Method and apparatus for storage of biological material

Abstract

To provide methods and apparatus for long-term storage of biological objects as well as other organic and inorganic aqueous materials.SOLUTION: Methods and apparatus use low temperature and elevated pressure to depress a freezing and melting temperature of water and aqueous solutions, to induce suspended animation in materials including but not limited to biological material, soluble molecules, organic and inorganic compounds. Disposing such materials in a pressure vessel and increasing the pressure to about 210 MPa depresses the freezing and melting temperature of water, biological matter, and materials in aqueous solution, to about -22°C. Storage at low temperature under high pressure suspends metabolic activity and induces cryostasis. The methods and apparatus may be used for cryo-banking biological materials that cannot be frozen or vitrified, or otherwise preserved, including, but not limited to, cells, tissues, human organs for transplantation, and entire organisms.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of the filing date of U.S. Patent Application No. 16 / 501,918, filed July 5, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] The field of the invention is the long-term preservation and storage of delicate materials that are damaged by freezing. More particularly, the invention relates to the long-term preservation and storage of delicate materials, such as aqueous solutions and biological materials, below their freezing temperature by applying high pressure to prevent freezing at temperatures as low as -22°C. [Background technology]

[0003] Current techniques for tissue or organ preservation require perfusion and storage at body temperature or within the hypothermic range of 4°C or above. While these methods are effective for storing and transporting organs for a few days, they are not suitable for long-term (i.e., multi-week, multi-month, or multi-year) biobanking. The need for transplantable organs is increasing, and countless people die each year while waiting for an organ transplant. This situation can be partially improved by better preserving organs during transport, but such progress is only incremental. With the realization of regenerative technologies for 3D printing, growing, and genetically modifying / immunomodulating organs for xenotransplantation, the need for transplantable organs will present new challenges. Organs from these sources will need to be stored until needed for transplantation because the processes used to manufacture them are time-consuming, a time that is unavailable to urgently needed patients. Furthermore, individuals may desire to have their own complete organs / tissues generated and preserved for future needs.

[0004] The effects of pressure at ambient temperatures on molecules, cells, and organisms have been studied, with results indicating that survival is possible even at extreme pressures. Some cells and organisms can remain viable at temperatures near absolute zero or outside the atmosphere. For over half a century, researchers have attempted to develop methods for freezing or vitrifying organs as a means of long-term preservation. All attempts have failed. There remains a need for long-term preservation of biological objects and other organic and inorganic aqueous materials. Summary of the Invention

[0005] One aspect of the present invention relates to a method for storing / preserving materials, including but not limited to water, organic and inorganic aqueous materials / substances / media, materials in aqueous suspension, aqueous solutions, aqueous mixtures, aqueous colloids, aqueous materials, biological materials, biologics, and biologically derived materials, at temperatures below their freezing or melting temperatures at ambient pressure by increasing pressure. By increasing the pressure applied to any or all of the materials in a pressure vessel, their freezing or melting temperatures are reduced. The storage temperature range at which the materials cannot freeze or vitrify ranges from -0.001°C to -21.985°C. The melting or freezing points of the materials are reduced by pressure over a pressure range from ambient pressure to 209.9 MPa, or approximately 210 MPa. Such biological materials may include, but are not limited to, organic molecules, molecular complexes, nucleic acids, sugars, amino acids, peptides, proteins, enzymes, organelles, organoids, cells, tissues, organs, and organisms.

[0006] In various embodiments, the method includes storing an aqueous material under pressure to prevent a phase transition to a solid and maintain it in a stable liquid state or in a metastable supercooled liquid state, wherein the stored material is water or water with an inorganic solute in an aqueous solution, or the stored material is water with an organic solute in an aqueous solution, or the stored material is water with organic and inorganic solutes in an aqueous solution, or the stored material is a mixture of water and organic material, or the stored material is water with a colloid, or the stored material is water in a mixture with either or both organic and / or inorganic material, or the stored material is Water in a mixture with biological material, or the material to be stored, is water in which biological material is present and / or suspended, containing organic and / or inorganic solutes, water in which biological material is present and / or suspended, containing organic and / or inorganic solutes and colloids, water in which biological material is present and / or suspended, or the material to be stored, is in a mixture with organic and / or inorganic compounds, containing both organic and / or inorganic solutes, colloids, and water in which biological material is present and / or suspended.

[0007] In one embodiment, the present invention provides a method for cooling the supercooling temperature (point) of organic and inorganic aqueous materials / substances / media, materials in aqueous suspension, aqueous solutions, aqueous mixtures, aqueous colloids, aqueous materials, biological materials, and biologically derived materials, including, but not limited to, organic and inorganic aqueous materials / substances / media, materials in aqueous suspension, aqueous solutions, aqueous mixtures, aqueous colloids, aqueous materials, biological materials, and biologically derived materials, to a temperature below their freezing temperature (i.e., melting temperature) at ambient pressure by increasing the pressure applied to the materials / substances. Thus, materials / substances can be supercooled and maintained in a metastable liquid state over a range of -0.001°C to -92°C. Supercooling occurs over a pressure range from ambient to 209.9 MPa. A stored material is supercooled if its storage temperature is below the pressure-decreased (pressure-dependent) freezing / melting point of the material. Biological materials may be, but are not limited to, organic molecules and molecular complexes, nucleic acids, sugars, amino acids, peptides, proteins, enzymes, biologics, organelles, organoids, cells, tissues, organs, and organisms.

[0008] In one embodiment, the present invention provides a method for lowering the freezing point of a material by adding a solute to the preservation medium and the material to be preserved, thereby further lowering the freezing temperature of the material, resulting in an additional freezing point depression of 1.86°C per mole of solute added, or a fraction or factor thereof, wherein the freezing point is lowered by 1.86°C per mole of solute added, or a fraction of 1.86°C per mole fraction.

[0009] In one embodiment, the present invention provides a method for depressing the freezing point of an aqueous medium under the conditions described herein by adjusting the colligative properties of an aqueous solution, mixture, colloid, or combination thereof by adding one or more moles or mole fractions of solutes to further reduce the freezing temperature of the aqueous medium. Further freezing point depression may be achieved by adding non-collective substances, including, but not limited to, antifreeze proteins, antifreeze sugars, ice-binding peptides, and other non-collective agents, which provide additional freezing point depression through ice inhibition or ice binding, thereby preventing, inhibiting, controlling, and / or separating ice crystal growth. The medium may or may not contain biological materials, including, but not limited to, organic molecules and molecular complexes, nucleic acids, sugars, amino acids, peptides, proteins, enzymes, biological agents, organelles, organoids, cells, tissues, and organisms. In various embodiments, the antifreeze protein may be derived from, for example, mealworm beetle (Tenebrio molitor), Antarctic fish (Type I, Type III), or perennial ryegrass (Lolium perenne).

[0010] Another aspect of the invention relates to a method for storing biological material, comprising placing the biological material in a pressure vessel, filling the pressure vessel with a driving liquid, evacuating the pressure vessel and sealing the pressure vessel, and using a pressure generator to increase pressure on the driving liquid and reduce the temperature inside the pressure vessel to below 0°C, wherein the pressure generator is used to apply a selected pressure to the driving liquid at a selected temperature, thereby maintaining the driving liquid in the pressure vessel in a stable liquid state, and applying the selected pressure to the driving liquid prevents freezing of the biological material at storage temperatures below 0°C.

[0011] In one embodiment, the method further includes placing the biological material in a sample bag along with a preservation solution, removing air from the sample bag, and sealing the sample bag, whereby the preservation solution and the driving solution are maintained in a stable liquid state.

[0012] In one embodiment, reducing the temperature and increasing the pressure includes increasing the pressure from ambient conditions in 200 psig (1.4 MPa) increments at 1000 psig / min (6.9 MPa) to about 30,000 psig (210 MPa) and reducing the temperature from ambient conditions to about -22°C.

[0013] In one embodiment, the biological material comprises one or more of an organic molecule, a molecular complex, a nucleic acid, a sugar, an amino acid, a peptide, a protein, an enzyme, an organelle, an organoid, a cell, a tissue, an organ, an organism, and an aqueous solution.

[0014] In one embodiment, the preservation solution comprises water and one or more of biological material, soluble molecules, organic and / or inorganic compounds, material in aqueous suspension, aqueous solution, aqueous mixture, aqueous colloid, aqueous material, and bio-derived material.

[0015] In one embodiment, the biological material comprises cells, tissues, organs, or whole organisms.

[0016] In one embodiment, the storage temperature is about -22°C.

[0017] In one embodiment, the applied pressure at storage temperature is about 30,000 psi (210 MPa).

[0018] In one embodiment, the storage temperature and applied pressure prevent freezing and cell damage by maintaining the cells in a metastable supercooled liquid state.

[0019] In one embodiment, the preservation solution comprises a solute, which includes one or more of antifreeze proteins, ice-binding proteins, antifreeze sugars, ice-binding sugars, ice-binding peptides, and other non-collective agents, that prevent, inhibit, control, or disrupt ice crystal growth and / or prevent ice nucleation.

[0020] In one embodiment, the driving fluid comprises propylene glycol or ethylene glycol, oil, petroleum oil, fish oil, mineral oil, vegetable oil, water, seawater, and any combination thereof.

[0021] In one embodiment, the selected storage temperature is from about -5°C to about -22°C.

[0022] Another aspect of the present invention relates to an apparatus for storing biological material, the apparatus comprising: a reservoir for containing a driving liquid; a pressure vessel having an internal well configured to receive the biological material, the pressure vessel operably connected to the reservoir for receiving the driving liquid from the reservoir; a pressure generator operably connected to the pressure vessel and the reservoir, the pressure generator applying pressure to the driving liquid; a pressure transducer indicative of the pressure of the driving liquid within the pressure vessel; a temperature sensor for sensing the temperature of the pressure vessel; and a cooling device configured to provide a controlled pressure vessel internal temperature below about 0°C, wherein at a selected pressure vessel temperature below about 0°C, the pressure generator applies a selected pressure to the driving liquid to maintain the driving liquid within the pressure vessel in a stable liquid state.

[0023] In one embodiment, the apparatus further comprises a data acquisition system (DAQ) that acquires data from one or more of the pressure transducer, the temperature sensor, the pressure generator, and the cooling equipment.

[0024] In one embodiment, the apparatus further comprises a controller operably connected to one or more of the pressure transducer, the temperature sensor, the pressure generator, and the cooling equipment, wherein the controller monitors and maintains at least one of a selected internal pressure vessel temperature and a selected pressure applied to the drive fluid in the pressure vessel.

[0025] In one embodiment, the pressure generator is automated and is mechanically, electrically, pneumatically, or hydraulically driven by a controller.

[0026] In one embodiment, the cooling device further comprises a heater, the heater comprising a temperature sensor and a temperature controller.

[0027] In one embodiment, the cooling device includes a proportional-integral-derivative (PID) control.

[0028] In one embodiment, the apparatus further comprises an evaporator.

[0029] In one embodiment, the device further comprises at least one valve that, when closed, allows isolation and removal of the pressure vessel from the device, such that the pressure vessel maintains the applied pressure of the driving fluid when removed from the device.

[0030] Another aspect of the present invention relates to a pressure vessel for storing biological material, the pressure vessel comprising: a housing having a cavity including a first portion and a sample well for receiving the biological material and a driving fluid, the first portion of the housing including an overflow channel opening to an exterior of the housing; and a lid including a first portion configured to engage the first portion of the housing, the position of the lid within the housing being adjustable over a range from the first position to a closed position, the lid including a second portion configured to partially fit within the sample well of the housing, the first portion of the lid including a port configured to interconnect with an external device, the lid including a driving fluid channel configured to direct driving fluid through the lid between the port and the sample well, wherein adjusting the lid to the closed position drains excess driving fluid from the sample well through the port and the overflow channel and the second portion of the lid seals the sample well, the pressure vessel configured to maintain an internal pressure of at least about 30,000 psi (210 MPa) of driving fluid in the sample well.

[0031] In one embodiment, pressure is applied to the driver fluid in the sample wells through a port by an external device.

[0032] In one embodiment, the pressure vessel further comprises at least one valve disposed between the port and the external device, which when closed isolates the pressure vessel from the external device and maintains the internal pressure of the sample well.

[0033] In one embodiment, the overflow channel is configured to receive a temperature sensor.

[0034] Another aspect of the invention relates to an apparatus for storing biological material at temperatures below 0° C. without freezing. In various embodiments, the apparatus includes a cooling / heating system for cooling and / or heating a fluid in a chamber containing a pressure vessel or a fluid flowing through a series of circuits in the wall of the pressure vessel or attached to the outside of the pressure vessel during or after pressurization, warming the fluid during or after depressurization and warming the pressure vessel, and one or more of the following apply:where the cooler and heater are separate components controlled manually, electrically, electronically, or by computer; where the cooler and heater are integrated into one component controlled manually, electrically, electronically, or by computer; where the cooler uses a reverse cycle for heating and is controlled manually, electrically, electronically, or by computer; where the cooler and / or heater uses a piston-type compressor, evaporator, and condenser; where the cooler and / or heater uses a reciprocating piston-type compressor, evaporator, and condenser and is controlled manually, electrically, electronically, or by computer; where the cooler / heater is thermoelectric and is controlled manually, electrically, electronically, or by computer; where the cooler / heater is a Stirling cooler, Stirling pulse tube cooler, and / or heater and is controlled manually, electrically, electronically, or by computer. where the cooler / heater is a sonic or ultrasonic device and is controlled either manually, electrically, electronically, or by computer; where the cooler operates by evaporative cooling (e.g., liquid nitrogen, dry ice) and is controlled either manually, electrically, electronically, or by computer; where heating and cooling is by radiative action and is controlled either manually, electrically, electronically, or by computer; where heating and cooling is by convection and is controlled either manually, electrically, electronically, or by computer; where heating and cooling is by induction and is controlled either manually, electrically, electronically, or by computer; where resistance is used for heating and is controlled either manually, electrically, electronically, or by computer; where lasers or masers are used for heating and / or cooling and are controlled either manually, electrically, electronically, or by computer.

[0035] In various embodiments, the device includes a control, a set of controls, one or more control systems, or a controller for initiating and / or maintaining or stopping its operation and for setting and / or adjusting the environment within the system as a whole and its components. In various embodiments, the temperature within the pressure vessel can be cooled or maintained by a cooling system having a temperature controller, and the temperature within the pressure vessel can be heated or maintained by a heating system using separate controllers. In various embodiments, the cooling controller and the heating controller can operate simultaneously, or a single temperature controller can be used to control the temperature during cooling and warming, where the temperature controller used during cooling can control the rate of temperature change, and where the temperature controller used during warming can control the rate of temperature change. In one embodiment, a single controller can be used to control the cooling and warming rates and the cooling and warming rates. In one embodiment, separate controllers can be used to control the pressurization rate or during pressurization to ballistically pressurize. In one embodiment, a controller can be used to control the depressurization rate or during pressurization to ballistically depressurize. In one embodiment, a single controller may be used to control the pressurization and depressurization and the rate of pressurization and depressurization. In one embodiment, a single controller may be used to control the temperature and rate of heating and cooling, and the single controller may also control the pressurization and depressurization and the rate of depressurization. Any or all of the aforementioned controls for pressure and temperature, or individually, may be mechanical, electrical, electronic, or computerized. Any or all of such controls may implement setpoint, rate of change, and / or time period at setpoint control for either or both temperature and pressure. The controller may have a temperature sensor that provides the controller with the current temperature inside the cooler and / or pressure vessel.The controller may have a pressure sensor, transducer, and / or gauge that provides the controller with the current pressure inside the pressure vessel, piping system, or portion thereof.

[0036] In one embodiment, the apparatus provides a temperature monitoring instrument by reading and / or recording the temperature from inside a cooler / heater, inside a pressure vessel, inside a pressure vessel wall, or from the surface of the pressure vessel in real time. Analog or digital temperature readings may be taken automatically at intervals or manually at intervals, and the readings may be recorded manually, mechanically, electrically, electronically, or by a computer. Temperature readings may be provided by sensors such as thermometers, thermistors, resistance thermal devices (RTDs), thermocouples, infrared sensors, infrared cameras, pyrometers, spring thermometers, liquid in column thermometers, or any other mechanical, chemical, liquid crystal, electrical, or electronic sensors. Data from any and / or all of the temperature sensors listed above may be used as input temperature information for the controller and control unit of the above embodiments.

[0037] In one embodiment, the device provides pressure monitoring equipment by reading and / or recording the pressure inside the pressure vessel, and / or the pressure in or from the pressure generator, and / or the pressure inside some or all of the piping system. Pressure readings are generated from pressure transducers, analog pressure gauges, and displayed in real time on analog and / or digital gauges. Data from the pressure gauges or pressure transducers may be recorded mechanically, electrically, electronically, or using a computer. Data from any and / or all of the pressure sensors listed above may be used as pressure information for the controller and controls of the above embodiments.

[0038] For a better understanding of the present invention and to show more clearly how the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which: [Brief explanation of the drawings]

[0039] [Figure 1] FIG. 1 is a phase diagram showing the pressure / temperature values ​​at which water remains in a stable liquid state, including the lowest temperature at which water remains in a stable liquid state and the corresponding pressure (designated "A"). [Figure 2] FIG. 2 shows one embodiment of an apparatus for storing biological material. [Figure 3] FIG. 3 shows an expanded view of one embodiment of a pressure vessel for containing biological material during long-term storage. [Figure 4] 4A-4E are schematic diagrams illustrating assembly of a pressure vessel, according to one embodiment. [Figure 5] 5A and 5B are plots illustrating pressure and temperature curves, respectively, for a biological material stored and for a biological material recovered from storage, according to one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0040] definition As used herein, "stasis" or "cryostasis" is used to describe a state in which metabolic and molecular activity ceases. Cryostasis more particularly relates to the sub-zero temperature and pressure ranges described herein.

[0041] "Suspended animation" refers to the same inactive state as described above.

[0042] "Material," "substance," and "object" are terms used interchangeably herein and refer to either biological or inorganic components that are difficult to preserve for long periods of time.

[0043] "Biological material" refers to any carbon-containing living or formerly viable matter or component thereof, including, but not limited to, molecules, proteins, cells, organelles, organoids, tissues, organs, and organisms.

[0044] "Aqueous material" is a general term that refers to any organic or inorganic matter that dissolves in, is suspended in, or contains water.

[0045] "Sub-zero" temperatures are used to refer to storage at any temperature below 0°C.

[0046] "Banking" or "biobanking" refers to the long-term preservation and storage of either biological or inorganic materials.

[0047] "Storage" and "preservation" are terms used interchangeably throughout this specification and refer to storing and maintaining materials in cryostasis.

[0048] As used herein, the term "approximately" indicates that the number that follows is an approximate number and is not limited to the exact number stated.

[0049] The singular forms "a," "an," and "the" include plural references unless specifically stated otherwise.

[0050] "Fluid" refers to gas, liquid, or a combination thereof, unless expressly stated otherwise.

[0051] "Supercooled" or "supercooled" refers to the metastable state of water below the melting temperature of water, 0°C, and atmospheric pressure.

[0052] The "collective" depression of the melting (freezing) temperature of water is defined by the number of molecules in solution: one mole of solute dissolved in one liter of water results in a melting point depression of 1.86°C.

[0053] The "non-collective" reduction in the melting (freezing) temperature of water is achieved by ice inhibitors or ice binders that prevent, inhibit, control, and / or separate ice crystal growth.

[0054] "Long-term" in the context of the present invention refers to any of the following periods: days, weeks, months, and years, unless expressly stated otherwise.

[0055] "Freezing point depression" (FPD) refers to lowering the melting (freezing) temperature of water below 0° C. This can be achieved by increasing pressure, supercooling, and / or adding colligative or non-colligative agents, as described herein.

[0056] As used herein, the term "UW® Solution," also known as University of Wisconsin Solution, refers to a preservation solution (Southard, JH et al., Transplantation Reviews 7(4): 176-190, 1993).

[0057] Embodiment Preservation of aqueous and biological materials that are sensitive to cryoinjury and / or freezing has presented a dilemma. Some molecules (e.g., DNA), cells (e.g., bovine sperm), and organisms (e.g., tardigrades, brine shrimp) can be successfully cryopreserved for years. However, most biological materials (e.g., mammalian organs) cannot survive freezing or long-term storage. The reasons for this are varied and relatively well understood. For example, during the phase change of water from liquid to solid (ice phase), the approximately 9% volume expansion causes physical damage to membranes, cells, and molecular machinery. This damage is exacerbated by cellular dehydration as a result of osmotic pressure imbalance and ice recrystallization during the thawing process. For biological materials with volumes numerically (dimensionlessly) larger than their surface area, such as human organs, achieving rapid and uniform freezing rates is difficult, if not impossible. In such cases, freezing begins rapidly from the outside, and then as the interior freezes, it expands and ruptures the outer layer, thus causing physical damage. The embodiments described herein aim to avoid the inherent problems associated with the phase change between liquid and solid by preventing the phase change. The devices and methods described herein are designed to prevent the phase change, whereby water and aqueous substances can be maintained in a stable liquid state for extended periods of time at temperatures below their melting points at atmospheric pressure due to increased pressure.

[0058] The embodiments described herein address the need for long-term storage and preservation of organs and other biological materials. Additionally, the embodiments described herein are suitable for long-term storage of, but not limited to, organic molecules, proteins, organelles, organoids, cells, tissues, organs, biologics, and pharmaceuticals, and initial studies indicate that the embodiments described herein can be used to store entire organisms in suspended animation, potentially facilitating interstellar travel. Some molecules, cells, and even organisms have been documented to be able to withstand extreme environmental conditions.

[0059] Embodiments described herein provide methods for cryogenically preserving aqueous materials using increased pressure to reduce the freeze / thaw temperature of water and / or aqueous materials. According to embodiments, a pressure generator is used to apply pressure to aqueous materials, biological materials, and the like, across the cryogenic temperature range used for preservation / storage. Initial applications include long-term storage of human organs for transplantation and biobanking. Embodiments provide devices for storing biological materials according to the methods described herein.

[0060] The purpose of the embodiments described herein is to provide a solution to the problem of long-term preservation of biological materials, such as human organs. The solution is to avoid freezing (phase change) and maintain delicate (i.e., non-freezable) materials in a stable liquid state at the lowest achievable temperature. This is achieved by applying pressure to aqueous substances, biological materials, and the like using a pressure generator across the cryogenic temperature range used for preservation / storage. The embodiments described herein induce a molecular / physiological "stasis" state by applying high pressure to reduce the freezing temperature (i.e., melting temperature) of water, biological objects, and other aqueous organic and inorganic materials. "Stasis" in the context of the present invention is more accurately defined as "cryostasis" because of the low temperatures required to induce this state. The embodiments described herein use a combination of pressure and temperature to facilitate long-term storage in cryostasis (e.g., multiple months, multiple years), providing a means for biobanking. The associated pressure can also induce a metastable, supercooled state that may be used for long-term preservation of aqueous materials. The embodiments described herein utilize the physicochemical properties of water and its interactions with pressure and temperature to maintain aqueous materials in a stable liquid state. One embodiment provides freezing-free storage at the lowest temperature and corresponding pressure at which water is in a stable liquid state (see FIG. 1). At pressures that produce the freeze / thaw temperature depression, molecular motion and metabolism are inhibited, resulting in cryostasis.

[0061] The present invention is based, at least in part, on the following hypothesis: Biological and other aqueous materials remain in a usable (functional) state longer the colder they are stored without freezing and thawing (i.e., without undergoing a phase transition) (i.e., the lower the storage temperature, the longer the viable storage duration). Therefore, the following question arises: Can the temperature of a living object be lowered sufficiently to induce cryostasis without freezing? For example, mammalian cells, tissues, organs, and organisms are aqueous, with roughly 300 millimoles of dissolved solutes. Based on colligative properties, these 300 millimoles of solutes result in a freezing point depression of 0.55°C of solution within mammalian tissue. A storage temperature of -0.55°C is not cold enough (i.e., only a few hours, let alone a few days) to adequately extend the usable life of an organ for transplantation. An alternative method is needed to achieve storage cold enough to preserve cells, tissues, organs, and organisms for months or years. The key to this method lies in the relationship between temperature and pressure.

[0062] Embodiments use high pressure (i.e., higher than the surrounding atmospheric pressure) to depress the freezing / melting points of water and aqueous solutions. The freezing point of pure water, and therefore all aqueous and biological materials, can be depressed by approximately 1°C per approximately 9.5 MPa (Daucik, K. et al., The International Association for the Properties of Water and Steam, IAPWSR14-08, 2011). For example, a pressure of approximately 210 MPa depresses the freezing point of water and aqueous solutions to approximately -22°C. Under these environmental conditions, molecular motion is reduced to the point where metabolic function is inhibited, resulting in a state of suspended animation, referred to herein as "cryostasis." As described herein, cells, tissues, organs, and organisms stored under high-pressure / low-temperature conditions for multiple days, weeks, or months maintain their function without showing signs of deterioration, apoptosis, or necrosis (see Table 1). The maximum storage period limits for biological and other aqueous materials under these described environmental conditions have not yet been determined, and there may be no definite time limit.

[0063] Broadly speaking, embodiments described herein result in unfrozen storage of biological and aqueous materials below 0°C by applying pressures greater than ambient pressure. Because the melting / freezing point of water decreases as pressure increases, biological and aqueous materials stored under pressures of approximately 210 MPa and temperatures of approximately −22°C or greater remain stable in a liquid state. FIG. 1 is a phase diagram of water illustrating the relationship between pressure and temperature and the melting curve (solid-liquid boundary line) depicting the pressure / temperature values ​​at which water remains stable in a liquid state. Some embodiments focus on the minimum temperature and corresponding pressure at which water remains stable in a liquid form. Below this minimum temperature and corresponding pressure, water becomes supercooled (supercooled) or forms ice III or ice Ih (see “A” in FIG. 1). Similarly, at pressures above the pressure corresponding to the minimum temperature for stable liquid water, water may become metastable and form ice III or ice Ih. These critical point parameters of pressure and temperature define the lowest temperature conditions that water, biological materials, and aqueous substances can be maintained in a liquid state without the possibility of freezing (phase change).According to embodiments, the pressure applied to biological materials and aqueous materials is increased accordingly as the temperature decreases, in order to prevent the phase change of liquid water and the formation of ice.As described herein, by storing biological materials such as cells, tissues, organelles, organoids, molecules, organs, and / or organisms under environmental conditions of high pressure (higher than atmospheric pressure) and a temperature below the freezing temperature (i.e., melting temperature) of water at atmospheric pressure (on earth), enzyme activity and overall metabolic activity are suppressed.As the temperature decreases and pressure increases, this suppression transitions to cryostasis, i.e., a state of suspended animation in which there is virtually no metabolic activity.

[0064] Thus, one aspect of the present invention relates to storing biological and other aqueous materials in a state of suspended animation, or cryostasis. By halting metabolism (aerobic and anaerobic), apoptosis, and / or necrosis during cryostasis, long-term preservation (i.e., banking) of organic and inorganic aqueous materials is achieved. The lower the storage temperature and the higher the pressure, the greater the depth of stasis.

[0065] Thus, the embodiments differ from prior art approaches that purport to result in the preservation or storage of biological materials using methods in which a reduced pressure is first applied to reduce the temperature, and no further reduced pressure is applied as the temperature is further reduced. Such prior art approaches rely on an observed increase in pressure that occurs as the temperature is further reduced. The observed increase in pressure in such prior art is said to prevent ice formation (phase change), thereby resulting in undamaged storage of the biological material. However, it is suggested herein that the observed increase in pressure can only be manifested by a phase change resulting in ice formation, with its attendant adverse effects on the biological material. In contrast, as discussed above, the embodiments described herein prevent the phase change and ice formation by continually increasing the applied pressure as the temperature is reduced.

[0066] Additionally, embodiments differ from prior art approaches that rely partially or entirely on the phase change of water from liquid to ice to generate high pressure in the storage compartment. Such prior approaches cannot control the pressure inside the storage compartment, resulting in damaging ice formation inside the storage compartment. In contrast, the embodiments described herein use a pressure generator and driving fluid to pressurize the pressure vessel, allowing for tight control of the pressure inside the pressure vessel and applying sufficient pressure to the driving fluid (which may be water) to prevent freezing. Embodiments using water as the driving fluid may be advantageous for use in storing biological materials, such as aquatic organisms (e.g., fresh water, salt water, etc.), in their natural medium.

[0067] Preservation of aqueous materials in a non-frozen state does not have to be limited to the use of pressure described above to depress the freezing (melting) point to approximately −22° C. Three means exist to provide further freezing point depression (FPD). 1) Supercooling: Aqueous materials can be supercooled under pressure, and the metastable liquid state can be maintained at least at -92°C. 2) Colligative Freezing Point Depression: Adding a water-soluble substance to water further depresses the freezing point of the solution to below approximately -22°C at approximately 210 PMa. The additional FDP is equivalent to 1.86°C per mole of colligatively acting solute. 3) Non-collapsitive freezing point depression: Non-collapsitive agents provide additional freezing point depression through ice inhibitors or ice binders, thereby preventing, inhibiting, controlling, and / or separating ice crystal growth. The three methods described above may be used individually or in combination to reduce the storage temperature of unfrozen material to below approximately −22° C. under approximately 210 MPa. In various embodiments, the storage temperature may be from about 0° C. to about −22° C., from about −5° C. to about −22° C., from about −10° C. to about −22° C., or from about −15° C. to about −22° C. Use of these techniques extends the preservation time of materials requiring cryostasis.

[0068] Environmental storage conditions of approximately 210 MPa or thereabouts and approximately −22° C. or thereabouts require a pressure vessel and equipment capable of generating pressure to pressurize and depressurize the pressure vessel. A vessel capable of reliably containing these pressures may be made of steel, stainless steel, titanium, or some other suitable material. The vessel must have a means for accessing and retrieving the stored material and a means for connecting a pressure generator to the vessel. The pressure generator (hydraulic, pneumatic, or otherwise) may be manually operated, and a timer or controller may be used to control the rate of pressurization and depressurization as appropriate. Alternatively, the pressure generator may be automatic and may be mechanically, pneumatically, hydraulically, or other actuated, and electrically, electronically, by computer, by analog mechanical, or other controller. One embodiment includes a hydraulic pressure generator.

[0069] The hydraulic pressure generator may be connected to the pressure vessel via a system of piping, valves, joints, fittings, pressure gauges, etc. that conducts the drive fluid. In such embodiments, a drive fluid reservoir may be used to hold the drive fluid. Examples of drive fluids include, but are not limited to, propylene glycol (PEG), ethylene glycol (EG), oil, petroleum oil, fish oil, mineral oil, vegetable oil, water, seawater, and any combination thereof.

[0070] To reduce or increase the temperature, the pressure vessel may be operatively connected to controlled cooling and heating equipment, systems, etc., containing a heat transfer medium. The heat transfer medium may be a fluid or a solid. For example, a cooling / heating system using a fluid as the heat transfer medium requires a vessel to contain the medium, supplied with the cooler / heater. The heater may be separate from the cooler and have its own temperature sensor and temperature controller, or these may be integrated.

[0071] A temperature controller may be used to control the cooler / heater based on temperature data provided by temperature sensors immersed in the heat transfer medium and / or inserted into the pressure vessel. The temperature controller may be computer software or may be a stand-alone controller, microprocessor, or other type of control. The sensors may be thermocouples, thermistors, RTDs (resistance temperature devices), or any other suitable equipment.

[0072] Cooling / heating systems that use fluids as heat transfer media require a mixing unit or some other device to continuously mix the transfer media. Mixing is important for an efficient and well-controlled heat transfer method, allowing for uniform temperature throughout the fluid enclosure and preventing thermocline. A pressure gauge or other measurement / monitoring device is used to monitor pressure. The pressure gauge can be, but is not limited to, an analog or digital gauge, or a pressure transducer connected to a display, or a data acquisition system (DAQ) attached to a computer that displays and records pressure, a controller, or the like. The temperature inside the pressure vessel and inside or near the fluid (e.g., air) within the enclosure is monitored by a temperature sensor (thermocouple, thermometer, thermistor, RTD, or other suitable device), and data strings may be displayed and / or recorded using a DAQ and computer system or other system. A thermometer or other temperature sensor may be immersed or partially immersed in the fluid within the enclosure to monitor temperature. The pressure vessel remains in the fluid during cooling and warming, as well as during the equilibration period.

[0073] The cooling / heating and pressure of the system may be integrated and controlled by a single controller using temperature and pressure sensors. Alternatively, the temperature system may be controlled during cooling / heating by a single controller using one or more temperature sensors, while the pressure generator operates separately using its own controller and sensor. The cooler / heater and pressure generator may each use their own sensor and controller. In one embodiment, all three components, i.e., heater, cooler, and pressure generator, are integrated into a single control, monitoring, and recording device. The entire high-pressure / low-temperature system control and monitoring device may be automated using a variety of control techniques using a variety of devices and methods.

[0074] In one embodiment (see FIG. 2), a fluid (e.g., air) is used as the heat transfer medium. The apparatus includes a pressure vessel 13 (FIG. 3) capable of reliably containing pressures up to at least 276 MPa, made from steel, stainless steel, titanium, or some other suitable material, with a removable top 21 and a port 27 for connecting a pressure generator 2 to the pressure vessel 13. The fluid-driven pressure generator 2 can be operated manually, optionally using separate timers to control the rates of pressurization and depressurization. Alternatively, the pressure generator can be mechanically, pneumatically, hydraulically, etc., actuated and controlled electrically, electronically, by computer, or by a mechanical analog controller. For example, the pressure generator may be mechanically driven and computer controlled.

[0075] The pressure generator may be connected to the pressure vessel by a system of pipes, valves, joints, fittings, pressure gauges, and hydraulic fluid reservoirs (see Figures 2 and 3). A system of controlled cooling and heating may be used to lower or raise the temperature of the pressure vessel 13. For cooling / heating systems that use a fluid (e.g., air) as the heat transfer medium, an insulated vessel 10 is required to house the cold / heat sink. The compressor and heat rejection unit may be housed in the same vessel outside the cooling / warming equipment, or they may be in separate enclosures and connected to the cooling equipment by insulated piping.

[0076] One embodiment of the mechanical refrigeration system uses a cylindrical reciprocating compressor, which eliminates power surges during startup, and utilizes PID (proportional-integral-derivative) control. The heater may be separate from the evaporator and have its own temperature sensor and temperature controller, or it may be integrated with the evaporator and share the same control unit. The temperature controller, utilizing PID control, controls the cooler / heater based on temperature data provided by a temperature sensor immersed in the heat transfer medium (fluid) or inserted into the pressure vessel. In one embodiment, PID control is used for temperature stability and RTD (resistance temperature device) sensors are used for accuracy and precision.

[0077] In one embodiment, a cooling system using a fluid as a heat transfer medium has an evaporator that is the same height as the linear volume of the pressure vessel's storage area and a mixer to provide a uniform temperature throughout the interior of the storage compartment. In one embodiment, access is from above, using a removable insulated top 8, thus creating a cryowell. The pressure vessel is inside the storage area during cooling and heating, pressurization and depressurization.

[0078] Pressure gauges and pressure transducers may be used to monitor the pressure. In one embodiment, the pressure transducers were connected to a data acquisition system (DAQ) connected to a computer that displayed and recorded the pressure. A thermistor 12 was immersed in the cold well, and a second thermistor 14 was inserted into the pressure vessel 13. Data from these temperature sensors was transferred (via the DAQ as described above) to a computer that displayed and recorded the data.

[0079] Tissue samples or organs may be obtained immediately postmortem, perfused, bagged, and sealed (see Example 2). Examples of perfusion and storage solutions include UW® Solution (Bridge to Life), CoStorSol®, Celsior®, Custodiol® HTK, Perfadex®, MACS® Tissue Storage Solution (Miltenyi Biotec), FW (Froedin-Wolgast), Sack', WMo-II, and Lifeport Liver Transporter Solution. Body heat may be removed by immersing the bagged sample in a solution pre-cooled to subzero temperatures. The tissue and / or organ may then be inserted into a pre-cooled pressure vessel filled with a working fluid, the pressure vessel closed, the air removed, and the contents pressurized and cooled using a pressure generator (see Example 3 and Figure 5). Items may be maintained in cryostasis for a predetermined period of time or until needed. Recovery may be achieved by warming the pressure vessel and then depressurizing (see Example 4 and Figure 6). It is understood that different types and sizes of stored materials (e.g., solutions, cells, organs, organisms, etc.) may require different pressure and temperature change rates, as well as different storage temperatures and pressures, both during initial storage and subsequent recovery. Tables 2 and 3 provide non-limiting examples of pressure and temperature change rates, as well as different storage temperatures and pressures, both during initial storage and subsequent recovery. For example, Figures 5A and 5B are plots showing pressure and temperature curves for storing and recovering biological material (porcine kidney cortex and medulla), respectively. In Figure 5B, "Temperature P" refers to the temperature of the refrigerated storage compartment inside the cooling equipment in which the pressure vessel is located, and "Temperature V" refers to the temperature of the pressure vessel.

[0080] A laboratory prototype was used to verify the effectiveness of this method and identify cooling and warming, pressurization, and decompression rates that would not adversely affect the biological materials. The benchtop instrument uses a PID-controlled cooling system to provide controlled cooling of the vertical walls of an insulated enclosure. The enclosure is open-topped and is covered with insulation during operation. The cooling system and controller are all housed within the same enclosure. Table 1 lists some of the materials stored, including the storage period and post-storage condition used.

[0081] Laboratory benchtop prototype equipment can be easily scaled up to accommodate entire organisms, such as humans, for interplanetary or interstellar space travel. Pressure vessels large enough to accommodate, but not limited to, kidneys, hearts, heart-lungs or lungs, livers, pancreases, or other human or mammalian organs, individually or in various combinations, are heavy, and some additional equipment may be required for organism storage. Overhead winches or cranes and / or forklifts or other heavy lifting means may be required to move vessels capable of maintaining temperatures as low as approximately 22°C and large, stable, walk-in or drive-in coolers.

[0082] The following examples further illustrate the present invention but are not intended to be limiting in any way. [Example]

[0083] Materials: Bagged and sealed renal cortical slices in UW® solution (see Example 1), a lidded pressure vessel (1 inch internal diameter, 6 inch internal well depth, 15 inch external) rated to 276 MPa, a pressure generator manual wheel capable of generating a hydraulic pressure of 210 MPa (available from High Pressure Equipment ("HIP"), Erie, PA, USA), a high-pressure piping system (available from HIP), valves (available from HIP), gauges (available from HIP), a pressure transducer (available from Omega Engineering, Saint-Eustache, Quebec, Canada), a working fluid reservoir, a 1:1 solution of propylene glycol and water referred to herein as the working fluid, a temperature ramping ultra-high stability cryogenic cooling POD 110 VAC (referred to herein as the "working POD") (the cooling device was supplied by Engel, Jupiter, FL, USA). The work POD was retrofitted with an Auber proportional-integral-derivative (PID) controller (available from Omega Engineering, Saint-Eustache, Quebec, Canada) and a resistance temperature device (RTD) sensor, a thermistor temperature sensor with a data acquisition and recording module (DAQ) (available from Vernier Inc., Beaverton, Oregon, USA), a 2-inch closed-cell foam insulation sheet sized to cover the work POD, a computer, an analog timer (available from GraLab Corporation, Centerville, Ohio, USA), a 30 cm Halsted forceps, a lid closure rod, a 5 / 8 inch open-end wrench, and an isothermal ultra-high stability cryogenic cooling POD 110 VAC (referred to herein as the "storage POD") with a PID controller, capable of operation down to -25°C, and a cradle for a pressure vessel. The temperature ramps of the working PODs were programmed using the instruction manual "SYL-2352P Ramp and Soak PID Temperature Controller, version 1.4 (Feb 2017)" published by Auber Instruments, Alphabetta, Georgia, USA.Pig kidney, 300 mM saline (NaCl + HO), 0.002 inch (mil) wall thickness x 1 L volume plastic bag, 3M NaCl in 6 L HO, 3.5 L plastic containers with lids, ultra-high stability cooling 12 VDC POD set at -5°C, ultra-high stability cooling 110 VAC POD set at -2°C, UW® Solution, 60 mL syringe with 20 gauge biopsy needle, lancet, scalpel, Tome Blade, scale (0.1 gram), digital thermometer (0.1°C resolution), 6 x 6 cm mil low density polyethylene plastic bag (available from International Plastics, Greenville, SC, USA), heat sealer. [Example 1]

[0084] Device for preserving biological material Referring to FIG. 2, one embodiment of a pressure-temperature apparatus is shown, including multiple components operatively connected to one another by pressure piping. Starting on the left side, a drive fluid reservoir 4 containing drive fluid is connected to a drive fluid isolation valve 3, which can be in an open position, allowing drive fluid to enter the piping, or in a closed position, preventing drive fluid flow. At this point in the line, a T-junction joins a pipe leading from a pressure generator 2, which includes an actuator, in this case, a handwheel 1. The pressure generator 2 is operatively connected so that turning the handwheel 1 in the appropriate direction can apply or remove pressure from the pipe. In other embodiments, the actuator may include a motor, servo, or other device capable of receiving a control signal (e.g., from a controller such as a microprocessor or computer) and adjusting the pressure provided by the pressure generator in accordance with the control signal, thereby allowing for partial or full automatic pressure control. Following the T-junction is a line containing a pressure gauge 5 (e.g., digital or analog) that displays a pressure reading. In embodiments with partially or fully automatic pressure control, the pressure gauge includes a pressure transducer that provides a pressure signal to the controller. The next component is a pressure generator isolation valve 7, which allows the upstream portion of the line generating the pressure to be isolated from the downstream portion at this point. Some embodiments may include a pressure transducer 6 that senses the pressure in the line and converts the pressure into a pressure signal that may be sent to a controller, microprocessor, computer, or the like. The line then enters a cooling section 10 having an insulating cover 8. The cooling section 10 may be operably connected to an appropriate controller to provide fully or partially automatic control of the temperature within the section. The line then leads to a pressure vessel isolation valve 9, which allows the pressure vessel 13 to be isolated from the pipeline. The line then connects to the pressure vessel 13, which contains the material to be stored and the drive fluid.Other components of cooling section 10 may include a cooler / heater 11 (optionally having an interface including, for example, a digital-to-analog converter (DAC) so that operation of heater / cooler 11 may be partially or fully automated using a controller), a temperature sensor 12 for cooling control, a temperature sensor 14 for monitoring the interior of the pressure vessel, and a circulation fan or agitator 15. The temperature sensor 12 for cooling control and the temperature sensor 14 for monitoring the interior of the pressure vessel may be implemented, for example, with a thermistor, and generate a corresponding temperature signal. The temperature signal may be sent to a controller, microprocessor, computer, etc. for monitoring and / or recording the temperature and for use in partially or fully automating the apparatus, as appropriate.

[0085] Thus, one embodiment includes a controller operably connected to one or more of the temperature sensor, cooling compartment, pressure transducer (or pressure gauge), pressure generator actuator, and heater / cooler, which may partially or fully automate the operation of the device. For example, the controller may control the cooling / heating and pressure of the system device. Alternatively, the temperature system may be controlled during cooling / heating by a single controller using one or more temperature sensors, while the pressure generator operates separately using its own controller and sensor. In one embodiment, the heating, cooling, and pressure generator are integrated into a single controller, which monitors, records, and regulates the pressure and temperature.

[0086] Referring to FIG. 3, an expanded view of one embodiment of pressure vessel 13 is shown, including pressure vessel top 21, retaining ring 22, O-ring seal 23, pressure vessel body 24, and overflow channel and thermistor well 25.

[0087] Figures 4A-4E show the assembly of pressure vessel 13 and pressure vessel top 21, sequentially, including the overflow of the driving fluid in overflow channel and thermistor well 25. When fully assembled (Figure 4E), the overflow channel and thermistor well 25 are sealed from the sample well 26 inside pressure vessel 13 and house the thermistor 14 for measuring the housing temperature. The thermistor 14 is positioned in the overflow channel and thermistor well 25 near the sample well containing the biological material and driving fluid. Placing the thermistor 14 closer to the sample well would require a hole near or within the pressure vessel pressurization cavity. Such a hole could cause the pressure vessel to fail upon pressurization. Due to the poor thermal conductivity of stainless steel, the distance separating the thermistor from the sample well can cause the actual temperature of the biological material to lag behind the temperature measured by the thermistor; however, this lag proves to be acceptable due to the slow cooling rate.

[0088] 5A and 5B are plots showing exemplary pressure and temperature curves for storing and recovering biological material (in this case, porcine kidney cortex and medulla), respectively. In FIG. 5B, "Temperature P" refers to the temperature inside the cooling compartment in which the pressure vessel is located, and "Temperature V" refers to the temperature of the pressure vessel as acquired by thermistors located in the pressure vessel's overflow channel and thermistor well. Of course, different types and sizes of stored material (e.g., solutions, cells, organs, organisms, etc.) may require different rates of pressure and temperature change, both during initial storage and subsequent recovery, and may also require different storage temperatures and pressures. [Example 2]

[0089] Preparation of archival porcine renal cortical biopsy sections Obtaining and preparing porcine kidneys Pig kidneys were obtained from Canadian Food Inspection Agency (CFIA)-certified slaughterhouses as soon as possible after death. Inspected kidneys were dissected by CFIA inspectors. Upon receipt, kidneys were excised, rinsed with 300 mM saline, perfused with UW® solution, rinsed with UW® solution, and placed in a 1 L plastic bag and sealed. The kidneys and UW® solution bag were immersed in 3 M saline at -5°C (plunge solution). The plunge solution was contained in a 3.5 L plastic tub within a 12 VDC POD. Each tub held up to three 150 gram kidneys, cooled to -1°C (thermal mass limit of the refrigerant volume and temperature). The lid of the tub was fitted over the end of the plastic bag and locked. The kidneys were placed in the plunge solution at -5°C for 45 minutes to 1 hour. One 6x6cm, 2mil plastic bag was labeled with the specimen (kidney) number. A 60cc syringe was fitted with a 20 gauge biopsy needle, filled with 50mL of -1°C UW® solution, and kept in an incubator until needed.

[0090] Obtaining kidney sections The kidneys were removed from the plunge solution, and biopsy sections were individually prepared. The bagged kidneys were removed from the plunge solution, and the kidneys were removed from the pouches. The internal kidney temperature was determined and recorded using a digital thermometer probe. Any residual fat or membranes were removed, and the kidney weight was determined and recorded. A longitudinal incision was made using a scalpel or tome blade, and cortical sections were removed. Cortical sections were 2–3 cm long and 1–1.5 cm wide. Cortical sections should not contain the medulla and should contain only one cut surface.

[0091] Extraction of cortical biopsy sections for archiving 7-10 mL of -1°C UW® solution was poured into a 6 x 6 cm, 2-mil plastic bag. The cortical sections were placed in the bag so that the cut surface was in contact with the wall of the bag, which was the boundary layer of UW® solution. Additional -1°C UW® solution was poured into the bag as needed to cover the cortical sections. The bag was closed and compressed, removing all air. The bag was sealed with a heat sealer, and excess plastic was trimmed off. The bag was placed in a -2°C cooling pod until all cortical sections for storage were prepared. [Example 3]

[0092] Storage process for storing biological materials at -18°C and 193 MPa preparation One day prior to storing the biological samples, the following steps were performed using an apparatus based on that shown in FIG. 2 and described in Example 1: The storage POD was set and maintained at -18°C. An empty pressure vessel 13 (see FIG. 2) was placed into the working POD 10. The temperature controller of the working POD was set to -2°C, and the internal temperature of the pressure vessel was ramped to -2°C over 6 hours. Once at -2°C, the pressure vessel 13 was allowed to stabilize for 8 hours. The Auber PID was programmed. The pressure vessel 13 was connected to the piping system. Two layers of 2-inch closed-cell foam insulation were placed on top of the working POD. A first thermistor 14 was inserted into the overflow channel / thermistor well 25 (see FIG. 3) of the pressure vessel 13. A second thermistor 12 was positioned inside the working POD next to the pressure vessel 13. The computer was turned on and connected to a data acquisition system (DAQ) and Vernier LabView software was configured to record readings every 10 seconds for 5000 minutes and recording was initiated.

[0093] The sample was placed in the working POD and ramped to -18°C and 193 MPa and stabilized. The following steps were performed on the day of sample collection. Pig kidney cortical biopsy samples were prepared and kept at -2°C as described in Example 2. Two closed-cell foam insulation sheets were removed from the top of the working POD. The first thermistor 14 was removed from 25, and the pressure vessel 13 was isolated from the piping system. The pressure vessel 13 was removed from the working POD and placed on its stand. The pressure vessel lid 21 was loosened and removed. Using Halsted forceps (0 cm), two bagged and sealed samples from the first set were placed side-by-side into the pressure vessel sample wells 26, and then two bagged and sealed samples from the second set were placed on top of the first set. Care was taken to leave enough space for the lid 21 to fit onto the pressure vessel 13 without touching the samples. The driving fluid was placed into the pressure vessel 13, and the lid 21 was closed by screwing it onto the pressure vessel 13 with the pressure vessel isolation valve 9 open. Excess driving fluid was then drained through the overflow channel / thermistor well 25 on the side of the pressure vessel 13 (see Figures 4A-4E). The overflow channel and thermistor well 25 were monitored until the driving fluid flowed smoothly and without bubbles. Once the lid 21 blocked the overflow channel, the driving fluid was drained from the top of the pressure vessel isolation valve 9 until no bubbles were observed. Using a strap wrench and lid closure rod, the lid 21 was tightened until it fit snugly onto the pressure vessel 13. The pressure vessel 13 was moved to the work POD and connected to the piping system. The fittings connecting the pressure vessel to the piping system were finger-tight. The driving fluid isolation valve 3, located upstream of the pressure generator 2, was opened. The pressure generator isolation valve 7, located downstream of the pressure generator 2, was also opened. The fitting collars on the pipe fittings connecting the piping system to the pressure vessel 13 were checked and tightened. The pipe fitting was inserted into the pressure vessel 13 and tightened by turning the threads one full turn. The fitting connecting the pressure vessel isolation valve 9 to the piping system (40 ft / lbs) was tightened until snug. The drive fluid reservoir isolation valve 3 was closed and a check was performed to ensure that the pressure generator isolation valve 7 and pressure vessel isolation valve 9 were open one full turn. The first thermistor 14 was re-inserted into the overflow channel and thermistor well 25.

[0094] The pressure inside the pressure vessel was programmed to increase and the temperature to decrease gradually (see, for example, Table 2). The controller of the working POD was programmed to ramp from -2°C to -18°C at a constant rate. Due to the heat transfer coefficient through the pressure vessel material (e.g., stainless steel), the tissue cools much more slowly than the working POD. A countdown Gra-Lab timer was set for 20 minutes and used to control the pressurization rate. Pressure Generator 1 was used to pressurize Pressure Vessel 13 to 30,000 psig (210 MPa) at a rate of 1,000 psig / min (6.9 MPa) with 200 psig (1.4 MPa) increases every 12 seconds. The system was allowed to ramp and soak for 12 hours. A loss of 2,000 psig (13.8 MPa) due to cooling was noted. The temperature and pressure were allowed to stabilize for 12 hours. At this time, the pressure was adjusted to 28,000 psig (193 MPa) and the system was allowed to stabilize for an additional 6 hours.

[0095] Transfer from working POD to storage POD, stored at -18°C and 193MPa Once the pressure vessel stabilized at -18°C and 193 MPa in the work POD, it was ready to be moved for storage in the storage POD, which was isothermal at -18°C. Pressure vessel isolation valve 9 was closed. Drive fluid reservoir isolation valve 3 was opened, and the piping system and pressure generator pressure was reduced to ambient pressure. The pipe fitting was removed from pressure vessel isolation valve 9 using a 5 / 8 inch open-end wrench. Drive fluid reservoir isolation valve 3 was closed. Temperature and pressure recording was stopped and the data was saved to a computer. Temperature sensor 14 was removed from pressure vessel 13. The top of the storage POD was opened. Pressure vessel 13 was lifted out of the work POD and placed in a cradle inside the storage POD, which was isothermal at -18°C. The top of the storage POD was closed. The samples in the pressure vessel were allowed to soak at -18°C for 10 days (Note: storage period is variable). [Example 4]

[0096] Recovery of biological material from storage POD at -18°C and 193 MPa to ambient temperature and pressure The samples were placed in a storage POD at -18°C and 193 MPa. When recovery of a stored sample was desired, the following steps were taken.

[0097] The following steps were performed 6 hours prior to recovery: The working POD was started and the controls were set to a temperature of -18°C for the working POD. Both layers of 2-inch thick closed-cell foam insulation were ensured to be placed on top of the working POD. The computer was started and a program (e.g., Graphical Analysis™ 4 available from Vernier, Inc., Beaverton, Oregon, USA) was launched to record temperature and pressure (e.g., sampling once every 10 seconds).

[0098] Six hours after the above steps, the following recovery protocol was implemented: The working POD was confirmed to have been isothermal at -18°C for more than four hours from the temperature data records. The pressure generator isolation valve 7 was opened. The drive fluid reservoir isolation valve 3 was closed. The cover of the storage POD was opened, and the pressure vessel assembly was removed and moved to the working POD. The base of the pressure vessel was placed in its stand at the bottom of the working POD. The piping system was connected to the pressure vessel isolation valve 9 and the fittings were rotated one full turn. The drive fluid reservoir isolation valve 3 was opened. The pressure vessel isolation valve vent hole was monitored until no air bubbles were present for 15 seconds. The fittings connecting the pressure vessel isolation valve 9 to the piping system were tightened. The drive fluid reservoir isolation valve 3 was closed. The piping system was pressurized to 193 MPa (28,000 psig) using pressure generator 1. The heat of compression was allowed to dissipate for 10 minutes. The pressure was adjusted to 193 MPa (28,000 psig). Pressure vessel isolation valve 9 was opened. A drop in system pressure below 171.1 MPa (24908 psig) was avoided as this could result in freezing and compromise specimen viability.

[0099] The working POD was ramped from -18°C to -2°C at a rate of 0.05°C / min (3.0°C / hr, 16°C ΔT total 5.5 hours). During warm-up, the internal pressure was observed to rise to 209 MPa (approximately 30,000 psig). The working POD was allowed to soak for a minimum of 1 hour. Using pressure generator manual wheel 1, the pressure vessel was depressurized at 1,000 psig / min (6.9 MPa) in increments of 200 psig (1.4 MPa) every 12 seconds for 30 minutes until ambient pressure was reached.

[0100] The pressure vessel 13 was disconnected from the piping system by loosening the fitting to the pressure vessel isolation valve 9. The -2°C pressure vessel 13 was opened by loosening its top 21 and removing the top from the vessel. Each of the four samples was removed from inside the vessel using a 30 cm hemostat.

[0101] Samples were stained using DAPI / PI (see Table 1 for full names) and analyzed for viability. The results are shown in Table 1. Unused portions of the stored kidney biopsy sections were frozen for subsequent caspase / adenosine triphosphate (ATP) analysis.

[0102] [Table 1-1] [Table 1-2]

[0103] [Table 2-1] [Table 2-2] [Table 2-3]

[0104] [Table 3-1] [Table 3-2] [Table 3-3]

[0105] Incorporation by Reference The contents of all cited documents are incorporated herein by reference in their entirety.

[0106] equivalent Although reference is made herein to particular embodiments, those skilled in the art will understand that other embodiments can be made utilizing the principles of the present invention which are within the spirit and scope of the present invention.

Claims

1. 1. A method for storing biological material, comprising: placing the biological material in a pressure vessel; filling the pressure vessel with a driving fluid; evacuating the pressure vessel and sealing the pressure vessel; increasing the pressure on the driving liquid using a pressure generator to reduce the temperature inside the pressure vessel to below 0°C; Including, applying a selected pressure to the driving fluid at a selected temperature using the pressure generator, thereby maintaining the driving fluid in a stable liquid state within the pressure vessel; The method wherein a selected pressure is applied to the driving fluid to prevent freezing of the biological material at storage temperatures below 0°C.

2. placing the biological material in a sample bag together with a preservation solution; removing air from the sample bag; sealing the sample bag; further comprising The method of claim 1 , wherein the storage solution and the driving solution are maintained in a stable liquid state.

3. 3. The method of claim 1 or 2, wherein the reducing the temperature and increasing the pressure comprises increasing the pressure from ambient conditions to about 30,000 psig (210 MPa) in 200 psig (1.4 MPa) increments at 1,000 psig / min (6.9 MPa) and reducing the temperature from ambient conditions to about -22°C.

4. 4. The method of any one of claims 1 to 3, wherein the biological material comprises one or more of organic molecules, molecular complexes, nucleic acids, sugars, amino acids, peptides, proteins, enzymes, organelles, organoids, cells, tissues, organs, organisms, and aqueous solutions.

5. 5. The method of any one of claims 2 to 4, wherein the preservation solution comprises water and one or more of biological material, soluble molecules, organic and / or inorganic compounds, material in aqueous suspension, aqueous solution, aqueous mixture, aqueous colloid, aqueous material, and bio-derived material.

6. The method of any one of claims 1 to 5, wherein the biological material comprises a cell, tissue, organ, or whole organism.

7. The method of any one of claims 1 to 6, wherein the storage temperature is about -22°C.

8. 8. The method of any one of claims 1 to 7, wherein at the storage temperature, the applied pressure is about 30,000 psi (210 MPa).

9. 9. The method of any one of claims 1 to 8, wherein the storage temperature and applied pressure maintain the cells in a metastable supercooled liquid state, thereby preventing freezing and cell damage.

10. The method of any one of claims 2 to 9, wherein the preservation solution comprises a solute.

11. 11. The method of claim 10, wherein the solute comprises one or more of antifreeze proteins, ice-binding proteins, antifreeze sugars, ice-binding sugars, ice-binding peptides, and other non-collective agents.

12. 12. The method of claim 10 or 11, wherein the solute prevents, inhibits, controls, or disrupts ice crystal growth and / or prevents ice nucleation.

13. The method of any one of claims 1 to 12, wherein the driving fluid comprises propylene glycol or ethylene glycol, oil, petroleum oil, fish oil, mineral oil, vegetable oil, water, seawater, and any combination thereof.

14. 14. The method of any one of claims 1 to 13, wherein the selected storage temperature is from about -5°C to about -22°C.

15. 1. A device for storing biological material, comprising: a reservoir for containing a driving fluid; a pressure vessel having an internal well configured to receive the biological material, the pressure vessel operably connected to the reservoir for receiving a drive fluid from the reservoir; a pressure generator operatively connected to the pressure vessel and the reservoir, the pressure generator applying pressure to the drive fluid; a pressure transducer indicating the pressure of the drive fluid within the pressure vessel; a temperature sensor that detects the temperature of the pressure vessel; a cooling device configured to provide a controlled pressure vessel internal temperature of less than about 0°C; and Equipped with The apparatus, wherein the pressure generator applies a selected pressure to the driving fluid at a selected pressure vessel temperature below about 0° C., such that the driving fluid within the pressure vessel is maintained in a stable liquid state.

16. 16. The apparatus of claim 15, further comprising a data acquisition system (DAQ) that acquires data from one or more of the pressure transducer, the temperature sensor, the pressure generator, and the cooling equipment.

17. a controller operably connected to one or more of the pressure transducer, the temperature sensor, the pressure generator, and the cooling equipment; 17. The apparatus of claim 15 or 16, wherein the controller monitors and maintains at least one of a selected internal pressure vessel temperature and a selected pressure applied to the drive fluid within the pressure vessel.

18. The apparatus of any one of claims 15 to 17, further comprising a pressure gauge.

19. 18. The apparatus of claim 17, wherein the pressure generator is automated and is mechanically, electrically, pneumatically, or hydraulically driven by the controller.

20. The apparatus of any one of claims 15 to 19, wherein the cooling device further comprises a heater.

21. 21. The apparatus of claim 20, wherein the heater comprises a temperature sensor and a temperature controller.

22. 22. Apparatus according to any one of claims 15 to 21, wherein the cooling device comprises a proportional-integral-derivative (PID) control.

23. The apparatus of any one of claims 15 to 22, further comprising an evaporator.

24. further comprising at least one valve that, when closed, allows isolation and removal of the pressure vessel from the apparatus; 24. An apparatus according to any one of claims 15 to 23, wherein the pressure vessel maintains the applied pressure of the driving liquid when removed from the apparatus.

25. An apparatus according to any one of claims 15 to 24, wherein the pressure vessel is made from a material selected from steel, stainless steel and titanium.

26. 26. The apparatus of any one of claims 15 to 25, wherein the pressure vessel is configured to withstand an internal pressure of at least about 30,000 psig (210 MPa).

27. 1. A pressure vessel for storing biological material, comprising: a housing having a cavity including a first portion and a sample well for receiving the biological material and a drive fluid; a housing, the first portion of the housing including an overflow channel opening to an exterior of the housing; a lid including a first portion configured to engage the first portion of the housing; wherein the position of the lid within the housing is adjustable from a first position to a closed position; the lid includes a second portion configured to partially fit within the sample well of the housing; the first portion of the lid includes a port configured to interconnect with an external device; the lid includes a drive fluid channel configured to direct the drive fluid through the lid between the port and the sample well; adjusting the lid to the closed position causes excess drive solution to drain from the sample well through the port and the overflow channel and the second portion of the lid to seal the sample well; A pressure vessel configured to maintain an internal pressure of at least about 30,000 psi (210 MPa) of driving fluid within the sample well.

28. 28. The pressure vessel of claim 27, wherein pressure is applied to the drive fluid in the sample well through the port by the external device.

29. further comprising at least one valve disposed between the port and the external device; 30. The pressure vessel of claim 28, wherein the at least one valve, when closed, isolates the pressure vessel from the external equipment and maintains internal pressure in the sample well.

30. A pressure vessel according to any one of claims 27 to 29, wherein the overflow channel is configured to receive a temperature sensor.

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