Systems, devices and methods for isochoric pressure-stabilized supercooling

Abstract

The present disclosure provides systems and devices for preserving unfrozen biological matter below its equilibrium melting temperature by applying pressure generated by isochoric freezing. The systems and devices include an isochoric chamber segmented into a first volume containing an aqueous solution with a first nucleation temperature and a second volume containing an aqueous solution with a second nucleation temperature, where the second volume increases when cooled below the second nucleation temperature, and also include a boundary separating the two volumes that transmits heat and pressure but not mass. Methods of using the systems and devices for preserving a biological matter without freezing at temperatures below its equilibrium melting point are also provided.

Description

Atty. Dkt: BCRH-003WO SYSTEMS, DEVICES AND METHODS FOR ISOCHORIC PRESSURE-STABILIZED SUPERCOOLING CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. Provisional Application No. 63 / 697,319, filed September 20, 2024, the disclosure of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present invention relates to systems, devices and methods for preserving biological matter, and more particularly, to systems, devices and methods for preserving unfrozen biological matter below its equilibrium melting temperature by applying pressure generated by isochoric freezing. 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 °C. 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 sub-zero 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 the 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 theAtty. Dkt: BCRH-003WO central challenges of cryopreservation, and methods and devices by which to achieve this avoidance are sought.

[0005] The equilibrium phase transition temperature of ordinary water at atmospheric pressure is 0 °C, however, water typically freezes below this point. As the initial freezing is an activated process (i.e. nucleation), the water may under certain conditions remain in a metastable liquid state many degrees below 0 °C for extended durations. This process is known as supercooling and has been proposed as a method of cryopreservation intended to avoid ice formation.

[0006] Supercooling produces a metastable state which implies that there is always some probability of ice nucleation. Ice nucleation is influenced by a number of factors such as the temperature (the further the temperature is below the equilibrium melting point the greater the likelihood for freezing becomes), mechanical vibration or shock simulation, heterogeneous interaction with solid or gaseous interfaces, and cavitation of gas bubbles within the liquid.

[0007] Attempts to reduce the probability of ice formation in supercooled systems (in order to increase stability at a given temperature or to enable supercooling to a lower temperature with the same stability) have led to a variety of methods. Because the probability for nucleation is a function of volume, one method aims to reduce the volume of water present. Another method uses electromagnetic fields to reduce the probability of freezing. Another method involves eliminating the interface between the liquid storage solution and air using an immiscible liquid phase. The immiscible liquid phase has a lower catalyzing potential than the free air interface, thereby reducing the probability of freezing occurring. Generally, these methods use physical means to stabilize the supercooled liquid.

[0008] Supercooling stability can often be quantified by the distance (number of degrees) a solution is cooled below its equilibrium phase transition temperature. For example, if water is cooled in a metastable liquid state to −5 °C, then the corresponding degree of supercooling is said to be equal to 5 degrees Centigrade. This state will theoretically be more stable than water cooled to −10 °C, at which point the degree of supercooling would be 10 degrees Centigrade. Thus, there exist other methods of reducing the likelihood of freezing involving reducing the equilibrium melting temperature of a solution in order to reduce the degree of supercooling.Atty. Dkt: BCRH-003WO

[0009] One method involves adding organic molecules (e.g., glycerol, trehalose, glucose, DMSO, propylene glycol, etc.) to the solution to depress the equilibrium liquidus temperature of the solution. It follows that by adding soluble organic molecules to a solution, which depresses the equilibrium liquidus temperature, the degrees of supercooling may be reduced. For example, physiological saline has an equilibrium melting point of approximately −0.5 °C. This solution may be cooled in a metastable liquid state to −5.5 °C, whereby the corresponding degrees of supercooling would be 5 °C. Thus, it may be cooled to a lower temperature yet with the same amount of supercooling and theoretically the same freezing probability. Alternatively, the saline may be cooled to the same temperature as the water, −5 °C, and have a lower degree of supercooling (4.5 °C instead of 5 °C), and may theoretically have a lower freezing probability.

[0010] In practice, an organ may be perfused with a cryoprotective solution containing organic solutes that lower the equilibrium phase transformation temperature of the organ, after which the organ may be stored in an ice-free state at sub-zero temperatures. This concept was applied to successfully cryopreserve a whole liver in a supercooled state at −4 °C. An inconvenience in the use of cryoprotectants to lower the freezing temperature is the need to perfuse and then remove the cryoprotectant from the biological organ.

[0011] The equilibrium liquidus temperature of a solution may also be lowered by increasing the hydrostatic pressure to which it is exposed. For example, application of about 60 MPa of pressure to ordinary water lowers its equilibrium melting temperature to −5 °C, application of about 110 MPa reduces it to about −10 °C, and application of about 190 MPa reduces it further to about −20 °C. The effect of pressure is thus equivalent to that of a chemical cryoprotectant with the advantage that it affects the entire region instantly. For example, a 4.3M solution of glycerol depresses the equilibrium melting point to approximately −5 °C. Thus, if a system containing ordinary water can normally be supercooled stably to −4 °C, then addition of 4.3M glycerol OR application of 60 MPa may reduce the temperature of stable supercooling to −9 °C.

[0012] Pressure and solutes may be used in tandem and their effects are often additive. Thus, another method of reducing the likelihood of freezing of a supercooled solution may be simultaneous addition of solutes and application of pressure. If 4.3 M glycerol and 60 MPa of pressure were to be applied to a system originally containing ordinary water that could beAtty. Dkt: BCRH-003WO supercooled stably to −4 °C, the combined effect could be to reduce the temperature of stable supercooling to −14 °C.

[0013] Beyond stabilizing the supercooled state by reducing the equilibrium melting point, solutes and pressure also have the potential to increase the interfacial free energy between the solution and ice. The ice nucleation rate is extremely sensitive to this parameter, such that increasing the ice-liquid interfacial free energy increases the critical nucleation barrier and reduces the nucleation rate, which has the effect that often solutes and / or pressure can impart stability onto the solution in excess of their according melting point depression.

[0014] Furthermore, pressure and solutes are known to increase the glass transition temperature of water and often also the viscosity of aqueous solutions. This has the effect of 1) reducing the rates necessary to avoid ice formation during cooling to and warming from below the glass transition temperature; and 2) increasing the temperature at which a solution can be maintained in a solid amorphous state. The schematic below depicts the effect of pressure on the liquidus, nucleation, and glass transition temperatures for a hypothetical aqueous solution. FIG.1 shows a schematic phase diagram of a hypothetical aqueous solution illustrating effect of pressure on the liquidus, nucleation, and glass transition temperatures. SUMMARY

[0015] The present disclosure provides systems and devices for preserving unfrozen biological matter below its equilibrium melting temperature by applying pressure generated by isochoric freezing. The systems and devices include an isochoric chamber segmented into a first volume containing an aqueous solution with a first nucleation temperature and a second volume containing an aqueous solution with a second nucleation temperature, where the second volume increases when cooled below the second nucleation temperature, and also include a boundary separating the two volumes that transmits heat and pressure but not mass. Methods of using the systems and devices for preserving a biological matter without freezing at temperatures below its equilibrium melting point are also provided.

[0016] Aspects of the present disclosure provide systems that reduce the probability of ice nucleation of an aqueous solution and / or biological matter that are cooled below theirAtty. Dkt: BCRH-003WO corresponding equilibrium melting point. The present disclosure further provides methods and devices enabling ice-free preservation of biological matter at temperatures below 0 °C.

[0017] Aspects of the present disclosure include systems for generating a pressure-stabilized supercooled state through the manipulation of temperature alone in a rigidly confined system, termed ‘isochoric baro-supercooling’. More specifically, isochoric baro-supercooling systems comprise an isochoric chamber segmented into at least two volumes with different nucleation temperatures. Methods to preserve biological matter in isochoric baro-supercooling systems include cooling the systems to a preservation temperature at which one of the two volumes will nucleate ice, thereby pressurizing the system, but the other volume, at the resultant temperature and pressure, will not nucleate ice, thereby achieving a pressurized supercooled state beneath its equilibrium melting point.

[0018] In some embodiments, the isochoric chamber is segmented into two volumes. The first volume contains biological matter to be preserved, and the second volume contains an aqueous solution with a nucleation temperature higher than the nucleation temperature of the first volume. The two volumes are separated by a barrier that transmits heat and pressure but not mass, such as flexible, impermeable membrane. The ratio of the two volumes is specified such that freezing of the second volume generates the desired magnitude of pressure.

[0019] In other embodiments, the isochoric chamber is segmented into three volumes. The first volume contains biological matter to be preserved; the second volume contains an aqueous solution with a nucleation temperature higher than the nucleation temperature of the first volume; and the third volume contains a material that does not expand as the temperature of the system is lowered. The volumes are separated by barriers that transmit heat and pressure, but not mass. One embodiment of such a barrier is a flexible, impermeable membrane. The size and composition of the volumes are specified such that freezing of the second volume generates the desired magnitude of pressure. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG.1 depicts a schematic phase diagram of a hypothetical aqueous solution illustrating effect of pressure on the liquidus, nucleation, and glass transition temperatures.Atty. Dkt: BCRH-003WO

[0021] FIGS.2A-12C depict example embodiments of the isochoric baro-supercooling devices and related devices for preservation of biological matter as detailed in the present disclosure.

[0022] FIGS.2A and 2B show a two-volume system. FIG.2A depicts a two-volume isochoric pressure-stabilized supercooling system. FIG.2B depicts pressure and ice phase fraction as a function of temperature for an isochoric system of ordinary water.

[0023] FIGS.3A and 3B show one embodiment of a three-volume system with solid material as third volume. FIG.3A depicts a three-volume isochoric pressure-stabilized supercooling system with the third volume comprising glass beads. FIG.3B depicts Pressure and ice phase fraction as a function of temperature for the three-volume isochoric system with ordinary water.

[0024] FIGS.4A and 4B show another embodiment of a three-volume system with solid material as third volume. FIG.4A depicts a three-volume isochoric pressure-stabilized supercooling system with the third volume comprising a solution that freezes at a lower temperature than the contents of the first and second volumes. FIG.4B depicts Pressure and ice phase fraction as a function of temperature for the three-volume isochoric system with ordinary water.

[0025] FIGS.5A and 5B show another embodiment of a three-volume system with solid material as third volume. FIG.5A depicts a three-volume isochoric pressure-stabilized supercooling system with the third volume further segmented into four sub-volumes. FIG.5B depicts Hypothetical cooling trajectory for the three-volume system. The individual sub- volumes comprising the third volume freeze at different temperatures and progressively increase the system pressure in a stepwise manner.

[0026] FIG.6 depicts one embodiment of a three-volume isochoric pressure-stabilized supercooling device for preservation of biological matter further comprising a temperature monitoring / control system and a pressure sensor.

[0027] FIGS.7A and 7B show embodiments of a three-volume system in which first and second volumes are thermally separated. FIG.7A depicts a three-volume isochoric pressure- stabilized supercooling system in which the first and second volumes are thermally separated yet maintained in hydrostatic communication. FIG.7B depicts a hypothetical cooling trajectory of the three-volume system depicting independent temperature control of the first and second volumes which enables the pressure to only develop at a desired time.Atty. Dkt: BCRH-003WO

[0028] FIG.8 shows pressure-temperature liquidus curve for ordinary water and ice.

[0029] FIG.9 depicts a schematic representation of the ability of pressure to depress the temperature of stable supercooling through depression of the equilibrium melting point.

[0030] FIG.10 depicts PTx liquidus surface for a binary glycerol:water solution with contour lines indicating the T-P liquidus for glycerol:water solutions of specific weight fractions.

[0031] FIG.11A depicts a temperature-concentration liquidus curve for binary NaCl:water solutions at atmospheric pressure. FIG.11B depicts the isochoric freezing trajectory for 0.9% w / w saline. FIG.11C depicts ice fraction as a function of temperature calculated for 0.9% w / w saline.

[0032] FIG.12A depicts a temperature-concentration liquidus curve for binary ethylene glycol- water solutions at atmospheric pressure. FIG.12B depicts isochoric freezing trajectory for 1M and 2M ethylene glycol. FIG.12C depicts ice fraction as a function of temperature calculated for 1M and 2M ethylene glycol. DETAILED DESCRIPTION 1. Introduction

[0033] Aspects of the present disclosure provide devices to reduce the likelihood of freezing in supercooled aqueous systems through isochoric freezing-generated pressure. Aspects of the present disclosure further provide devices and methods for preserving unfrozen biological matter below their equilibrium melting point in a pressurized environment generated through manipulation of temperature alone.

[0034] In some aspects, the present disclosure provide systems for inducing pressure- stabilized supercooling at a desired pressure and temperature. More specifically, the present disclosure provides generating a pressure-stabilized supercooled state through the manipulation of temperature alone in a rigidly confined system, termed “isochoric baro- supercooling.” The isochoric baro-supercooling systems comprise an isochoric chamber segmented into at least two volumes with different nucleation temperatures.

[0035] In other aspects, the present disclosure further provides isochoric baro-supercooling devices for preserving biological matter.Atty. Dkt: BCRH-003WO

[0036] In still other aspects, the present disclosure provide methods to preserve biological matter in isochoric baro-supercooling systems of the present disclosure, the methods comprise cooling the systems to a preservation temperature at which one of the two volumes will nucleate ice, thereby pressurizing the system, but the other volume, at the resultant temperature and pressure, will not nucleate ice, thereby achieving a pressurized supercooled state beneath its equilibrium melting point.

[0037] There are related prior art as followings:

[0038] i) Isobaric equilibrium pressurized cryopreservation

[0039] Pressure has been used to enable sub-zero Centigrade preservation in the liquid state at or above the equilibrium melting point of the biological matter. This has been achieved using systems that employ manual or motorized mechanical pumps to pressurize and depressurize the preservation system. In these systems the biological matter is in thermodynamic contact with a pressure reservoir and therefore the temperature and pressure within the system are to a great extent decoupled. Isobaric equilibrium pressurized cryopreservation is described in Takahashi, et al. “Functional integrity of the rat liver after sub- zero preservation under high pressure,” Transplantation Proceedings, Volume 32, Issue 7, November 2000, Pages 1634-1636, which is incorporated herein by reference.

[0040] ii) Isochoric equilibrium pressurized cryopreservation

[0041] Other devices have been proposed that confine biological matter in a rigid, principally air-free isochoric chamber. In these systems, the confined growth of ice generates pressure within the isochoric system. Because the aqueous material is denied access to a pressure reservoir and its volume is restricted, the temperature and pressure are coupled and vary along a trajectory following global equilibrium states. In other words, the biological matter exists at the equilibrium melting point. As water can only exist in a metastable state down to roughly −22 °C (at ~210 MPa), these techniques have largely been restricted to a temperature range of about −22 °C to 0 °C. Solutes may be added to water to depress the equilibrium melting point and may extend the equilibrium stability range to lower temperatures. Critically, these works teach isochoric freezing as a method for achieving a globally stable equilibrium specifically to avoid lowering the temperature of biological matter below its equilibrium melting point. It is described in B. Rubinsky, “The thermodynamic principles of isochoricAtty. Dkt: BCRH-003WO cryopreservation,” Cryobiology, Volume 50, Issue 2, April 2005, Pages 121-138, which is incorporated herein by reference.

[0042] iii) Ice-free isochoric supercooling

[0043] Separately from pressurized equilibrium isochoric processes, isochoric devices and methods have been proposed to stabilize supercooled aqueous solutions below their equilibrium melting point. Various works have taught the process of isochoric supercooling for avoiding ice, as well as avoiding the pressure generated by ice growth within isochoric systems during cryopreservation at temperatures generally above −20 °C. Critically, isochoric processes in the prior art teach one of two approaches – either allowing the system to nucleate ice and holding any stored biologic at thermodynamic equilibrium at high pressure, or disallowing the nucleation of ice in the system (and thus disallowing any pressurization) and holding the biologic in a metastable supercooled state at approximately atmospheric pressure. Within the state of the art to date, these two methods are mutually exclusive, insofar as isochoric supercooling methods teach explicitly to avoid ice formation and pressurization in the system at all costs, while isochoric freezing methods teach explicitly to avoid supercooling at all costs, i.e. to nucleate ice in the system and achieve thermodynamic equilibrium. Isochoric supercooling is described in US20220325937A1, US20230404067A1, US20240023543A1, and US20230189795A1, which are incorporated herein by reference.

[0044] iv) isobaric pressurized vitrification

[0045] Certain cryopreservation processes have the goal of cooling biological matter to cryogenic temperatures while avoiding ice formation as the aqueous system transforms into a glass. Various devices and methods have been devised to mechanically apply pressure to biological matter during cooling to cryogenic temperature. These processes take advantage of pressure depressing the equilibrium melting point, increasing the liquid-ice interfacial free energy, reducing molecular diffusivity, and increasing the glass transition temperature in order to facilitate vitrification and thawing without ice formation. Critically, these processes teach the independent control of temperature and pressure. Isobaric pressurized vitrification is described in US4559298A and DE102011115467A1, which are incorporated herein by reference.Atty. Dkt: BCRH-003WO

[0046] v) Self-pressurized vitrification

[0047] Related devices have even leveraged the confined growth of ice within rigid principally air-free systems to increase pressure and facilitate vitrification during rapid cooling. This process is often termed self-pressurized rapid freezing (SPRF). These systems were originally developed to preserve cellular ultrastructure for microscopic imaging and were later developed to enable reversible cryopreservation. Notably, as the name suggests, SPRF processes aim to rapidly cool the entire system in a cryogenic fluid. This ignores the potential to modulate the pressurization process through intentional control of temperature. Further, SPRF systems often consist of only a single homogeneous volume meaning that ice grows in the direct vicinity of the biological matter. This may lead to potential inadvertent ice formation in the biological matter. Other SPRF systems have been proposed that physically separate the biological matter. These systems separate the biological matter from the freezing volume by sliding gaskets or plugs. While these aspects may provide physical separation between the biological matter and ice, they have the effect of restricting the application of pressure. Whereas with a flexible membrane, pressure is applied uniformly over the entire volume, the sliding gaskets and plugs enforce application of pressure from one or two directions. Furthermore, prior art in this domain has not taught methods by which to manipulate or control the amount of pressurization experienced at a given sub-zero temperature, leading to processes and devices with insufficiently granular control of the pressurization and subsequent preservation process.

[0048] Notably, SPRF processes teach how to employ the self-pressurized technique to facilitate vitrification and do not address the possibility of reducing the probability of freezing at high sub-zero Centigrade temperatures. For example, Leunissen teaches that self- pressurized rapid freezing can only be used to avoid ice formation at temperatures less than 251 K.

[0049] Rolle, et al. (2023) teach self-pressurized rapid freezing in a <1mm diameter fine copper capillary whereby they separate the cavity into two volumes, one containing the vitrifying sample and the other containing a DMSO:water solution whose concentration can be modulated to alter the pressure that is generated during quench cooling. Critically, the method of Rolle, et al. does not teach the importance of the sizes of the individual volumes, theAtty. Dkt: BCRH-003WO importance of the composition of the primary volume, the importance of the geometrical configuration of the sub-volumes, nor the importance of the spatially and temporally varying thermal transport for affecting the pressure that develops. Additionally, the method of Rolle, et al. does not teach the control of temperature and only presents the possibility of quenching cooling micron-scale capillary tubes.

[0050] Self-pressurized vitrification is described in US20120210734A1, US20090011505A1 and Konrad Rolle et al., “Self-pressurised rapid freezing at arbitrary cryoprotectant concentrations” J. Microsc.2023;292:27–36, which are incorporated herein by reference. 2. Definitions

[0051] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, 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.

[0052] 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, between 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.

[0053] 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.Atty. Dkt: BCRH-003WO

[0054] As used herein, the term “isochoric freezing” refers to the freezing of aqueous solutions in a constant volume chamber or in a rigid chamber. In isochoric freezing, the phase transition encompasses the entire volume when cooling below the liquidus line and a substantial portion of the volume remains unfrozen to the triple point temperatures and pressures and is associated with an increase in pressure.

[0055] As used herein, the term “isobaric freezing” refers to a freezing process at constant pressure. In isobaric freezing, the entire system freezes as soon as the temperature is below the liquidus line intersection with the constant pressure line.

[0056] As used herein, the term “equilibrium melting point,” “equilibrium melting temperature,” “equilibrium phase transition temperature,” “equilibrium liquidus temperature” and “liquidus temperature” are interchangeably used herein, and refers to the temperature at which solid and liquid phases of a substance are in thermodynamic equilibrium at a given pressure.

[0057] As used herein, the term “freezing point,” “freezing temperature” and “nucleation temperature” are interchangeably used herein and refers to the temperature at which a solid phase nucleates from a liquid phase. Because nucleation is an activated process and thus requires a driving force to occur, the nucleation temperature of a substance is always and must always be lower than the equilibrium melting point of the same substance.

[0058] 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 sub-zero centigrade temperatures. As such, the high entropy solution may be used for preserving unfrozen biological matter at sub-zero centigrade temperature without ice formation.

[0059] 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. 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,Atty. Dkt: BCRH-003WO 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.

[0060] 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.

[0061] 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).

[0062] It is understood that aspects and embodiments of the present disclosure described herein include “comprising,” “consisting,” and “consisting essentially of” aspects and embodiments.

[0063] 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 embodimentsAtty. Dkt: BCRH-003WO 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.

[0064] 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. 3. Systems for inducing pressure-stabilized supercooling at a desired pressure and temperature

[0065] Aspects of the present disclosure provide systems for inducing pressure-stabilized supercooling at a desired pressure and temperature. More specifically, the present disclosure provides systems for generating a pressure-stabilized supercooled state through the manipulation of temperature alone in a rigidly confined system, termed “isochoric baro- supercooling.” The isochoric baro-supercooling systems comprise an isochoric chamber segmented into at least two volumes with different nucleation temperatures. 3.1. the isochoric chamber segmented into two volumes

[0066] In some aspects, the present disclosure provides a system for inducing pressure- stabilized supercooling at a desired pressure and temperature, comprising: a) an isochoric chamber segmented into two volumes; b) a first volume containing an aqueous solution with a first nucleation temperature (^^^^,^); c) a second volume containing an aqueous solution with a second nucleation temperature (^^^^,^), wherein the second volume increases when cooled below the second nucleation temperature (^^^^,^); and d) a boundary separating the two volumes that transmits heat and pressure but not mass.

[0067] The terms "isochoric chamber," "rigid chamber" and "constant volume chamber" are used interchangeably herein. In the isochoric freezing, a substantial portion of the volume remains unfrozen which is associated with an increase in pressure. The process of isochoricAtty. Dkt: BCRH-003WO freezing occurs along the liquidus line in a temperature-pressure phase diagram as the temperature is dropped because the volume in an isochoric chamber is constant. The isochoric freezing technique is described in US2007 / 0042337A1 and PCT / US24 / 33896, which are incorporated by reference herein.

[0068] In some embodiments, the fluid in the isochoric chamber is aqueous solution or pure water. In the isochoric freezing, if ice nucleates in the constant volume chamber, the pressure of the system increases, and the nucleation can be detected by the pressure gauge. For example, U.S. Patent Publication No.2007-0042337A1, incorporated by reference herein, which discusses isochoric freezing system and method for cryopreservation of a biological sample.

[0069] In some embodiments, the isochoric chamber is segmented into two volumes. In certain embodiments, the first volume contains biological matter to be preserved, and the second volume contains an aqueous solution with a nucleation temperature higher than the nucleation temperature of the first volume.

[0070] In some embodiments, the two volumes are separated by a boundary that transmits heat and pressure but not mass, such as flexible, impermeable membrane. For example, the boundary can be a barrier made by polyethylene sterile bag. In some embodiments, the boundary is selectively permeable and allows transfer of non-water molecules. In other embodiments, the non-water molecules are dissolved gases or solutes.

[0071] In some embodiments, the ratio of the two volumes is specified such that freezing of the second volume generates the desired magnitude of pressure. In some embodiments, the desired magnitude of pressure is between about 1 and about 300 MPa. In certain embodiments, the desired magnitude of pressure is about 1 MPa to about 250 MPa, about 10 MPa to about 300 MPa, about 20 MPa to about 250 MPa, about 30 MPa to about 250 MPa, about 40 MPa to about 250 MPa, about 40 MPa to about 200 MPa, about 50 MPa to about 170 MPa or about 50 MPa to about 150 MPa. In certain embodiments, the desired magnitude of pressure is about 1 MPa, about 5 MPa, about 10 MPa, about 20 MPa, about 25 MPa, about 30 MPa, about 35 MPa, about 40 MPa, about 45 MPa, about 50 MPa, about 55 MPa, about 60 MPa, about 65 MPa, about 70 MPa, about 75 MPa, about 80 MPa, about 85 MPa, about 90 MPa, about 95MPa, about 100 MPa, about 110 MPa, about 120 MPa, about 130 MPa, aboutAtty. Dkt: BCRH-003WO 140 MPa, about 150 MPa, about 160 MPa, about 170 MPa, about 180 MPa, about 190 MPa, about 200 MPa, about 210 MPa, about 220 MPa, about 230 MPa, about 240 MPa, about 250 MPa, about 260 MPa, about 270 MPa, about 280 MPa, about 290 MPa, or about 300 MPa.

[0072] In some embodiments, a first volume contains an aqueous solution with a first nucleation temperature (^^^^,^) and a second volume contains an aqueous solution with a second nucleation temperature (^^^^,^). In certain embodiments, the first nucleation temperature and the second nucleation temperature are different. In certain embodiments, the second nucleation temperature is higher than the first nucleation temperature (^^^^,^> ^^^^,^) and the second volume increases when cooled below the second nucleation temperature (^^^^,^). In some embodiments, the second nucleation temperature is higherthan the first nucleation temperature (^^^^,^ > ^^^^,^).

[0073] In some embodiments, the second volume is thermally separated from the first volume.

[0074] In some embodiments, the second volume is further segmented into multiple sub- volumes each with different compositions such that they freeze at different temperatures. In certain embodiments, the second volume is further segmented into multiple sub-volumes such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 sub-volumes and each sub-volume contains different compositions such that they freeze at different temperatures.

[0075] In some embodiments, one of the volumes nucleates ice, thereby pressuring the system and the other volume at a resultant temperature and pressure does not nucleate ice, thereby achieving a pressurized supercooled state beneath equilibrium melting point.

[0076] In some embodiments, size, composition, and configuration of the first and second volumes are chosen such that the desired pressure is generated by the freezing of the second volume at the desired temperature, and the likelihood of ice formation in the first volume is thereby reduced at the same temperature.

[0077] In some embodiments, the first volume contains biological matter to be preserved, and the second volume contains an aqueous solution with the second nucleation temperature, wherein the second nucleation temperature is higher than the first nucleation temperature of the first volume.Atty. Dkt: BCRH-003WO

[0078] In some embodiments, the fluid in the second volume is aqueous solution or pure water with or without organic molecules therein. In some embodiments, the aqueous solution that is primarily water may contain other chemicals so that the freezing point of the water- based solution may be modified as required for a specific application. For example, the water- based solution may contain salt. In certain embodiments, the external solution is water. In some embodiments the isochoric chamber is filled with a fluid that does not freeze at the storage temperature, such as a solution of glycerol.

[0079] In some embodiments, the biological matter is immersed in an aqueous solution. In some embodiments, the biological matter is immersed in an aqueous solution with which it was perfused. In some embodiments, the biological matter is immersed in a solution different from the aqueous solution with which it was perfused.

[0080] In some embodiments, the solution with which the biological matter is perfused is a high-entropy solution. In some embodiments, the solution in which the biological matter is immersed is a high-entropy solution. 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 sub-zero centigrade temperatures. As such, the high entropy solution may be used for preserving unfrozen biological matter at sub-zero centigrade temperature without ice formation.

[0081] In some embodiments, the biological matter is immersed in a water-immiscible non- aqueous solution.

[0082] In some embodiments, the temperature at which the biological matter is stored is lower than the equilibrium melting temperature of the biological matter.

[0083] In some embodiments, the biological matter is cooled and stored without ice formation.

[0084] In some embodiments, the cooling of the system is non-uniform.

[0085] In some embodiments, preservation time of the present disclosure is a length of time for preservation in the system of the present disclosure. In certain embodiments, the preservation time is a length of time from hours to days, hours to weeks, hours to months, days to months, days to weeks, weeks to months, or months to years. In some embodiments,Atty. Dkt: BCRH-003WO the biological matter is stored / preserved for a duration between about 1 hour and about 1000 years.

[0086] In certain embodiments, the preservation time is more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, more than 11 months, or more than 12 months.

[0087] In certain embodiments, the preservation time is more than 1 week, more than 2 weeks, more than 3 weeks, more than 4 weeks, more than 5 weeks, more than 6 weeks, more than 7 weeks, more than 8 weeks, more than 9 weeks, more than 10 weeks, more than 15 weeks, more than 20 weeks, more than 30 weeks, more than 40 weeks, more than 50 weeks, more than 100 weeks.

[0088] In some embodiments, the preservation time is more than 1 day. In some embodiments, the preservation time is 1 day to 80 days. In some embodiments, the preservation time is 1 day to 50 days. In some embodiments, the preservation time is 1 day to 14 days. In certain embodiments, the preservation time is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 70 days, 80 days, 90 days, or 100 days or more.

[0089] In certain embodiments, the preservation time of the present disclosure is 1 hour or more, 24 hours or more. In certain embodiments, the preservation time of the present disclosure is 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, or 48 hours or more.

[0090] In some embodiments, the second volume contains ice nucleating agents. The nucleating agent forms an ice crystal, thereby a biological matter can be preserved in the unfrozen part of the volume. The ice crystal formed by the nucleating agent generates pressures of the isochoric chamber, and the enhanced pressures hinder further formation of ice. In some embodiments, the nucleating agent can be a structural element of the isochoricAtty. Dkt: BCRH-003WO freezing device. For example, the nucleating agent can be walls of the isochoric chamber itself, or walls of the matter container itself. In other embodiments, the nucleating agent can be any agent that promotes the formation of ice crystal in the isochoric chamber. For example, the ice nucleating agent is, but not limited to, minute solid particles, such as dust or food particles, large molecules, ice-nucleating proteins. In some embodiments, the nucleating agent is placed in the bottom of the isochoric chamber, and the chamber is completely filled with an aqueous solution. In some embodiments, a biological matter is placed in a container in an isochoric chamber that allows transfer of only pressure and heat but not mass and the isochoric chamber is filled with a fluid and nucleation agents that cause the fluid to freeze before the biological matter in the container freezes.

[0091] In some embodiments, the pressure that is generated is between about 1 and about 300 MPa. In certain embodiments, the pressure that is generated is about 1 MPa to about 250 MPa, about 10 MPa to about 300 MPa, about 20 MPa to about 250 MPa, about 30 MPa to about 250 MPa, about 40 MPa to about 250 MPa, about 40 MPa to about 200 MPa, about 50 MPa to about 170 MPa or about 50 MPa to about 150 MPa. In certain embodiments, the pressure that is generated is about 1 MPa, about 5 MPa, about 10 MPa, about 20 MPa, about 25 MPa, about 30 MPa, about 35 MPa, about 40 MPa, about 45 MPa, about 50 MPa, about 55 MPa, about 60 MPa, about 65 MPa, about 70 MPa, about 75 MPa, about 80 MPa, about 85 MPa, about 90 MPa, about 95MPa, about 100 MPa, about 110 MPa, about 120 MPa, about 130 MPa, about 140 MPa, about 150 MPa, about 160 MPa, about 170 MPa, about 180 MPa, about 190 MPa, about 200 MPa, about 210 MPa, about 220 MPa, about 230 MPa, about 240 MPa, about 250 MPa, about 260 MPa, about 270 MPa, about 280 MPa, about 290 MPa, or about 300 MPa.

[0092] In some embodiments, the pressure that is generated depends on the volume of the freezable volume, the composition of the freezable volume and other volumes, the storage temperature, and the temperature history (e.g., cooling rate).

[0093] In some embodiments, the pressure does not irreversibly damage the biological matter.

[0094] In some embodiments, the generated pressure lowers the equilibrium melting temperature of the biological material in the first volume.Atty. Dkt: BCRH-003WO

[0095] In some embodiments, the generated pressure raises the glass transition temperature of the biological material in the first volume.

[0096] In some embodiments, the pressure lowers the cooling or warming rates required to avoid ice formation during cooling to or warming from the storage temperature.

[0097] In some embodiments, the cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 1000 K / s. In certain embodiments, the cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 900 K / s, between about 0.1 K / hr and about 800 K / s, between about 0.1 K / hr and about 700 K / s, between about 0.1 K / hr and about 600 K / s, between about 0.1 K / hr and about 500 K / s, between about 0.1 K / hr and about 400 K / s, between about 0.1 K / hr and about 300 K / s, between about 0.1 K / hr and about 200 K / s¸ between about 0.1 K / hr and about 100 K / s, between about 1 K / hr and about 1000 K / s, between about 10 K / hr and about 1000 K / s, between about 100 K / hr and about 1000 K / s, between about 100 K / hr and about 500 K / s, or between about 100 K / hr and about 250 K / s.

[0098] In some embodiments, the warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 1000 K / s. In certain embodiments, the warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 900 K / s, between about 0.1 K / hr and about 800 K / s, between about 0.1 K / hr and about 700 K / s, between about 0.1 K / hr and about 600 K / s, between about 0.1 K / hr and about 500 K / s, between about 0.1 K / hr and about 400 K / s, between about 0.1 K / hr and about 300 K / s, between about 0.1 K / hr and about 200 K / s¸ between about 0.1 K / hr and about 100 K / s, between about 1 K / hr and about 1000 K / s, between about 10 K / hr and about 1000 K / s, between about 100 K / hr and about 1000 K / s, between about 100 K / hr and about 500 K / s, or between about 100 K / hr and about 250 K / s.

[0099] In some embodiments, the cooling or warming rates are not constant.

[0100] 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.Atty. Dkt: BCRH-003WO

[0101] In some embodiments, the system further comprises a temperature controller configured to monitor and control the temperature of the system. In certain embodiments, the temperature controller is configured to cool the system of the present disclosure or heat the system of the present disclosure. In certain embodiments, the temperature controller comprises a cooling system and a heating system. In certain embodiments, the temperature controller is any device or system configured to cool the fluid in the isochoric chamber below 0 °C and warm the fluid in the isochoric chamber above 0 °C. For example, the cooling system is a conventional freezer, and the warming system is a conventional heater.

[0102] In some embodiments, the cooling temperature is above about −200 °C and below the equilibrium melting point of the contents of the first volume.

[0103] In some embodiments, the temperature control system is further configured to warm the fluid in the isochoric chamber above 0 °C. In some cases, the warming temperature is above 0 °C to 5 °C, above 0 °C to 6 °C, above 0 °C to 7 °C, above 0 °C to 8 °C, above 0 °C to 9 °C, or above 0 °C to 10 °C. The warming or thawing conditions are well known in the art. The conventional warming or thawing conditions can be applied herein.

[0104] In some embodiments, the system of the present disclosure generates a pressure-stabilized supercooled state through the manipulation of temperature alone in the isochoric system.

[0105] In some embodiments, the system optionally comprises a pressure controller configured to monitor the pressure within the system. 3.2. the isochoric chamber is segmented into three volumes

[0106] In some aspects, the present disclosure provides a system for inducing pressure- stabilized supercooling at a desired pressure and temperature, comprising a) an isochoric system segmented into three volumes; b) a first volume containing an aqueous solution with a first nucleation temperature (^^^^,^); c) a second volume containing an aqueous solution with a second nucleation temperature (^^^^,^), and the second volume increases when cooled below the second nucleation temperature (^^^^,^); d) a third volume filling the remaining volume of the isochoric system not occupied by the first and second volumes and comprising a material whose volume does not substantially increase at the desired temperature; and e)Atty. Dkt: BCRH-003WO boundaries separating the first, the second, and the third volumes that transmit heat and pressure but not mass.

[0107] The terms "isochoric chamber," "rigid chamber" and "constant volume chamber" are used interchangeably herein. In the isochoric freezing, a substantial portion of the volume remains unfrozen which is associated with an increase in pressure. The process of isochoric freezing occurs along the liquidus line in a temperature-pressure phase diagram as the temperature is dropped because the volume in an isochoric chamber is constant. The isochoric freezing technique is described in US2007 / 0042337A1 and PCT / US24 / 33896, which are incorporated by reference herein. The Isochoric freezing is described in "3.1 the isochoric chamber segmented into two volumes" and is incorporated into this section.

[0108] In some embodiments, the fluid in the isochoric chamber is aqueous solution or pure water. In the isochoric freezing, if ice nucleates in the constant volume chamber, the pressure of the system increases, and the nucleation can be detected by the pressure gauge. For example, U.S. Patent Publication No.2007-0042337A1, incorporated by reference herein, which discusses isochoric freezing system and method for cryopreservation of a biological sample.

[0109] In some embodiments, the isochoric chamber is segmented into three volumes. In some embodiments, the first volume contains biological matter to be preserved; the second volume contains an aqueous solution with a nucleation temperature higher than the nucleation temperature of the first volume; and the third volume contains a material that does not expand as the temperature of the system is lowered. The volumes are separated by barriers that transmit heat and pressure, but not mass. One embodiment of such a barrier is a flexible, impermeable membrane. The size and composition of the volumes are specified such that freezing of the second volume generates the desired magnitude of pressure.

[0110] In some embodiments, a first volume contains an aqueous solution with a first nucleation temperature (^^^^,^), and a second volume contains an aqueous solution with a second nucleation temperature (^^^^,^). In some embodiments, the second nucleationtemperature is higher than the first nucleation temperature (^^^^,^ > ^^^^,^).Atty. Dkt: BCRH-003WO

[0111] In some embodiments, the third volume contains an aqueous solution that freezes at a lower temperature than the contents of the first and second volumes. In other embodiments, the third volume contains non-aqueous fluid or solid material.

[0112] In some embodiments, the second volume is thermally separated from the first volume. In other embodiments, the second volume is thermally separated from the third volume.

[0113] In some embodiments, the boundaries are selectively permeable and allow transfer of non-water molecules. For example, the non-water molecules are dissolved gases or solutes.

[0114] In some embodiments, the second volume is further segmented into multiple sub-volumes each with different compositions such that they freeze at different temperatures. In certain embodiments, the second volume is further segmented into multiple sub-volumes such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 sub-volumes and each sub-volume contains different compositions such that they freeze at different temperatures.

[0115] In some embodiments, one of the volumes nucleates ice, thereby pressuring the system and the other volume at a resultant temperature and pressure does not nucleate ice, thereby achieving a pressurized supercooled state beneath equilibrium melting point.

[0116] In some embodiments, size, composition, and configuration of the first, the second, and the third volumes are chosen such that the desired pressure is generated by the freezing of the second volume at the desired temperature, and the likelihood of ice formation in the first volume is thereby reduced at the same temperature.

[0117] In some embodiments, the first volume contains biological matter to be preserved; the second volume contains an aqueous solution with the second nucleation temperature, wherein the second nucleation temperature is higher than the nucleation temperature of the first volume; and the third volume contains a material that does not expand as the temperature of the system is lowered.

[0118] In some embodiments, the fluid in the second volume is aqueous solution or pure water with or without organic molecules therein. In some embodiments, the aqueous solution that is primarily water may contain other chemicals so that the freezing point of theAtty. Dkt: BCRH-003WO water-based solution may be modified as required for a specific application. For example, the water-based solution may contain salt. In certain embodiments, the external solution is water. In some embodiments the isochoric chamber is filled with a fluid that does not freeze at the storage temperature, such as a solution of glycerol.

[0119] In some embodiments, the biological matter is immersed in an aqueous solution. In some embodiments, the biological matter is immersed in an aqueous solution with which it was perfused. In some embodiments, the biological matter is immersed in a solution different from the aqueous solution with which it was perfused.

[0120] In some embodiments, the solution in which the biological matter is immersed is a high-entropy solution. In some embodiments, the solution with which the biological matter is perfused is a high-entropy solution. 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 sub-zero centigrade temperatures. As such, the high entropy solution may be used for preserving unfrozen biological matter at sub-zero centigrade temperature without ice formation. The high entropy solution is described in US patent application no.63 / 670,528, which is incorporated by reference herein.

[0121] In some embodiments, the biological matter is immersed in a water-immiscible non-aqueous solution.

[0122] In some embodiments, the temperature at which the biological matter is stored is lower than the equilibrium melting temperature of the biological matter.

[0123] In some embodiments, the biological matter is cooled and stored without ice formation.

[0124] In some embodiments, the cooling of the system is non-uniform.

[0125] In some embodiments, the biological matter can be stored for a duration from hours to days, hours to weeks, hours to months, days to months, days to weeks, weeks to months, or months to years. In some embodiments, the biological matter is stored / preserved for a duration between about 1 hour and about 1000 years.

[0126] In certain embodiments, the preservation time is more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6Atty. Dkt: BCRH-003WO months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, more than 11 months, or more than 12 months.

[0127] In certain embodiments, the preservation time is more than 1 week, more than 2 weeks, more than 3 weeks, more than 4 weeks, more than 5 weeks, more than 6 weeks, more than 7 weeks, more than 8 weeks, more than 9 weeks, more than 10 weeks, more than 15 weeks, more than 20 weeks, more than 30 weeks, more than 40 weeks, more than 50 weeks, more than 100 weeks.

[0128] In some embodiments, the preservation time is more than 1 day. In some embodiments, the preservation time is 1 day to 80 days. In some embodiments, the preservation time is 1 day to 50 days. In some embodiments, the preservation time is 1 day to 14 days. In certain embodiments, the preservation time is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 70 days, 80 days, 90 days, or 100 days or more.

[0129] In certain embodiments, the preservation time of the present disclosure is 1 hour or more, 24 hours or more. In certain embodiments, the preservation time of the present disclosure is 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, or 48 hours or more.

[0130] In some embodiments, the second volume contains ice nucleating agents. The nucleating agent forms an ice crystal, thereby a biological matter can be preserved in the unfrozen part of the volume. The ice crystal formed by the nucleating agent generates pressures of the isochoric chamber, and the enhanced pressures hinder further formation of ice. In some embodiments, the nucleating agent can be a structural element of the isochoric freezing device. For example, the nucleating agent can be walls of the isochoric chamber itself, or walls of the matter container itself. In other embodiments, the nucleating agent can be any agent that promotes the formation of ice crystal in the isochoric chamber. For example, the ice nucleating agent is, but not limited to, minute solid particles, such as dust or food particles,Atty. Dkt: BCRH-003WO large molecules, ice-nucleating proteins. In some embodiments, the nucleating agent is placed in the bottom of the isochoric chamber, and the chamber is completely filled with an aqueous solution. In some embodiments, a biological matter is placed in a container in an isochoric chamber that allows transfer of only pressure and heat but not mass and the isochoric chamber is filled with a fluid and nucleation agents that cause the fluid to freeze before the biological matter in the container freezes.

[0131] In some embodiments, the pressure that is generated is between about 1 and about 300 MPa. In certain embodiments, the pressure that is generated is about 1 MPa to about 250 MPa, about 10 MPa to about 300 MPa, about 20 MPa to about 250 MPa, about 30 MPa to about 250 MPa, about 40 MPa to about 250 MPa, about 40 MPa to about 200 MPa, about 50 MPa to about 170 MPa or about 50 MPa to about 150 MPa. In certain embodiments, the pressure that is generated is about 1 MPa, about 5 MPa, about 10 MPa, about 20 MPa, about 25 MPa, about 30 MPa, about 35 MPa, about 40 MPa, about 45 MPa, about 50 MPa, about 55 MPa, about 60 MPa, about 65 MPa, about 70 MPa, about 75 MPa, about 80 MPa, about 85 MPa, about 90 MPa, about 95MPa, about 100 MPa, about 110 MPa, about 120 MPa, about 130 MPa, about 140 MPa, about 150 MPa, about 160 MPa, about 170 MPa, about 180 MPa, about 190 MPa, about 200 MPa, about 210 MPa, about 220 MPa, about 230 MPa, about 240 MPa, about 250 MPa, about 260 MPa, about 270 MPa, about 280 MPa, about 290 MPa, or about 300 MPa.

[0132] In some embodiments, the pressure that is generated depends on the volume of the freezable volume, the composition of the freezable volume and other volumes, the storage temperature, and the temperature history (e.g., cooling rate).

[0133] In some embodiments, the pressure does not irreversibly damage the biological matter.

[0134] In some embodiments, the generated pressure lowers the equilibrium melting temperature of the biological material in the first volume.

[0135] In some embodiments, the generated pressure raises the glass transition temperature of the biological material in the first volume.

[0136] In some embodiments, the pressure lowers the cooling or warming rates required to avoid ice formation during cooling to or warming from the storage temperature.Atty. Dkt: BCRH-003WO

[0137] In some embodiments, the cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 1000 K / s. In certain embodiments, the cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 900 K / s, between about 0.1 K / hr and about 800 K / s, between about 0.1 K / hr and about 700 K / s, between about 0.1 K / hr and about 600 K / s, between about 0.1 K / hr and about 500 K / s, between about 0.1 K / hr and about 400 K / s, between about 0.1 K / hr and about 300 K / s, between about 0.1 K / hr and about 200 K / s¸ between about 0.1 K / hr and about 100 K / s, between about 1 K / hr and about 1000 K / s, between about 10 K / hr and about 1000 K / s, between about 100 K / hr and about 1000 K / s, between about 100 K / hr and about 500 K / s, or between about 100 K / hr and about 250 K / s.

[0138] In some embodiments, the warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 1000 K / s. In certain embodiments, the warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 900 K / s, between about 0.1 K / hr and about 800 K / s, between about 0.1 K / hr and about 700 K / s, between about 0.1 K / hr and about 600 K / s, between about 0.1 K / hr and about 500 K / s, between about 0.1 K / hr and about 400 K / s, between about 0.1 K / hr and about 300 K / s, between about 0.1 K / hr and about 200 K / s¸ between about 0.1 K / hr and about 100 K / s, between about 1 K / hr and about 1000 K / s, between about 10 K / hr and about 1000 K / s, between about 100 K / hr and about 1000 K / s, between about 100 K / hr and about 500 K / s, or between about 100 K / hr and about 250 K / s.

[0139] In some embodiments, the cooling or warming rates are not constant.

[0140] 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.

[0141] In some embodiments, the system further comprises a temperature controller configured to monitor and control the temperature of the system. In certain embodiments, the temperature controller is configured to cool the system of the present disclosure or heat the system of the present disclosure. In certain embodiments, the temperature controller comprises a cooling system and a heating system. In certain embodiments, the temperatureAtty. Dkt: BCRH-003WO controller is any device or system configured to cool the fluid in the isochoric chamber below 0 °C and warm the fluid in the isochoric chamber above 0 °C. For example, the cooling system is a conventional freezer, and the warming system is a conventional heater.

[0142] In some embodiments, the cooling temperature is above about −200 °C and below the equilibrium melting point of the contents of the first volume.

[0143] In some embodiments, the temperature control system is further configured to warm the fluid in the isochoric chamber above 0 °C. In some cases, the warming temperature is above 0 °C to 5 °C, above 0 °C to 6 °C, above 0 °C to 7 °C, above 0 °C to 8 °C, above 0 °C to 9 °C, or above 0 °C to 10 °C. The warming or thawing conditions are well known in the art. The conventional warming or thawing conditions can be applied herein.

[0144] In some embodiments, the system optionally comprises a pressure controller configured to monitor the pressure within the system.

[0145] In some embodiments, the generated pressure raises the glass transition temperature of the biological material in the first volume.

[0146] In some embodiments, the system generates a pressure-stabilized supercooled state through the manipulation of temperature alone in the isochoric system. 4. Device for preserving a biological matter comprising the system of the present disclosure

[0147] Aspects of the present disclosure provide a device for preserving a biological matter without freezing at temperatures below its equilibrium melting point, the device comprises a) the pressure-stabilized supercooling system of the present disclosure; b) a biological matter placed within the first volume of the system of the present disclosure; c) a temperature controller configured to monitor and control the temperature of the system; and d) a pressure controller configured to monitor the pressure within the system.

[0148] The pressure-stabilized supercooling system of the present disclosure is precisely described in the above section (“3. Systems for inducing pressure-stabilized supercooling at a desired pressure and temperature”) and is incorporated into this section.

[0149] In some embodiments, the biological matter is immersed in an aqueous solution. In some embodiments, the biological matter is immersed in an aqueous solution with which it was perfused. In some embodiments, the biological matter is immersed in a solutionAtty. Dkt: BCRH-003WO different from the aqueous solution with which it was perfused. In some embodiments, the biological matter is immersed in a water-immiscible non-aqueous solution.

[0150] In some embodiments, the solution with which the biological matter is perfused is a high-entropy solution. In some embodiments, the solution in which the biological matter is immersed is a high-entropy solution. 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 sub-zero centigrade temperatures. As such, the high entropy solution may be used for preserving unfrozen biological matter at sub-zero centigrade temperature without ice formation.

[0151] In some embodiments, the temperature at which the biological matter is stored is lower than the equilibrium melting temperature of the biological matter.

[0152] In some embodiments, the biological matter is cooled and stored without ice formation in the system of the present disclosure.

[0153] In some embodiments, the cooling of the system is non-uniform.

[0154] In some embodiments, preservation time of the present disclosure is a length of time for preservation in the system of the present disclosure. In certain embodiments, the preservation time is a length of time from hours to days, hours to weeks, hours to months, days to months, days to weeks, weeks to months, or months to years. In some embodiments, the biological matter is stored / preserved for a duration between about 1 hour and about 1000 years.

[0155] In certain embodiments, the preservation time is more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, more than 11 months, or more than 12 months.

[0156] In certain embodiments, the preservation time is more than 1 week, more than 2 weeks, more than 3 weeks, more than 4 weeks, more than 5 weeks, more than 6 weeks, more than 7 weeks, more than 8 weeks, more than 9 weeks, more than 10 weeks, more than 15 weeks, more than 20 weeks, more than 30 weeks, more than 40 weeks, more than 50 weeks, more than 100 weeks.Atty. Dkt: BCRH-003WO

[0157] In some embodiments, the preservation time is more than 1 day. In some embodiments, the preservation time is 1 day to 80 days. In some embodiments, the preservation time is 1 day to 50 days. In some embodiments, the preservation time is 1 day to 14 days. In certain embodiments, the preservation time is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 70 days, 80 days, 90 days, or 100 days or more.

[0158] In certain embodiments, the preservation time of the present disclosure is 1 hour or more, 24 hours or more. In certain embodiments, the preservation time of the present disclosure is 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, or 48 hours or more.

[0159] In some embodiments, the pressure that is generated is between about 1 and about 300 MPa. In certain embodiments, the pressure that is generated is about 1 MPa to about 250 MPa, about 10 MPa to about 300 MPa, about 20 MPa to about 250 MPa, about 30 MPa to about 250 MPa, about 40 MPa to about 250 MPa, about 40 MPa to about 200 MPa, about 50 MPa to about 170 MPa or about 50 MPa to about 150 MPa. In certain embodiments, the pressure that is generated is about 1 MPa, about 5 MPa, about 10 MPa, about 20 MPa, about 25 MPa, about 30 MPa, about 35 MPa, about 40 MPa, about 45 MPa, about 50 MPa, about 55 MPa, about 60 MPa, about 65 MPa, about 70 MPa, about 75 MPa, about 80 MPa, about 85 MPa, about 90 MPa, about 95MPa, about 100 MPa, about 110 MPa, about 120 MPa, about 130 MPa, about 140 MPa, about 150 MPa, about 160 MPa, about 170 MPa, about 180 MPa, about 190 MPa, about 200 MPa, about 210 MPa, about 220 MPa, about 230 MPa, about 240 MPa, about 250 MPa, about 260 MPa, about 270 MPa, about 280 MPa, about 290 MPa, or about 300 MPa.

[0160] In some embodiments, the pressure that is generated depends on the volume of the freezable volume, the composition of the freezable volume and other volumes, the storage temperature, and the temperature history (e.g., cooling rate).Atty. Dkt: BCRH-003WO

[0161] In some embodiments, the pressure does not irreversibly damage the biological matter.

[0162] In some embodiments, the generated pressure lowers the equilibrium melting temperature of the biological material in the first volume.

[0163] In some embodiments, the generated pressure raises the glass transition temperature of the biological material in the first volume.

[0164] In some embodiments, the pressure lowers the cooling or warming rates required to avoid ice formation during cooling to or warming from the storage temperature.

[0165] In some embodiments, the cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 1000 K / s. In certain embodiments, the cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 900 K / s, between about 0.1 K / hr and about 800 K / s, between about 0.1 K / hr and about 700 K / s, between about 0.1 K / hr and about 600 K / s, between about 0.1 K / hr and about 500 K / s, between about 0.1 K / hr and about 400 K / s, between about 0.1 K / hr and about 300 K / s, between about 0.1 K / hr and about 200 K / s¸ between about 0.1 K / hr and about 100 K / s, between about 1 K / hr and about 1000 K / s, between about 10 K / hr and about 1000 K / s, between about 100 K / hr and about 1000 K / s, between about 100 K / hr and about 500 K / s, or between about 100 K / hr and about 250 K / s.

[0166] In some embodiments, the warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 1000 K / s. In certain embodiments, the warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 900 K / s, between about 0.1 K / hr and about 800 K / s, between about 0.1 K / hr and about 700 K / s, between about 0.1 K / hr and about 600 K / s, between about 0.1 K / hr and about 500 K / s, between about 0.1 K / hr and about 400 K / s, between about 0.1 K / hr and about 300 K / s, between about 0.1 K / hr and about 200 K / s¸ between about 0.1 K / hr and about 100 K / s, between about 1 K / hr and about 1000 K / s, between about 10 K / hr and about 1000 K / s, between about 100 K / hr and about 1000 K / s, between about 100 K / hr and about 500 K / s, or between about 100 K / hr and about 250 K / s.

[0167] In some embodiments, the cooling or warming rates are not constant.Atty. Dkt: BCRH-003WO

[0168] 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.

[0169] In some embodiments, the system further comprises a temperature controller configured to monitor and control the temperature of the system. In certain embodiments, the temperature controller is configured to cool the system of the present disclosure or heat the system of the present disclosure. In certain embodiments, the temperature controller comprises a cooling system and a heating system. In certain embodiments, the temperature controller is any device or system configured to cool the fluid in the isochoric chamber below 0 °C and warm the fluid in the isochoric chamber above 0 °C. For example, the cooling system is a conventional freezer, and the warming system is a conventional heater.

[0170] In some embodiments, the cooling temperature is above about −200°C and below the equilibrium melting point of the contents of the first volume.

[0171] In some embodiments, the temperature control system is further configured to warm the fluid in the isochoric chamber above 0 °C. In some cases, the warming temperature is above 0 °C to 5 °C, above 0 °C to 6 °C, above 0 °C to 7 °C, above 0 °C to 8 °C, above 0 °C to 9 °C, or above 0 °C to 10 °C. The warming or thawing conditions are well known in the art. The conventional warming or thawing conditions can be applied herein.

[0172] In some embodiments, the device of the present disclosure generates a pressure-stabilized supercooled state through the manipulation of temperature alone in the isochoric system.

[0173] In some embodiments, the device comprises a pressure controller configured to monitor the pressure within the system.

[0174] In some embodiments, the device comprises a temperature controller configured to monitor and control the temperature of the system. In some embodiments, the temperature controller is configured to cool the fluid in the isochoric chamber and warm the fluid in the isochoric chamber above 0 °C. In some embodiments, the temperature controller may comprise a cooling system and a warming system. For example, the cooling system is a conventional freezer. For example, the warming system is a conventional heater.Atty. Dkt: BCRH-003WO

[0175] In some embodiments, the cooling temperature is above about −200 °C and below the equilibrium melting point of the contents of the first volume.

[0176] In some embodiments, the warming temperature is above 0 °C to 5 °C, above 0 °C to 6 °C, above 0 °C to 7 °C, above 0 °C to 8 °C, above 0 °C to 9 °C, or above 0 °C to 10 °C. The warming or thawing conditions are well known in the art. The conventional warming or thawing conditions can be applied herein. 5. A method of preserving a biological matter

[0177] In some aspects, the present disclosure provides a method of preserving a biological matter without freezing at temperatures below its equilibrium melting point, the method comprises a) placing the biological matter within the first volume of the pressure- stabilized supercooling system of the present disclosure; b) cooling the system to a temperature lower than the equilibrium melting point of the biological matter and lower than the nucleation temperature of the second volume (^^^^,^); and c) storing the biological matter without freezing in a pressurized state at a temperature above about −200 °C and below the equilibrium melting point of the contents of the first volume (^^^^,^).

[0178] The pressure-stabilized supercooling system of the present disclosure is precisely described in the above section (“3. Systems for inducing pressure-stabilized supercooling at a desired pressure and temperature”) and is incorporated into this section.

[0179] In some embodiments, the biological matter can be directly placed in the first volume of the pressure-stabilized supercooling system of the present disclosure. In other embodiments, the biological matter can be placed in a container that allows transfer of pressure and heat but not mass, and the container can be directly placed in first volume of the pressure-stabilized supercooling system of the present disclosure.

[0180] In some embodiments, the temperature at which the biological matter is stored is lower than the equilibrium melting temperature of the biological matter.

[0181] In some embodiments, the biological matter is cooled and stored without ice formation.

[0182] In some embodiments, the biological matter is stored for a duration from hours to days, hours to weeks, hours to months, days to months, days to weeks, weeks to months, orAtty. Dkt: BCRH-003WO months to years. In some embodiments, the biological matter is stored for a duration between about 1 hour and about 1000 years.

[0183] In certain embodiments, the preservation time is more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, more than 11 months, or more than 12 months.

[0184] In certain embodiments, the preservation time is more than 1 week, more than 2 weeks, more than 3 weeks, more than 4 weeks, more than 5 weeks, more than 6 weeks, more than 7 weeks, more than 8 weeks, more than 9 weeks, more than 10 weeks, more than 15 weeks, more than 20 weeks, more than 30 weeks, more than 40 weeks, more than 50 weeks, more than 100 weeks.

[0185] In some embodiments, the preservation time is more than 1 day. In some embodiments, the preservation time is 1 day to 80 days. In some embodiments, the preservation time is 1 day to 50 days. In some embodiments, the preservation time is 1 day to 14 days. In certain embodiments, the preservation time is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 70 days, 80 days, 90 days, or 100 days or more.

[0186] In certain embodiments, the preservation time of the present disclosure is 1 hour or more, 24 hours or more. In certain embodiments, the preservation time of the present disclosure is 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, or 48 hours or more.

[0187] In some embodiments, the pressure that is generated is between about 1 and about 300 MPa. In certain embodiments, the pressure that is generated is about 1 MPa to about 250 MPa, about 10 MPa to about 300 MPa, about 20 MPa to about 250 MPa, about 30 MPa to about 250 MPa, about 40 MPa to about 250 MPa, about 40 MPa to about 200 MPa, about 50 MPa to about 170 MPa or about 50 MPa to about 150 MPa. In certain embodiments,Atty. Dkt: BCRH-003WO the pressure that is generated is about 1 MPa, about 5 MPa, about 10 MPa, about 20 MPa, about 25 MPa, about 30 MPa, about 35 MPa, about 40 MPa, about 45 MPa, about 50 MPa, about 55 MPa, about 60 MPa, about 65 MPa, about 70 MPa, about 75 MPa, about 80 MPa, about 85 MPa, about 90 MPa, about 95MPa, about 100 MPa, about 110 MPa, about 120 MPa, about 130 MPa, about 140 MPa, about 150 MPa, about 160 MPa, about 170 MPa, about 180 MPa, about 190 MPa, about 200 MPa, about 210 MPa, about 220 MPa, about 230 MPa, about 240 MPa, about 250 MPa, about 260 MPa, about 270 MPa, about 280 MPa, about 290 MPa, or about 300 MPa.

[0188] In some embodiments, the pressure that is generated depends on the volume of the freezable volume, the composition of the freezable volume and other volumes, the storage temperature, and the temperature history (e.g., cooling rate).

[0189] In some embodiments, the pressure does not irreversibly damage the biological matter.

[0190] In some embodiments, the generated pressure lowers the equilibrium melting temperature of the biological material in the first volume.

[0191] In some embodiments, the generated pressure raises the glass transition temperature of the biological material in the first volume.

[0192] In some embodiments, the pressure lowers the cooling or warming rates required to avoid ice formation during cooling to or warming from the storage temperature.

[0193] In some embodiments, the cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 1000 K / s. In certain embodiments, the cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 900 K / s, between about 0.1 K / hr and about 800 K / s, between about 0.1 K / hr and about 700 K / s, between about 0.1 K / hr and about 600 K / s, between about 0.1 K / hr and about 500 K / s, between about 0.1 K / hr and about 400 K / s, between about 0.1 K / hr and about 300 K / s, between about 0.1 K / hr and about 200 K / s¸ between about 0.1 K / hr and about 100 K / s, between about 1 K / hr and about 1000 K / s, between about 10 K / hr and about 1000 K / s, between about 100 K / hr and about 1000 K / s, between about 100 K / hr and about 500 K / s, or between about 100 K / hr and about 250 K / s.Atty. Dkt: BCRH-003WO

[0194] In some embodiments, the warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 1000 K / s. In certain embodiments, the warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 900 K / s, between about 0.1 K / hr and about 800 K / s, between about 0.1 K / hr and about 700 K / s, between about 0.1 K / hr and about 600 K / s, between about 0.1 K / hr and about 500 K / s, between about 0.1 K / hr and about 400 K / s, between about 0.1 K / hr and about 300 K / s, between about 0.1 K / hr and about 200 K / s¸ between about 0.1 K / hr and about 100 K / s, between about 1 K / hr and about 1000 K / s, between about 10 K / hr and about 1000 K / s, between about 100 K / hr and about 1000 K / s, between about 100 K / hr and about 500 K / s, or between about 100 K / hr and about 250 K / s.

[0195] In some embodiments, the cooling or warming rates are not constant.

[0196] 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.

[0197] In some embodiments, the method comprises cooling the system to a temperature lower than the equilibrium melting point of the biological matter and lower than the nucleation temperature of the second volume (^^^^,^). In certain embodiments, the temperature controller is any device or system configured to cool the fluid in the isochoric chamber below 0 °C and warm the fluid in the isochoric chamber above 0 °C. For example, the cooling system is a conventional freezer, and the warming system is a conventional heater.

[0198] In some embodiments, the cooling temperature is above about −200 °C and below the equilibrium melting point of the contents of the first volume.

[0199] In some embodiments, the method comprises storing the biological matter without freezing in a pressurized state at a temperature above about −200 °C and below the equilibrium melting point of the contents of the first volume (^^^^,^).

[0200] In other aspects, the present disclosure provides a method by which to determine the desired volumes, compositions, configuration, and temperature history needed to achieve the desired pressure-stabilized supercooling condition at desired temperature using thermodynamic calculations.Atty. Dkt: BCRH-003WO

[0201] In other aspects, the present disclosure provides a method by which to determine the desired volumes, compositions, configuration, and temperature history needed to achieve the desired pressure-stabilized supercooling condition at a desired temperature using iterative thermodynamic experiments. 6. Examples of Non-Limiting Aspects of the Disclosure

[0202] 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:

[0203] Aspect 1. A system for inducing pressure-stabilized supercooling at a desired pressure and temperature, comprising: an isochoric chamber segmented into two volumes; a first volume containing an aqueous solution with a first nucleation temperature (^^^^,^); a second volume containing an aqueous solution with a second nucleation temperature (^^^^,^), wherein the second volume increases when cooled below the second nucleation temperature (^^^^,^); and a boundary separating the two volumes that transmits heat and pressure but not mass.

[0204] Aspect 2. The system of Aspect 1, wherein the first nucleation temperature and the second nucleation temperature are different.

[0205] Aspect 3. The system of Aspect 1 or 2, wherein the second nucleationtemperature is higher than the first nucleation temperature (^^^^,^ > ^^^^,^).

[0206] Aspect 4. The system of any one of Aspects 1-3, wherein the boundary is selectively permeable and allows transfer of non-water molecules.Atty. Dkt: BCRH-003WO

[0207] Aspect 5. The system of Aspect 4, wherein the non-water molecules are dissolved gases or solutes.

[0208] Aspect 6. The system of any one of Aspects 1-5, wherein the second volume is thermally separated from the first volume.

[0209] Aspect 7. The system of any one of Aspects 1-6, wherein the second volume is further segmented into multiple sub-volumes each with different compositions such that they freeze at different temperatures.

[0210] Aspect 8. The system of any one of Aspects 1-7, wherein one of the volumes nucleates ice, thereby pressuring the system and the other volume at a resultant temperature and pressure does not nucleate ice, thereby achieving a pressurized supercooled state beneath equilibrium melting point.

[0211] Aspect 9. The system of any one of Aspects 1-8, wherein size, composition, and configuration of the first and second volumes are chosen such that the desired pressure is generated by the freezing of the second volume at the desired temperature, and the likelihood of ice formation in the first volume is thereby reduced at the same temperature.

[0212] Aspect 10. The system of any one of Aspects 1-9, wherein the first volume contains biological matter to be preserved, and the second volume contains an aqueous solution with the second nucleation temperature, wherein the second nucleation temperature is higher than the first nucleation temperature of the first volume.

[0213] Aspect 11. The system of Aspect 10, wherein the biological matter is immersed in an aqueous solution.

[0214] Aspect 12. The system of Aspect 10, wherein the biological matter is immersed in an aqueous solution with which it was perfused.

[0215] Aspect 13. The system of Aspect 10, wherein the biological matter is immersed in a solution different from the aqueous solution with which it was perfused.

[0216] Aspect 14. The system of Aspect 12 or 13, wherein the solution with which the biological matter is perfused is a high-entropy solution.

[0217] Aspect 15. The system of Aspect 11, wherein the solution in which the biological matter is immersed is a high-entropy solution.Atty. Dkt: BCRH-003WO

[0218] Aspect 16. The system of Aspect 10, wherein the biological matter is immersed in a water-immiscible non-aqueous solution.

[0219] Aspect 17. The system of any one of Aspects 10-16, wherein the temperature at which the biological matter is stored is lower than the equilibrium melting temperature of the biological matter.

[0220] Aspect 18. The system of any one of Aspects 10-17, wherein the biological matter is cooled and stored without ice formation.

[0221] Aspect 19. The system of any one of Aspects 10-18, wherein the cooling of the system is non-uniform.

[0222] Aspect 20. The system of any one of Aspects 10-18, wherein the biological matter is stored for a duration between about 1 hour and about 1000 years.

[0223] Aspect 21. The system of any one of Aspects 1-20, wherein the second volume contains ice nucleating agents.

[0224] Aspect 22. The system of any one of Aspects 1-21, wherein the pressure that is generated is between 1 and 300 MPa.

[0225] Aspect 23. The system of any one of Aspects 1-22, wherein the pressure that is generated depends on the volume of the freezable volume, the composition of the freezable volume and other volumes, the storage temperature, and the temperature history (e.g., cooling rate).

[0226] Aspect 24. The system of any one of Aspects 1-23, wherein the pressure does not irreversibly damage the biological matter.

[0227] Aspect 25. The system of any one of Aspects 1-24, wherein the generated pressure lowers the equilibrium melting temperature of the biological material in the first volume.

[0228] Aspect 26. The system of Aspects 1-25, wherein the generated pressure raises the glass transition temperature of the biological material in the first volume.

[0229] Aspect 27. The system of any one of Aspects 1-26, wherein the pressure lowers the cooling or warming rates required to avoid ice formation during cooling to or warming from the storage temperature.Atty. Dkt: BCRH-003WO

[0230] Aspect 28. The system of any one of Aspects 1-27, wherein cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 1000 K / s.

[0231] Aspect 29. The system of any one of Aspects 1-27, wherein warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 1000 K / s.

[0232] Aspect 30. The system of Aspect 28 or 29, wherein the cooling or warming rates are not constant.

[0233] Aspect 31. The system of Aspect 10, 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.

[0234] Aspect 32. The system of any one of Aspects 1-31, wherein the system further comprises a temperature controller configured to monitor and control the temperature of the system.

[0235] Aspect 33. The system of any one of Aspects 1-32, wherein the system optionally comprises a pressure controller configured to monitor the pressure within the system.

[0236] Aspect 34. The system of any one of claims 1-33, where the system generates a pressure-stabilized supercooled state through the manipulation of temperature alone in the isochoric system.

[0237] Aspect 35. A system for inducing pressure-stabilized supercooling at a desired pressure and temperature, comprising: an isochoric system segmented into three volumes; a first volume containing an aqueous solution with a first nucleation temperature (^^^^,^); a second volume containing an aqueous solution with a second nucleation temperature (^^^^,^), and the second volume increases when cooled below the second nucleation temperature (^^^^,^);Atty. Dkt: BCRH-003WO a third volume filling the remaining volume of the isochoric system not occupied by the first and second volumes and comprising a material whose volume does not substantially increase at the desired temperature; and boundaries separating the first, the second, and the third volumes that transmit heat and pressure but not mass.

[0238] Aspect 36. The system of Aspect 35, wherein the first nucleation temperature and the second nucleation temperature are different.

[0239] Aspect 37. The system of Aspect 35 or 36, wherein the second nucleationtemperature is higher than the first nucleation temperature (^^^^,^ > ^^^^,^).

[0240] Aspect 38. The system of any one of Aspects 35-37, wherein the third volume contains an aqueous solution that freezes at a lower temperature than the contents of the first and second volumes.

[0241] Aspect 39. The system of any one of Aspects 35-38, wherein the boundaries are selectively permeable and allows transfer of non-water molecules.

[0242] Aspect 40. The system of Aspect 39, wherein the non-water molecules are dissolved gases or solutes.

[0243] Aspect 41. The system of any one of Aspects 35-40, wherein the second volume is thermally separated from the first volume.

[0244] Aspect 42. The system of any one of Aspects 35-41, wherein the second volume is further segmented into multiple sub-volumes each with different compositions such that they freeze at different temperatures.

[0245] Aspect 43. The system of any one of Aspects 35-42, wherein the third volume contains an aqueous solution that freezes at a lower temperature than the contents of the first and second volumes.

[0246] Aspect 44. The system of any one of Aspects 35-42, wherein the third volume contains non-aqueous fluid or solid material.

[0247] Aspect 45. The system of any one of Aspects 35-44, wherein size, composition, and configuration of the first, the second, and the third volumes are chosen such that the desired pressure is generated by the freezing of the second volume at the desiredAtty. Dkt: BCRH-003WO temperature, and the likelihood of ice formation in the first volume is thereby reduced at the same temperature.

[0248] Aspect 46. The system of any one of Aspects 35-45, wherein the first volume contains a biological matter to be preserved; the second volume contains an aqueous solution with the second nucleation temperature, wherein the second nucleation temperature is higher than the nucleation temperature of the first volume; and the third volume contains a material that does not expand as the temperature of the system is lowered.

[0249] Aspect 47. The system of Aspect 46, wherein the biological matter is immersed in an aqueous solution.

[0250] Aspect 48. The system of Aspect 46, wherein the biological matter is immersed in an aqueous solution with which it was perfused.

[0251] Aspect 49. The system of Aspect 46, wherein the biological matter is immersed in a solution different from the aqueous solution with which it was perfused.

[0252] Aspect 50. The system of any one of Aspects 46-49, wherein the solution with which the biological matter is perfused is a high-entropy solution.

[0253] Aspect 51. The system of any one of Aspects 46-49, wherein the solution in which the biological matter is immersed is a high-entropy solution.

[0254] Aspect 52. The system of Aspect 46, wherein the biological matter is immersed in a water-immiscible non-aqueous solution.

[0255] Aspect 53. The system of any one of Aspects 35-52, wherein the temperature at which the biological matter is stored is lower than the equilibrium melting temperature of the biological matter.

[0256] Aspect 54. The system of any one of Aspects 35-53, wherein the biological matter is cooled and stored without ice formation.

[0257] Aspect 55. The system of any one of Aspects 35-54, wherein the cooling of the system is non-uniform.

[0258] Aspect 56. The system of any one of Aspects 46-55, wherein the biological matter is stored for a duration between about 1 hour and about 1000 years.

[0259] Aspect 57. The system of any one of Aspects 35-56, wherein the second volume contains ice nucleating agents.Atty. Dkt: BCRH-003WO

[0260] Aspect 58. The system of any one of Aspects 35-57, wherein the pressure that is generated is between 1 and 300 MPa.

[0261] Aspect 59. The system of any one of Aspects 35-58, wherein the pressure that is generated depends on the volume of the freezable volume, the composition of the freezable volume and other volumes, the storage temperature, and the temperature history (e.g., cooling rate).

[0262] Aspect 60. The system of any one of Aspects 46-59, wherein the pressure does not irreversibly damage the biological matter.

[0263] Aspect 61. The system of any one of Aspects 35-60, wherein the generated pressure lowers the equilibrium melting temperature of the biological material in the first volume.

[0264] Aspect 62. The system of any one of Aspects 35-61, wherein the generated pressure raises the glass transition temperature of the biological material in the first volume.

[0265] Aspect 63. The system of any one of Aspects 35-62, wherein the pressure lowers the cooling or warming rates required to avoid ice formation during cooling to or warming from the storage temperature.

[0266] Aspect 64. The system of Aspect 63, wherein the cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 1000 K / s.

[0267] Aspect 65. The system of Aspect 64, wherein the warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 1000 K / s.

[0268] Aspect 66. The system of Aspect 64 or 65, wherein the cooling or warming rates are not constant.

[0269] Aspect 67. The system of any one of Aspects 46-66, 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.Atty. Dkt: BCRH-003WO

[0270] Aspect 68. The system of any one of Aspects 35-67, wherein the system further comprises a temperature controller configured to monitor and control the temperature of the system.

[0271] Aspect 69. The system of any one of Aspects 35-68, wherein the system optionally comprises a pressure controller configured to monitor the pressure within the system.

[0272] Aspect 70. The system of any one of Aspects 35-69, where the system generates a pressure-stabilized supercooled state through the manipulation of temperature alone in the isochoric system.

[0273] Aspect 71. A device for preserving a biological matter without freezing at temperatures below its equilibrium melting point, the device comprises: the pressure-stabilized supercooling system of Aspects 1-70; a biological matter placed within the first volume of the system of Aspects 1-70; a temperature controller configured to monitor and control the temperature of the system; and a pressure controller configured to monitor the pressure within the system.

[0274] Aspect 72. The device of Aspect 71, wherein the biological matter is immersed in an aqueous solution.

[0275] Aspect 73. The device of Aspect 71, wherein the biological matter is immersed in an aqueous solution with which it was perfused.

[0276] Aspect 74. The device of Aspect 71, wherein the biological matter is immersed in a solution different from the aqueous solution with which it was perfused.

[0277] Aspect 75. The device of Aspect 71, wherein the solution with which the biological matter is perfused is a high-entropy solution.

[0278] Aspect 76. The device of Aspect 71, wherein the solution in which the biological matter is immersed is a high-entropy solution.

[0279] Aspect 77. The device of Aspect 71, wherein the biological matter is immersed in a water-immiscible non-aqueous solution.Atty. Dkt: BCRH-003WO

[0280] Aspect 78. The device of any one of Aspects 71-77, wherein the temperature at which the biological matter is stored is lower than the equilibrium melting temperature of the biological matter.

[0281] Aspect 79. The device of any one of Aspects 71-78, wherein the biological matter is cooled and stored without ice formation.

[0282] Aspect 80. The device of any one of Aspects 71-79, wherein the cooling of the system is non-uniform.

[0283] Aspect 81. The device of any one of Aspects 71-80, wherein the biological matter is stored for a duration between about 1 hour and about 1000 years.

[0284] Aspect 82. The device of any one of Aspects 71-81, wherein the second volume contains ice nucleating agents.

[0285] Aspect 83. The device of any one of Aspects 71-82, wherein the pressure that is generated is between 1 and 300 MPa.

[0286] Aspect 84. The device of any one of Aspects 71-83, wherein the pressure that is generated depends on the volume of the freezable volume, the composition of the freezable volume and other volumes, the storage temperature, and the temperature history (e.g., cooling rate).

[0287] Aspect 85. The device of any one of Aspects 71-84, wherein the pressure does not irreversibly damage the biological matter.

[0288] Aspect 86. The device of any one of Aspects 71-85, wherein the generated pressure lowers the equilibrium melting temperature of the biological material in the first volume.

[0289] Aspect 87. The device of any one of Aspects 71-85, where the device generates a pressure-stabilized supercooled state through the manipulation of temperature alone in the isochoric system.

[0290] Aspect 88. A method of preserving a biological matter without freezing at temperatures below its equilibrium melting point, the method comprises: placing the biological matter within the first volume of the pressure-stabilized supercooling system of Aspects 1-70;Atty. Dkt: BCRH-003WO cooling the system to a temperature lower than the equilibrium melting point of the biological matter and lower than the nucleation temperature of the second volume (^^^^,^); and storing the biological matter without freezing in a pressurized state at a temperature above about −200 °C and below the equilibrium melting point of the contents of the first volume (^^^^,^).

[0291] Aspect 89. The method of Aspect 88, wherein the temperature at which the biological matter is stored is lower than the equilibrium melting temperature of the biological matter.

[0292] Aspect 90. The method of Aspect 88 or 89, wherein the biological matter is cooled and stored without ice formation.

[0293] Aspect 91. The method of any one of Aspects 88-90, wherein the biological matter is stored for a duration between about 1 hour and about 1000 years.

[0294] Aspect 92. The method of any one of Aspects 88-91, wherein the pressure that is generated is between 1 and 300 MPa.

[0295] Aspect 93. The method of any one of Aspects 88-92, wherein the pressure does not irreversibly damage the biological matter.

[0296] Aspect 94. The method of any one of Aspects 88-93, wherein the generated pressure lowers the equilibrium melting temperature of the biological material in the first volume.

[0297] Aspect 95. The method of any one of Aspects 88-94, wherein the generated pressure raises the glass transition temperature of the biological material in the first volume.

[0298] Aspect 96. The method of any one of Aspects 88-95, wherein the pressure lowers the cooling or warming rates required to avoid ice formation during cooling to or warming from the storage temperature.

[0299] Aspect 97. The method of any one of Aspects 88-96, wherein the cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 1000 K / s.Atty. Dkt: BCRH-003WO

[0300] Aspect 98. The method of any one of Aspects 88-96, wherein the warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 1000 K / s.

[0301] Aspect 99. The method of Aspect 97 or 98, wherein the cooling or warming rates are not constant.

[0302] Aspect 100. The method of any one of Aspects 88-99, 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.

[0303] Aspect 101. A method by which to determine the desired volumes, compositions, configuration, and temperature history needed to achieve the desired pressure- stabilized supercooling condition at desired temperature using thermodynamic calculations.

[0304] Aspect 102. A method by which to determine the desired volumes, compositions, configuration, and temperature history needed to achieve the desired pressure- stabilized supercooling condition at a desired temperature using iterative thermodynamic experiments. EXAMPLES

[0305] 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 invention 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.

[0306] All publications and patent applications cited in this specification are incorporated by reference herein as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0307] 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 theAtty. Dkt: BCRH-003WO 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. Example 1. Two-volume system

[0308] FIGS.2A and 2B show a two-volume system. FIG.2A depicts a two-volume isochoric pressure-stabilized supercooling system. FIG.2B depicts pressure and ice phase fraction as a function of temperature for an isochoric system of ordinary water.

[0309] Aspects of the present disclosure introduce a system for inducing pressure- stabilized supercooling at a desired pressure and temperature, comprising:

[0310] 1) an isochoric system segmented into two volumes;

[0311] 2) the first volume containing an aqueous solution with nucleation temperature ^^^^,^;

[0312] 3) the second volume containing an aqueous solution with a nucleationtemperature ^^^^,^ > ^^^^,^, and whose volume increases when cooled below ^^^^,^; and

[0313] 4) a boundary separating the two volumes that transmits heat and pressure but not mass.

[0314] Wherein the size, composition, and configuration of the volumes are chosen such that the desired pressure is generated by the freezing of the second volume at the desired temperature, and the likelihood of ice formation in the first volume is thereby reduced at the same temperature. The size and composition required to generate the desired pressure at the desired temperature can be determined by thermodynamic calculation or determined by iterative thermodynamic experiments.

[0315] In one embodiment, the isochoric system is segmented into two volumes. The first volume holds the biological matter to be preserved and is separated from the second volume by a flexible boundary (e.g., plastic bag) that transmits pressure and heat but not mass. The second volume fills the remainder of the system. This second volume consists of an aqueous solution that has a higher nucleation temperature than the contents of the first volume. This has the effect that when the temperature of the system is lowered below 0 °C, ice forms within the second volume, and as a result increases the hydrostatic pressure within theAtty. Dkt: BCRH-003WO system. By increasing the pressure within the system, the stability of the supercooled state of the first system is enhanced due to the pressure lowering the equilibrium liquidus temperature and increasing the interfacial free energy between the solution and an incipient ice nucleus.

[0316] To illustrate this further, an embodiment of this example may have two volumes containing ordinary water. The first volume takes up 75% of the volume of the isochoric system, and the second volume takes up the remaining 25%. At atmospheric pressure, the first volume might have a nucleation temperature of −3 °C and the second volume, which may contain ice nucleating particles, has a nucleation temperature of −1.5 °C. As the temperature of the system is lowered below −1.5 °C, ice forms within the second volume and the pressure within the system increases. According to the pressure-temperature-volume phase diagram for water, when the temperature of the system reaches −5 °C, the entire second volume should be ice and the pressure within the system should have risen to about 60 MPa. As the equilibrium liquidus temperature of the solution within the first volume has been reduced by about 5 °C, we may expect that it will be able to supercool at least this same distance below the normal temperature of nucleation (i.e., −3 °C).

[0317] As the volume of the system that transforms into ice is the primary determinant of the pressure that develops, the ratio of the two volumes must be defined so that the entire second volume transforms to ice at the desired storage temperature. Thus, if it is only desired to apply 30 MPa of pressure onto the biological matter, then the second volume must take up 15% of the system volume, with the first volume taking up the remaining 85%. In this way, in order to achieve different pressures, the volume of both compartments must be varied.

[0318] In certain embodiments, the boundary or boundaries separating the volumes transmit pressure and heat, but not mass. In certain embodiments, these boundaries may be comprised of flexible, impermeable membranes. In other embodiments, these boundaries may be comprised of plastic bags, rubber bags, vacuum sealed bags, or other polymer barriers. Example 2. Three-volume system with solid material as third volume

[0319] Aspects of the present disclosure further introduce a system for inducing pressure-stabilized supercooling at a desired pressure and temperature, comprising:

[0320] 1) an isochoric system segmented into three volumes;Atty. Dkt: BCRH-003WO

[0321] 2) the first volume containing an aqueous solution with nucleation temperature ^^^^,^;

[0322] 3) the second volume containing an aqueous solution with a nucleationtemperature ^^^^,^ > ^^^^,^, and whose volume increases when cooled below ^^^^,^;

[0323] 4) the third volume filling the remaining volume of the isochoric system not occupied by the first and second volumes and comprising a material whose volume does not substantially increase when the temperature is reduced; and

[0324] 5) boundaries separating the individual volumes that transfer heat and pressure but to not transfer mass.

[0325] Wherein the size, composition, and configuration of the volumes are chosen such that the desired pressure is generated by the freezing of the second volume at the desired temperature, and the likelihood of ice formation in the first volume is thereby reduced at the same temperature. The size and composition required to generate the desired pressure at the desired temperature can be determined by thermodynamic calculation or determined by iterative thermodynamic experiments.

[0326] As clarified in the first example, in certain embodiments, the volume of the system that transforms into ice is the primary determinant of the pressure that develops. Thus, the ratio of the two volumes must be defined so that the entire second volume transforms to ice at the desired storage temperature. Often, it is desirable to maintain the size of the first volume as small as possible or to vary its volume independently. In order to do this, the remaining portion of the isochoric chamber (occupied by the second, freezable volume) can be filled partially with solid material in order to displace liquid and give the user another method by which to vary the ratio of the two liquid volumes. This scenario is depicted in the figure below wherein glass beads are used to displace fluid in the isochoric chamber.

[0327] In certain embodiments, the boundary or boundaries separating the volumes transmit pressure and heat, but not mass. In certain embodiments, these boundaries may be comprised of flexible, impermeable membranes. In other embodiments, these boundaries may be comprised of plastic bags, rubber bags, vacuum sealed bags, or other polymer barriers.

[0328] FIGS.3A and 3B show one embodiment of a three-volume system with solid material as third volume. FIG.3A depicts a three-volume isochoric pressure-stabilizedAtty. Dkt: BCRH-003WO supercooling system with the third volume comprising glass beads. FIG.3B depicts Pressure and ice phase fraction as a function of temperature for the three-volume isochoric system with ordinary water.

[0329] A further embodiment involves a three-volume system with aqueous solution as third volume. It may be desirable to vary the first and second volumes independently to achieve the specific ratio, V2 / Vsys, which produces the desired magnitude of pressure. Instead of using solid material to displace volume within the isochoric system, another liquid may be used which has a nucleation temperature lower than the desired storage temperature. For example, if the desired storage temperature is −4 °C, a 15% w / w ethanol:water solution may be used as the third volume as it has an atmospheric pressure equilibrium liquidus temperature of about −6.5 °C. In this embodiment both first and second volumes are contained within flexible membranes (e.g., bags) that transmit pressure and heat but not mass, and the non-freezable fluid fills the remaining volume of the isochoric system.

[0330] In other embodiments, the fluid that comprises the third volume may, in addition to being an aqueous solution that has a lower freezing temperature than the storage temperature, may be a non-aqueous fluid such as mineral oil or a perfluorocarbon fluid, or may be a combination of solid and liquid materials.

[0331] FIGS.4A and 4B show another embodiment of a three-volume system with solid material as third volume. FIG.4A depicts a three-volume isochoric pressure-stabilized supercooling system with the third volume comprising a solution that freezes at a lower temperature than the contents of the first and second volumes. FIG.4B depicts Pressure and ice phase fraction as a function of temperature for the three-volume isochoric system with ordinary water.

[0332] Yet additional aspects of this disclosure introduce a three-volume isochoric baro-supercooling system in which the second volume, which freezes and raises the pressure within the isochoric system, is further segmented into multiple sub-volumes. Each of these sub-volumes may have unique volumes, compositions, and nucleation temperatures (however all are greater than the nucleation temperature of the first volume, third volume, and storage temperature). This has the intended effect that as the temperature of the system is lowered, the pressure increases in a stepwise manner. The precise nature of the stepwise increase inAtty. Dkt: BCRH-003WO pressure is controlled by certain properties of the individual sub-volumes including but not limited to: the nucleation temperature, the volume, the chemical composition, the location within the isochoric chamber.

[0333] In one embodiment, depicted in the figure below, the second volume is segmented into four sub-volumes (V2,A, V2,B, V2,C, and V2,D). The nucleation temperature of V2,Ais greater than the nucleation temperature of V2,Bwhich is greater than the nucleation temperature of V2,C, which is further greater than the nucleation temperature of V2,D. As the temperature of the system is reduced, the sub-volumes freeze in succession, each generating a finite increase in pressure.

[0334] In certain embodiments, the nucleation temperature of the volumes is tuned by heterogeneous nucleation catalysts such as addition of antifreeze proteins.

[0335] FIGS.5A and 5B show another embodiment of a three-volume system with solid material as third volume. FIG.5A depicts a three-volume isochoric pressure-stabilized supercooling system with the third volume further segmented into four sub-volumes. FIG.5B depicts Hypothetical cooling trajectory for the three-volume system. The individual sub- volumes comprising the third volume freeze at different temperatures and progressively increase the system pressure in a stepwise manner. Example 3. A cryopreservation device

[0336] Aspects of the present disclosure introduce a device for preserving a biological matter without freezing at temperatures below its equilibrium melting point, the device comprising:

[0337] 1) Either a pressure-stabilized supercooling system, comprising: a. an isochoric system segmented into two volumes; b. the first volume containing an aqueous solution with nucleation temperature ^^^^,^; c. the second volume containing an aqueous solution with a nucleation temperature ^^^^,^ > ^^^^,^, and whose volume increases when cooled below^^^^,^; andAtty. Dkt: BCRH-003WO d. a boundary separating the two volumes that transmits heat and pressure but not mass.

[0338] Wherein the size, composition, and configuration of the volumes are chosen such that the desired pressure is generated by the freezing of the second volume at the desired temperature, and the likelihood of ice formation in the first volume is thereby reduced at the same temperature.

[0339] 2) Or a pressure-stabilized supercooling system, comprising a. a. An isochoric system segmented into three volumes; b. the first volume containing an aqueous solution with nucleation temperature ^^^^,^; c. the second volume containing an aqueous solution with a nucleation temperature ^^^^,^ > ^^^^,^, and whose volume increases when cooled below^^^^,^; d. the third volume filling the remaining volume of the isochoric system not occupied by the first and second volumes and comprising a material whose volume does not substantially increase when the temperature is reduced; and e. boundaries separating the individual volumes that transmit pressure and heat but not mass.

[0340] Wherein the size, composition, and configuration of the volumes are chosen such that the desired pressure is generated by the freezing of the second volume at the desired temperature, and the likelihood of ice formation in the first volume is thereby reduced at the same temperature.

[0341] 3) A biological matter placed within the first volume of the pressure-stabilized supercooling system;

[0342] 4) A means to monitor and control the temperature of the system; and

[0343] 5) A means to monitor the pressure within the system.

[0344] One embodiment of a device for preserving biological matter at temperatures below its equilibrium melting point is depicted in the figure. This certain device comprises an isochoric chamber segmented into three volumes. The first volume contains the biological matter immersed in an aqueous solution. The second volume contains an aqueous solutionAtty. Dkt: BCRH-003WO with a nucleation temperature higher than the nucleation temperature of the contents of the first volume and higher than the desired storage temperature. As the system is cooled to the storage temperature this volume freezes, thereby increasing the pressure within the isochoric system. The third volume contains an aqueous solution that has a nucleation temperature lower than the desired storage temperature. The volumes are separated by boundaries that transmit heat and pressure but not mass, such as flexible, impermeable membranes, an example of which includes polyethylene bags. The device further comprises a temperature monitoring and control system to lower the temperature of the system to the storage temperature, maintain the system at the storage temperature for the desired storage duration, and rewarm the system above the melting point of the biological matter (a recirculating cooling bath can achieve this purpose). The device further comprises a pressure sensor to monitor the pressure that develops within the isochoric chamber over the duration of the preservation.

[0345] FIG.6 depicts one embodiment of a three-volume isochoric pressure-stabilized supercooling device for preservation of biological matter further comprising a temperature monitoring / control system and a pressure sensor. Example 4. Three-volume system in which first and second volumes are thermally separated.

[0346] It may be desirable to manipulate the temperatures of the first and second volumes independently in order to control the timing and rate of phase change within the second volume relative to the temperature of the first volume. Aspects of the present invention disclose a system for inducing pressure-stabilized supercooling at a desired pressure and temperature, comprising:

[0347] 1) An isochoric system segmented into three volumes;

[0348] 2) The first volume containing an aqueous solution with nucleation temperature ^^^^,^;

[0349] 3) The second volume containing an aqueous solution with a nucleationtemperature ^^^^,^ > ^^^^,^, and whose volume increases when cooled below ^^^^,^;Atty. Dkt: BCRH-003WO

[0350] 4) The third volume filling the remaining volume of the isochoric system not occupied by the first and second volumes and comprising a material whose volume does not substantially increase when the temperature is reduced; and

[0351] 5) Boundaries separating the individual volumes that transmit heat and pressure but not mass.

[0352] Wherein the size, composition, and configuration of the volumes are chosen such that the desired pressure is generated by the freezing of the second volume at the desired temperature, the likelihood of ice formation in the first volume is thereby reduced at the same temperature, and additionally the second volume is thermally separated from the first volume.

[0353] In one embodiment, the first supercooled volume and second freezable pressurizing volume are separated thermally yet are maintained in hydrostatic communication so that the pressure still equilibrates uniformly throughout the isochoric chamber. This may be achieved, for example, by connecting two larger volumes via a smaller conduit which reduces the cross sectional area through which heat can diffuse. One exemplary benefit of this configuration is that pressure can be applied to selectively raise the glass transition temperature of the first volume’s contents at a desired time such as when the temperature of the first volume is above the glass transition temperature under initial pressure conditions but below the glass transition temperature at elevated pressures.

[0354] FIG.7 shows some embodiments of a three-volume system in which first and second volumes are thermally separated. FIG.7A depicts a three-volume isochoric pressure- stabilized supercooling system in which the first and second volumes are thermally separated yet maintained in hydrostatic communication. FIG.7B depicts a hypothetical cooling trajectory of the three-volume system depicting independent temperature control of the first and second volumes which enables the pressure to only develop at a desired time. Example 5. Method for determining system volumes and compositions through thermodynamic calculations

[0355] Aspects of the present disclosure introduce methods to determine the volumes, compositions and configuration of an isochoric baro-supercooling system required to produceAtty. Dkt: BCRH-003WO a desired pressure at a desired storage temperature. The method involves developing a thermodynamic model for the composite system as a function of the volumes and compositions of the individual segmented volumes. These parameters are then computed for the desired pressure and desired temperature conditions.

[0356] Regarding specifying the configuration of the segmented volumes, transient heat transfer may lead to development of temperature gradients during cooling en route to the ultimate desired temperature. To ensure that phase transformations occur as desired, it may be necessary to incorporate temporal heat transfer into the thermodynamic calculations.

[0357] In one embodiment, a method to compute the ratio between the second volume and the system volume, as related to a two-volume pressure-assisted supercooling system in which both volumes contain pure water is as follows. First, the pressure-temperature water-ice liquidus is depicted in FIG.8.

[0358] FIG.8 shows pressure-temperature liquidus curve for ordinary water and ice.

[0359] Next, a desired storage temperature and pressure is selected that is beneath the liquidus curve such that liquid water at the specified PT coordinates would be metastablysupercooled (e.g., ^^^^^^^^ = −10 °C and ^^^^^^^^ = 50 MPa). Then the specific volumes ofwater and ice at this temperature and pressure are calculated. For T = −10 °C and P = 50 MPa, ^^^^^^= 0.9764 ml / g and ^^^^= 1.0829 ml / g. The specific volume of the system must then be specified. For isochoric systems, this value is approximately fixed by the temperature at which the system is sealed. For a system comprised only of water and sealed at, for example, a temperature of 4°C, ^^^^^^^= 1.0 ml / g. Next the volume of the system occupied by ice, ^^, at 50 MPa and −10 °C is calculated by the relation

[0360] ^^ = 1 − !"#$%&%'#(*'#+an ice fraction of 0.222 is obtained. Thus, the second volume will occupy 22.2% of the system at the desired storage temperature and pressure. As the volume fraction may change slightly during cooling from the assembly state to the storage state, it may be preferred to also compute the corresponding volume fraction at the assembly condition (for example, 4 °C). To determine this, the mass of the ice (i.e., second volume) must be calculatedAtty. Dkt: BCRH-003WO

[0362] ,^^^= -.⋅0%&%'#(!"#fraction occupied by the second volume at the assemblyby

[0364] ^1=%&%'#(^!"#%&%'#( = ,^^^the segmented volumes may not comprise solely puremay These solutes may have specific desirable cryoprotective properties and otherwise may modulate the temperature-pressure response of the composite system. For example, adding glycerol to the second volume will reduce the pressure generated. In these cases, the thermodynamic phase behavior and thermo- volumetric properties of each phase must be known. Relevant phase behavior includes, but is not limited to, the pressure and concentration dependence of the liquidus temperature.

[0366] In yet additional embodiments, the system may be segmented into three volumes in which the third volume provides an additional degree of freedom for specifying the sizes and compositions of the other volumes. Calculations in this scenario must consider the thermo-volumetric properties of the material comprising the third volume.

[0367] One basic utility of this technique is that it enables extension of the normal supercooling range. For example, if water at 0.1 MPa is found to supercool stably at −3 °C without freezing, then pressure can shift this temperature downwards. The nucleation rate, and thus also the freezing probability, can be approximated as functions of the degrees below the melting point, and so if a pressure of 40 MPa is applied to the same system, shifting the melting point by 3.2 °C, it may be expected to supercool stably at −6.2 °C. Likewise if 80 MPa is applied to the same system, shifting the melting point by 6.9 °C, it may be expected to supercool stably at −9.9 °C. This premise is depicted in FIG.9. FIG.9 depicts a schematic representation of the ability of pressure to depress the temperature of stable supercooling through depression of the equilibrium melting point.

[0368] The same holds for aqueous solutions. Below is the PTx liquidus surface for a binary glycerol:water solution, which may be used as a guide for designing pressure-stabilized supercooling processes. The contour lines indicate the T-P liquidus for glycerol:water solutions of specific weight fractions. FIG.10 depicts PTx liquidus surface for a binary glycerol:waterAtty. Dkt: BCRH-003WO solution with contour lines indicating the T-P liquidus for glycerol:water solutions of specific weight fractions.

[0369] The freezing probability for a given system is described by Poisson statistics as

[0370] ^2 = 1 −345 (−6(^, ^) ⋅ 7)

[0371] wherein 6 is the nucleation rate, which is a function of pressure, P, and temperature, T, and solution composition, x_i, and t is the duration the system is held in the supercooled state. If nucleation rate is approximated by the relation

[0372] 6(^, ^) = 8(^^(^, 4^) − ^)^ = 89^(^, ^, 4^)^

[0373] wherein 8 and : are empirical parameters and ^^is the melting point, a stability metric, ;, may be defined as

[0374] ;(^, ^, 4^) = ^^(^, 4^) − ^ = 9^(^, ^, 4^).

[0375] Thus, for a given system, any state with the same ;(^, ^, 4^) should have thesame supercooling stability. Classical nucleation theory can provideinsight. The ice nucleation rate as predicted by CNT is

[0376] 6 = 61345 <− =>∗ @ABCprefactor, 9D∗is the nucleation barrier and EFis the Boltzman constant.61is not as sensitive to temperature or pressure as the terms in the exponent. The nucleation barrier is predicted as

[0378] 9D∗ ∝ HIK is the interfacial free energy between water and ice, and 9L is the chemical potential difference between water and a growing ice cluster. The interfacial free energy is further predicted by the Turnbull model as

[0380] K ∝ 9M^

[0381] wherein 9M^is the enthalpy of fusion between water and ice. This relation strictly only holds for planar interfaces at equilibrium but has been found to hold well even for curved interfaces out of equilibrium. The chemical potential difference (i.e., thermodynamic driving force) below the melting point can be approximated as

[0382] 9L ≈ =O(B=BAtty. Dkt: BCRH-003WO

[0383] Assembling these terms together we can define a new stability metric (in this non-limiting case for ordinary water) as

[0384] ;(^, ^) = BI=BP=O(B(Q

[0385] to arbitrary aqueous solutions through incorporation of ainterfacial free energy and thermodynamic driving force. Supporting data necessary for these computations may include pressure-temperature liquidus curves, isochoric pressure-temperature trajectories as well as correlations between ice fraction and temperature. Below is the temperature-concentration liquidus curve for binary NaCl:water solutions at atmospheric pressure, the isochoric freezing trajectory for 0.9% w / w saline, as well as ice fraction as a function of temperature calculated for 0.9% w / w saline. FIG.11A depicts a temperature-concentration liquidus curve for binary NaCl:water solutions at atmospheric pressure. FIG.11B depicts the isochoric freezing trajectory for 0.9% w / w saline. FIG.11C depicts ice fraction as a function of temperature calculated for 0.9% w / w saline.

[0386] Depicted below is the temperature-concentration liquidus curve for binary ethylene glycol-water solutions at atmospheric pressure, the isochoric freezing trajectory for 1M and 2M ethylene glycol, as well as ice fraction as a function of temperature calculated for 1M and 2M ethylene glycol. FIG.12A depicts a temperature-concentration liquidus curve for binary ethylene glycol-water solutions at atmospheric pressure. FIG.12B depicts isochoric freezing trajectory for 1M and 2M ethylene glycol. FIG.12C depicts ice fraction as a function of temperature calculated for 1M and 2M ethylene glycol. Example 6. Method for determining system volumes and compositions through iterative thermodynamic experiments

[0387] Instead of determining the necessary volumes, compositions, and configuration of the segmented volumes that generate the desired pressure at the desired temperature by thermodynamic calculations, aspects of the present disclosure also introduce methods to achieve the same through iterative trial-and-error experiments. The method comprises 1) making an initial guess at the necessary volumes and compositions, 2) assembling the isochoric system in a specific configuration, 3) cooling the isochoric system to the desired temperature,Atty. Dkt: BCRH-003WO 4) recording the pressure that develops, 5) adjusting the size and compositions of the volumes if the developed pressure is different from the desired pressure and / or positioning the volumes in a different configuration, 6) reassembling the isochoric system, 7) cooling the isochoric system to the desired temperature, 8) recording the pressure that develops, and 9) if this pressure still deviates more than is deemed acceptable rom the desired pressure then adjust the volumes and compositions of the segmented volumes again, 10) repeat this process until the developed pressure is within acceptable deviation window about the desired pressure.

[0388] There are multiple ways to adjust the volumes and compositions in the scenario wherein a pressure develops which deviates from the desired pressure. For example if the pressure is less than desired, then the size of the second volume can be increased; if a solution is used as the second volume, its concentration can be reduced; if the isochoric system is segmented into three volumes, a less compressible material can be used as the third volume or can be replaced with a material whose volume contracts to a lesser degree as the temperature is lowered; or if any material other than ordinary water is used in any of the segmented volumes then the temperature at which the system is sealed can be reduced. If the pressure is higher than desired, then the size of the second volume can be reduced; if a solution is used as the second volume, its concentration can be increased; or if the isochoric system is segmented into three volumes, a more compressible material can be used as the third volume or can be replaced with a material whose volume contracts to a greater degree as the temperature is lowered; or if any material other than ordinary water is used in any of the segmented volumes then the temperature at which the system is sealed can be increased. The configuration of the segmented volumes may not affect the global thermodynamic state, however, it is conceivable that the aqueous solution comprising the second volume may possess sluggish kinetics that prevent or at least slow its approach to equilibrium, in this scenario the system may be configured such that the second volume is cooled more quickly or over cooled. Additionally, in larger systems, thermal gradients inevitably develop during transient cooling. Thus, to ensure that the second volume freezes before the first volume, it may be necessary to configure the system such that the second volume receives faster localized cooling en route to the desired steady temperature.Atty. Dkt: BCRH-003WO

[0389] In one embodiment, a user may like to preserve a heart immersed in UW solution at a temperature of −10 °C and pressure of 50 MPa. The user would further like to utilize a three-volume system in which the first volume contains the heart and immersion solution, the second volume contains ordinary water, and the third volume contains an aqueous solution of 20% w / w ethanol, which has a melting point at atmospheric pressure of about −10.5 °C. The user would like to assemble the system as shown in FIG.6 and discussed in Example 3. The volume of the first system is fixed when the bag is sealed, and the user would like to determine the necessary volume of the second volume.

[0390] This volume can be determined by trial-and-error as follows. An initial guess of the volume is made, and the system is assembled accordingly. The temperature of the system is then reduced to the desired storage temperature of −10 °C, during which the fluid in the second volume freezes and raises the pressure within the system. Once the system has come to steady-state, the value of the pressure developed is recorded. If the pressure is greater than the desired pressure of 50 MPa, then the user shall reduce the volume of water in the second volume. If the pressure is lower than the desired pressure of 50 MPa, then the user shall increase the volume of water in the second volume. In doing so, the volume of the first volume is likely unchanged and fluid from the third volume is modulated to compensate for the modification to the second volume. The user then reassembles the system, repeats the cooling process, records the pressure that develops, and re-evaluates whether the desired pressure is generated. This process is repeated until the conditions are found which produce the desired pressure at the desired storage temperature.

[0391] While the present invenSon 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 subsStuted without deparSng from the true spirit and scope of the invenSon. In addiSon, many modificaSons may be made to adapt a parScular situaSon, material, composiSon of maUer, process, process step or steps, to the objecSve, spirit and scope of the present invenSon. All such modificaSons are intended to be within the scope of the claims appended hereto.

Claims

Atty. Dkt: BCRH-003WO CLAIMS What is claimed is:

1. A system for inducing pressure-stabilized supercooling at a desired pressure and temperature, comprising: a. an isochoric chamber segmented into two volumes; b. a first volume containing an aqueous solution with a first nucleation temperature (^^^^,^); c. a second volume containing an aqueous solution with a second nucleation temperature (^^^^,^), wherein the second volume increases when cooled below the second nucleation temperature (^^^^,^); and d. a boundary separating the two volumes that transmits heat and pressure but not mass.

2. The system of claim 1, wherein the first nucleation temperature and the second nucleation temperature are different.

3. The system of claim 1 or 2, wherein the second nucleation temperature is higher than the first nucleation temperature (^^^^,^ > ^^^^,^).

4. The system of any one of claims 1-3, wherein the boundary is selectively permeable and allows transfer of non-water molecules.

5. The system of claim 4, wherein the non-water molecules are dissolved gases or solutes.

6. The system of any one of claims 1-5, wherein the second volume is thermally separated from the first volume.Atty. Dkt: BCRH-003WO 7. The system of any one of claims 1-6, wherein the second volume is further segmented into multiple sub-volumes each with different compositions such that they freeze at different temperatures.

8. The system of any one of claims 1-7, wherein one of the volumes nucleates ice, thereby pressuring the system and the other volume at a resultant temperature and pressure does not nucleate ice, thereby achieving a pressurized supercooled state beneath equilibrium melting point.

9. The system of any one of claims 1-8, wherein size, composition, and configuration of the first and second volumes are chosen such that the desired pressure is generated by the freezing of the second volume at the desired temperature, and the likelihood of ice formation in the first volume is thereby reduced at the same temperature.

10. The system of any one of claims 1-9, wherein the first volume contains biological matter to be preserved, and the second volume contains an aqueous solution with the second nucleation temperature, wherein the second nucleation temperature is higher than the first nucleation temperature of the first volume.

11. The system of claim 10, wherein the biological matter is immersed in an aqueous solution.

12. The system of claim 10, wherein the biological matter is immersed in an aqueous solution with which it was perfused.

13. The system of claim 10, wherein the biological matter is immersed in a solution different from the aqueous solution with which it was perfused.

14. The system of claim 12 or 13, wherein the solution with which the biological matter is perfused is a high-entropy solution.Atty. Dkt: BCRH-003WO 15. The system of claim 11, wherein the solution in which the biological matter is immersed is a high-entropy solution.

16. The system of claim 10, wherein the biological matter is immersed in a water- immiscible non-aqueous solution.

17. The system of any one of claims 10-16, wherein the temperature at which the biological matter is stored is lower than the equilibrium melting temperature of the biological matter.

18. The system of any one of claims 10-17, wherein the biological matter is cooled and stored without ice formation.

19. The system of any one of claims 10-18, wherein the cooling of the system is non- uniform.

20. The system of any one of claims 10-18, wherein the biological matter is stored for a duration between about 1 hour and about 1000 years.

21. The system of any one of claims 1-20, wherein the second volume contains ice nucleating agents.

22. The system of any one of claims 1-21, wherein the pressure that is generated is between 1 and 300 MPa.

23. The system of any one of claims 1-22, wherein the pressure that is generated depends on the volume of the freezable volume, the composition of the freezable volume and other volumes, the storage temperature, and the temperature history (e.g., cooling rate).Atty. Dkt: BCRH-003WO 24. The system of any one of claims 1-23, wherein the pressure does not irreversibly damage the biological matter.

25. The system of any one of claims 1-24, wherein the generated pressure lowers the equilibrium melting temperature of the biological material in the first volume.

26. The system of any one of claims 1-25, wherein the generated pressure raises the glass transition temperature of the biological material in the first volume.

27. The system of any one of claims 1-26, wherein the pressure lowers the cooling or warming rates required to avoid ice formation during cooling to or warming from the storage temperature.

28. The system of any one of claims 1-27, wherein cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 1000 K / s.

29. The system of any one of claims 1-27, wherein warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 1000 K / s.

30. The system of claim 28 or 29, wherein the cooling or warming rates are not constant.

31. The system of claim 10, 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.Atty. Dkt: BCRH-003WO 32. The system of any one of claims 1-31, wherein the system further comprises a temperature controller configured to monitor and control the temperature of the system.

33. The system of any one of claims 1-32, wherein the system optionally comprises a pressure controller configured to monitor the pressure within the system.

34. The system of any one of claims 1-33, where the system generates a pressure-stabilized supercooled state through the manipulation of temperature alone in the isochoric system.

35. A system for inducing pressure-stabilized supercooling at a desired pressure and temperature, comprising: a. an isochoric system segmented into three volumes; b. a first volume containing an aqueous solution with a first nucleation temperature (^^^^,^); c. a second volume containing an aqueous solution with a second nucleation temperature (^^^^,^), and the second volume increases when cooled below the second nucleation temperature (^^^^,^); d. a third volume filling the remaining volume of the isochoric system not occupied by the first and second volumes and comprising a material whose volume does not substantially increase at the desired temperature; and e. boundaries separating the first, the second, and the third volumes that transmit heat and pressure but not mass.

36. The system of claim 35, wherein the first nucleation temperature and the second nucleation temperature are different.

37. The system of claim 35 or 36, wherein the second nucleation temperature is higher than the first nucleation temperature (^^^^,^ > ^^^^,^).Atty. Dkt: BCRH-003WO 38. The system of any one of claims 35-37, wherein the third volume contains an aqueous solution that freezes at a lower temperature than the contents of the first and second volumes.

39. The system of any one of claims 35-38, wherein the boundaries are selectively permeable and allows transfer of non-water molecules.

40. The system of claim 39, wherein the non-water molecules are dissolved gases or solutes.

41. The system of any one of claims 35-40, wherein the second volume is thermally separated from the first volume.

42. The system of any one of claims 35-41, wherein the second volume is further segmented into multiple sub-volumes each with different compositions such that they freeze at different temperatures.

43. The system of any one of claims 35-42, wherein the third volume contains an aqueous solution that freezes at a lower temperature than the contents of the first and second volumes.

44. The system of any one of claims 35-42, wherein the third volume contains non-aqueous fluid or solid material.

45. The system of any one of claims 35-44, wherein size, composition, and configuration of the first, the second, and the third volumes are chosen such that the desired pressure is generated by the freezing of the second volume at the desired temperature, and the likelihood of ice formation in the first volume is thereby reduced at the same temperature.Atty. Dkt: BCRH-003WO 46. The system of any one of claims 35-45, wherein the first volume contains a biological matter to be preserved; the second volume contains an aqueous solution with the second nucleation temperature, wherein the second nucleation temperature is higher than the nucleation temperature of the first volume; and the third volume contains a material that does not expand as the temperature of the system is lowered.

47. The system of claim 46, wherein the biological matter is immersed in an aqueous solution.

48. The system of claim 46, wherein the biological matter is immersed in an aqueous solution with which it was perfused.

49. The system of claim 46, wherein the biological matter is immersed in a solution different from the aqueous solution with which it was perfused.

50. The system of any one of claims 46-49, wherein the solution with which the biological matter is perfused is a high-entropy solution.

51. The system of any one of claims 46-49, wherein the solution in which the biological matter is immersed is a high-entropy solution.

52. The system of claim 46, wherein the biological matter is immersed in a water- immiscible non-aqueous solution.

53. The system of any one of claims 35-52, wherein the temperature at which the biological matter is stored is lower than the equilibrium melting temperature of the biological matter.Atty. Dkt: BCRH-003WO 54. The system of any one of claims 35-53, wherein the biological matter is cooled and stored without ice formation.

55. The system of any one of claims 35-54, wherein the cooling of the system is non- uniform.

56. The system of any one of claims 46-55, wherein the biological matter is stored for a duration between about 1 hour and about 1000 years.

57. The system of any one of claims 35-56, wherein the second volume contains ice nucleating agents.

58. The system of any one of claims 35-57, wherein the pressure that is generated is between 1 and 300 MPa.

59. The system of any one of claims 35-58, wherein the pressure that is generated depends on the volume of the freezable volume, the composition of the freezable volume and other volumes, the storage temperature, and the temperature history (e.g., cooling rate).

60. The system of any one of claims 46-59, wherein the pressure does not irreversibly damage the biological matter.

61. The system of any one of claims 35-60, wherein the generated pressure lowers the equilibrium melting temperature of the biological material in the first volume.

62. The system of any one of claims 35-61, wherein the generated pressure raises the glass transition temperature of the biological material in the first volume.Atty. Dkt: BCRH-003WO 63. The system of any one of claims 35-62, wherein the pressure lowers the cooling or warming rates required to avoid ice formation during cooling to or warming from the storage temperature.

64. The system of claim 63, wherein the cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 1000 K / s.

65. The system of claim 64, wherein the warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 1000 K / s.

66. The system of claim 64 or 65, wherein the cooling or warming rates are not constant.

67. The system of any one of claims 46-66, 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.

68. The system of any one of claims 35-67, wherein the system further comprises a temperature controller configured to monitor and control the temperature of the system.

69. The system of any one of claims 35-68, wherein the system optionally comprises a pressure controller configured to monitor the pressure within the system.

70. The system of any one of claims 35-69, where the system generates a pressure- stabilized supercooled state through the manipulation of temperature alone in the isochoric system.Atty. Dkt: BCRH-003WO 71. A device for preserving a biological matter without freezing at temperatures below its equilibrium melting point, the device comprises: a. the pressure-stabilized supercooling system of claims 1-70; b. a biological matter placed within the first volume of the system of claims 1-70; c. a temperature controller configured to monitor and control the temperature of the system; and d. a pressure controller configured to monitor the pressure within the system.

72. The device of claim 71, wherein the biological matter is immersed in an aqueous solution.

73. The device of claim 71, wherein the biological matter is immersed in an aqueous solution with which it was perfused.

74. The device of claim 71, wherein the biological matter is immersed in a solution different from the aqueous solution with which it was perfused.

75. The device of claim 71, wherein the solution with which the biological matter is perfused is a high-entropy solution.

76. The device of claim 71, wherein the solution in which the biological matter is immersed is a high-entropy solution.

77. The device of claim 71, wherein the biological matter is immersed in a water-immiscible non-aqueous solution.

78. The device of any one of claims 71-77, wherein the temperature at which the biological matter is stored is lower than the equilibrium melting temperature of the biological matter.Atty. Dkt: BCRH-003WO 79. The device of any one of claims 71-78, wherein the biological matter is cooled and stored without ice formation.

80. The device of any one of claims 71-79, wherein the cooling of the system is non- uniform.

81. The device of any one of claims 71-80, wherein the biological matter is stored for a duration between about 1 hour and about 1000 years.

82. The device of any one of claims 71-81, wherein the second volume contains ice nucleating agents.

83. The device of any one of claims 71-82, wherein the pressure that is generated is between 1 and 300 MPa.

84. The device of any one of claims 71-83, wherein the pressure that is generated depends on the volume of the freezable volume, the composition of the freezable volume and other volumes, the storage temperature, and the temperature history (e.g., cooling rate).

85. The device of any one of claims 71-84, wherein the pressure does not irreversibly damage the biological matter.

86. The device of any one of claims 71-85, wherein the generated pressure lowers the equilibrium melting temperature of the biological material in the first volume.

87. The device of any one of claims 71-85, where the device generates a pressure-stabilized supercooled state through the manipulation of temperature alone in the isochoric system.Atty. Dkt: BCRH-003WO 88. A method of preserving a biological matter without freezing at temperatures below its equilibrium melting point, the method comprises: a. placing the biological matter within the first volume of the pressure-stabilized supercooling system of claims 1-70; b. cooling the system to a temperature lower than the equilibrium melting point of the biological matter and lower than the nucleation temperature of the second volume (^^^^,^); and c. storing the biological matter without freezing in a pressurized state at a temperature above about −200 °C and below the equilibrium melting point of the contents of the first volume (^^^^,^).

89. The method of claim 88, wherein the temperature at which the biological matter is stored is lower than the equilibrium melting temperature of the biological matter.

90. The method of claim 88 or 89, wherein the biological matter is cooled and stored without ice formation.

91. The method of any one of claims 88-90, wherein the biological matter is stored for a duration between about 1 hour and about 1000 years.

92. The method of any one of claims 88-91, wherein the pressure that is generated is between 1 and 300 MPa.

93. The method of any one of claims 88-92, wherein the pressure does not irreversibly damage the biological matter.

94. The method of any one of claims 88-93, wherein the generated pressure lowers the equilibrium melting temperature of the biological material in the first volume.Atty. Dkt: BCRH-003WO 95. The method of any one of claims 88-94, wherein the generated pressure raises the glass transition temperature of the biological material in the first volume.

96. The method of any one of claims 88-95, wherein the pressure lowers the cooling or warming rates required to avoid ice formation during cooling to or warming from the storage temperature.

97. The method of any one of claims 88-96, wherein the cooling rate required to avoid ice formation during cooling to the storage temperature is between about 0.1 K / hr and about 1000 K / s.

98. The method of any one of claims 88-96, wherein the warming rate required to avoid ice formation during warming from the storage temperature is between about 0.1 K / hr and about 1000 K / s.

99. The method of claim 97 or 98, wherein the cooling or warming rates are not constant.

100. The method of any one of claims 88-99, 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.

101. A method by which to determine the desired volumes, compositions, configuration, and temperature history needed to achieve the desired pressure- stabilized supercooling condition at desired temperature using thermodynamic calculations.

102. A method by which to determine the desired volumes, compositions, configuration, and temperature history needed to achieve the desired pressure-Atty. Dkt: BCRH-003WO stabilized supercooling condition at a desired temperature using iterative thermodynamic experiments.

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