 125-136, wherein the biological matter and the high entropy solution are cooled by using liquid nitrogen. 138. The method of any one of aspects 125-137, wherein the biological matter and the high entropy solution are cooled and / or stored in a refrigerator or a freezer. 139. The method of any one of aspects 125-138, wherein the biological matter is a human cell, a human tissue, a human organ, a whole human body, a non-human cell, a non-human tissue, a non-human organ, a whole non-human body, an organism, a biomedical product, an agricultural product, a food product, a fruit, or a beverage. 140. The method of aspect 139, wherein the human tissue or organ is a kidney, a liver, a heart, a lung, a brain, a limb, or skin. 141. The method of aspect 140, wherein the whole human body is preserved at a temperature less than 0 °C to reduce or suspend metabolism. EXAMPLES

[0253] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present inventionAttorney Reference: BCHR-001WO and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric (e.g. at 101 kPa of pressure).

[0254] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0255] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims. 1. Application to experimental data

[0256] In this section, homogeneous nucleation data for 15+ binary aqueous solutions at a range of concentrations and across classes of organic solutes, including classical cryoprotective agents (CPAs), glycols, sugars, alcohols, sugar alcohols, etc. were collected from literature. In order to underline the importance and suitability of solute size dependence in the entropy model of the present disclosure, solutes were chosen across as wide a range of molar volumes as possible, ranging from methanol (the smallest) to sucrose (the largest). Solute molar masses, mass densities, and molar volumes are given in Table 1.

[0257] Table 1. Solute molar masses, densities, and molar volumes, provided at room temperature and atmospheric pressure. Solute Molar Mass (g / mol) Density (g / ml) Molar Volume (ml / mol)Attorney Reference: BCHR-001WO Hexylene glycol 118.17 0.92 128.03 Glucose 180.06 1.56 115.42

[0258] Molar volume is the molar mass of a compound divided by its density. For example, the molar mass in g / mol can be divided by its density in g / ml to obtain a molar mass with units of ml / mol.

[0259] Aggregated homogeneous nucleation data are plotted as a function of molar concentration in FIG. 1A. Then, the size-dependent (Flory) entropy of mixing is calculated foreach of these solutions, according to the formulations ∆^^ = −^^^^ ln ^^ + ∑^^ ^^ ln^^^^^ andTh is replotted as a function of ∆^^in FIG. 1B. Across solute types and concentrations, the homogeneous nucleation temperatures of the considered solutions collapse onto a single curve of entropy of mixing, demonstrating the dominance of solution entropy over the nucleation process. For the predictive use, a second-order polynomial fit of these data, Th(ΔSm) = −2.3210 ΔS2m + 1.4325 ΔSm + 232.1309, which agrees to an R2of 0.94 (Th and ΔSm retain the units shown in FIG. 1A), are provided.Attorney Reference: BCHR-001WO

[0260] In order to further motivate the inventor’s choice of entropy descriptor, homogeneous nucleation vs ideal entropy of mixing (ΔSmix, ideal = −R(xw ln xw +Σi xi ln xi)) is plotted in FIG. 1C, which, failing to account for any manner of difference between solutes, fails to produce account for any manner of difference between solutes, and fails to produce any substantive predictive power. Finally, in order to confirm that the proposed entropic mechanism is dominating the observed behavior, as opposed to some coincident yet distinct effect related strictly to solute size, homogeneous nucleation temperature is also plotted against the volume fraction of water. As expected, given the functional form of the Flory entropy and the mol fractions considered herein, the volume fraction is a good predictor of nucleation temperature as compared to the ideal entropy of mixing but remains inferior to the entropy of mixing shown in FIG. 1B.

[0261] It is important to note further that for these calculations, given the unavailability of temperature-dependent excess volumes for many of the solutions, room-temperature pure- phase solute molar volumes were employed in the calculation of the entropy. These values provide an acceptable estimation for the solutes considered herein, as excess volumes for binary aqueous solutions of organic and / or non-ionic solutes are generally on the order of <1 cm3 / mol, and thermal changes in volume over the 100 K temperature band shown here are on the order of 5 % or less for the entire solution, with the relative volume fraction almost certainly changing less.

[0262] The widening of the temperature spread with increasing entropy is likely in part a function of increasing deviation from the estimated room-temperature volume fraction in solution, but that this deviation does not affect our overarching demonstration. 2. Implications on physical understanding, ice nucleation modeling

[0263] FIGS. 1A-1D demonstrate the dominance of solution entropy over the homogeneous nucleation temperature of ice, which, critically, yields an extremely simple analytical lever by which to estimate the nucleation temperature of an aqueous binary solution based only on its individual constituents. For the non-ionic solutions considered here, other current theories nigh-universally require some experimental knowledge (often the empirical activity coefficient at some reference temperature) of the solution at hand in order to estimate the appropriate nucleation temperature, limiting their utility in predictive contexts. The theory presented here provides remarkable predictive power with knowledge only of the individualAttorney Reference: BCHR-001WO molar masses and volumes of the pure components at room temperature and pressure, data that are broadly available for most materials miscible with water. This ability to accurately predict homogeneous nucleation temperatures as a function of readily available solute properties may save researchers significant experimental effort in future characterization of binary aqueous solutions, with applications in the study of supercooling- and vitrification-based cryopreservation processes, agricultural cold storage processes, atmospheric nucleation processes, etc.

[0264] More generally, the physical interpretation of the role of entropy provided here establishes a theoretical foundation from which to enable the rational design and predictive synthesis of aqueous solutions with desired nucleation behaviors. For example, in the burgeoning field of organ and tissue cryopreservation, the relative stability of aqueous solutions against ice nucleation is often the single most important physical factor driving solution choice. In supercooled biopreservation, solutions are sought that may remain in a prolonged metastable liquid state at temperatures several degrees below their melting points; in vitrified biopreservation, solutions are sought whose homogeneous nucleation temperatures are so low that glass formation can be all but guaranteed. The last several decades of solution chemistry design for low-temperature biopreservation have proceeded in a nigh-totally empirical fashion and, given the experimental intensiveness of quantifying the nucleation behaviors of new solutes, have relied on a small selection of well-characterized “cryoprotectant” solutes. The entropic theory herein provides a method by which to rapidly screen new potential solutes of biological interest for their nucleation capacities and provide a rational physical framework from which to approach solution design. Armed with a quantitative means of predicting general nucleation behaviors, future solution design efforts may also be able to optimize supercooling stability / nucleation avoidance against biological toxicity or other desired parameters, opening new pathways toward systematic engineering design of biopreservation solutions and protocols. 3. Extension to high entropy solutions

[0265] The link between increasing entropy and decreasing ice nucleation probability establishes the foundation from which to design high entropy aqueous solutions capable of significantly avoiding freezing at sub-0°C temperatures, and thereby enabling ice-free cryopreservation of biological matter. Such solutions may be designed either by computationally optimizing the amounts of each of 2 or more non-water components in solution so as to maximize the entropy of mixing, or by including a sufficient number of principal non-Attorney Reference: BCHR-001WO water components, about 10 to 100, so as to reach a sufficient entropy of mixing without optimization of individual component amounts. Principal components are defined as non-water components of molar concentrations within a factor of ten of the most abundant non-water component, which thereby substantially contribute to the entropy of mixing.

[0266] Current aqueous cryopreservation solutions employ at most 5-6 principal components, and furthermore do not computationally optimize the amounts of these components to maximize entropy of mixing. This doubled lack of attention to designing sufficient entropy to significantly suppress ice formation explains in large part why successful cryopreservation of human or large-animal organs and large tissues has never been achieved using published solutions. 4. Example 1

[0267] A preferable application of the methods of present disclosure is presented below.

[0268] First, a high-entropy solution is designed and synthesized. It is then characterized to ensure that it will not freeze at a particularly desirable sub-0°C preservation temperature (-80 °C). It is then placed in contact with a complex biological sample, cooled to the desired temperature without freezing, and re-warmed. The biological sample is then evaluated under a microscope and by histological evaluation to confirm that no freezing has occurred within.

[0269] To design the high entropy solution in accordance with the methods of the present disclosure, a number and group of components are chosen. Each of the components is desirable for not being acutely toxic or for being commonly employed as a food additive, skin care additive, or similar. Aiming for high entropy and minimal mol fractions of each of the individual components in solution, a 10 component solution (n = 10) is chosen and the solution is comprised of the following component: water (molar volume about 18.0 mL / mol); hexylene glycol (molar volume about 128.03 mL / mol); sorbitol (molar volume about 122.26 mL / mol); glucose (molar volume about 115.42 mL / mol); xylitol (molar volume about 100.10 mL / mol); butylene glycol (molar volume about 89.64 mL / mol); propylene glycol (molar volume about 73.16 mL / mol); glycerol (molar volume about 73.03 mL / mol); ethanol (molar volume about 58.32 mL / mol); ethylene glycol (molar volume about 55.92 mL / mol).

[0270] Next, a total mol fraction of water ^^ = 0.80 consistent with minimal toxicity,as evidenced by many cryoprotective non-high entropy solutions published previously in the scientific literature, is chosen.Attorney Reference: BCHR-001WO

[0271] Next, the equation ∆^^ = −^^^^ ln ^^ + ∑^^= ^^ ln^^^^^ to describe theentropy of mixing is used, wherein ^^and ^^are respectively the mol and volume fractions of water, which is taken as the 1st of n components, and ^^and ^^are the mol and volume fractions of each of the non-water components, taken as the 2nd n-th components, such that(^ + ∑^^= ^ = 1) and ^^ + ∑^^=> ^ = 1)^^ ^ ^ . The mol and volume fractions of each ofthe n components in solution are related by the molar volumes mv of each constituent, such that^^ = ?@ ^A@?I^AIJ∑B@K^ ?@ ^A@ and ^^ = ?I ^AI?I^AIJ∑B . Holding ^^ as constant at ^^ = 0.80, a@K^ ?@ ^A@standardto calculate the values of the component mol fractions ^^that minimize the ∆^^equation, producing a solution of the following composition:

[0272] Table 2. COMPONENT MOL FRACTION VOLUME FRACTION 7 3 5 8 3 9 4 4 6 4

[0273] The Table 2 embodiment is related to embodiments that fulfill the following criteria: there are 6 or more PNWCCs, each of the 6 or more PNWCCs is an organic compound with a molar volume ranging from 40 ml / mol to 400 ml / mol, each of the 6 or more PNWCCs is an organic compound selected from the group consisting of glycols, sugar alcohols, methylated organic compounds, methoxylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers, and the molar volume of the high entropy solution is 30 ml / mol or more.Attorney Reference: BCHR-001WO

[0274] The absolute value of the calculated entropy of mixing for this solution is about 11.93 J / mol K. The mol fractions of each non-water component ascend in the same order as the molar volumes of each component.

[0275] In order to initially confirm that the solution will not freeze at sub-0°C temperatures, differential scanning calorimetry tests were performed on 15 mg samples of the solution at cooling and warming rates of 10 °C / minute. These tests showed no freezing. The glass transition temperature of the solution, such as about -95°C, were also demonstrated.

[0276] With this initial validation, increasingly large volumes of solution were tested at increasingly low cooling and warming rates. Solutions from 10 mL to 1L in volume were tested at cooling and warming rates from 5 °C / min to 0.1 °C / min, cooling to and from approximately - 80 °C by placing the sample (in a glass beaker) in dry ice 24 hours, and then the sample were removed and allowed to warm in air. For each test, after the 24 hours on dry ice, the sample (at - 80 °C) was visually evaluated for ice formation, and then continuously visually monitored for ice formation during warming thereafter. In all tests performed, no freezing was observed. -80 °C is above the glass transition temperature of the solution (-95 °C), demonstrating the unique ability of the methods of the present disclosure to produce solutions that do not freeze even in the non-glassy sub-0°C state, well below the conventional range for aqueous supercooling solutions (down to -20°C).

[0277] Also, the tests described above were repeated using submersion in liquid nitrogen (-196 °C) instead of dry ice (-80 °C) and once again no freezing was observed. It was confirmed that the solution does also not freeze during cooling to temperatures beneath the glass transition temperature of the solution (-95 °C).

[0278] With the stability of the solution against freezing to a range of useful sub-0°C temperatures, the solution was tested for the preservation of a biological sample without freezing. To this end, two pig kidneys were procured from a local slaughterhouse. The solution was re-mixed using the University of Wisconsin organ preservation solution (a physiological- osmolality saline solution commonly used in clinical organ preservation) as the base instead of pure water. Cannulating the abdominal aorta, simple gravity perfusion was used to perfuse the solution into each kidney. In order to minimize potential stresses related to osmotic shock or vascular constriction or dilation, the kidneys were perfused in multiple concentration steps, i.e., starting with diluted forms of the solution. 25%-, 50%-, 75%-, and 100%-strength solution were used in these steps, where 25%-strength solution is prepared by mixing the solution withAttorney Reference: BCHR-001WO additional base University of Wisconsin solution in a ratio of 1:3 by volume, 50%-strength solution is prepared by mixing the solution with additional base University of Wisconsin solution in a ratio of 1:2 by volume, 75%-strength solution is prepared by mixing the solution with additional base University of Wisconsin solution in a ratio of 3:1 by volume, and 100%- strength is the solution as prepared. Approximately 250 mL of each of these four concentrations, chilled to about 4 °C in a refrigerator, were perfused through each kidney. Then, each kidney was submerged in full-strength solution in polyethylene bags, the bags were sealed and placed in a large insulated tub of dry ice at -80 °C, such that the volume of dry ice was much larger than that of the bags. The bags and kidneys were left at -80 °C on dry ice overnight. After approximately 24 hours, one bag was removed from the dry ice and evaluated. First the temperature was confirmed to be -80 °C by placing a thermocouple into the solution surrounding the kidney in the bag. Then, the kidney was removed from the bag (still at -80 °C) and placed on a chilled steel tray sitting on a bed of dry ice, also at about -80 °C. Then, using a surgical blade also pre-chilled to -80 °C, the kidney was dissected to inspect for ice formation. If the cryopreservation were performed at a temperature less than the glass transition temperature, smooth slicing of the kidney would be impossible, as the kidney would be in the solid, brittle, glassy state. Likewise, were the kidney frozen, it would be impossible to smoothly slice. Instead, the kidney remained in a semi-solid state with a mechanical constitution similar to chilled butter, its interior liquid contents being in a highly viscous, low-temperature unfrozen and unvitrified state. Upon dissection, no ice formation was detected anywhere in the kidney, including in the difficult-to-preserve medulla. The kidney slices were then allowed to warm in air while monitored, and again to ice formation was detected. The second kidney and bag were removed from the dry ice tub and initially evaluated for ice formation (none was detected). Then, instead of opening the bag and dissecting the kidney, the kidney was allowed to warm in air at a rate of about 0.5 – 1 °C / min to 0 °C, while monitored continuously for visual ice formation (none was detected). After reaching 0 °C, the kidney was removed from the bag and once again cannulated for perfusion. Then, gravity perfusion was used to remove the solution from the kidney, again using concentration steps (this time using only two steps, 50%-strength solution and pure University of Wisconsin solution). Upon completion of the final perfusion step with pure University of Wisconsin solution, the kidney was then sliced and fixed in neutral buffered formalin. These kidney slices were then stained with hemotoxylin and eosin and evaluated histologically. No evidence of ice formation nor acute toxicity was observed inAttorney Reference: BCHR-001WO histological evaluation of any part of the kidney, including slices from the medulla and the cortex.

[0279] This experiment provides a useful example of the methods, compositions, and devices claimed herein. 5. Example 2

[0280] In order to further demonstrate the unique capacity of the high entropy solutions disclosed herein to facilitate ice free preservation of biological matter at a wide range of sub- 0°C temperatures, and at the slow cooling and warming rates achievable without specialized machinery, the high entropy solution composition described in Example 1 above were also compared to a host of standard solutions used for ice-free cryopreservation by vitrification.

[0281] To this end, four published vitrification solutions were synthesized: VEG (1,3), DP6 (2), VS41A (3,4), and VS55 (2,4). Each of these solutions has a glass transition temperature between about -115°C and about -130°C and is generally indicated for use in the ice-free cryopreservation of biological matter through the process of vitrification.

[0282] To demonstrate the limits of their stability relative to a high entropy solution synthesized in accordance with the methods and compositions disclosed herein, 15 mL of each solution was placed in a standard mL polyethylene media vial; buried each in dry ice pellets, inducing an average cooling rate of approximately 5°C / minute; the solutions were left at approximately -80°C; then were removed and inspected visually.

[0283] Images of each solution immediately after removal from -80 °C conditions on dry ice are shown in FIG. 2. For all four of these solutions, ice was observed to have formed to some degree, with VEG and DP6 showing ice throughout the entire 15 mL volume and VS55 and VS41A showing ice in approximately 20 % of the volume. As described in Example 1, by comparison, the exemplar high entropy solutions disclosed in the present disclosure show no ice formation whatsoever, even after 24 hours at -80 °C, and even when perfused into biological matter which may otherwise substantially increase the likelihood of ice nucleation.

[0284] To further illustrate the distinction between high entropy solutions and standard vitrification solutions in current use, this comparative test was also repeated with fresh samples, but using liquid nitrogen at -196 °C as the cryogen, instead of dry ice at -80 °C. This test yielded cooling rates of 20 °C / minute to 30 °C / minute.Attorney Reference: BCHR-001WO

[0285] The results of this test are shown in FIG. 3 and demonstrate that with faster cooling rates and a storage temperature beneath the glass transition temperature range of each solution (-115°C to -130°C), each of the conventional solutions tested can indeed achieve ice- free conditions (though these cooling rates produce excessive cracking of the solution, introducing a parallel challenge for effective cryopreservation of biological matter). However, when allowed to warm at approximately 5°C / min in air, each of these solutions then nucleated ice upon warming.

[0286] These tests demonstrate the characteristic differences between the invention of the present disclosure and the current state of the art in ice-free cryopreservation. Current ice- free cryopreservation solutions nigh-universally exhibit strong dependence on cooling and warming rate to avoid ice formation and require storage at temperatures beneath their glass transition temperatures. The high-entropy solutions of the present disclosure do not exhibit strong dependence on cooling or warming rate, being coolable and warmable at rates ranging from less than 0.1 °C to greater 10 °C without freezing, and they may be stored at temperatures above and below their glass transition temperatures without freezing. 6. Example 3

[0287] A preferable application of the methods of the present disclosure is presented below.

[0288] First, a high-entropy solution is designed and synthesized. It is then characterized to ensure that it will not freeze at a particularly desirable sub-0°C preservation temperature (e.g., -120 °C). It is then placed in contact with a complex biological sample, cooled to the desired temperature without freezing, and re-warmed. The biological sample is evaluated visually at multiple points in the cooling and warming process to confirm that no freezing has occurred within.

[0289] To design the high entropy solution in accordance with the methods of the present disclosure, a number and group of components are chosen. Each of the components is desirable for not being acutely toxic or for being commonly employed as a food additive, skin care additive, or similar, and select components are additionally desirable for high permeability, low viscosity, anti-oxidant properties, or osmoprotective properties. Aiming for high entropy and minimal mol fractions of each of the individual components in solution, a solution with 10Attorney Reference: BCHR-001WO principal non-water components and water (n = 11) is chosen comprising the following principal non-water components: hexylene glycol (molar volume about 128.03 mL / mol); sorbitol (molar volume about 122.26 mL / mol); betaine (molar volume about 117.15 mL / mol); xylitol (molar volume about 100.10 mL / mol); dimethyl sulfoxide (molar volume about 71.03 mL / mol); propylene glycol (molar volume about 73.16 mL / mol); glycerol (molar volume about 73.03 mL / mol); ethanol (molar volume about 58.32 mL / mol); ethylene glycol (molar volume about 55.92 mL / mol); and diglyme (molar volume about 141.96 mL / mol). Three additional non- principal non-water components were also added, which support biological function and minimize ischemic reperfusion injury after cryopreservation, but are not necessary contributors to the entropic suppression of ice formation: Vitamin-E and ascorbic acid, which are reactive oxygen species scavengers and anti-oxidants, and polyethylene glycol (35 kilo-Dalton molecular weight), which is a cell membrane stabilizer.

[0290] Next, a total mol fraction of water ^^ = 0.75 consistent with minimal toxicity,as evidenced by many cryoprotective non-high entropy solutions published previously in the scientific literature, are chosen.

[0291] Next, the concentrations of the principal components are selected. With 10 principal non-water components, the solution entropy is sufficiently high to suppress ice formation without computational optimization of the amount of each component. As such, other information may be used to select the amounts of each component, without sacrificing the high entropy nature of the solution. Hexylene glycol is selected as the most abundant principal component as it is highly permeating; dimethyl sulfoxide is selected as the second most abundant component as it possesses a low viscosity and enables better perfusion delivery; diglyme is selected as the third most abundant principal component as it exhibits low toxicity in in vitro cellular toxicity screens; and betaine is selected as the fourth most abundant principal component as it exhibits antioxidant and osmoprotective properties. The concentrations of the remaining principal components contribute principally to increasing entropy and thereby stability of the solution against ice formation, and are selected such that the mol fraction of the least abundant principal component is greater than or equal to one tenth the mol fraction of the most abundant principal component, producing a solution of the following composition:

[0292] Table 3. COMPONENT MOL FRACTION VOLUME FRACTIONAttorney Reference: BCHR-001WO Water 0.75 0.3426 Hexylene Glycol 0.0521 0.1693 5 6 2 5 5 4 2 9 3

[0293] The absolute value of the calculated entropy of mixing for this solution, using theequation ∆^^ = −^^^^ ln ^^ + ∑^^= ^^ ln^^^^^, is about 12.08 J / mol K.

[0294] In order to initially confirm that the solution will not freeze at sub-0°C temperatures, differential scanning calorimetry tests were performed on 15 mg samples of the solution at cooling and warming rates of 10 °C / minute. These tests showed no freezing. The glass transition temperature of the solution, such as about -105°C, was also demonstrated.

[0295] With this initial validation, increasingly large volumes of solution were tested at increasingly low cooling and warming rates. Solutions from 10 mL to 1L in volume were tested at cooling and warming rates from 5 °C / min to 0.1 °C / min, cooling to and warming from temperatures beneath the glass transition temperature of the solution (around -105 °C). For each test, the sample was continuously visually monitored for ice formation during cooling and warming. In all tests performed, no freezing was observed.

[0296] With the stability of the solution against freezing to a range of useful sub-0°C temperatures, the solution was tested for the preservation of a biological sample without freezing. To this end, two pig kidneys were procured from a local slaughterhouse. The solution was re-mixed using the University of Wisconsin organ preservation solution (a physiological- osmolality saline solution commonly used in clinical organ preservation) as the base instead of pure water, and to this solution the non-principal components were added. Vitamin E and ascorbic acid were each added at concentrations of 10 mM (about 0.01 mol%) and polyethyleneglycol was added last at a concentration of 1g per liter (about 5 × 10.O mol%). Cannulating theabdominal aorta, machine perfusion was used to perfuse the solution into each kidney, at an initial flow rate of about 10 mL per minute and an average pressure of about 40 mmHg. In orderAttorney Reference: BCHR-001WO to minimize potential stresses related to osmotic shock or vascular constriction or dilation, the kidneys were perfused in multiple concentration steps, i.e., starting with diluted forms of the solution. 25%-, 50%-, 75%-, and 100%-strength solution were used in these steps, where 25%- strength solution is prepared by mixing the solution with additional base University of Wisconsin solution in a ratio of 1:3 by volume, 50%-strength solution is prepared by mixing the solution with additional base University of Wisconsin solution in a ratio of 1:2 by volume, 75%- strength solution is prepared by mixing the solution with additional base University of Wisconsin solution in a ratio of 3:1 by volume, and 100%-strength is the solution as prepared. Approximately 250 mL of each of these four concentrations, chilled to about 4 °C in a refrigerator, were perfused through each kidney. Then, the kidney is incised along the coronal (frontal) plane through the hilum, separating it into anterior and posterior halves, thereby exposing the renal cortex, medulla, pyramids, calyces, and renal pelvis. Then, the two halves are reflected open and positioned with their cut (medial) surfaces flush against a borosilicate glass plate, exposing the internal architecture in direct contact with the surface of the plate. The specimens are then placed into a -80 °C freezer and the temperature of the kidney (monitored with a probe embedded into one of the halves) is allowed to reach approximately -80 °C over the course of a few hours. Once the temperature has reached steady state, the specimens are transferred into a -140 °C ultra-cold freezer to further reduce their temperature below the glass transition temperature (about -105 °C) at a rate of about 0.25 °C / min. After the temperature has equilibrated, the specimens are visually inspected through the glass pane for evidence of ice formation within the inner tissue architecture. Upon confirmation of the ice-free vitrified state, the kidney specimens are transferred back into the -80 °C freezer to begin the controlled process of rewarming above the glass transition temperature. Once the kidney temperature has rewarmed to -80 °C, the specimens are again visually inspected for evidence of ice formation. Then, the specimens are transferred (within a Styrofoam box) into a -20 °C freezer to initiate the controlled process of rewarming above the melting point. Throughout this process, the specimens are periodically inspected for evidence of ice formation. Once the temperature of the specimens has safely rewarmed to 0 °C, the test is concluded.

[0297] This experiment provides a useful example of the methods, compositions, and devices claimed herein. 7. Example 4

[0298] A preferable application of the methods of present disclosure is presented below.Attorney Reference: BCHR-001WO

[0299] First, a high-entropy solution is designed and synthesized. It is then characterized to ensure that it will not freeze at a particularly desirable sub-0°C preservation temperature (e.g., -140 °C). It is then placed in contact with a complex biological sample, cooled to the desired temperature without freezing, and rewarmed. The biological sample is visually inspected after cooling to and warming from below the glass transition temperature to ensure that no freezing has occurred within.

[0300] To design the high entropy solution in accordance with the methods of the present disclosure, a number and group of components are chosen. Each of the components is desirable for not being acutely toxic or for being commonly employed as a food additive, skin care additive, or similar, and select components are additionally desirable for high permeability, low viscosity, anti-oxidant properties, or osmoprotective properties. Aiming for high entropy and minimal mol fractions of each of the individual components in solution, a solution with 40 principal components (n = 40) is chosen comprising the following principal components: 2- isopropoxyethanol (molar volume about 114.45 mL / mol); 2-methoxy-1-propanol (molar volume about 96.08 mL / mol); 2-methoxyethanol (molar volume about 78.87 mL / mol); 2-methyl-2,4- pentanediol (molar volume about 128.03 mL / mol); 3-methoxy-1-butanol (molar volume about 112.23 mL / mol); 3-methoxy-3-methylbutan-1-ol (molar volume about 127.48 mL / mol); diacetone alcohol (molar volume about 123.84 mL / mol); diglyme (molar volume about 141.96 mL / mol); dimethylacetamide (molar volume about 92.39 mL / mol); methyl (s)-(-)-lactate (molar volume about 95.25 mL / mol); n-ethyl-n-methylformamide (molar volume about 99.23 mL / mol); tetraethylene glycol dimethyl ether (molar volume about 220.30 mL / mol); tetrahydro-4h-pyran- 4-ol (molar volume about 95.36 mL / mol); tetrahydrofurfuryl alcohol (molar volume about 96.86 mL / mol); triethylene glycol diacetate (molar volume about 209.66 mL / mol); triglyme (molar volume about 180.03 mL / mol); 1,5-pentanediol (molar volume about 141.00 mL / mol); n,n- dimethylpropionamide (molar volume about 109.95 mL / mol); 3-methoxy-1,2-propanediol (molar volume about 96.19 mL / mol); 3-methyl-1,3-butanediol (molar volume about 106.71 mL / mol); 2-methyl-1,3-propanediol (molar volume about 88.79 mL / mol); 2,3-butanediol (molar volume about 86.24 mL / mol); 2-methylpropane-1,2-diol (molar volume about 90.12 mL / mol); propionamide (molar volume about 66.45 mL / mol); formamide (molar volume about 39.75 mL / mol); 1-(2-hydroxyethyl)-2-pyrrolidinone (molar volume about 113.00 mL / mol); 1,2- propanediol (molar volume about 73.16 mL / mol); dimethyl sulfoxide (molar volume about 70.97 mL / mol); 1,3-propanediol (molar volume about 71.78 mL / mol); triethanolamine (molarAttorney Reference: BCHR-001WO volume about 132.02 mL / mol); n-(2-hydroxyethyl)acetamide (molar volume about 92.07 mL / mol); diethylene glycol (molar volume about 94.75 mL / mol); ethylene glycol (molar volume about 55.92 mL / mol); n-methylacetamide (molar volume about 78.00 mL / mol); tetraethylene glycol (molar volume about 173.42 mL / mol); triethylene glycol (molar volume about 133.48 mL / mol); 1,2,4-butanetriol (molar volume about 89.18 mL / mol); 1,3- dihydroxyacetone (molar volume about 69.29 mL / mol); acetamide (molar volume about 50.92 mL / mol); glycerol (molar volume about 73.03 mL / mol).

[0301] Next, the proportions of the principal components are selected. With such a large number of components the entropy of the solution dominates, and individual component toxicities, viscosities, and permeabilities contribute negligibly to the ensemble behavior. The 40 components are therefore mixed in equiatomic proportion.

[0302] Next, a total mol fraction of water ^^ = 0.76 is chosen, consistent with minimaltoxicity, as evidenced by many cryoprotective non-high entropy solutions published previously in the scientific literature, producing a solution with the following composition:

[0303] Table 4. COMPONENT MOL FRACTION VOLUME FRACTIONAttorney Reference: BCHR-001WO Triethylene Glycol Diacetate 0.006 1.2579 Triglyme 0.006 1.0802

[0304] The absolute value of the calculated entropy of mixing for this solution, using theequation ∆^^ = −^^^^ ln ^^ + ∑^^= ^^ ln^^^^^, is about 15.0 J / mol K.

[0305] Five additional non-principal components are also added, which support biological function and minimize ischemic reperfusion injury after cryopreservation, but are notAttorney Reference: BCHR-001WO necessary contributors to the entropic suppression of ice formation: Vitamin-E, ascorbic acid, dexamethasone, insulin, and polyethylene glycol (35 kilo-Dalton molecular weight).

[0306] Upon preparation of this solution, the pH is adjusted as necessary using HEPES buffer and NaOH to achieve a target pH of 7.4 at the perfusion temperature (about 4 °C).

[0307] In order to initially confirm that the solution will not freeze at sub-0°C temperatures, differential scanning calorimetry tests are performed on 15 mg samples of the solution at cooling and warming rates of 10 °C / minute. These tests show no freezing. The glass transition temperature of the solution, such as about -85 °C, is also demonstrated.

[0308] With this initial validation, increasingly large volumes of solution are tested at increasingly low cooling and warming rates. Solutions from 10 mL to 1L in volume are tested at cooling and warming rates from 5 °C / min to 0.1 °C / min, cooling to and warming from temperatures beneath the glass transition temperature of the solution (around -85 °C). For each test, the sample is continuously visually monitored for ice formation during cooling and warming. In all tests performed, no freezing is observed.

[0309] With the stability of the solution against freezing to a range of useful sub-0°C temperatures verified, the solution is tested for the preservation of a biological sample without freezing. To this end, a porcine liver is procured from a local slaughterhouse. The solution is re- mixed using the University of Wisconsin machine perfusion solution (a physiological- osmolality saline solution commonly used in clinical perfusion-based organ preservation) as the base instead of pure water, and to this solution the non-principal components are added. Vitamin E and ascorbic acid are each added at concentrations of 10 mM (about 0.01 mol%), in additionto 200 units / L of insulin, 25 mg / L of dexamethasone (about 1.15 × 10.Q mol%), andpolyethylene glycol is added last at a concentration of 1g per liter (about 5 × 10.O mol%).Cannulating the portal vein and the hepatic artery, machine perfusion is used to perfuse the solution into the liver, at an initial flow rate of about 60 mL per minute. In order to minimize potential stresses related to osmotic shock or vascular constriction or dilation, the concentration of the perfusate is gradually ramped up starting from 0% strength and increasing to full strength (volumetric dilution) over the course of 150 minutes. A thermocouple is then inserted into the portal vein and the liver is then placed in a polyethylene bag containing the full-strength solution. The bag is then sealed and placed in a large, insulated tub of dry ice at -80 °C, such that the volume of dry ice is much larger than that of the bag. The liver is left on dry ice to allow the temperature of the liver to decrease in a controlled fashion to about -80 °C. After theAttorney Reference: BCHR-001WO temperature reaches steady state, the liver is transferred to a -140 °C freezer to further reduce the temperature of the liver below the glass transition temperature (about -85 °C). After the temperature of the liver reaches a temperature of -140 °C, the liver is transferred back into a tub of dry ice in order to rewarm the liver in a controlled fashion back above the glass transition temperature. Once the temperature of the liver reaches -80 °C, the liver was segmented by bisecting through the thickest portion of each lobe, and the tissue was visually inspected for evidence of ice formation. No ice formation was observed in any portion of the tissue including the most difficult to perfuse and least vascularized regions. The test was subsequently terminated with the conclusion that the high-entropy solution successfully prevented ice formation in the liver during cooling to and warming from below the glass transition temperature.

[0310] This experiment provides a useful example of the methods, compositions, and devices claimed herein.

[0311] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

Attorney Reference: BCHR-001WO CLAIMS What is claimed is:

1. A high entropy solution comprising: a. water; b. two or more non-water chemical components with different physical or thermodynamic properties; and c. an entropy of mixing equal to or greater than 7 J / (mol K), wherein the high entropy solution does not freeze at a temperature between 0 °C and about -200 °C.

2. The high entropy solution of claim 1, wherein the number of non-water chemical components is between 2 and 100.

3. The high entropy solution of claim 2 or 3, wherein the entropy of mixing increases with the number of the non-water chemical components.

4. The high entropy solution of any one of claims 1-3, wherein the non-water chemical components are non-water carbon-based components with molar volumes between about 40 mL / mol and about 400 mL / mol.

5. The high entropy solution of claim 4, wherein the non-water chemical components are non-water carbon-based components with molar volumes between about 40 mL / mol and about 150 mL / mol.Attorney Reference: BCHR-001WO 6. The high entropy solution of any one of claims 1-5, wherein molar volumes of non-water chemical components are different from one another, and molar volumes of non-water chemical components are different from molar volume of the water.

7. The high entropy solution of any one of claims 1-6, wherein the entropy of mixing increases when the average molar volume of the non-water chemical components increases.

8. The high entropy solution of any one of claims 1-7, wherein the average molar volume of the high entropy solution is greater than 18 ml / mol.

9. The high entropy solution of claim 8, wherein the average molar volume of the high entropy solution is greater than 30 ml / mol.

10. The high entropy solution of any one of claims 1-9, wherein the entropy of mixing of the high entropy solution is at least 11 J / (mol K).

11. The high entropy solution of any one of claims 1-10, wherein mol fractions of the non- water chemical components are different from one another, and mol fractions of the non- water chemical components are different from mol fraction of water.

12. The high entropy solution of any one of claims 1-11, wherein the mol fractions of the non-water chemical components ascend in magnitude with ascending molar volumes of the non-water chemical components.

13. The high entropy solution of any one of claims 1-12, wherein mol fraction of the water is between 0.65 and 0.99.Attorney Reference: BCHR-001WO 14. The high entropy solution of any one of claims 1-13, wherein mol fraction of the water is constant while mol fractions of the non-water chemical components vary to increase the entropy of mixing.

15. The high entropy solution of any one of claims 1-14, wherein the number of the non- water chemical components is equal to or greater than 3, and the mol fraction of at least one of the non-water chemical components is constant and mol fractions of at least two of the non-water chemical components vary to increase the entropy of mixing.

16. The high entropy solution of any one of claims 1-15, wherein the entropy of mixing in the liquid phase is calculated by entropy of mixing equation: ^ wherein ^^and^^and ^^are respectively mol and volume fractions of each of n non-water chemical components; n is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^is entropy of mixing.

17. The high entropy solution of any one of claims 1-16, wherein the entropy of mixing in the liquid phase is calculated by ideal entropy of mixing equation: ^wherein ^^of n non-water chemical components; n is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^.^&(F#is ideal entropy of mixing.Attorney Reference: BCHR-001WO 18. The high entropy solution of claim 17, wherein the mol fractions and the volume fractions of the non-water chemical components, the mol fraction and the volume fraction of the water, and the number of the non-water chemical components are determined to increase the entropy of mixing.

19. The high entropy solution of claim 17 or 18, wherein the mol fractions and the volume fractions of the non-water chemical components and the mol fraction and the volume fraction of the water are necessary to find or approach a local or global extremum in the entropy of mixing equation.

20. The high entropy solution of any one of claims 1-19, wherein the number of non-water chemical components is equal to or greater than 3 and the high entropy solution comprises: a. water with molar volume about 18 mL / mol; b. a first group comprising one or more non-water chemical components in molar volume range between about 40 mL / mol and about 75 mL / mol; c. a second group comprising one or more non-water chemical components in molar volume range between about 75 mL / mol and about 100 mL / mol; and d. a third group comprising one or more non-water chemical components in molar volume range between about 100 mL / mol and about 150 mL / mol.

21. The high entropy solution of claim 20, wherein the molar volumes of the non-water chemical components in the first group are different from one another, the molar volumes of the non-water chemical components in the second group are different from one another, and the molar volumes of the non-water chemical components in the third group are different from one another.Attorney Reference: BCHR-001WO 22. The high entropy solution of any one of claims 1-21, wherein the water is replaced by a saline solution, a hypothermic organ preservation solution, or a base solution containing electrolytes.

23. The high entropy solution of claim 22, wherein the saline solution is selected from the group consisting of University of Wisconsin solution, Celsior® solution, Custodiol® HTK solution, Del Nidos solution, LM5 solution, B2 solution, phosphate buffered saline solution (PBS solution), Belzer Machine Perfusion Solution (Belzer MPS), filtered seawater, Lactated Ringers solution, isotonic saline solution containing 0.9 percent sodium chloride (salt) and hypotonic saline solution containing 0.45 percent sodium chloride.

24. The high entropy solution of any one of claims 1-23, wherein any of the non-water chemical components is selected from glycols, sugar alcohols, methylated organic compounds, methoxylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers.

25. The high entropy solution of claim 24, wherein any of the non-water chemical components is selected from ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di-ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol, threitol, ethanol, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose, trehalose, 1,2,4-butanetriol, 1,2,6-hexanetriol, 1,2-butanediol, 1,2-hexanediol, 1,2-pentanediol, 1,2-propanediol, 1,3-butanediol, 1,3-diaminopropane, 1,3-dihydroxyacetone, 1,3-dimethlurea, 1,3-propanediol, 1,4-butanediol, 1,5- pentanediol, 1,6-hexanediol, 1-2-hydroxyethyl-2-pyrrolidinone, 1-amino-2-propanol, 1- methyl-pyrrolidinone, 1-propanol, 2,3-butanediol, 2,5-dimethyl-2,5-hexanediol, 2-Attorney Reference: BCHR-001WO hydroxymethyl-1,3-propanediol, 2-isopropoxyethanol, 2-Methoxyethanol, 2-methyl-1,3- propanediol, 2-methyl-2,4-pentanediol, 2-propanol, 2-pyrrolidone, 3-amino-1-propanol, 3-methoxy-1,2-propanediol, 3-methoxy-1-butanol, 3-methoxy-1-propanol, 3-methyl-1,3- butanediol, Acetamide, Acetic acid, Acetone, Allantoin, Aspartame, Betaine, Citric acid, Diacetone alcohol, Diacetyl, Di-ethylene-glycol, Diethylene-glycol-diethyl-ether, Diethylene-glycol-dimethyl-ether, Diethylene-glycol-monoethyl-ether, Diethylene- glycol-monomethyl-ether, Diglycerol, Dimethyl isosorbide, Dimethyl sulfone, Dimethyl sulfoxide, Dipropylene-glycol-dimethyl-ether, Dipropylene-glycol-monomethyl-ether, Erythritol, Ethanol, Ethanolamine, Ethylene diamine, Ethylene glycol, Ethylene-glycol- diethyl-ether, Ethylene-glycol-dimethyl-ether, Ethylene-glycol-monoethyl-ether, Ethylene-glycol-monopropyl-ether, Formamide, Formic acid, Fructose, Galatose, Glucose, Glycerol, Glycerol formal, Glycine, Hydroxy-acetone, Isoascorbic acid, Isosorbide, Lactic acid, L-Arginine, Malic acid, Maltitol, Maltose, Mannitol, Meglumine, Methanol, Methyl-(S)-(-)-lactate, N,N-dimethylacetamide, N,N- dimethylformamide, N,N-dimethylpropionamide, N-Acetylethanolamine, N- acetylglycine, N-Formylmorpholine, Nicotinamide, N-methyl-2-pyrrolidone, N- methylacetamide, N-Methyldiethanolamine, N-methylformamide, Oxalic acid, PEG- 200, PEG-300, PEG-400, Proline, Propionamide, Propylene-glycol-monomethyl-ether, Propylene-glycol-monopropyl-ether, Pyridine, Raffinose, Sorbitol, Sucrose, Sulfolane, Tartaric acid, Taurine, Tert-butanol, Tetraethylene glycol, Tetraethylene-glycol- dimethyl-ether, Tetrahydro-4-pyranol, Tetrahydrofurfuryl alcohol, Trehalose, Triethanolamine, Tri-ethylene-glycol, Triethylene-glycol-dimethyl-ether, Triethylene- glycol-monobutyl-ether, Triethylene-glycol-monoethyl-ether, Triethylene-glycol- monomethyl-ether, Triglycerol, Trimethylamine n-oxide, Trimethylolpropane, Tripropylene-glycol-monomethyl-ether, TRIS, and Urea.Attorney Reference: BCHR-001WO 26. The high entropy solution of any one of claims 1-25, comprising: a. water at a mol fraction between 0.65 and 0.99; b. hexylene glycol at a mol fraction greater than all other non-water chemical components; c. sorbitol at a mol fraction less than the mol fraction of the hexylene glycol but greater than all other non-water chemical components; d. glucose at a mol fraction less than the mol fractions of the sorbitol and the hexylene glycol but greater than all other non-water chemical components; e. xylitol at a mol fraction less than the mol fractions of the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; f. butylene glycol at a mol fraction less than the mol fractions of the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non- water chemical components; g. propylene glycol at a mol fraction less than the mol fractions of the butylene glycol, the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; h. glycerol at a mol fraction less than the mol fractions of the propylene glycol, the butylene glycol, the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; i. ethanol at a mol fraction less than the mol fractions of the glycerol, the butylene glycol, the xylitol, the glucose, the sorbitol, and the hexylene glycol, but greater than all other non-water chemical components; j. ethylene glycol at a mol fraction less than all other non-water chemical components; andAttorney Reference: BCHR-001WO k. an entropy of mixing equal to or greater than 7 J / (mol K) as calculated using the entropy of mixing equation that accepts both mol fractions and volume fractions as arguments.

27. The high entropy solution of any one of claims 1-26, wherein the high entropy solution further comprises trehalose, sucrose, polyethylene glycol, polyvinyl alcohol, or another polymer with molar volume greater than 150 mL / mol in an amount less than 50% by mass.

28. The high entropy solution of any one of claims 1-27, wherein the high entropy solution further comprises a surfactant in an amount less than 50% by mass.

29. The high entropy solution of any one of claims 1-28, wherein the non-water chemical components are biocompatible.

30. The high entropy solution of any one of claims 1-29, wherein the non-water chemical components are non-toxic.

31. The high entropy solution of any one of claims 1-30, further comprising a pH adjusting compound.

32. The high entropy solution of any one of claims 1-31, further comprising a vasodilation inducing compound.

33. The high entropy solution of any one of claims 1-32, further comprising a compound providing oncotic support to an organ.Attorney Reference: BCHR-001WO 34. A method of producing a high entropy solution, the method comprising: a. selecting two or more non-water chemical components with different physical or thermodynamic properties and water; b. determining each amount of the non-water chemical components and amount of the water to increase an entropy of mixing; and c. mixing the non-water chemical components and the water with the determined amounts to produce the high entropy solution, wherein the high entropy solution does not freeze at a temperature between 0 °C and about -200 °C.

35. The method of any one of claim 34, wherein the entropy of mixing in the high entropy solution is equal to or greater than 7 J / (mol K).

36. The method of any one of claims 34-35, wherein the number of the non-water chemical components is between 2 and 100.

37. The method of any one of claims 34-36, wherein the entropy of mixing increases with the number of the non-water chemical components.

38. The method of any one of claims 34-37, wherein the non-water chemical components are non-water carbon-based components with molar volumes between about 40 mL / mol and about 400 mL / mol.

39. The method of claim 38, wherein the non-water chemical components are non-water carbon-based components with molar volumes between about 40 mL / mol and about 150 mL / mol.Attorney Reference: BCHR-001WO 40. The method of any one of claims 34-39, wherein molar volumes of non-water chemical components are different from one another, and molar volumes of non-water chemical components are different from molar volume of the water.

41. The method of any one of claims 34-40, wherein mol fractions of the non-water chemical components are different from one another, and mol fractions of the non-water chemical components are different from mol fraction of water.

42. The method of any one of claims 34-41, wherein the mol fractions of the non-water chemical components ascend in magnitude with ascending molar volumes of the non- water chemical components.

43. The method of any one of claims 34-42, wherein mol fraction of the water is between 0.65 and 0.

99.

44. The method of any one of claims 34-43, wherein mol fraction of the water is constant while mol fractions of the non-water chemical components vary to increase the entropy of mixing.

45. The method of any one of claims 34-44, wherein the number of the non-water chemical components is equal to or greater than 3, and mol fraction of at least one of the non- water chemical components are constant and mol fractions of at least two of the non- water chemical components vary to increase the entropy of mixing.

46. The method of any one of claims 34-45, wherein the number of non-water chemical components is equal to or greater than 3 and the high entropy solution comprises: a. water with molar volume about 18 mL / mol;Attorney Reference: BCHR-001WO b. a first group comprising one or more non-water chemical components in molar volume range between about 40 mL / mol and about 75 mL / mol; c. a second group comprising one or more non-water chemical components in molar volume range between about 75 mL / mol and about 100 mL / mol; and d. a third group comprising one or more non-water chemical components in molar volume range between about 100 mL / mol and about 150 mL / mol.

47. The method of claim 34-46, wherein the molar volumes of the non-water chemical components in the first group are different from one another; the molar volumes of the non-water chemical components in the second group are different from one another; and the molar volumes of the non-water chemical components in the third group are different from one another.

48. The method of any one of claims 34-47, wherein the water is replaced by a physiological saline solution, a hypothermic organ preservation solution, or a base solution containing electrolytes.

49. The method of any one of claims 34-48, wherein any of the non-water chemical components is selected from glycols, sugar alcohols, methylated organic compounds, methoxylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers.

50. The method of claim 49, wherein any of the non-water chemical components is selected from ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di-ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol, threitol, ethanol, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose,Attorney Reference: BCHR-001WO trehalose, of ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di- ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol, threitol, ethanol, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose, trehalose, 1,2,4-butanetriol, 1,2,6-hexanetriol, 1,2-butanediol, 1,2-hexanediol, 1,2-pentanediol, 1,2-propanediol, 1,3-butanediol, 1,3-diaminopropane, 1,3- dihydroxyacetone, 1,3-dimethlurea, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1-2-hydroxyethyl-2-pyrrolidinone, 1-amino-2-propanol, 1-methyl- pyrrolidinone, 1-propanol, 2,3-butanediol, 2,5-dimethyl-2,5-hexanediol, 2- hydroxymethyl-1,3-propanediol, 2-isopropoxyethanol, 2-Methoxyethanol, 2-methyl-1,3- propanediol, 2-methyl-2,4-pentanediol, 2-propanol, 2-pyrrolidone, 3-amino-1-propanol, 3-methoxy-1,2-propanediol, 3-methoxy-1-butanol, 3-methoxy-1-propanol, 3-methyl-1,3- butanediol, Acetamide, Acetic acid, Acetone, Allantoin, Aspartame, Betaine, Citric acid, Diacetone alcohol, Diacetyl, Di-ethylene-glycol, Diethylene-glycol-diethyl-ether, Diethylene-glycol-dimethyl-ether, Diethylene-glycol-monoethyl-ether, Diethylene- glycol-monomethyl-ether, Diglycerol, Dimethyl isosorbide, Dimethyl sulfone, Dimethyl sulfoxide, Dipropylene-glycol-dimethyl-ether, Dipropylene-glycol-monomethyl-ether, Erythritol, Ethanol, Ethanolamine, Ethylene diamine, Ethylene glycol, Ethylene-glycol- diethyl-ether, Ethylene-glycol-dimethyl-ether, Ethylene-glycol-monoethyl-ether, Ethylene-glycol-monopropyl-ether, Formamide, Formic acid, Fructose, Galatose, Glucose, Glycerol, Glycerol formal, Glycine, Hydroxy-acetone, Isoascorbic acid, Isosorbide, Lactic acid, L-Arginine, Malic acid, Maltitol, Maltose, Mannitol, Meglumine, Methanol, Methyl-(S)-(-)-lactate, N,N-dimethylacetamide, N,N- dimethylformamide, N,N-dimethylpropionamide, N-Acetylethanolamine, N- acetylglycine, N-Formylmorpholine, Nicotinamide, N-methyl-2-pyrrolidone, N- methylacetamide, N-Methyldiethanolamine, N-methylformamide, Oxalic acid, PEG- 200, PEG-300, PEG-400, Proline, Propionamide, Propylene-glycol-monomethyl-ether,Attorney Reference: BCHR-001WO Propylene-glycol-monopropyl-ether, Pyridine, Raffinose, Sorbitol, Sucrose, Sulfolane, Tartaric acid, Taurine, Tert-butanol, Tetraethylene glycol, Tetraethylene-glycol- dimethyl-ether, Tetrahydro-4-pyranol, Tetrahydrofurfuryl alcohol, Trehalose, Triethanolamine, Tri-ethylene-glycol, Triethylene-glycol-dimethyl-ether, Triethylene- glycol-monobutyl-ether, Triethylene-glycol-monoethyl-ether, Triethylene-glycol- monomethyl-ether, Triglycerol, Trimethylamine n-oxide, Trimethylolpropane, Tripropylene-glycol-monomethyl-ether, TRIS, and Urea.

51. The method of any one of claims 34-50, wherein the non-water chemical components are biocompatible or non-toxic.

52. The method of any one of claims 34-51, herein the entropy of mixing in the liquid phase is calculated by entropy of mixing equation: ^ wherein ^^and^^and ^^are respectively mol and volume fractions of each of n non-water chemicaln is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^is entropy of mixing.

53. The high entropy solution of any one of claims 34-51, wherein the entropy of mixing in the liquid phase is calculated by ideal entropy of mixing equation: ^wherein ^^of n non-water chemical components; n is the number of the non-water chemical components and n is anAttorney Reference: BCHR-001WO integer equal to or greater than 2; R is the ideal gas constant; and ∆^^.^&(F#is ideal entropy of mixing.

54. The method of claim 34-53, wherein the mol fractions and the volume fractions of the non-water chemical components, the mol fraction and the volume fraction of the water, and the number of the non-water chemical components are determined to increase the entropy of mixing.

55. The method of any one of claims 34-54, wherein the mol fractions and the volume fractions of the non-water chemical components and the mol fraction and the volume fraction of the water are necessary to find or approach a local or global extremum in the entropy of mixing equation.

56. The method of any one of claims 34-55, wherein optimization of the entropy of mixing equation is performed by using a computational optimization method.

57. The method of claim 56, wherein the computational optimization method includes a brute force optimization method, a binary search optimization method, a convex optimization method, a stochastic optimization method, or a particle swarm optimization method.

58. A high entropy solution with principal non-water chemical components (PNWCCs), the high entropy solution comprising: a. water; and b. at least 10 PNWCCs, wherein the mol fraction of the most abundant PNWCC is not more than 10 times the mol fraction of the least abundant PNWCC.Attorney Reference: BCHR-001WO 59. The high entropy solution of claim 58, wherein the at least 10 PNWCCs is 10 to about 100 PNWCCs.

60. The high entropy solution of any one of claims 58-59, further comprising at least 1 non- principal non-water component (N-PNWCCs).

61. The high entropy solution of claim 60, wherein the at least 1 N-PNWCC is 1 to about 100 N-PNWCCs.

62. The high entropy solution of any one of claims 60-61, wherein each amount of N- PNWCC is such that the most abundant N-PNWCC is less than one tenth the mol fraction of the most abundant PNWCC.

63. The high entropy solution of any one of claims 58-62, wherein the PNWCCs are non- water carbon-based components with molar volumes between about 40 mL / mol and about 400 mL / mol.

64. The high entropy solution of any one of claims 58-63, wherein the PNWCCs are non- water carbon-based components with molar volumes between about 40 mL / mol and about 150 mL / mol.

65. The high entropy solution of any one of claims 58-64, wherein the average molar volume of the high entropy solution is greater than 18 ml / mol.

66. The high entropy solution of claim 65, wherein the average molar volume of the high entropy solution is greater than 30 ml / mol.Attorney Reference: BCHR-001WO 67. The high entropy solution of any one of claims 58-66, wherein the entropy of mixing of the high entropy solution is at least 11 J / (mol K).

68. The high entropy solution of any one of claims 58-67, wherein molar volumes of PNWCCs are different from one another, and molar volumes of PNWCCs are different from molar volume of the water.

69. The high entropy solution of any one of claims 58-68, wherein the weighted sample standard deviation of the molar volumes of the PNWCCs ranges from 1 ml / mol to 100 ml / mol, such as 5 ml / mol to 50 ml / mol or 10 ml / mol to 25 ml / mol.

70. The high entropy solution of any one of claims 58-69, wherein the entropy of mixing increases when the average molar volume of the PNWCCs increase.

71. The high entropy solution of any one of claims 58-70, wherein mol fractions of the PNWCCs are different from one another, and mol fractions of the PNWCCs are different from mol fraction of water.

72. The high entropy solution of any one of claims 58-71, wherein the mol fractions of the PNWCCs ascend in magnitude with ascending molar volumes of the PNWCCs.

73. The high entropy solution of any one of claims 58-72, wherein mol fraction of the water is between 0.65 and 0.

99.

74. The high entropy solution of any one of claims 58-73, wherein mol fraction of the water is constant while mol fractions of the PNWCCs vary to increase the entropy of mixing.Attorney Reference: BCHR-001WO 75. The high entropy solution of any one of claims 58-74, wherein the entropy of mixing in the liquid phase is calculated by entropy of mixing equation: ^ ∆^^ = −^ C^^ ln ^^ + D ^^ln^^^^Ewherein ^^and^^and ^^are respectively mol and volume fractions of each of n PNWCCs; n is the number of the PNWCCs and n is an integer equal to or greater than 10; R is the ideal gas constant; and ∆^^is entropy of mixing.

76. The high entropy solution of any one of claims 58-74, wherein the entropy of mixing in the liquid phase is calculated by ideal entropy of mixing equation: ^ wherein ^^of n PNWCCs; n is the number of the PNWCCs and n is an integer equal to or greater than 10; R is the ideal gas constant; and ∆^^.^&(F#is ideal entropy of mixing.

77. The high entropy solution of any one of claims 58-76, wherein the mol fractions and the volume fractions of the PNWCCs, the mol fraction and the volume fraction of the water, and the number of the PNWCCs are determined to increase the entropy of mixing.

78. The high entropy solution of any one of claims 58-77, wherein the mol fractions and the volume fractions of the PNWCCs and the mol fraction and the volume fraction of the water are necessary to find or approach a local or global extremum in the entropy of mixing equation.Attorney Reference: BCHR-001WO 79. The high entropy solution of any one of claims 58-78, wherein the number of PNWCCs is equal to or greater than 10 and the high entropy solution comprises: a. water with molar volume about 18 mL / mol; b. a first group comprising one or more PNWCCs in molar volume range between about 40 mL / mol and about 75 mL / mol; c. a second group comprising one or more PNWCCs in molar volume range between about 75 mL / mol and about 100 mL / mol; and d. a third group comprising one or more PNWCCs in molar volume range between about 100 mL / mol and about 150 mL / mol.

80. The high entropy solution of any one of claims 58-79, wherein the molar volumes of the PNWCCs in the first group are different from one another, the molar volumes of the PNWCCs in the second group are different from one another, and the molar volumes of the PNWCCs in the third group are different from one another.

81. The high entropy solution of any one of claims 58-80, wherein the water is replaced by a saline solution, a hypothermic organ preservation solution, or a base solution containing electrolytes.

82. The high entropy solution of any one of claims 58-81, wherein the saline solution is selected from the group consisting of University of Wisconsin solution, Celsior® solution, Custodiol® HTK solution, Del Nidos solution, LM5 solution, B2 solution, phosphate buffered saline solution (PBS solution), Belzer Machine Perfusion Solution (Belzer MPS), filtered seawater, Lactated Ringers solution, isotonic saline solution containing 0.9 percent sodium chloride (salt) and hypotonic saline solution containing 0.45 percent sodium chloride.Attorney Reference: BCHR-001WO 83. The high entropy solution of any one of claims 58-82, wherein any of the PNWCCs is selected from glycols, sugar alcohols, methylated organic compounds, methoxylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers.

84. The high entropy solution of any one of claims 58-83, wherein any of the PNWCCs is selected from ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di- ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol, threitol, ethanol, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose, trehalose, 1,2,4-butanetriol, 1,2,6-hexanetriol, 1,2-butanediol, 1,2-hexanediol, 1,2-pentanediol, 1,2-propanediol, 1,3-butanediol, 1,3-diaminopropane, 1,3- dihydroxyacetone, 1,3-dimethlurea, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1-2-hydroxyethyl-2-pyrrolidinone, 1-amino-2-propanol, 1-methyl- pyrrolidinone, 1-propanol, 2,3-butanediol, 2,5-dimethyl-2,5-hexanediol, 2- hydroxymethyl-1,3-propanediol, 2-isopropoxyethanol, 2-Methoxyethanol, 2-methyl-1,3- propanediol, 2-methyl-2,4-pentanediol, 2-propanol, 2-pyrrolidone, 3-amino-1-propanol, 3-methoxy-1,2-propanediol, 3-methoxy-1-butanol, 3-methoxy-1-propanol, 3-methyl-1,3- butanediol, Acetamide, Acetic acid, Acetone, Allantoin, Aspartame, Betaine, Citric acid, Diacetone alcohol, Diacetyl, Di-ethylene-glycol, Diethylene-glycol-diethyl-ether, Diethylene-glycol-dimethyl-ether, Diethylene-glycol-monoethyl-ether, Diethylene- glycol-monomethyl-ether, Diglycerol, Dimethyl isosorbide, Dimethyl sulfone, Dimethyl sulfoxide, Dipropylene-glycol-dimethyl-ether, Dipropylene-glycol-monomethyl-ether, Erythritol, Ethanol, Ethanolamine, Ethylene diamine, Ethylene glycol, Ethylene-glycol- diethyl-ether, Ethylene-glycol-dimethyl-ether, Ethylene-glycol-monoethyl-ether, Ethylene-glycol-monopropyl-ether, Formamide, Formic acid, Fructose, Galatose, Glucose, Glycerol, Glycerol formal, Glycine, Hydroxy-acetone, Isoascorbic acid,Attorney Reference: BCHR-001WO Isosorbide, Lactic acid, L-Arginine, Malic acid, Maltitol, Maltose, Mannitol, Meglumine, Methanol, Methyl-(S)-(-)-lactate, N,N-dimethylacetamide, N,N- dimethylformamide, N,N-dimethylpropionamide, N-Acetylethanolamine, N- acetylglycine, N-Formylmorpholine, Nicotinamide, N-methyl-2-pyrrolidone, N- methylacetamide, N-Methyldiethanolamine, N-methylformamide, Oxalic acid, PEG- 200, PEG-300, PEG-400, Proline, Propionamide, Propylene-glycol-monomethyl-ether, Propylene-glycol-monopropyl-ether, Pyridine, Raffinose, Sorbitol, Sucrose, Sulfolane, Tartaric acid, Taurine, Tert-butanol, Tetraethylene glycol, Tetraethylene-glycol- dimethyl-ether, Tetrahydro-4-pyranol, Tetrahydrofurfuryl alcohol, Trehalose, Triethanolamine, Tri-ethylene-glycol, Triethylene-glycol-dimethyl-ether, Triethylene- glycol-monobutyl-ether, Triethylene-glycol-monoethyl-ether, Triethylene-glycol- monomethyl-ether, Triglycerol, Trimethylamine n-oxide, Trimethylolpropane, Tripropylene-glycol-monomethyl-ether, TRIS, and Urea.

85. The high entropy solution of any one of claims 58-84, wherein the high entropy solution further comprises trehalose, sucrose, polyethylene glycol, polyvinyl alcohol, or another polymer with molar volume greater than 150 mL / mol in an amount less than 50% by mass.

86. The high entropy solution of any one of claims 58-85, wherein the high entropy solution further comprises a surfactant in an amount less than 50% by mass.

87. The high entropy solution of any one of claims 58-86, wherein the non-water chemical components are biocompatible, non-toxic, or a combination thereof.

88. The high entropy solution of any one of claims 58-87, further comprising a pH adjusting compound.Attorney Reference: BCHR-001WO 89. The high entropy solution of any one of claims 58-88, further comprising a vasodilation inducing compound.

90. The high entropy solution of any one of claims 58-89, further comprising a compound providing oncotic support to an organ.

91. A method of producing a high entropy solution with principal non-water chemical components (PNWCCs), the method comprising: a) selecting at least 10 PNWCCs, optionally selecting one or more non-principal non-water components (N-PNWCCs), and water; b) determining each amount of PNWCCs such that the mol fraction of the most abundant PNWCCs is not more than 10 times the mol fraction of the least abundant PNWCCs, thereby resulting in a sufficient entropy of mixing to suppress ice formation based on number of PNWCCs; and c) if one or more N-PNWCCs were selected, determining each amount of N- PNWCCs such that the most abundant N-PNWCCs is less than one tenth the mol fraction of the most abundant PNWCC; and d) mixing the PNWCCs, the N-PNWCCs, and the water with the determined amounts to produce the high entropy solution.

92. A high entropy solution comprising: water; and 2 or more principal non-water chemical components (PNWCCs), wherein the high entropy solution has an entropy of mixing of 7 J / (mol K) or more, wherein the high entropy solution does not freeze when cooling from 0 °C to a lower temperature or when warming from the lower temperature to 0 °C, wherein the lower temperature is -80 °C, wherein the cooling and warming are both performed at a rate of 1 °C / min and a pressure of 101 kPa.Attorney Reference: BCHR-001WO 93. The high entropy solution of claim 92, wherein the 2 or more PNWCCs is 4 or more or more PNWCCs, such as 6 or more, 8 or more, 10 or more, or 12 or more.

94. The high entropy solution of any one of claims 92-93, wherein the total mol fraction of the PNWCCs is 0.1 or more, such as 0.2 or more, 0.3 or more, or 0.4 or more.

95. The high entropy solution of any one of claims 92-94, wherein the mol fraction of each PNWCC ranges from 0.001 to 0.

015.

96. The high entropy solution of any one of claims 92-95, wherein at least 1 PNWCC is an organic compound with a molar volume ranging from about 40 ml / mol to about 400 ml / mol, such as 60 ml / mol to 300 ml / mol, such as at least 2 PNWCCs, at least 3 PNWCCs, at least 4 PNWCCs, at least 5 PNWCCs, at least 6 PNWCCs, or at least 7 PNWCCs.

97. The high entropy solution of any one of claims 92-96, wherein at least 1 PNWCC is selected from the group consisting of glycols, sugar alcohols, methylated organic compounds, methoxylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers, such as at least 2 PNWCCs, at least 3 PNWCCs, at least 5 PNWCCs, or at least 7 PNWCCs.

98. The high entropy solution of any one of claims 92-97, wherein at least 1 PNWCC is selected from the group consisting of ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, di-ethylene glycol, polyethylene glycol, propanediol, glycerol, sorbitol, xylitol, erythritol, mannitol, dulcitol, arabitol, ribitol, threitol, ethanol, methanol, dimethylsulfoxide, polyvinyl alcohol, acetamide, formamide, glycine, proline, betaine, glucose, fructose, sucrose, trehalose, 1,2,4-butanetriol, 1,2,6-hexanetriol, 1,2-butanediol, 1,2-hexanediol, 1,2-pentanediol, 1,2-propanediol, 1,3-butanediol, 1,3-diaminopropane, 1,3-dihydroxyacetone, 1,3-dimethlurea, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1-2-hydroxyethyl-2- pyrrolidinone, 1-amino-2-propanol, 1-methyl-pyrrolidinone, 1-propanol, 2,3-butanediol, 2,5- dimethyl-2,5-hexanediol, 2-hydroxymethyl-1,3-propanediol, 2-isopropoxyethanol, 2- Methoxyethanol, 2-methyl-1,3-propanediol, 2-methyl-2,4-pentanediol, 2-propanol, 2- pyrrolidone, 3-amino-1-propanol, 3-methoxy-1,2-propanediol, 3-methoxy-1-butanol, 3- methoxy-1-propanol, 3-methyl-1,3-butanediol, Acetamide, Acetic acid, Acetone, Allantoin, Aspartame, Betaine, Citric acid, Diacetone alcohol, Diacetyl, Di-ethylene-glycol, Diethylene-Attorney Reference: BCHR-001WO glycol-diethyl-ether, Diethylene-glycol-dimethyl-ether, Diethylene-glycol-monoethyl-ether, Diethylene-glycol-monomethyl-ether, Diglycerol, Dimethyl isosorbide, Dimethyl sulfone, Dimethyl sulfoxide, Dipropylene-glycol-dimethyl-ether, Dipropylene-glycol-monomethyl-ether, Erythritol, Ethanol, Ethanolamine, Ethylene diamine, Ethylene glycol, Ethylene-glycol-diethyl- ether, Ethylene-glycol-dimethyl-ether, Ethylene-glycol-monoethyl-ether, Ethylene-glycol- monopropyl-ether, Formamide, Formic acid, Fructose, Galatose, Glucose, Glycerol, Glycerol formal, Glycine, Hydroxy-acetone, Isoascorbic acid, Isosorbide, Lactic acid, L-Arginine, Malic acid, Maltitol, Maltose, Mannitol, Meglumine, Methanol, Methyl-(S)-(-)-lactate, N,N- dimethylacetamide, N,N-dimethylformamide, N,N-dimethylpropionamide, N- Acetylethanolamine, N-acetylglycine, N-Formylmorpholine, Nicotinamide, N-methyl-2- pyrrolidone, N-methylacetamide, N-Methyldiethanolamine, N-methylformamide, Oxalic acid, PEG-200, PEG-300, PEG-400, Proline, Propionamide, Propylene-glycol-monomethyl-ether, Propylene-glycol-monopropyl-ether, Pyridine, Raffinose, Sorbitol, Sucrose, Sulfolane, Tartaric acid, Taurine, Tert-butanol, Tetraethylene glycol, Tetraethylene-glycol-dimethyl-ether, Tetrahydro-4-pyranol, Tetrahydrofurfuryl alcohol, Trehalose, Triethanolamine, Tri-ethylene- glycol, Triethylene-glycol-dimethyl-ether, Triethylene-glycol-monobutyl-ether, Triethylene- glycol-monoethyl-ether, Triethylene-glycol-monomethyl-ether, Triglycerol, Trimethylamine n- oxide, Trimethylolpropane, Tripropylene-glycol-monomethyl-ether, TRIS, and Urea, such as at least 2 PNWCCs, at least 3 PNWCCs, at least 4 PNWCCs, at least 5 PNWCCs, at least 6 PNWCCs, or at least 7 PNWCCs.

99. The high entropy solution of any one of claims 92-98, wherein the weighted sample standard deviation of the molar volumes of the PNWCCs ranges from 1 ml / mol to 100 ml / mol, such as 5 ml / mol to 50 ml / mol or 10 ml / mol to 25 ml / mol.

100. The high entropy solution of any one of claims 92-99, wherein the average molar volume of the high entropy solution is 25 ml / mol or more, such as 30 ml / mol or more, 35 ml / mol or more, 40 ml / mol or more, 45 ml / mol or more, 50 ml / mol or more, or 60 ml / mol or more.

101. The high entropy solution of any one of claims 92-100, wherein the lower temperature is -100 °C, -120 °C, -140 °C, -160 °C, -180 °C, or -200 °C.Attorney Reference: BCHR-001WO 102. The high entropy solution of any one of claims 92-101, wherein the high entropy solution has an entropy of mixing of 8 J / (mol K) or more, such as 9 or more, 10 or more, or 11 or more.

103. The high entropy solution of any one of claims 92-102, wherein the entropy of mixing is entropy of mixing according to the equation: ^ ∆^^ = −^ ln ^^ + D ^^wherein ^^and^^and ^^are respectively mol and volume fractions of each of n non-water chemical n is thenumber of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^is entropy of mixing.

104. The high entropy solution of any one of claims 92-102, wherein the entropy of mixing is ideal entropy of mixing according to the equation: ^ wherein ^^of n non-water chemical components; n is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^.^&(F#is ideal entropy of mixing.

105. The high entropy solution of claim 92, wherein: the 2 or more PNWCCs are 6 or more PNWCCs, each of the 6 or more PNWCCs is an organic compound with a molar volume ranging from 40 ml / mol to 400 ml / mol, each of the 6 or more PNWCCs is an organic compound selected from the group consisting of glycols, sugar alcohols, methylated organic compounds, methoxylated organic compounds, sugars, polyols, alcohols, amides, amines, amino acids, and organic polymers, and the molar volume of the high entropy solution is 30 ml / mol or more.Attorney Reference: BCHR-001WO 106. The high entropy solution of claim 105, wherein the high entropy solution has an entropy of mixing of 11 J / (mol K) or more according to the equation: ^ =−^ C^^ ln ^^ + D ^^ln^^^^E wherein ^^of n non-water chemical components; n is the number of the non-water chemical components and n is an integer equal to or greater than 2; R is the ideal gas constant; and ∆^^.^&(F#is ideal entropy of mixing.

107. A device for preserving a biological matter without freezing at sub-0°C temperatures, the device comprising: a. the high entropy solution of any one of the preceding claims; b. a biological matter in contact with the high entropy solution; and c. a cooling system configured to cool the biological matter and high entropy solution to below 0 °C.

108. The device of claim 107, wherein the high entropy solution and the biological matter are placed within a chamber.

109. The device of claim 108, wherein the chamber is an isochoric chamber under isochoric conditions.

110. The device of any one of claims 108-109, wherein the isochoric chamber is hermetically sealed.Attorney Reference: BCHR-001WO 111. The device of any one of claims 107-110, wherein the biological matter is loaded with the high entropy solution by a perfusion process, a diffusion process, a submersion process, or a convection process.

112. The device of claim 111, wherein the perfusion process is powered by gravity, application of pressure to a transfusion bag, a pump, or a machine perfusion device.

113. The device of any one of claims 107-112, wherein the biological matter is loaded with the high entropy solution at hypothermic temperatures between about 20 °C and about -20 °C.

114. The device of any one of claims 107-113, wherein the biological matter is loaded with the high entropy solution and submerged in the high entropy solution.

115. The device of any one of claims 107-114, wherein the cooling system uses dry ice to cool the biological matter and the high entropy solution to below 0 °C.

116. The device of any one of claims 107-115, wherein the cooling system uses liquid nitrogen to cool the biological matter and high entropy solution to below 0 °C.

117. The device of any one of claims 107-116, wherein the cooling system is a freezer or a refrigerator to cool and / or store the biological matter and the high entropy solution.

118. The device of any one of claims 107-117, wherein the cooling system controls the rate of cooling of the biological matter and the high entropy solution, wherein the rate of cooling is between about 0.01°C / min and about 10 °C / min.Attorney Reference: BCHR-001WO 119. The device of any one of claims 107-118, wherein the biological matter and the high entropy solution are cooled to a temperature above the glass transition temperature of the high entropy solution without significant ice formation because 1% or less by mass of the high entropy solution is ice.

120. The device of any one of claims 107-119, wherein the biological matter and the high entropy solution are cooled to a temperature equal to or beneath the glass transition temperature of the high entropy solution without significant ice formation because about 1% or less by mass of the high entropy solution is ice.

121. The device of any one of claims 107-120, wherein the biological matter and the high entropy solution are first cooled to about -80 °C and then stored at a temperature between about -80 °C and about -200 °C.

122. The device of any one of claims 107-121, wherein the biological matter is a human cell, a human tissue, a human organ, a whole human body, a non-human cell, a non-human tissue, a non-human organ, a whole non-human body, an organism, a biomedical product, an agricultural product, a food product, a fruit, or a beverage.

123. The device of claim 122, wherein the human tissue or organ is a kidney, a liver, a heart, a lung, a brain, a limb, or skin.

124. The device of claim 122, wherein the whole human body is preserved at a temperature less than 0 °C to reduce or suspend metabolism.

125. A method of preserving a biological matter without freezing at sub-0°C temperatures, the method comprising:Attorney Reference: BCHR-001WO a. placing the biological matter in contact with the high entropy solution of any of the preceding claims, or the high entropy solution produced by a method of any one of the preceding claims; b. cooling the biological matter and the high entropy solution to a temperature lower than 0°C; and c. storing the biological matter without ice formation at a temperature between 0 °C and about -200 °C.

126. The method of claim 125, wherein the biological matter is loaded with the high entropy solution by a perfusion process, a diffusion process, a submersion process, or a convection process.

127. The method of claim 126, wherein the perfusion process is powered by gravity, application of pressure to a transfusion bag, a pump, or a machine perfusion device.

128. The method of any one of claims 125-127, wherein the biological matter is loaded with the high entropy solution at hypothermic temperatures between about 20 °C and about -20 °C.

129. The method of any one of claims 125-128, wherein the biological matter is loaded with the high entropy solution and submerged in the high entropy solution.

130. The method of any one of claims 125-129, wherein the biological matter and the high entropy solution are cooled to a temperature above the glass transition temperature of the high entropy solution without ice formation.Attorney Reference: BCHR-001WO 131. The method of any one of claims 125-130, wherein the biological matter and the high entropy solution are cooled to a temperature equal to or beneath the glass transition temperature of the high entropy solution without ice formation.

132. The method of any one of claims 125-131, wherein the biological matter and the high entropy solution are cooled to about -80 °C.

133. The method of any one of claims 125-132, wherein the biological matter and high entropy solution are first cooled to about -80 °C, then optionally transported at about -80 °C, then stored at a temperature between about -130 °C and about -200 °C.

134. The method of any one of claims 125-133, wherein the biological matter is cooled at a rate between about 0.01°C / min and about 10 °C / min.

135. The method of any one of claims 125-134, wherein the biological matter and the high entropy solution are cooled by using dry ice.

136. The method of any one of claims 125-135, where the biological matter and the high entropy solution are transported or stored on dry ice.

137. The method of any one of claims 125-136, wherein the biological matter and the high entropy solution are cooled by using liquid nitrogen.

138. The method of any one of claims 125-137, wherein the biological matter and the high entropy solution are cooled and / or stored in a refrigerator or a freezer.Attorney Reference: BCHR-001WO 139. The method of any one of claims 125-138, wherein the biological matter is a human cell, a human tissue, a human organ, a whole human body, a non-human cell, a non-human tissue, a non-human organ, a whole non-human body, an organism, a biomedical product, an agricultural product, a food product, a fruit, or a beverage.

140. The method of claim 139, wherein the human tissue or organ is a kidney, a liver, a heart, a lung, a brain, a limb, or skin.

141. The method of claim 140, wherein the whole human body is preserved at a temperature less than 0 °C to reduce or suspend metabolism.

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