Patent Description:
A liquid such as water is considered to be thermodynamically "supercooled" when it is brought to a temperature beneath its equilibrium freezing point but remains in a liquid state. This process is extremely useful in the medical field, enabling long-term preservation of sensitive biological materials such as tissues and organs by reducing their temperature and arresting their metabolism while simultaneously protecting them from damaging ice formation. However, supercooling is a thermodynamically metastable process; a liquid in a supercooled state may destabilize and convert to its solid state (e.g. it may freeze) upon even mild agitation of the system, or due to spontaneous processes [<NUM>]. Thus, supercooling techniques are currently limited to use in extremely controlled and stationary laboratory environments. New methods and devices for enhancing the stability of supercooling are required in order to make supercooled systems transportable, reliable for long term storage, and clinically relevant.

Disruption of the metastable supercooled state and the ensuing ice nucleation can be caused by a variety of factors, including mechanical or vibrational stimulation [<NUM>], ultrasonic stimulation [<NUM>-<NUM>], random thermal microscopic density fluctuations [<NUM>], fluid-fluid interface instabilities, heterogeneous interaction with solid surfaces or gaseous interfaces [<NUM>,<NUM>], and cavitation of gas bubbles within the liquid [<NUM>,<NUM>,<NUM>]. Thus, in order to maximize the stability of a supercooled systems, devices and methods must be developed which simultaneously combat all of these methods of destabilization.

The majority of past technological efforts aiming to increase the stability of supercooled water have addressed only one of these destabilization mechanisms, surface/interfacial interactions between the water and its surroundings. For example, Usta et al. developed a method of sealing the free surface of supercooled water with an immiscible liquid such as an oil, which they claim reduces the likelihood of nucleation by removing air as a heterogeneous nucleation site (<CIT>). Similarly, Aizenberg et al. has developed a variety of porous surface coatings impregnated with hydrophobic liquids (typically perfluorinated substances) in order to reduce heterogeneous ice nucleation on container surfaces (<CIT>). Two notable exceptions to these surface/interfacial efforts are reported in the literature. One includes an attempt to enhance supercooling through the use of magnetic or electric fields (<CIT>). The second employs constant volume systems to reduce the probability of random ice nucleation (<CIT>). However, while all of the referenced works attempt to reduce the likelihood of ice nucleation, none of these studies specifically address the myriad mechanisms of destabilization associated with transportation and long term clinical use, including mechanical stimulation, ultrasonic stimulation, cavitation, fluid-fluid interface instabilities, increased probability for density-fluctuations, etc. For supercooling to become a practical method for preservation, these aspects cannot be ignored.

In the present disclosure, the inventors present methods and devices based on newly realized thermodynamic, kinetic, and fluid dynamic phenomena to dramatically enhance the long term stability of supercooled systems exposed to a wide variety of external agitations, including vibration, acute impact, ultrasonication, and thermal fluctuation. The authors describe how these methods and devices can be used for the fail-safe, transportable, and long term clinical-grade preservation of sensitive biological matter such as organic molecules, tissues, organs and organisms. Furthermore, while these methods are described principally in the context of preservation of biological matter such as organic molecules, tissues, organs and organisms, they are equally relevant to all contexts requiring stable and predictable supercooling, including various metallurgical processes [<NUM>,<NUM>], semiconductor processing [<NUM>], cold storage of food products [<NUM>], etc..

Preciado and Rubinsky [<NUM>] disclose a pressure vessel designed to validate that isochoric (constant-volume) preservation automatically minimizes the pressure of a given temperature. The vessel is used, e.g., to examine the effect of an isochoric environment on freezing point nucleation in an aqueous antifreeze protein solution and to generate pressure-temperature phase diagrams for various cryoprotectant solutions. The vessel chamber was instrumented with a pressure transducer to continuously measure the internal pressure of the chamber, and two thermistors were provided to evaluate the inner temperature of the chamber. The vessel was cooled to -<NUM> using a cold bath while monitoring temperature and pressure. The beginning of a pressure rise was used to determine the time point of nucleation. Once the pressure began to rise, the vessel was removed from the cold bath and thawed under warm water.

In international patent publication <CIT> a process for temperature and pressure controlled cryopreservation of samples by using isochoric systems is described, including a process for identifying whether a biological sample has or has not undergone a vitrification and/or devitrification cryopreservation process.

The invention provides methods and devices for the high-stability supercooling of aqueous solutions or suspensions, and the preservation of biological matter therein Provided are methods and devices as defined in the appended claims for maintaining long term high-stability and transportable thermodynamic supercooling of an aqueous medium in a constant-volume (isochoric) system, in which biological materials may be stored at temperatures beneath the equilibrium freezing point of the medium without ice formation.

In a first aspect, the invention provides a method of inducing high-stability and optionally transportable supercooling, the method comprising:.

In a further aspect, the invention provides a device for inducing high-stability and optionally transportable supercooling of aqueous media, the device comprising:.

The aqueous medium within the container may optionally be water or an aqueous solution containing organic molecules or chemical cryoprotectants. The biological materials may optionally be human or non-human cells, molecules, multicellular constructs, tissues, organs, or full organisms. The container may be optionally made of metal ceramic, or any other rigid material. Isochoric storage methods and devices can be used to enhance the long term supercooling stability and the ability to withstand system perturbations.

A key element in this invention is the realization that because pressure changes with the formation of a first ice nucleus, the isochoric container features intrinsic real-time ice nucleation detection capabilities that can be used to ensure continued stability of the supercooled medium in the presence of an ice nucleation event. Nucleation detection is achieved by incorporating or connecting a means to monitor pressure such as a digital or analogue pressure transducer or gauge or an electrical resistance source into the container. We have found that under principally-air-free isochoric supercooling an ice nucleation event results in an increase in the hydrostatic pressure, unlike in other supercooling systems. Such a pressure increase will propagate through the system as a sound wave, and is thus detectable at the speed of sound in water (approximately <NUM>/s), and can be used to detect an ice nucleation event in real time. This high-speed real-time detection of nucleation is not possible when detecting nucleation using temperature monitoring, as heat diffuses through water at a much slower characteristic rate (approximately <NUM>-<NUM> m/s).

The container further features on-board or off-board means to deliver energy to the supercooled fluid to cause the dissolution or melting of a growing or stable ice nucleus or crystal. In a preferred embodiment, a heater may be used to melt any ice that may form and reset the state of supercooling, ensuring total safety of the preserved biologic. Compared to other supercooling conditions, ice forms much more slowly under air-free isochoric conditions due to reduced thermodynamic driving forces. The combination of slow ice formation and real-time nucleation detection enables rapid elimination of ice crystals via immediate controlled heating. Power for the heating may be supplied by an on-board or off-board power supply or battery.

The container features on-board or off-board control capabilities, which may employ a microprocessor, computer, or other programmable processing device to monitor the pressure reading from the pressure transducer and activate the energy delivery module (heating) should the pressure reading surpass a given threshold value. The controller will continuously monitor the pressure and turn off the energy delivery module (heating) when the pressure returns to beneath the threshold value, or another value as deemed appropriate, indicating that all or most ice has melted and that the state of stable supercooling may be resumed. This ensures that the temperature in the system is elevated only to just above the melting point of the medium and the stored biologic remains cold. The container is then allowed to return to the desired preservation temperature and supercooling is resumed. This control capability ensures total safety of the preserved biologic, even if the supercooled system should become briefly unstable.

In some embodiments, if the supercooled media destabilizes and ice begins to form for any reason, it may be allowed to grow and preservation may be continued with or without interruption.

The container may be cooled by immersion in an external cooling bath or by on-board cooling, and the cooling process may also be optionally controlled by the programmable processing device mentioned previously. Cooling of the container may be provided by cooled liquid, gas or vapor, by refrigeration, by phase-change material, by thermoelectric or peltier cooler, by stirling cooler, or by any arbitrary cooling mechanism.

The container may feature additional measures to protect the aqueous medium from cavitation caused by vibrations, which can cause unwanted ice nucleation. These vibrations may be encountered during flight, ground-transport, or general use. Protective measures may include a sleeve, coating, mount, or other external feature made of a vibration-reducing material such as neoprene or other rubbers, or may include springs or other mechanical features for vibration reduction.

The container may feature additional measures to protect the supercooled medium from temperature changes, which may destabilize the system and cause nucleation, or which may negatively affect the stored biologic. Such measures may optionally include a thermally insulating sheath, sleeve, or coating; a surrounding phase-change material; a vacuum-insulated panel, material, or chamber; or a secondary container or apparatus of any kind intended to thermally insulate the primary container.

The container may feature additional measures to further protect against heterogeneous nucleation at internal liquid-solid interfaces, including hydrophobic or superhydrophobic surfaces or surface coatings, including but not limited to polytetrafluoroethylene-based and perfluorocarbon-based substances.

The aqueous medium employed may optionally feature chemical cryoprotectants to modulate the range of temperatures in which the system is supercooled or an increase in the degree and stability of supercooling at a given preservation temperature, such as dimethyl sulfoxide, ethylene glycol, polyethylene glycol, <NUM>-OMG, glycerol, antifreeze proteins etc. The aqueous medium may also include any other solute or combinations of solutes which reduce the freezing point of the solution, such as trehalose, glucose, fructose, mannitol, betaine, glycine, etc..

The container, housing a supercooled media and/or preserved biologic, may be stored at any temperature between <NUM> and -<NUM>, including -<NUM>, -<NUM>, -<NUM>, -<NUM>, -<NUM>, -<NUM>, -<NUM> - -<NUM>,. The container may also be cooled at any arbitrary rate, including <<NUM> per minute, <<NUM> per minute, <<NUM> per minutes, <<NUM> per minute, <<NUM> per minute,. <<NUM> per minute, etc..

The stored biologic may be preserved within the container for any length of time, including but not limited to <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> days, <NUM> days, <NUM> days, <NUM> days, <NUM> days, <NUM> days, <NUM> weeks, <NUM> weeks, <NUM> month, <NUM> months, <NUM> months, <NUM> months, <NUM> year, <NUM> years, <NUM> years, <NUM> years, <NUM> years, <NUM> years.

The container may store biologics of any type or scale, including organic molecules, cells, blood, bone marrow, blood vessels, pancreatic islets, reproductive tissues, skin, etc. It may store full organs such as hearts, livers, kidneys, lungs, pancreases, spleens, etc.; other biologics such as eyes, full or partial limbs, fingers or toes, etc.; engineered tissues such as 3D microtissue constructs, liver-on-a-chip constructs, lung-on-a-chip constructs, heart-on-a-chip constructs, etc.; full organisms such as zebrafish, coral, nematodes, or other marine or land-dwelling animals; and foodstuffs such as cherries, berries, potatoes, tomatoes, fish, beef, etc..

The container may be made of any rigid material, including metals such as steel and alloys thereof, aluminum and alloys thereof, titanium and alloys thereof, copper and alloys thereof, etc.; ceramic materials; plastics such as acrylic, polyvinyl chloride, polymethylmethacrylate, polyurethane, etc.; composites such as carbon fiber reinforced polymers (CFRP) or glass fiber reinforced polymers (GFRP); or any combination thereof. This material may be subjected to one or multiple surface treatments, such as anodizing, nickel-plating, zinc-plating, etc., for the purposes of preventing corrosion, preventing heterogeneous ice nucleation, maintaining biocompatibility, etc..

The container may be made air-tight by a sealing mechanism, including rubber o-rings, spring energized o-rings, metal-on-metal contact, rubber gaskets, metal gaskets, etc. The closure of the container may make use of a threaded cap, a threaded plug, a clamped lid, a bolted lid, a mechanically-retained plate or plug, etc. Within the primary container, preserved biologics may optionally also be stored in a secondary container, such as a bag, balloon, covered vial or tube, or other vessel with at least one flexible surface capable of transmitting hydrostatic pressure from its surroundings to its internal contents. This secondary container may also be filled with an aqueous solution, be completely or mostly free of air, and sealed. This secondary containment will protect the biologics from osmotic damage in the event that ice forms in the principal supercooled media (for however brief or long a period).

Biologics preserved within the container may also optionally be coated with or immersed in a cross-linked hydrogel, such as sodium alginate or hyaluronic acid cross-linked with calcium or other ionic, oxidative, or covalent cross-linkers. This cross-linked gel will protect the biologics from potential osmotic damage during stable supercooling or during periods of ice nucleation. This hydrogel may be impregnated with an organ preservation solution or any other manner of aqueous solution in the interest of maintaining osmotic balance, delivering drugs, enhancing anti-freezing effects, etc..

The solution within the container may optionally be seawater, and the preserved biologics may optionally be marine organisms or matter. These biologics may optionally be collected directly from the ocean.

In addition, the invention is also useful for providing aqueous environments that remain liquid at sub-zero centigrade temperatures, and thus may be used to facilitate temperature-controlled chemical reactions, polymerization, gelation, or other thermal or chemical processes.

Effective preservation of complex organ and tissue systems is essential to a wide range of <NUM>st century medical and research efforts[<NUM>], including expanding access to lifesaving organ transplantations, enabling the storage and transportation of engineered tissues for drug-testing, etc. While classical approaches to preservation have often included high doses of cryoprotectant chemicals to avoid damage from ice formation, a new generation of protocols is leveraging thermodynamic supercooling to dramatically enhance the duration and quality of biopreservation while minimizing cryoprotectant concentrations[<NUM>,<NUM>-<NUM>].

Although this approach has produced strong early biological results in the laboratory, the reduction of these protocols to practice in a clinical or industry setting faces a fundamental limitation: thermodynamic stability [<NUM>]. Supercooling is a metastable thermodynamic state, in which a substance remains liquid at temperatures beneath its freezing point due to a lack of sufficient kinetic stimuli. Upon even slight agitations, a supercooled system can instantaneously and destructively freeze, returning to thermodynamic equilibrium and destroying any preserved biologics. This can also happen randomly after sufficiently long periods of preservation, because the probability for ice nucleation is a function of time. Thus, in order to develop supercooling preservation protocols that are practicable outside a highly controlled laboratory environment, long term, transportable, and clinically convenient, new methods but be sought to enhance the stability of supercooled systems.

We present here an isochoric (constant-volume) supercooling method which greatly enhances the stability of supercooled water in the face of a range of mechanical and thermal disturbances by minimizing the effects of many different ice nucleation mechanisms. Without being bound by theory, we also present several possible explanations concerning the fundamental mechanisms contributing to this enhancement, unifying factors that stem from thermodynamics, fluid dynamics, and kinetics. The results herein have been put to immediate use in the preservation of sensitive biological matter, to excellent effect.

Nucleation of a stable ice phase from supercooled (metastable) water occurs when a perturbation within the system proves sufficiently large to drive the free energy of a cluster of liquid molecules over the nucleation barrier [<NUM>]. Such perturbations can stem from the constant random microscopic fluctuations undergone by any system with finite temperature, or from micro- or macroscopic mechanical or thermal agitation [<NUM>,<NUM>,<NUM>]. Thus, for a supercooling-based preservation technique to become practical or clinically relevant, it must maintain stability not only when experiencing microscopic fluctuations, but also when experiencing the macroscopic agitations that characterize practical use and mobility, including, long term preservation, motion, macroscopic vibration, impact with rigid surfaces, temperature swings, etc..

Most supercooling preservation protocols operate under isothermal (constant temperature) and isobaric (constant pressure) conditions. According to statistical thermodynamics, systems in contact with a temperature reservoir (such as a cooling bath) and a pressure reservoir (the atmosphere) are free to fluctuate in energy and volume [<NUM>] (or density if mass is constant), the extensive conjugates of temperature and pressure (<FIG>, left). Thus, systems under isobaric conditions are constantly undergoing microscopic density fluctuations due to the random motion of particles, which can lead to the formation of ice clusters that meet and exceed the critical size required for nucleation. Furthermore, when exposed to macroscopic perturbations, isobaric systems in contact with the atmosphere are susceptible to bulk fluid motion and bulk mixing with air, which can also lead to nucleation through cavitation or the introduction of new nucleation sites [<NUM>,<NUM>,<NUM>].

Isochoric (constant-volume) systems, by their very definition, do not microscopically fluctuate in density [<NUM>] (<FIG>, left), and restrict bulk motion of the contained liquid. Furthermore, we have shown that both the process of ice nucleation and growth and the fundamental water-ice phase equilibria are different under isochoric conditions: As seen by comparing the T-P and T-V phase diagrams for pure water (<FIG>/B, right), nucleation at constant pressure yields complete freezing, while nucleation at constant volume yields only partial freezing, resulting in a two-phase water-ice equilibrium. This ultimate two-phase equilibrium has myriad useful consequences, and we have demonstrated theoretically that amongst these consequences may be heightened nucleation barriers and reduced thermodynamic driving forces for nucleation (see Example <NUM> to follow). Additionally, in isochoric systems, all fluid-fluid interfaces are eliminated and bulk motion of the water is totally constrained; this drastically minimizes the chance of cavitation events that may initiate ice nucleation, eliminates the opportunity for fluid-fluid interface instabilities that may initiate ice nucleation, and removes any opportunity for heterogeneous nucleation of ice on a bubble or surface of a gaseous or secondary liquid phase.

The sum total of these thermodynamic, kinetic, and fluid-dynamic considerations demonstrate that isochoric conditions augment supercooling through myriad different yet complementary means, the combination of which provides a level of stability and protection against ice nucleation that is both unprecedented and unclaimed in other technologies. Based on these considerations, which are further clarified in Examples to follow, we claim that the methods and devices described in this disclosure, which universally employ isochoric conditions, can be used to yield enhanced supercooling stability.

The invention provides methods and devices as defined by the appended claims for maintaining long term high-stability and optionally transportable thermodynamic supercooling of aqueous media in a constant-volume (isochoric) system, in which biological materials may be stored at temperatures beneath the equilibrium freezing point of the media without ice formation.

The most basic configuration of this device is presented schematically in <FIG> (not according to the invention). In this and future figures, the term "bulk gas phase" is taken to mean bubbles or other configurations of one or multiple undissolved gaseous phases comprising a thermodynamically appreciable volume of the system, which may optionally be equal to ><NUM>%, ><NUM>%, ><NUM>%, or ><NUM>%. Any dissolved gases are taken to be a component of the supercooled aqueous media. The rigid container indicated may be constructed of any rigid material, such as a metal or metallic alloy, glass, ceramic, polymer, plastic, hard rubber, composite material, or any combination thereof. The rigid container may also be constructed in any arbitrary geometry, including cylindrical, spherical, rectilinear, and any combination thereof. The air-tight closure may be constructed using any arbitrary sealing or retention mechanism, such as an o-ring or gasket-based seal, metal-on-metal contact seal, threaded seal, etc..

The invention is optimally suited for the preservation of biological matter, which is achieved by: placing aqueous media and biologic(s) in a rigid container: removing all or most bulk gas phase from the container; sealing the container with an air-tight closure, thus inducing isochoric conditions; preventing cavitation, fluid-fluid interface effects and instabilities, bulk motion of the media, or density fluctuations by inducing isochoric conditions; preventing ice nucleation by preventing cavitation, fluid-fluid interface effects and instabilities, bulk motion of the media, or density fluctuations. The aqueous media within the container may optionally be water or an aqueous solution containing organic molecules or chemical cryoprotectants. The preserved biologics may optionally be human or non-human cells, multicellular constructs, tissues, organs, or full organisms. Isochoric storage methods and devices can be used to enhance the long term preservation and supercooling stability and the ability to withstand system perturbations.

In other preferred embodiments, any number of sensing and control implements may be incorporated into an isochoric supercooling device. <FIG> depicts schematically one preferred embodiment of the device in which pressure monitoring capabilities, active control capabilities, energy delivery capabilities, and cooling capabilities are incorporated.

Given the fact that ice is less dense than liquid water, at constant-volume, the nucleation of ice will cause an immediate increase in hydrostatic pressure. Because pressure changes with the formation of a first ice nucleus, isochoric containers feature intrinsic real-time ice nucleation detection capabilities that can be used to ensure continued stability of the supercooled media. Nucleation detection is achieved by incorporating or connecting a means to monitor pressure, such as a digital or analogue pressure transducer or gauge or an electrical resistance source, into, onto, or in communication with the container. We have found that under isochoric supercooling, an ice nucleation event results in an easily detectable increase in the hydrostatic pressure, unlike in other non-isochoric supercooling systems. Such a pressure increase will propagate through the system as a sound wave, and is thus detectable at the speed of sound in water (approximately <NUM>/s), enabling real-time detection of nucleation events. This high-speed real-time detection of nucleation is not possible when attempting to detect nucleation using temperature monitoring, as heat diffuses through water at a much slower characteristic rate (approximately <NUM>^-<NUM>/s), and can be used not only to monitor stability but also to trigger various re-stabilizing actions, such as controlled melting of emerging ice crystals.

The container further features on-board or off-board means to deliver energy to the supercooled fluid to cause the dissolution or melting of a growing or stable ice nucleus or crystal. In a preferred embodiment, a heater situated within or external to the container may be used to melt any ice that may form and reset the state of supercooling, ensuring total safety of the preserved biologic. Compared to other supercooling conditions, ice forms much more slowly under air-free isochoric conditions due to reduced thermodynamic driving forces. The combination of slow ice formation and real-time nucleation detection enables rapid elimination of ice crystals via immediate controlled heating. Power for the heating may be supplied by an on-board or off-board power supply or battery. Means used to provide the energy required for melting or dissolution of the ice phase are described in the claims and include joule heating, inductive heating, ultrasonic heating, electromagnetic heating, etc..

The container features on-board or off-board control capabilities, which may in preferred embodiments employ a microprocessor, computer, or other programmable processing device to monitor the pressure reading from the pressure transducer and activate the energy delivery module (heating) should the pressure reading surpass a given threshold value. The controller continuously monitors the pressure and turn off the energy delivery module (heating) when the pressure returns to beneath the threshold value, or another value as deemed appropriate, indicating that all or most ice has melted and that the state of stable supercooling may be resumed. This ensures that the temperature in the system is elevated only to just above the freezing point of the medium and the stored biologic remains cold and preserved. The container is then allowed to return to the desired preservation temperature and supercooling is resumed. This control capability ensures total safety of the preserved biologic, even if the supercooled system should become briefly unstable, and is unique to isochoric systems and unprecedented in the field of supercooling.

In some additional embodiments, if the supercooled media destabilizes and ice begins to form for any reason, it may be also be allowed to grow and preservation may be continued with or without interruption. Isochoric conditions offer an additional layer of protection for biologics in the case of destabilization, because only part of the system will convert to ice after nucleation, resulting in a stable ice-water two-phase equilibrium. In accordance with the T-V phase diagram provided in <FIG>. Thus, in some embodiments, the biologic can continue ice-free or partially-frozen preservation within the isochoric container after ice has nucleated and been allowed to grow. This secondary protection of initially-supercooled biologics is also unique to isochoric systems.

In some embodiments, a secondary container may be used to house one or multiple primary isochoric containers. In some embodiments, a secondary container may be used to house one or multiple primary isochoric containers for the purposes of transportation. In some embodiments, the secondary container may include measures to protect the housed primary container(s) from temperature changes, vibration, ultrasonic stimulation, contamination, desterilization, or any other kind of disturbance. In some embodiments, a secondary container may be used which specifically enhances the suitably of the primary container for air, ground, or sea transportation.

In some embodiments, a single device may be built which includes more than one isochoric container. In some embodiments, two or more of these isochoric containers may be sealed by the same or different mechanisms. In some embodiments, the container may be cooled by immersion in an external cooling bath or by on-board cooling, and the cooling process may also be optionally controlled by the programmable processing device mentioned previously. Cooling of the container may be provided by cooled liquid, gas or vapor, by refrigeration, by phase-change material, by thermoelectric or peltier cooler, by stirling cooler, or by any arbitrary cooling mechanism. In some embodiments, cooling of the container may be active, as provided for example by refrigeration, and in other embodiments it may be passive, as provided for example by a phase change material such as ice or a eutectic salt.

In some embodiments, the container may feature additional measures to protect the supercooled aqueous media from cavitation caused by vibrations, which can cause unwanted ice nucleation. These vibrations may be encountered during flight, ground-transport, or general use. Protective measures may include a sleeve, coating, mount, or other external feature made of a vibration-reducing material such as neoprene or other rubbers, or may include springs or other mechanical features for vibration reduction. In some embodiments, a primary isochoric container may be placed in a secondary container for the purpose of vibration reduction, isolation, or protection.

In some embodiments, the container may feature additional measures to protect the supercooled medium from temperature changes, which may destabilize the system and cause nucleation, or which may negatively affect the stored biologic. Such measures may optionally include a thermally insulating sheath, sleeve, or coating; a surrounding phase-change material; a vacuum-insulated panel, material, or chamber; or a secondary container or apparatus of any kind intended to thermally insulate the primary container.

In some embodiments, the container may feature additional measures to further protect against heterogeneous nucleation at internal liquid-solid interfaces, including hydrophobic or superhydrophobic surfaces or surface coatings, including but not limited to polytetrafluoroethylene-based and perfluorocarbon-based substances.

In some embodiments, the aqueous media employed may optionally feature chemical cryoprotectants to modulate the range of temperatures in which the system is supercooled or an increase in the degree and stability of supercooling at a given preservation temperature. In some embodiments, such cryoprotectants may include dimethyl sulfoxide, ethylene glycol, polyethylene glycol, <NUM>-OMG, glycerol, etc. The aqueous media may also include any other solute or combinations of solutes which reduce the freezing point of the solution, including but not limited to trehalose, glucose, fructose, mannitol, betaine, glycine, sodium, calcium, potassium, magnesium, combinations thereof, and etc..

In some embodiments, the container may be stored at any temperature between <NUM> and -<NUM>, including - <NUM>, -<NUM>, -<NUM>, -<NUM>, -<NUM>, -<NUM>, -<NUM> - -<NUM>,. The container may also be cooled at any arbitrary rate, including <<NUM> per minute, <<NUM> per minute, <<NUM> per minutes, <<NUM> per minute, <<NUM> per minute,. <<NUM> per minute, etc..

In some embodiments, the volume capacity of the container may be <<NUM> microliter, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>, <<NUM>,<NUM>.

In some embodiments, the stored biologic may be preserved within the container for any length of time, including but not limited to <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> hours, <NUM> days, <NUM> days, <NUM> days, <NUM> days, <NUM> days, <NUM> days, <NUM> weeks, <NUM> weeks, <NUM> month, <NUM> months, <NUM> months, <NUM> months, <NUM> year, <NUM> years, <NUM> years, <NUM> years, <NUM> years, <NUM> years.

In some embodiments the container may store biologics of any type or scale, including organic molecules, cells, blood, bone marrow, blood vessels, pancreatic islets, reproductive tissues, skin, etc. It may store full organs such as hearts, livers, kidneys, lungs, pancreases, spleens, etc.; other biologics such as eyes, full or partial limbs, fingers or toes, etc.; engineered tissues such as 3D microtissue constructs, liver-on-a-chip constructs, lung-on-a-chip constructs, heart-on-a-chip constructs, etc.; full organisms such as zebrafish, coral, nematodes, or other marine or land-dwelling animals; and foodstuffs such as cherries, berries, potatoes, tomatoes, fish, beef, etc..

In some embodiments, preserved biologics may be perfused with or in the aqueous media prior to preservation. In other embodiments, biologics may undergo some manner of conditioning prior to preservation, including normothermic or hypothermic machine perfusion, passive or active perfusion with or immersion in an aqueous solution of any kind.

In some embodiments container may be made of any rigid material, including metals such as steel and alloys thereof, aluminum and alloys thereof, titanium and alloys thereof, copper and alloys thereof, etc.; ceramic materials; plastics such as acrylic, polyvinyl chloride, polymethylmethacrylate, polyurethane, etc.; composites such as carbon fiber reinforced polymers (CFRP) or glass fiber reinforced polymers (GFRP); or any combination thereof. In some embodiments, the container may be made from a composite-overwrapped pressure vessel. In some embodiments, this material may be subjected to one or multiple surface treatments, such as anodizing, nickel-plating, zinc-plating, etc., for the purposes of preventing corrosion, preventing heterogeneous ice nucleation, maintaining biocompatibility, etc..

In some embodiments, the container may be made air-tight by a sealing mechanism, including rubber o-rings, spring energized o-rings, metal-on-metal contact, rubber gaskets, metal gaskets, etc. In some embodiments, the closure of the container may make use of a threaded cap, a threaded plug, a clamped lid, a bolted lid, a mechanically-retained plate or plug, etc..

In some embodiments, within the primary container, preserved biologics may optionally also be stored in a secondary container, such as a bag, balloon, covered vial or tube, or other vessel with at least one flexible surface capable of transmitting hydrostatic pressure from its surroundings to its internal contents. This secondary container may also be filled with an aqueous solution, be completely or mostly free of bulk gas phase, and sealed. This secondary aqueous solution may be the same as or different from the aqueous solution within the primary container. This secondary containment will protect the biologics from osmotic damage in the event that ice forms in the principal supercooled media (for however brief or long a period). In some embodiments, the secondary containment may preserve one or multiple biologics, and in some embodiments one or multiple secondary containers may be deployed within the primary container.

In some embodiments, biologics preserved within the container may also optionally be coated with or immersed in a cross-linked hydrogel, such as sodium alginate or hyaluronic acid cross-linked with calcium or other ionic, oxidative, or covalent cross-linkers. This cross-linked gel will protect the biologics from potential osmotic damage during stable supercooling or during periods of ice nucleation. This hydrogel may be impregnated with an organ preservation solution or any other manner of aqueous solution in the interest of maintaining osmotic balance, delivering drugs, enhancing anti-freezing effects, etc..

In some embodiments, the solution within the container may optionally be seawater, and the preserved biologics may optionally be marine organisms or matter. In some embodiments, these biologics may optionally be collected directly from the ocean.

In some embodiments, the container, aqueous media, or biologic may be exposed to electric or magnetic fields. These fields may be of a static, oscillating, or pulsed nature. The combination of electric or magnetic fields with isochoric conditions may further enhance supercooling stability in the enclosed aqueous media.

In some embodiments, the container may be fabricated from a transparent rigid material. This container may optionally be used to study or monitor the internal contents or behaviors of the container, including but not limited to the behavior of preserved biologics or of phase transitions that may occur during a destabilization or ice nucleation event. In some embodiments, the container may be integrated into a microscope platform, allowing microscopic examination of the contents within. In some embodiments, the container may be constructed in geometries at the millimeter or micron length scale for these purposes.

In some embodiments, containers may be constructed at the millimeter or micron length scale for any purpose, including but not limited to the preservation of individual cells or groups of cells, rapid cooling for the purposes of maintaining supercooling until the glass transition temperature of the aqueous media, study of microscale thermophysical properties or effects under isochoric conditions, etc..

In addition, the invention is also useful for providing aqueous environments that remain liquid at sub-zero centigrade temperatures, and thus may be used to facilitate temperature-controlled chemical reactions, polymerization, gelation, or other thermal or chemical processes. The invention may also be used to control supercooling of liquid metals or semiconductors for the eventual purposes of controlled crystallization.

The invention is further illustrated in the following examples, which do not limit the scope of the invention described in the claims.

Without being bound to the theory, we will introduce here a conceptual theoretical analysis of supercooled thermodynamic systems under isochoric and conventional isobaric conditions. This theoretical section is included only for clarity and completeness.

The phase transitions of water are generally described using natural variables of temperature and pressure, which correspond to the Gibbs thermodynamic potential G(T,P). Consider the freezing of pure water in an undeformable container (that is, at constant volume). Water expands upon freezing to ice-<NUM>, and should the container have a lower specific volume than that of ice-<NUM>, the contents of the system will never be able to freeze entirely, resulting in a two-phase water-ice equilibrium. The standard T-P phase diagram for pure water includes only single-phase regions, so this anticipated two-phase equilibrium of water and ice under isochoric conditions cannot be quantified in a straightforward fashion from the traditional water phase diagram. By retaining pressure as a natural variable, one is forced to analyze this two-phase equilibrium as occurring between two distinct entities-the ice and the water-and one must solve for mechanical equilibrium by balancing the bulk moduli of the solid and liquid phases against the hydrostatic pressure that emerges as the ice forms and expands [<NUM>]. While the equilibrium states of the system can indeed be predicted using this approach, the mathematical formulation proves awkward, and must be continuously re-solved as temperature and pressure are altered.

While the Gibbs potential is appropriate under most physical contexts, it is only one of many possible Legendre transforms of the internal energy, U(S,V). Should a situation arise in which the Gibbs free energy ceases to be convenient, it should be replaced in favor of a better-suited potential. For the case of freezing under isochoric conditions, a more elegant thermodynamic approach is to exchange pressure as a natural variable in favor of volume, thereby leveraging the Helmholtz thermodynamic potential for this analysis. In this description, the thermodynamic boundary conditions are reframed around the containing vessel, describing the entire two-phase water-ice system at once.

Herein we use the Helmholtz free energy to derive the equilibrium phase behaviors of water held in a constant volume system at subzero temperatures. Our derivation yields the T-V phase diagram for water and ice, featuring a prominent two-phase equilibrium region, analogous to those found in binary eutectic phase diagrams. We also derive a classical nucleation theory for ice under isochoric conditions, which reveals fundamental differences between the kinetics of freezing under constant volume versus constant pressure. In isochoric systems, we reveal that the energetic barrier to nucleation is higher, and that there exists a critical absolute volume threshold (on the order of microns), under which nucleation of a single ice nucleus becomes kinetically prohibited. Our analyses here provide a classical foundation from which to analyze the thermodynamics and kinetics of freezing in isochoric systems; establishing context to interpret the phenomenology of water and ice under these important boundary conditions.

Phase diagrams are constructed by a projection of the lowest free-energy phases onto axes of the natural thermodynamic variables. The choice of natural variables governs the geometry of the free-energy surfaces, and thereby the phase coexistence behavior in the resulting phase diagram.

In order for a homogeneous single-phase substance to be stable, its internal energy surface, U, must be positive-definite; ∂<NUM>U/∂X<NUM> > <NUM>, where X are the extensive thermodynamic variables X = S, V, N, etc. When it is not convenient to use an extensive natural variable, a new thermodynamic potential can be constructed with intensive natural variables, Y = T, P, µ, etc, by a Legendre transformation of the extensive variable with respect to its intensive conjugate, U - (∂U/∂X)X = U-XY [<NUM>]. Following a Legendre transformation, the curvature of the free-energy surface becomes concave-down in the corresponding intensive thermodynamic variable(s) [<NUM>], or otherwise retains the convex-up curvature of U in the extensive variable(s) [<NUM>].

The Gibbs potential has intensive natural variables of temperature and pressure, and thus Gibbs free-energy surfaces are concave-down in both T and P. Projection of the lowest Gibbs free-energy phase onto the temperature and pressure axes recovers the standard T-P phase diagram, as shown for H<NUM>O in <FIG>, constructed using thermodynamic data from the International Association for the Properties of Water and Steam (IAPWS) [<NUM>,<NUM>]. For a single-component system like H<NUM>O, phase coexistence is governed by the intersection of these concave-down free-energy surfaces, resulting in a <NUM>-dimensional phase-coexistence line in the T-P plane.

In an isochoric system however, the natural variables should be temperature and volume, rather than pressure, which corresponds to the Helmholtz thermodynamic potential, F. We can construct F(T, V) for water and ice-<NUM> by a Legendre transform of the Gibbs free energy data as:<MAT>.

Because V is extensive and T is intensive, F is convex-up in volume, and concave-down in temperature.

The convexity of the Helmholtz free-energy as a function of volume fundamentally changes the nature of phase coexistence in the T- V space. The lowest free-energy envelope now includes tangent lines between the convex Fwater(V) and Fice(V) curves, which signify a 2D two-phase equilibrium region, as opposed to a 1D phase-coexistence line. Gibbs called these tangent lines "Lines of Dissipated Energy" [<NUM>], along which a single-phase homogeneous substance can reduce its free-energy by forming a heterogeneous mixture of two phases. These tangent lines are analogous to those used in the convex hull construction of binary eutectic phase diagrams, which are constructed from the Gibbs free energy, G(T,x)[<NUM>]. The convex hull construction can be applied to both G(x) and F(V) because both composition and volume are extensive variables, and thus their free-energy surfaces are convex-up.

By projecting the lowest-energy convex hull formed by the Fwater and Fice surfaces in <FIG>, we construct the T-V phase diagram for water, shown in <FIG>. To the best of our knowledge, this phase diagram has not been reported previously in the literature. The T-V phase diagram of water features a two-phase equilibrium region, where the equilibrium phase fraction can be solved using the Lever rule, in the same manner employed for T-x binary eutectic phase diagrams[<NUM>]. For the reader's reference, the phase fraction as a function of temperature and system specific volume is shown in <FIG>.

At a given temperature, the slope of these tangent lines, (∂F/∂V)T, provides the pressure that the two-phase mixture exerts onto the constant-volume container. We mark these pressure isoclines on the T-V phase diagram in <FIG>. Note that in the equilibrium two-phase region, the tangent lines connect the water and ice-<NUM> free energy curves, implying that water and ice experience the same pressure, which is indeed a requirement for mechanical equilibrium.

The kinetics of ice nucleation in isochoric systems: Physical motivations.

The equilibrium T-V phase diagram produced in <FIG> is specific-volume dependent, as the phase-coexistence regions define thermodynamic equilibrium regardless of the amount of material present. We next show that the kinetics of nucleation in isochoric systems are additionally dependent on the absolute volume of the system container, and we leverage both dependencies to derive a new isochoric theory of nucleation. Consider the process of isochoric freezing, in which a closed, fixed-volume container filled with pure water is brought below <NUM> to a metastable supercooled state. Before ice nucleates, the supercooled water will experience some pressure P<NUM>, which is given by the slope of the tangent line ∂Fwaer/∂v at the specific volume of the container, as marked on <FIG>. When ice-<NUM> nucleates, the nascent nucleus will exert further pressure on the water and the container, and because the total system volume cannot change, the container will exert equal and opposite pressure back on both the water and the ice nucleus. This reduces the specific volume of ice and water, and increases their specific free energy according to the F(v) curves shown in <FIG>. We name the energy required for this pressurization of the system and densification of the initial liquid phase the "isochoric growth penalty", which can be interpreted as an energetic penalty that the solid phase must pay in order to grow within a system of constrained absolute and specific volume.

Intuitively, the magnitude of this penalty must vary with the absolute volume of the system; growth of a single ice nucleus confined in an ocean will cause no appreciable effect, but growth of the same nucleus in a nanoscale container may compress the remaining water significantly.

The pressure within the system is therefore a function of the relative phase fraction of ice that has grown. Because water and ice remain in constant mechanical equilibrium, the pressure experienced by both phases will be equal. Recalling that the pressure within a given phase at a given specific volume is described by the slope of the line tangent to its F(v) curve at that volume, this physical constraint can be illustrated by a "parallel tangent construction" as shown in <FIG>. , whereby the specific volumes and energies of each phase for a given phase-fraction of ice are identified by the points on the F(v) curves for water and ice that yield parallel tangents between the phases.

The use of this parallel tangent construction to track continuity of pressure between phases is analogous to the tangent construction originally used by Gibbs to describe continuity of chemical potential between phases in binary systems at constant temperature and pressure [<NUM>]. During the growth of ice in an isochoric system (and the accompanying densification of both phases), the tangent lines will remain parallel while gradually increasing in slope until the two lines merge and form the common tangent, which marks the two-phase equilibrium state shown in <FIG> and provides the equilibrium pressure Pequilibrium.

In the preceding section, it was established that in an isochoric system, the specific free energies of water and ice do not remain constant during the process of nucleation and growth, instead shifting dynamically along their respective F(v)|T curves according to the parallel tangent construction. In order to mathematically describe this behavior, an expression relating the specific volumes (and thus free energies) of the phases as a function of ice nucleus growth is needed.

Given a closed isochoric system, mass and volume must be conserved <MAT> <MAT> which further implies conservation of specific volume. However, we note that the conservation of specific volume does not take the form vsystem = vice + vwater as might be expected, because the system specific volume is not itself an inherently conserved quantity; it is instead conserved as a simple mathematical consequence of the conservation of the system mass and volume, and is thus given by definition as: <MAT>.

The requirement of equal pressure throughout the system provides the final constraint. Leveraging the parallel tangent logic, the pressures of ice and water during the non-equilibrium nucleation process can be tracked along the F - v curves by their derivatives: <MAT>.

In the Supporting Information, we use these four constraints to determine the specific volumes of each phase as a function of nucleus volume (vwater(Vice), vwater(Vice)) along the reaction coordinate of the nucleation process. These specific volumes thereby give the specific free energy for both water and ice,.

Fwater (vwater(Vice)), Fice(vice(Vice)) as a function of the ice nucleus volume, enabling derivation of the nucleation barrier.

We consider now two states that the isochoric system may occupy (<FIG>). For clarity, we will describe the total free energy of the system with the variable Φ, and the specific Helmholtz free energies of each phase as Fwater and Fice. The subscripts <NUM> and <NUM> will be used to denote the values of parameters in States <NUM> and <NUM>.

In State <NUM>, the entire system is in the liquid phase, and its free energy is thus given by: <MAT> in which Fwater<NUM> = Fwater(vwater<NUM> = vsystem). In State <NUM>, an ice-<NUM> nucleus of absolute volume Vice has formed, dividing the system into two phases with specific volumes vwater(Vice) and vice(Vice). Defining the ice phase fraction of the system as: <MAT> and incorporating a standard interfacial free energy term γ which scales with the surface area of the ice nucleus Aice, the total free energy of State <NUM> is given by: <MAT> in which Fwater<NUM> = Fwater (vwater<NUM> = vwater(Vice)) and Fice<NUM> = Fice (vice<NUM> = vice(Vice)). Rearranging these equations and grouping specific free energy terms by phase, the free energy change ΔØ upon formation of a nucleus is given by: <MAT>.

The two energy difference terms in eqn. (<NUM>) have distinct and meaningful physical significance. (Fwater<NUM>-Fwater<NUM>) describes the "isochoric growth penalty", or the energy required to pressurize the system and densify the water phase, which the emergent ice nucleus must provide in order to grow. This term will always be positive. ( Fice<NUM> - Fwater<NUM> ), which will always be negative, gives the bulk free energy difference between the phases at their present specific volumes, which is weighted by the phase fraction f in order to capture the two-phase nature of the equilibrium system.

The free energy change accompanying the formation of an ice nucleus in an isochoric system can thus be reduced to the following: <MAT>.

The interfacial and bulk free energy terms in eqn. (<NUM>) are roughly analogous to those found in classical nucleation theory; the former will scale with the surface area of the nucleus in the positive direction, the latter with its volume or mass in the negative. However, it is crucial to note that within the Gibbs formulation, the bulk free energy difference between water and ice is considered constant- whereas in an isochoric system, it varies as a function of the ice phase fraction, decreasing in magnitude as the ice grows in a reflection of the fact that the ultimate thermodynamic destination of the system is a state of two-phase water-ice equilibrium, not complete freezing.

The isochoric growth penalty term is unique to systems of constrained volume, and will be shown next to fundamentally alter the nucleation behavior.

In <FIG>, we plot the total free energy change ΔΦ alongside the three separate energy contributions; the interfacial energy, the bulk energy, and the isochoric growth penalty. Our calculations for <FIG> describe a single ice-<NUM> nucleus of spherical geometry in a system of absolute volume VS ~ <NUM> x <NUM>-<NUM> m<NUM> at a temperature of -<NUM> (additional parameters available in SI).

<FIG> features additional ΔØ curves for the same temperature but varying absolute system volumes VS. These plots reveal a fundamental difference between ice formation at constant volume and ice formation at constant pressure: in the classical Gibbs formulation, the ΔG (radius) curve features one critical point, while in an isochoric system there are two critical points.

Classically, the critical point of the free energy curve is a maximum and defines the nucleation barrier, or the energetic barrier after which continued ice growth will lower the free energy of the system indefinitely until the entirety has changed phase. In the isochoric case however, ice growth is not indefinite-it must cease upon reaching the equilibrium phase fraction, in accordance with the T-V phase diagram (<FIG>). This limitation is captured kinetically by the isochoric growth term, and thus the ΔΦcurves can possess two critical points; the first a maximum at which the bulk driving force for phase transition overcomes the penalty of forming a new phase interface, and the second a minimum at which the isochoric growth energy overcomes the bulk driving force.

Importantly, because the isochoric growth term is a function of the absolute system volume (scaling as Vice/Vsys), its contribution vanishes at the infinite volume limit, consistent with intuition. In this case, which we label the Gibbs Limit in <FIG>, the ΔØ free energy curve will be identical to that found using the classical Gibbs formulation, featuring only an initial maximum.

Conversely, as the system volume decreases the relative contribution of the isochoric growth term increases, both introducing the second critical point (corresponding to the phase fraction limitation) and increasing the critical radius of the nucleation barrier. This increase is captured in <FIG>, given as a function of absolute system volume for various sub-freezing temperatures. Note that at temperatures close to the freezing point, the effect of the volume constraint on isochoric nucleation can be significant even under relatively large system volumes-on the order of microns.

Our derivation further reveals the existence of a discrete absolute system volume, which we term the critical confinement volume, below which the second critical point will reach an energy equal to the first, erasing the inflection point between them and yielding a nucleus free energy curve that increases monotonically with radius. A representative free energy curve at this absolute volume threshold is labeled as the Helmholtz Limit in <FIG>. These critical confinement volumes are also marked on the critical radius curves in <FIG>, indicating the system volume at which the critical radius for nucleation would become infinite. These critical system volumes are then plotted independently against temperature in <FIG>, resulting in a "kinetic phase diagram" for freezing water under confined volumes. Our kinetic phase diagram reveals a unique implication for systems of constant volume: there exists a volumetric regime in which nucleation of ice-<NUM> from the supercooled liquid state is kinetically impossible.

Notably, the existence of ice within this regime is not thermodynamically prohibited-comparing <FIG> with <FIG> it can be seen that these critical ice nucleus volumes are orders of magnitude smaller than the equilibrium phase fraction limit. These results indicate that ice could theoretically exist at these system volumes (e.g. if an ice crystal was artificially seeded in the system and the volume was then constrained), but that supercooled water simply has no kinetic pathway to freezing in a sufficiently confined isochoric system.

The kinetic analysis provided herein only describes the formation of only the first ice-<NUM> nucleus in an infinitely rigid container (e.g. a container of truly constant volume), and is subject to further simplifying assumptions, including the assumption of spherical nuclei and of known interfacial energy. However, the insights revealed provide the conceptual foundations for a wide range of further study, and outline the limiting energetic behaviors of ice nuclei in isochoric systems. We anticipate that in systems that deviate from the conditions described herein, ice nucleation kinetics will lie somewhere between the identified Gibbs and Helmholtz limits. For example, systems of nanoscale volume found in biological matter may experience a reduced isochoric growth penalty due to some degree of flexibility within the container, but the excess energy required to grow in a constrained volume is still a physically pertinent feature. Conversely, within the rigid containers described in this invention, the assumption of infinite rigidity is likely acceptable, but the macroscopic volumes are too large to appreciate the effect of a single ice nucleus. However, continuity of pressure within the system remains, forcing every growing nucleus to interact with every other via pressure, and thus a macroscopic effect on the nucleation kinetics will still be observed. This has useful implications, such as enhanced or high-stability supercooling due to heightened nucleation barriers, and ensemble-level analysis built from the single-nucleus isochoric nucleation theory must be studied in the future. In summary, regardless of the experimental details of the system, if it is operating under constant-volume conditions, the nucleation barrier equation will feature a positive isochoric growth term, increasing the energetic barrier that must be crossed in order to grow a bulk ice phase and decreasing the likelihood of initial nucleation.

In this example, we find that isochoric conditions and chambers can significantly improve the long term stability and ability to withstand perturbation of a supercooled solution, over other techniques for maintaining supercooling.

In this example we supercooled deionized water in identical rigid chambers under three sets of conditions:.

All systems were initially supercooled to -<NUM> +/- <NUM> in a constant-temperature circulating bath and then exposed to various macroscopic perturbations, including drop-impact from a height of 1ft onto a hard acrylic surface, <NUM> vibrational loading on a rotary shaking table, ultrasonication in a cooled bath at <NUM>, and continuous thermal cycling between <NUM> and -6C for <NUM> hours (experimental details available in Methods below). Nucleation was evaluated visually, and the nucleation frequency was recorded as the number of chambers per group that experienced ice formation. All tests were conducted in n = <NUM> trials of N = <NUM> chambers, and repeated in two sizes (<NUM> and <NUM>) of borosilicate glass media bottles with rigid threaded polypropylene caps. In order to ensure the relevance of these tests to preservation protocols of interest, which invariably involve the introduction of other potential nucleation sites into the system, a PDMS-on-glass chip was also added to each container, representative of the lab-on-a-chip systems used to house engineered tissue constructs[<NUM>].

Chamber preparation - Isobaric chambers were filled to approximately <NUM>% volume with de-ionized water and capped, leaving a layer of air approximately <NUM> in height atop the liquid. Oil-sealed isobaric chambers were filled to the same level, then sealed via syringe with a layer of mineral oil (Sigma-Aldrich, USA) approximately <NUM> in height. As per the protocol outlined in previous studies [<NUM>] was taken to ensure that the entire water-air interface was eliminated. For assembly of isochoric chambers, a rubber plug approximately <NUM> in volume was press-fit into the cap of each chamber, in order to displace volume as the cap was turned onto the threads of the chamber and ensure that no air remained upon sealing of the system. After sealing, isochoric chambers were turned upside down and shaken in order to visually verify that no air remained present in the system. In all three assembly cases, DI water was chilled to <NUM> prior to filling, and the chamber was ultrasonicated after the initial pour in order to the remove any trapped air bubbles. A <NUM> x <NUM> x <NUM> PDMS-on-glass chip was also added to each system to ensure that observed effects were not products of the specific containers being employed.

For all non-thermal disturbance scenarios, chambers were first supercooled to -<NUM> +/- <NUM> in a programmable constant-temperature circulating chiller bath (PolyScience, USA) for four hours. They were then removed and immediately exposed to one of the following disturbances.

Impact: Chambers were dropped from a height of one foot onto a hard acrylic plate of <NUM>" thickness. As per the schematic in Figure 2A, a clear acrylic tube of slightly larger diameter than the chambers was used to ensure a straight and repeatable drop trajectory.

Vibration: Chambers were mounted to a covered rotary shaking table (ThermoFisher, USA) and shaken for <NUM> seconds at a rate of <NUM> rpm and a rotary radius of <NUM>, yielding acceleration magnitudes of approximately <NUM>. Chambers were mounted in an insulating foam rack, and the atmosphere within the covered shaking table was maintained at -<NUM> +/- <NUM> via circulation of cold CO2 vapor. During initial experimental design, the temperature inside the chambers was confirmed via thermocouple to remain consistent within <NUM> over the <NUM> second shaking period.

Ultrasonication: Chambers were moved directly from the circulating chiller to an ultrasonic bath (Fisher Scientific, USA), submerged completely, and sonicated at <NUM> for <NUM> seconds. The bath was filled with <NUM>% w/v NaCl solution pre-chilled to -<NUM> to ensure temperature consistency.

Chambers were submerged fully in the -<NUM> bath directly following assembly. The chilling bath then was programmed to ramp continuously between <NUM> and -<NUM> on a one hour period for <NUM> hours (constituting twelve cycles between the two temperatures), maintaining an average temperature of -<NUM>. This range was chosen to reflect the temperature oscillation encountered in standard on/off vapor-compression refrigeration units. After <NUM> hours, chambers were carefully removed and evaluated for ice nucleation.

In all disturbance scenarios, ice nucleation was evaluated visually, as shown in Fig. 2B, and recorded as a binary pass/fail for the purposes of calculating the nucleation frequency.

In order to enable clear photo capture of the interface behaviors displayed in <FIG>, alternative containers made of optically-clear virgin polystyrene with a rectilinear profile were used (T75 cell culture flask, ThermoFisher, USA). <NUM> of DI water was added to each container, with careful avoidance of air bubbles during filling. <NUM> of mineral oil was then added to one container, completely sealing the free water surface. The two chambers were then vibrated on a vertical-action vortex mixer (ThermoFisher, USA) and video of the interface behavior was captured at 1080p resolution and a speed of <NUM> frames-per-seconds on a Nikon D3400 camera.

Each experimental group, defined as the chambers exposed to a given disturbance (e.g. impact, vibration, ultrasonication, thermal) at a given container volume (e.g. <NUM> or <NUM>) under a given loading condition (e.g. isobaric, isobaric oil-sealed, or isochoric), was comprised of <NUM> chambers divided into n = <NUM> groups of N = <NUM> chambers. Values plotted in the results represent means, while error bars represent standard deviations. Statistically significant differences between groups were evaluated using paired-sample t-tests computed using MATLAB, with a standard significance threshold of P < <NUM>.

The nucleation frequency as a function of disturbance type is plotted for all three chamber configurations in Fig. 5A, and comparisons between chamber configurations for each disturbance type are presented individually in Fig. 5B-E for statistical evaluation. As demonstrated in Fig. 5A, isochoric conditions afford greatly enhanced supercooling stability across all perturbation types, at both volume scales. Notably, isochoric supercooling at a <NUM> volume remained stable in roughly <NUM>% of trials when exposed to ultrasonication, which is the most universal and sure-fire trigger of ice nucleation currently known [<NUM>,<NUM>,<NUM>], and remained stable in all trials when exposed to vibrational loading comparable to that encountered during commercial flight. To the best of the inventors knowledge, no technology has ever been presented that purports to be capable of resisting ultrasonic-induced nucleation in pure water, or of demonstrating this degree of stability at bulk volumes across a wide range of disturbance types. This finding demonstrates an extreme degree of stability and provides proof to our claim that isochoric conditions enable high stability supercooling. Standard isobaric conditions yielded the least stability by comparison, while oil-sealing provided statistically significant stability enhancements relative to standard isobaric conditions during exposure to macroscopic vibrational loading and acute impact, but did not significantly affect resistance to ultrasonic or thermal perturbation. As a whole, as demonstrated inFig. 5A, isochoric supercooling provides drastically enhanced stability compared to other methods.

Mechanical stimuli have long been known to induce ice nucleation [<NUM>], and the last century of research into the topic has clarified that cavitation is the most prominent responsible mechanism [<NUM>,<NUM>,<NUM>,<NUM>]. While cavitation is frequently associated with ultrasonication, it is also caused by all manner of shockwaves [<NUM>] and by vibrational surface effects such as the Faraday instability [<NUM>], which occur at bulk fluid-fluid interfaces.

Our results obtained in this study demonstrate that isochoric supercooling is significantly more stable than its isobaric counterparts when exposed to mechanical stimuli of any kind, and we thus suggest, without wishing to be theoretically bound, that a fundamental mechanism driving this isochoric stability is a reduced likelihood of cavitation. By totally constraining the liquid volume, isochoric conditions eliminate opportunities for cavitation from effects that require bulk fluid-fluid interfaces (such as the Faraday instability or analogous effects), and eliminate opportunities for cavitation from bulk motion of the stored water. They furthermore present two thermodynamic obstacles to cavitation from shockwaves or ultrasonication: firstly, because there is no bulk air anywhere in the system, cavitation must occur in dissolved air that is first forced out of solution with the supercooled water; secondly, the formation of a low-density air bubble in a constrained volume of water will create a positive pressure, increasing its energetic barrier to formation. While theoretical analysis of this latter effect is outside the scope of this work, it is directly analogous in concept to the increased energy barriers produced by the formation of ice in a constrained volume (as described in Example <NUM>).

In total, the superior supercooling stability experienced in isochoric systems is a composite effect, reflective of the complex thermodynamic and kinetic factors driving ice nucleation in systems of bulk volume.

Thermodynamic factors such as the reduction or elimination of microscopic density fluctuations and the increase of the ice nucleation barrier under isochoric conditions contribute [<NUM>, Example <NUM>]; the elimination of all fluid-fluid interfaces contributes [<NUM>]; and an increased overall resistance to cavitation plays a central role. The experimental reality of enhanced high-stability supercooling under isochoric conditions can be employed immediately for low-risk preservation and transportation of sensitive biological matter.

In some embodiments of the system shown in schematic in <FIG> (not according to the invention), the base constant-volume container used to achieve high-stability isochoric supercooling may be designed as detailed in the engineering drawings featured in <FIG>. It may also be designed in any other arbitrary fashion so as to ensure rigid containment of the liquid within and effective air-sealing.

This design makes use of a two-part cap-and-plug closure to ensure robust o-ring sealing and effective optional transmission of hydrostatic pressure to a pressure transducer that can be connected via one of the <NUM>/<NUM>-<NUM>-UNF high-pressure threaded connections.

Temperature monitoring implements, implements for the measurement of dielectric properties within the supercooled solution, or any other manner of wired or unwired probe or apparatus may be run through the secondary posterior threaded connecting port.

Each of the parts detailed in <FIG> may be constructed of any rigid material, including metallic materials such as but not limited to Aluminum <NUM>, Titanium Grade <NUM>, <NUM> Stainless Steel, composites such as but not limited to carbon- or glass-fiber reinforced polymers, ceramics, hard plastics, etc..

Each of the parts may additionally be treated or coated in any manner in order to achieve a variety of improving effects, including but not limited to corrosion resistance, biocompatibility, hydrophobicity, ice nucleation resistance or anti-nucleation effects, aesthetic improvement, etc..

The inventors have fabricated these devices from Aluminum <NUM>, Titanium Grade <NUM>, and <NUM> Stainless Steel, and several combinations thereof, and have verified that this design can effectively hold seal at pressures up to <NUM> megapascals and temperatures as low as -<NUM> when an appropriate wall thickness is chosen.

In some embodiments of the system shown in schematic in <FIG> (not according to the invention), a device configured as detailed in <FIG> may be employed. This device leverages high-stability isochoric supercooling to maintain a preserved media and biologic in a supercooled state, and additionally features real-time pressure-based nucleation detection to continuously monitor stability. It also features an external controller and an external Joule heating element, and employs an external cooling bath for the maintaining of the desired supercooling temperature.

In this configuration, if ice nucleation occurs for any reason, an increase in pressure will be detected by the digital pressure transducer. The digital pressure transducer transmits to a microcontroller, and if the detected pressure value surpasses an assigned threshold value (typically a small amount greater than the resting value of the system), the microcontroller will activate the heater in order to raise the temperature of the system to just above its freezing point, thereby eliminating any ice formation and allowing the system to re-supercool.

The return of the pressure to its former resting value will signal to the pressure transducer that all of the ice in the system has been melted, and that the heater should be shut off and the system allowed to re-supercool. Such a configuration was built by the inventors and experimentally validated. A custom fabricated pressure vessel built of Grade V titanium was used as the isochoric container, pressure was monitored using a digital pressure transducer, Joule heating was provided by a coiled copper wire, and control was provided by a laptop computer running MATLAB technical computing software. Pressure was plotted in real time, alongside heater activity. The employed supercooled media was pure deionized water, free of air bubbles. The entire container assembly was immersed in a constant-temperature cooling bath held at -<NUM>.

In <FIG>, a sample data output generated using this device is provided. As labeled, the initial state of stable supercooling is indicated by no change in the pressure reading (solid line). Upon artificial excitation of nucleation, the pressure rises sharply, and current is sent to the relay controlling the heater (dotted line), activating the Joule heating. In this example, heat was supplied at a rate of <NUM> watts and no PID control was used. As heating continues, the pressure rise is arrested, and the pressure begins to decrease as the small amount of interior ice is melted. When the pressure returns to within a small percentage of its original steady value, the heater turns off. The system is then observed to re-enter a stable supercooled state at -<NUM> and remain there for the remaining six hours of the experiment.

Based on the pressure and the compressibility of water and ice-<NUM>, it was calculated that ice inhabited approximately <NUM>% of the container volume at the peak of its growth. Thus this example demonstrates that not only does the technology described in this enclosure enable high-stability supercooling, but in the unlikely event of destabilization and ice nucleation, pressure-based nucleation detection and responsive heating can be employed to arrest ice growth before it can overcome an appreciable portion of the container volume, thus universally protecting any stored biologics.

Claim 1:
A method of inducing high-stability and optionally transportable supercooling, the method comprising:
providing a device comprising a rigid container containing biological matter in an aqueous media;
ensuring the removal of essentially all bulk gas phase from the contained aqueous media;
sealing the container with a rigid air-tight closure, wherein the percentage of the volume of bulk gas phase remaining in the aqueous media upon sealing of the container is less than <NUM>%; and
cooling the aqueous media to a temperature below <NUM> under isochoric thermodynamic conditions,
wherein the container further comprises an on-board or off-board pressure sensing implement, a control implement and an energy delivery implement,
wherein the pressure sensing implement measures or detects pressure, wherein a change in pressure is used to detect formation or elimination of ice within the aqueous media,
wherein the energy delivery implement uses an energy delivery mechanism selected from the group consisting of conductive heating, convective heating, radiative heating, inductive heating, Joule heating, electromagnetic heating, radio heating, and ultrasonic heating,
wherein the energy delivery implement delivers energy to melt, dissolve, or otherwise eliminate ice nuclei or crystals, and
wherein upon detection of a change in pressure, a routine is triggered within the control implement, wherein the control implement continuously monitors the pressure, and the routine activates the energy delivery implement when the pressure reading surpasses a given first threshold value, and turns off the energy delivery implement when the pressure returns to beneath the first threshold value or a different threshold value.