Method and apparatus for reducing the probability of ice nucleation during storage of biological substances in an isovolume system

Abstract

A method for reducing the probability of ice nucleation during the storage of a biological material in an isochoric system by placing the biological material in a flexible impermeable inner container, adding an inner solution having a melting point higher than the desired storage temperature, removing bulk gas from the inner container and sealing the inner container, placing the inner container in a rigid non-insulating outer container, filling the space between the inner container and the outer container with an outer solution, removing bulk gas from the outer container and sealing the outer container, cooling the system to the desired storage temperature, maintaining the desired storage temperature over the desired storage period, heating the system to a temperature higher than the desired storage temperature, opening the outer and inner containers, and removing the biological material. The outer solution has a melting point lower than the equilibrium melting points of the biological material and the inner solution.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Patent Application No. 63 / 351,825, filed on June 14, 2022, the content of which is incorporated herein by reference in its entirety, pursuant to 35 U.S.C.§119(e).

[0002] 1. Field of the Invention The present invention generally relates to methods and systems for preserving biological materials, and more particularly, to methods and devices for preserving unfrozen biological materials at temperatures below 0 degrees Celsius by reducing the probability of ice nucleation in isovolume supercooling preservation or isovolume vitrification preservation.

Background Art

[0003] 2. Description of the Related Art The preservation of biological materials such as molecules, cells, complex organs or organisms, tissues, or foods is essential for current medical and research applications, as well as the food and pharmaceutical industries (1 - 3). The life process is a temperature - dependent chemical reaction, and the storage time of biological materials can be extended by storing the materials at increasingly lower temperatures. Conventional near - normal body temperature preservation for a wide range of biological materials is carried out at 4°C or near it. Preservation at even lower temperatures may further extend the storage period, but this further extension is often hindered by the formation of ice at temperatures below 0 degrees Celsius, which results in chemical and mechanical effects that have been found to be harmful to biological materials.

[0004] The present invention is designed to reduce the probability of ice nucleation during two forms of sub - zero preservation of biological materials, namely isovolume vitrification and isovolume supercooling. These two techniques are more fully described below.

[0005] A. Vitrification Cryopreservation of biological materials by vitrification has been known since the early 20th century (4). The basic principle of vitrification-based preservation is to bring biological materials to extremely low temperatures without ice formation, as a result of which the water in the biological materials becomes a glass, that is, a liquid with a very high viscosity that makes ice formation impossible on a long time scale. The success of the vitrification process depends on first avoiding ice nucleation during the process of cooling the biological material below its glass transition temperature or to a temperature at which its viscosity reaches above 10 13 poise, and reheating it again. The probability of ice nucleation in any system depends on the chemical or solute concentration of the system (the higher the concentration, the less likely nucleation is to occur), the viscosity of the system (the higher the viscosity, the less likely nucleation is to occur), the volume of the system (the higher the volume, the more likely nucleation is to occur), and the number and effectiveness of heterogeneous nucleation sites within the system (the more heterogeneous nucleation sites, the more likely nucleation is to occur). The vitrification process is also an essentially metastable thermodynamic process, which means that ice nucleation is not thermodynamically impossible in the vitrified state, but rather is very unlikely to occur. Considering this metastability, new techniques have been developed for the preservation of biological materials by vitrification in order to reduce the probability of ice nucleation during the vitrification process.

[0006] The basic principle of the preservation of biological materials by vitrification is described in (5)(6). The success of embryo vitrification was reported in (7). Attempts to vitrify larger volumes of tissue have also been reported (8). The disadvantages of biological material preservation by vitrification include the technical difficulty of introducing into biological materials the high concentrations of chemical additives usually required to avoid ice nucleation, the biological toxicity of these additives, and the technical difficulty of removing these additives after preservation. At the concentrations required for unconditional success in vitrification, that is, vitrification that does not depend on the cooling rate or other processing parameters, the chemical additives are severely toxic and perfusion of large biological materials can be difficult. Currently, the preservation of single cells by vitrification has become routine, but the preservation of large-volume organs has not yet been achieved.

[0007] Several patents relate to the use of vitrification for the preservation of biological materials. One example is U.S. Patent No. 4,559,298 (Fahy, 1985), which provides "a method for successfully cryopreserving biological materials including whole organs, organ sections, tissues and cells in a non-frozen (vitrified) state, the method comprising cooling the biological material to be preserved, in the presence of a non-toxic vitrifiable protective solution, under pressure, to at least its glass transition temperature, to vitrify the solution without substantial nucleation or ice crystal growth and without significant damage to the biological material. This invention also provides non-toxic protective glass solid solutions useful for the cryopreservation of biological materials." Attempts to improve vitrification have mainly focused on developing new compositions of solutions that promote vitrification at lower concentrations and lower toxicity when introduced into biological materials.

[0008] Recently, a new technique called "isochoric vitrification" has been introduced. This technique appears to promote vitrification at lower concentrations and / or lower cooling and warming rates. In isochoric vitrification, the biological material and the surrounding solution in osmotic equilibrium with the biological material are confined within a rigid chamber in the absence of a large amount of air. In U.S. Patent Application Publication No. 20200178518 (Rubinsky et al.), the inventors explain that (i) it is possible to determine whether a given solution undergoes vitrification by monitoring the temperature and pressure inside the chamber, and (ii) this monitoring can be used to ensure the success of vitrification of the biological material within the chamber.

[0009] B. Supercooling Supercooling is another method of cryopreservation aimed at avoiding ice formation and is used to store biological materials at temperatures above their glass transition temperature. The term "supercooling" broadly describes the process by which an aqueous solution can exist in a metastable liquid state at a temperature lower than the thermodynamic melting temperature of the solution. Similar to vitrification, the metastability of supercooling means that the probability of ice nucleation always exists to some extent, which can typically be avoided on a time scale of days to months at conventional temperatures in the range of 0 °C to -20 °C. Ice nucleation in supercooling systems is affected by several factors. The general likelihood of nucleation is affected by the same factors that influence vitrification (system chemistry, system viscosity, system volume, heterogeneous nucleation sites). Furthermore, ice nucleation can be directly initiated in supercooling systems by mechanical or vibrational stimuli, ultrasonic stimuli, instability at the fluid-fluid interface, heterogeneous interactions with solid surfaces or gas interfaces, and cavitation of bubbles within the liquid. Nevertheless, the preservation of biological materials by supercooling has been reported and used successfully (9).

[0010] Attempts to reduce the probability of ice formation in supercooling systems have led to the development of various methods. Since the probability of nucleation is a direct function of the volume of water within the system, one method aims to reduce the volume of water within the cell (10). Another method aims to use an electromagnetic field to reduce the probability of nucleation (11). For this purpose, antifreeze proteins have also been used (12), (13), (14 - 16).

[0011] Another method for supercooling involves removing the interface between the liquid storage solution and air using immiscible liquid phases. The air-solution interface of a solution containing biological material is covered with a hydrocarbon oil such as mineral oil, olive oil or paraffin oil, or an alcohol and an alkane, all of which reduce the probability of heterogeneous (or surface-based) ice formation at the air / solution interface (17). A further method for reducing the probability of ice formation in supercooled biological materials involves confining the material and any accompanying storage solution within a rigid, airtight isochoric chamber. The advantages of isochoric supercooling storage extend to applications involving both heterogeneous and homogeneous (volume-based) ice nucleation (18)(19).

[0012] Several patents and patent applications aim to increase the stability of water in the supercooled metastable state (i.e., reduce the probability of ice formation) by removing unfavourable surfaces or interfaces in contact with biological materials or accompanying storage solutions, thereby reducing heterogeneous ice nucleation. For example, as previously described, Usta et al. have developed a method of sealing the free surface of supercooled water with an immiscible liquid (such as an oil), which they claim reduces the probability of nucleation by removing air as a heterogeneous nucleation site. International Publication No. WO 2021 / 158203. Similarly, Aizenberg et al. have developed various porous surface coatings impregnated with hydrophobic liquids (typically perfluorinated materials) to reduce heterogeneous ice nucleation on container surfaces. U.S. Patent No. 9,932,484 (2018). A method of enhancing supercooling by the use of a magnetic or electric field is reported in Jun et al.'s U.S. Patent No. 10,111,452 (2018).

[0013] The use of an isochoric (constant volume) system to reduce the probability of homogeneous ice nucleation has been reported in U.S. Patent Application Publication No. 20070042337 (Rubinsky et al.). The use of a constant volume (isochoric) system to reduce the probability of heterogeneous ice nucleation has been reported in International Patent Application No. PCT / US21 / 12863. Detailed information regarding isochoric preservation can be found in the 2006 Ph.D. thesis of Pedro Alejandro Perez, titled "Thermodynamics and Heat Transfer analysis for isochoric cyropreservation" (20), from the University of California, Berkeley.

[0014] C. Objectives of the Invention The present invention is directed to a method and apparatus for reducing the probability of ice nucleation during isochoric preservation. The present invention relates to the preservation of biological materials by isochoric vitrification and isochoric supercooling. More specifically, the present invention provides a method and apparatus for reducing the probability of ice nucleation within a biological material by preserving the biological material in a vitrified or partially vitrified state at a temperature lower than the glass transition temperature of the biological material and the solution in which it is held. Further, the present invention provides a method and apparatus for reducing the probability of ice nucleation of a biological material in an isochoric system by preserving it in a supercooled state at a temperature lower than the equilibrium melting point of the biological material and the solution in which it is held. In either case, the present invention reduces the probability of ice nucleation by (1) reducing the liquid volume within an isochoric system that is susceptible to the effects of ice nucleation, and (2) ensuring that the reduced volume contacts only a heterogeneous material or surface that is less likely to stimulate heterogeneous nucleation to the same extent or more than the walls of the isochoric chamber itself.

Summary of the Invention

[0015] A method for reducing the probability of ice nucleation during the storage of biological substances in an isovolume system, the method comprising: placing the biological substance in a flexible and impermeable inner container; adding an inner solution having a melting point higher than the desired storage temperature to the inner container; removing bulk gas from the inner container; sealing the inner container; placing the inner container in a rigid and non-insulating outer container to form a space between the outer surface of the inner container and the inner surface of the outer container; filling the space between the outer surface of the inner container and the inner surface of the outer container with an outer solution, wherein the biological substance and the inner solution each have an equilibrium melting point, and the outer solution has a melting point lower than the equilibrium melting point of the biological substance and lower than the equilibrium melting point of the inner solution; removing bulk gas from the outer container; sealing the outer container; cooling the inner container, the outer container, the biological substance, the inner solution, and the outer solution to the desired storage temperature; maintaining the inner container, the outer container, the biological substance, the inner solution, and the outer solution at the desired storage temperature for a desired storage period; heating the inner container, the outer container, the biological substance, the inner solution, and the outer solution to a temperature higher than the desired storage temperature; opening the outer container and the inner container; and removing the biological substance from the inner container.

[0016] In an alternative embodiment, the present invention is a method for reducing the probability of ice nucleation during the storage of biological materials in an isovolume system, the method comprising the steps of: disposing the biological material in a flexible and impermeable inner container; removing bulk gas from the inner container; sealing the inner container; disposing the inner container within a rigid and non-insulating outer container to form a space between the outer surface of the inner container and the inner surface of the outer container; filling the space between the outer surface of the inner container and the inner surface of the outer container with an outer solution, wherein the biological material has a melting point and the outer solution has a melting point lower than the melting point of the biological material; removing bulk gas from the outer container; sealing the outer container; cooling the inner container, the outer container, the biological material, and the outer solution to a desired storage temperature; maintaining the inner container, the outer container, the biological material, and the outer solution at the desired storage temperature for a desired storage period; heating the inner container, the outer container, the biological material, and the outer solution to a temperature higher than the desired storage temperature; opening the outer container and the inner container; and removing the biological material from the inner container.

[0017] In a preferred embodiment, the desired storage temperature is 0 degrees Celsius or lower. In another preferred embodiment, the desired storage temperature is lower than the equilibrium melting point of the biological material and lower than the equilibrium melting point of the inner solution, and the desired storage temperature is higher than the melting point of the outer solution. In yet another preferred embodiment, the desired storage temperature is lower than the melting point of the biological material and the desired storage temperature is higher than the melting point of the outer solution.

[0018] In a preferred embodiment, the biological material and the inner solution each have a glass transition temperature, the outer solution has a glass transition temperature, the desired storage temperature is lower than the glass transition temperature of the biological material and lower than the glass transition temperature of the inner solution, the desired storage temperature is lower than the glass transition temperature of the outer solution, the glass transition temperature of the outer solution is higher than the glass transition temperature of the biological material and higher than the glass transition temperature of the inner solution. In an alternative embodiment, the biological material has a glass transition temperature, the outer solution has a glass transition temperature, the desired storage temperature is less than the glass transition temperature of the biological material, the desired storage temperature is lower than the glass transition temperature of the outer solution, and the glass transition temperature of the outer solution is higher than the glass transition temperature of the biological material.

[0019] The inner container is preferably composed of a hydrophobic polymer material. In one embodiment, the inner solution is composed of an aqueous solution containing an organic molecule at a first concentration. In another embodiment, the inner solution is composed of an aqueous solution containing a chemical cryoprotectant at a first concentration. Preferably, the outer solution is composed of an aqueous solution containing an organic molecule at a second concentration, and the second concentration of the organic molecule in the outer solution is higher than the first concentration of the organic molecule in the inner solution. Preferably, the outer solution is composed of an aqueous solution containing a chemical cryoprotectant at a second concentration, and the second concentration of the chemical cryoprotectant in the outer solution is higher than the first concentration of the organic molecule in the inner solution.

[0020] The method optionally includes a further step of perfusing a biological material together with an inner solution. In one embodiment, the step of cooling the inner container, the outer container, the biological material, the inner solution, and the outer solution to a desired storage temperature and the step of heating the inner container, the outer container, the biological material, the inner solution, and the outer solution to a temperature higher than the desired storage temperature are both performed at a rate within the range of 0.01 °C / min to 10 °C / min. In another embodiment, the step of cooling the inner container, the outer container, the biological material, the inner solution, and the outer solution to a desired storage temperature and the step of heating the inner container, the outer container, the biological material, the inner solution, and the outer solution to a temperature higher than the desired storage temperature are both performed at a rate within the range of 1 °C / min to 1000 °C / min. In an alternative embodiment, the step of cooling the inner container, the outer container, the biological material, and the outer solution to a desired storage temperature and the step of heating the inner container, the outer container, the biological material, and the outer solution to a temperature higher than the desired storage temperature are both performed at a rate within the range of 0.01 °C / min to 10 °C / min. In another alternative embodiment, the step of cooling the inner container, the outer container, the biological material, and the outer solution to a desired storage temperature and the step of heating the inner container, the outer container, the biological material, and the outer solution to a temperature higher than the desired storage temperature are both performed at a rate within the range of 1 °C / min to 1000 °C / min.

[0021] The inner container may be composed of a flexible material that directly contacts the outer surface of the biological material and does not allow mass transfer. The flexible material may be a tissue adhesive.

[0022] Furthermore, the present invention is an apparatus for reducing the probability of ice nucleation during the storage of biological substances in an isochoric system, comprising: an outer container that is rigid and non-insulating, and is provided with a seal configured to perform airtight and liquidtight sealing; an inner container positioned within the outer container, the inner container being flexible but unable to transmit mass; an inner solution within the inner container, the inner solution having an equilibrium melting point exceeding a desired storage temperature below 0°C; and an outer solution within the outer container and outside the inner container, the outer solution being composed of a liquid having an equilibrium melting point lower than the desired storage temperature below 0°C. Alternatively, the present invention is an apparatus for reducing the probability of ice nucleation during the storage of biological substances in an isochoric system, comprising: an outer container that is rigid and non-insulating, and is provided with a seal configured to perform airtight and liquidtight sealing; an inner container positioned within the outer container, the inner container being flexible but unable to transmit mass; an inner solution within the inner container, the inner solution having an equilibrium melting point exceeding a desired storage temperature below 0°C; and an outer solution within the outer container and outside the inner container, the outer solution being configured to undergo vitrification at the desired storage temperature below 0°C.

[0023] In an alternative configuration, the present invention is an apparatus for reducing the probability of ice nucleation during the storage of biological materials in an isovolumetric system, the apparatus comprising: an outer container that is rigid and non-insulating, the outer container comprising a seal configured to effect an airtight and liquidtight seal; at least two inner containers positioned within the outer container, the at least two inner containers being flexible but unable to transmit mass; an inner solution within each inner container, the inner solution having an equilibrium melting point that exceeds a desired storage temperature below 0 °C; and an outer solution within the outer container and outside of the inner containers, the outer solution being composed of a liquid having an equilibrium melting point lower than the desired storage temperature below 0 °C. Alternatively, the present invention is an apparatus for reducing the probability of ice nucleation during the storage of biological materials in an isovolumetric system, the apparatus comprising: an outer container that is rigid and non-insulating, the outer container comprising a seal configured to effect an airtight and liquidtight seal; at least two inner containers positioned within the outer container, the at least two inner containers being flexible but unable to transmit mass; an inner solution within each inner container, the inner solution having an equilibrium melting point that exceeds a desired storage temperature below 0 °C; and an outer solution within the outer container and outside of the inner containers, the outer solution being configured to undergo vitrification at the desired storage temperature below 0 °C.

[0024] The apparatus of the present invention preferably further comprises means for controlling the temperature of the apparatus, means for monitoring the temperature of the outer container, means for monitoring the pressure within the outer container, and an external processor configured to communicate with the means for controlling the temperature, the means for monitoring the temperature, and the means for monitoring the pressure. In a preferred embodiment, each of the inner containers is composed of low density polyethylene. The outer container is preferably composed of a transparent rigid material. Preferably, the present invention further comprises means for protecting the apparatus from vibration. BRIEF DESCRIPTION OF THE DRAWINGS

[0025]

Figure 1

Figure 2

Figure 3

Figure 4

Embodiments for Carrying Out the Invention

[0026] A. Overview Conventional preservation by isovolume vitrification or isovolume supercooling involves placing biological substances and the surrounding aqueous solution in a rigid container that transfers heat (i.e., is not designed to provide a thermal barrier) but does not transfer pressure (i.e., is rigid) or mass (i.e., is impermeable), removing excess air from the system, sealing the container so that the system is no longer in contact with the atmosphere or any other pressure reservoir, and monitoring the temperature and pressure within the chamber. Such rigid containers are typically used for preservation by isovolume freezing (21), (20), isovolume supercooling (22 - 24), and isovolume vitrification (25), and the pressure read within the system for a given temperature indicates whether ice nucleation has occurred (and, if so, to what extent).

[0027] In current methods and devices for preservation by isovolume supercooling or isovolume vitrification, the solution occupying the entire volume of the rigid isovolume chamber is exposed to the probability of nucleation, and this volume can be excessive with respect to the volume of the biological substances being preserved because the rigid chamber itself cannot conform to the arbitrary shape of the biological substances stored inside.

[0028] The present invention aims to reduce the probability of nucleation in an isochoric storage system by using two containers instead of one, filled with two solutions having specific thermodynamic relationships, namely, a sealed outer container composed of a conventional rigid isochoric chamber, and a sealed inner container that cannot transfer mass (i.e., is impermeable) but can transfer pressure (i.e., is flexible). Specifically, the solution and / or biological substance within the inner container, referred to as the "inner solution," has a higher melting point than the solution within the space between the outer wall of the inner container and the inner wall of the outer container, referred to as the "outer solution." At the desired storage temperature, the inner solution is susceptible to ice nucleation, while the outer solution is less so.

[0029] The present invention reduces the probability of ice nucleation during isochoric storage by restricting the volume within a system susceptible to the effects of ice nucleation such that the volume comes into contact with a surface that is more or less likely to stimulate heterogeneous ice nucleation than the walls of the chamber itself. In relation to storage by isochoric supercooling, the outer solution is thermodynamically stable in liquid form down to the storage temperature. In relation to storage by isochoric vitrification, the outer solution need not be thermodynamically stable, but must not nucleate ice during the process of cooling to or warming from a temperature below the glass transition temperature of the biological substance being stored.

[0030] Accordingly, in the present invention, the total volume susceptible to the effects of ice nucleation during these isochoric storage processes is restricted to the volume of the inner container only, thereby reducing the total probability of ice nucleation within the system. Furthermore, since the inner container is not subject to the same rigidity requirements as the outer container, it can be constructed from any of a wide range of flexible materials that do not provide an effective substrate for heterogeneous ice nucleation, such as common plastics or polymers derived from hydrocarbons or fluorine compounds (but not limited thereto).

[0031] B. Detailed Description of the Figures Figure 1 is a flowchart showing the initial steps of a preferred embodiment of the method of the present invention. First, regardless of the presence or absence of an inner solution around a melting point higher than the desired storage temperature, the biological material is placed within an inner container that can transmit pressure but cannot transmit mass (101). Next, all or most of the bulk gas phase is removed from the inner container (102). Next, the inner container is sealed (103). Preferably, the inner container is composed of a material known to have an insufficient heterogeneous ice nucleation ability, such as (but not limited to) polytetrafluoroethylene, polyethylene, or another hydrophobic polymeric material.

[0032] Figure 2 is a flowchart showing the intermediate steps of a preferred embodiment of the method of the present invention. After the steps shown in Figure 1 are completed, the inner container with the biological material prepared as described above is placed within a rigid outer container that can transmit heat (i.e., is non-insulated) (201). Next, the space between the inner container and the outer container is filled with an outer solution that (a) has a melting point lower than both the aqueous content within the biological material and any inner solution, and (b) is less affected by ice nucleation at the desired storage temperature (202). Next, all or most of the bulk gas phase is removed from the outer container (203). Next, the outer container is sealed (204).

[0033] FIG. 3 is a flowchart showing the final steps of a preferred embodiment of the method of the present invention. First, a composite system composed of a sealed inner chamber and an outer chamber is cooled (301) to a desired storage temperature below 0° C. Next, the composite system is maintained at this temperature for a desired storage period (302). Next, the temperature of the composite system is reheated to a temperature higher than 0° C. (303). Next, the chamber is opened and the biological material is removed (304). In some embodiments, when applying the present invention to the preservation of biological materials by isovolume supercooling, the storage temperature is lower than both the melting points of the aqueous contents and any inner solution within the biological material and higher than the melting point of the outer solution. In other embodiments, when applying the present invention to the preservation of biological materials by isovolume vitrification, the storage temperature is lower than the glass transition temperatures of both the aqueous contents and any inner solution within the biological material and also lower than the glass transition temperature of the outer solution.

[0034] Figure 4 is a schematic cross-sectional view showing the core components of a preferred embodiment of the apparatus of the present invention. The apparatus has an outer container 401 having a seal 402 that is rigid, conducts heat (i.e., is non-insulating), and can provide an airtight and liquidtight seal, an inner container 403 within the outer container that can transmit pressure (i.e., is flexible) but cannot transmit mass, an inner solution 404 within the inner container that can store biological material 405 internally and has an equilibrium melting point higher than the desired storage temperature below 0 °C, thereby making it susceptible to ice nucleation, and a separate outer solution 406 within the outer container and outside the inner container, which may or may not be essentially aqueous and is composed of a liquid having an equilibrium melting point lower than the desired storage temperature below 0 °C or, otherwise, not undergoing a first-order phase change at the same desired storage temperature. The apparatus also optionally includes external means for temperature control and cooling / warming with respect to the system 407, such as a circulating liquid, gas, or vapor bath, a refrigerator, a phase change material, a thermoelectric or Peltier module, a Stirling cooler, or a resistive heater, means for monitoring the temperature of the system 408, such as a thermocouple, a resistor, or a thermometer, means for monitoring the pressure within the outer container 409, such as a digital pressure transducer, a pressure gauge, a pressure-sensitive optical port, or a strain gauge, and a control system 410, such as a computer or a microprocessor, that communicates with the temperature and / or pressure measurement means and the temperature control and cooling / warming means.

[0035] The inner container 403 houses the biological material 405 to be stored. In some embodiments of the present invention, the inner solution 404 within the inner container 403 is composed of water or an aqueous solution containing added organic molecules or chemical cryoprotectants. These additives can determine the temperature range in which the system can be supercooled without ice nucleation, or enhance the stability of supercooling at a given storage temperature. They can also raise the glass transition temperature of the solution to enhance ease of vitrification, lower the melting or freezing point of the solution, and / or minimize toxicity to the biological material. Such chemical additives include, but are not limited to, dimethyl sulfoxide, ethylene glycol, polyethylene glycol, 3-OMG, glycerol, antifreeze proteins, ice recrystallization inhibitors, synthetic or organic ice modifiers, sugars, sugar alcohols, amino acids, salts, and the like.

[0036] The outer solution 406 may be composed of an aqueous solution incorporating these same additives, although at a higher concentration to make the outer solution less susceptible to the effects of ice nucleation at the desired storage temperature. For example, an aqueous solution of 49% (weight / weight) dimethyl sulfoxide is known to vitrify (i.e., avoid ice formation and form a glass) under any cooling and warming conditions. Thus, these solutions represent preferred embodiments of the outer solution 406 for applications involving isovolume vitrification. Further, since the inner container 403 housing the biological material 405 does not transfer mass, the liquid constituting the outer solution 406 need not be aqueous, biocompatible, or of minimal toxicity. As an example, for the use of storing human organs via isovolume supercooling at a desired storage temperature of -10°C, the inner solution 404 may be a conventional aqueous organ preservation solution that easily freezes at a temperature of less than about -0.5°C, such as Custodiol™, and the outer solution 406 may be a perfluorocarbon or hydrocarbon liquid having a melting point of less than -10°C.

[0037] By way of illustration and not limitation, the biological material 405 may be composed of human or non-human cells, organic molecules, multicellular constructs, tissues, organs, whole organisms and / or foodstuffs including, but not limited to, stem cells, blood, bone marrow, blood vessels, pancreatic islets, reproductive tissue, skin, etc.; heart, liver, kidney, lung, pancreas, spleen, etc.; eye, all or part of a limb, finger or toe, brain, spinal column, dorsal ganglion, nerve tissue, etc.; artificial tissues such as 3D microtissue constructs, liver-on-a-chip constructs, lung-on-a-chip constructs, heart-on-a-chip constructs, etc.; whole organisms such as zebrafish, coral, nematodes, or other marine or land animals; and / or foods such as strawberries, berries, potatoes, tomatoes, fish, beef, etc.

[0038] The biological material 405 may be perfused with or in the inner solution 404 prior to storage. The biological material may also be subjected to quality improvement in any manner including, but not limited to, normothermic or hypothermic machine perfusion, passive or active perfusion with a liquid, or immersion in any kind of liquid prior to storage.

[0039] In some embodiments, a plurality of separate and / or different inner containers 403 having separate and / or different inner solutions 404 and separate and / or different biological materials 405 are housed within the outer container 401. Each inner container and inner solution is subject to the same requirements and thermodynamic relationships to the outer solution as described for a single inner container.

[0040] The outer container 401 and all of its contents may be stored at one or more temperatures between 0°C and -273°C for any amount of time (302), and may be cooled (301) and / or heated (303) at any rate. In some embodiments, when the biological material 405 to be stored is a human organ, the desired storage temperature may be in the range of 0°C to -20°C to ensure avoidance of nucleation from a supercooled state, and the desired cooling and heating rates may be in the range of 0.01°C / min to 10°C / min so as not to avoid damage from overly rapid temperature changes, and an isovolume supercooling technique may be used. In other embodiments, when the biological material 405 to be stored is a reproductive material such as a cell, sperm, oocyte or embryo, or an organism such as a coral, the desired storage temperature may be in the range of -80°C to -196°C to promote the glass transition process, and the desired cooling and heating rates may be in the range of 1°C / min to 1000°C / min to ensure avoidance of ice nucleation during the vitrification process, and an isovolume glass solidification technique may be used.

[0041] In a preferred embodiment of the apparatus shown in FIG. 4, the outer container 401 is cooled by an external cooling and / or heating system 407 outside the outer container, but an internal cooling and / or heating system may also be used, examples of which include an internal heat exchanger tube or an internal phase change material. In all cases, the cooling and / or heating system 407 that regulates the temperature of the outer container 401 may be active (i.e., requires an input of thermodynamic work), as in the case of a refrigerator or a circulation bath, or passive (i.e., proceeds spontaneously), as in the case of a phase change material such as ice or eutectic salt.

[0042] The outer container 401 may be equipped with an instrument for measuring or inferring the pressure within 409, such as a pressure transducer, a pressure gauge, a pressure-sensitive optical port, or a strain gauge. This instrument can be used to monitor the pressure at continuous or discrete points during the processes of cooling (301), storage (302), or heating (303). The increase in pressure can be used to determine that ice has nucleated within the system, and a control system 410 that communicates with the temperature control means 407 and the pressure measurement instrument 409 can cause a change in temperature based on such readings from the pressure measurement instrument. For example, when the biological material 405 within the device is a human heart intended for transplantation and this heart is being stored (302) at a temperature of -4°C, if an increase in pressure is detected (indicating ice nucleation within the closed system), the control system 410 may issue a command to the temperature control instrument 407 to immediately heat the system 303. Also, the control system 410 can be used to change or adjust the temperature of the system in response to any change in the measured or inferred pressure within the system, this is because in an isochoric system, temperature and pressure are coupled.

[0043] The outer container 401 or the inner container 403 may be characterized by further means for protecting the internal liquid from vibration, including a sleeve, coating, mount, or other external mechanism made of a vibration-reducing material such as neoprene or other rubber; a spring or other mechanical mechanism for vibration reduction; and / or a combination thereof. Vibration that may be encountered during flight, ground transportation, or general use can cause undesirable ice nucleation.

[0044] Ice nucleation can also be stimulated by undesirable or uncontrolled changes in temperature, which can also have an adverse effect on the stored biological material 405. Thus, the outer container 401 and / or the inner container 403 may be characterized by further means for protecting the stored supercooled or vitrified biological material 405 from undesirable temperature changes, including an insulating sheath, sleeve, or coating; a surrounding phase change material; a vacuum insulation panel, material, or chamber; and / or other insulation measures. Further, the inner container 403 may also be characterized by further means for specifically protecting against heterogeneous ice nucleation at the solid-liquid interface between the inner container 403 and the inner solution 404, including but not limited to a hydrophobic or superhydrophobic surface or surface coating, examples of which include polytetrafluoroethylene-based, hydrocarbon-based, and / or perfluorocarbon-based materials.

[0045] The outer container 401 and the inner container 403 may each contain any volume, and a wide range of volumes may be desired based on the biological material 405 to be stored. For example, for cryopreserving mesenchymal stem cells by isovolume vitrification, both containers may contain volumes in the range of 1 microliter to 10 mL. In contrast, for cryopreserving the human liver by isovolume supercooling, these containers may contain volumes in the range of 1 L to 20 L. Further, for high-throughput storage of small biological materials such as cell suspensions or engineered tissues, a large outer container 401 on the order of 1 to 10 L can be paired with hundreds or thousands of small inner containers on the order of 1 to 10 mL. For bulk agricultural applications, particularly those aimed at preserving food during transportation, an outer container on the order of 20 to 1000 L may also be desirable.

[0046] The outer container 401 may be fabricated from one or more suitable rigid materials. These may include metals such as steel and its alloys, aluminum and its alloys, titanium and its alloys, copper and its alloys; ceramic materials; plastics such as acrylic, polyvinyl chloride, polymethyl methacrylate, polyurethane; composite materials such as carbon fiber reinforced polymer (CFRP), glass fiber reinforced polymer (GFRP); and / or any combination thereof. These materials may also be subjected to one or more surface treatments such as anodization, nickel plating, zinc plating, etc. for purposes such as preventing corrosion, preventing heterogeneous ice nucleation, and maintaining biocompatibility. The choice of materials and surface coatings is a function of the biological substance 405 being stored and the intended use, as is the case with many other aspects of the present invention.

[0047] The outer container 401 may also be made in whole or in part of a transparent rigid material such as polycarbonate or sapphire, which may be used to study or monitor the internal contents or behavior of the container during cooling (301), storage (302), or warming (303) of the system, including but not limited to the stored biologic or any possible phase transition behavior. In some embodiments, a completely or partially transparent outer container is incorporated into a microscope platform to enable microscopy of the contents contained therein. The container may also be geometrically configured on a millimeter or micrometer length scale for these purposes.

[0048] The inner container 403 may be composed in whole or in part of a material that transmits pressure but not mass, such as low density polyethylene (LDPE). In some embodiments, the inner container 403 that houses the biological substance 405 may be a bag, balloon, vial, or tube covered by a flexible material and / or another container that includes at least one flexible surface that is sealable and capable of transmitting pressure from its surroundings to its internal contents.

[0049] The seal 402 that enables airtight sealing of the outer container 401 may include one or more sealing mechanisms, some of which may include rubber O-rings, spring-biased O-rings, metal-to-metal contact parts, rubber gaskets, metal gaskets, and the like. The inner container 403 may optionally be sealed by one or more ridge closures similar to a Ziploc (trademark) bag (U.S. Patent No. 7,137,736: Closure Device for a Re-closable Pouch), or by a screw cap, screw plug, clamp lid, bolted lid, mechanically held plate or plug, press film, knot, and / or another sealing mechanism. The inner container may also be composed of one or more vacuum-sealed bags and / or heat-sealed bags.

[0050] If the individual inner solution 404 does not surround the biological material 405, the inner container 403 may be composed of a flexible material that directly contacts the surface of the biological material that does not permit mass transfer. This container may be composed of a coating of petrolatum and / or a cross-linked hydrogel such as sodium alginate or hyaluronic acid cross-linked with calcium or other ionic, oxidative, or covalent cross-linking agents. This coating itself may be impregnated with an organ preservation solution or any other form of aqueous solution for purposes such as maintaining osmotic balance, drug delivery, enhancing cryoprotective effects, and the like. The inner container may also be composed of a tissue adhesive, examples of which include fibrin glue, cyanoacrylate, and urethane prepolymer. The uses of adhesives on living tissues range from soft (connective) tissue adhesion to hard (calcified) tissue adhesion. The adhesive may be in the form of a liquid, paste, or thin film. A list of such adhesives can be found in Bhagat et al. (26).

[0051] C. Examples To demonstrate the concept of the present invention, a device was manufactured according to the general design of FIG. 4 and tested in the preservation of biological materials by isovolume supercooling. A comprehensive description of the results, methods, and devices used in this study is presented in detail in reference (27).

[0052] In this example, the biological substance stored was a porcine liver, which was successfully stored at -2°C for 48 hours without ice nucleation by the general method of FIGS. 1-3. After rewarming and removal from the chamber, the liver was evaluated by a qualified surgeon and found to be healthy. Histological samples were also taken to demonstrate the structural integrity of the stored tissue.

[0053] In these successful tests, the outer container consisted of a cylindrical stainless steel container with an inner diameter of 300 mm and an internal height of 150 mm, sealed via a rubber O-ring. The inner container in which the liver was stored consisted of a flexible hydrophobic low-density polyethylene bag, sealed using heat sealing and reinforced with plastic clamps.

[0054] The outer solution consisted of a 3 molar NaCl solution having an equilibrium melting point far below the desired storage temperature of -2°C and thus being less susceptible to the effects of ice nucleation. The inner solution consisted of Custodiol™, a physiological saline having an osmotic pressure of approximately 300 mM, which is used as a clinical standard in the preservation of livers and other internal organs for transplantation. Since the equilibrium melting point of Custodiol™ is approximately -0.5°C, it was kept in a supercooled state at the storage temperature and was susceptible to the effects of ice nucleation.

[0055] The outer container was also equipped with a thermocouple for continuously monitoring the temperature and a digital pressure transducer for continuously monitoring the pressure. An increase in pressure within the sealed isochoric system indicates ice nucleation and expansion, and thus the pressure readings were used to continuously evaluate the state of the system, i.e., to verify that no ice nucleation occurred. Using this apparatus and the general method of FIGS. 1-3, no ice nucleation occurred in any of the tests and a healthy preserved liver was obtained.

[0056] To isolate the beneficial effects of the methods disclosed herein, in additional tests, the outer container was completely filled with saline and the liver was placed directly into this container without using a separate inner container and solution. This approach is a conventional approach previously disclosed in the literature and is prior art surrounding the isochoric preservation of biological materials, but it maximizes the probability of harmful ice nucleation within the system. As expected, in all tests, this approach resulted in ice nucleation and freezing of the liver, irreversibly damaging the liver.

[0057] References 1. S.Giwa,et al.,The promise of organ and tissue preservation to transform medicine.Nat.Biotechnol.35,530-542(2017). 2. M.J.Taylor,B.P.Weegman,S.C.Baicu,S.E.Giwa,New Approaches to Cryopreservation of Cells,Tissues,and Organs.Transfus.Med.Hemotherapy 46,197-215(2019). 3. J.K.Lewis,et al.,The Grand Challenges of Organ Banking:Proceedings from the first global summit on complex tissue cryopreservation.Cryobiology 72,169-182(2016). 4. B.J.Luyet,E.L.Hodapp,Revival of frog’s spermatozoa vitrified in liquid air.Proc.Soc.Exp.Biol.Med.39,433-434(1938). 5. G.M.Fahy,D.R.MacFarlane,C.A.Angell,H.T.Meryman,Vitrification as an approach to cryopreservation.Cryobiology 21,407-26(1984). 6. G.M.Fahy,et al.,Cryopreservation of organs by vitrification:Perspectives and recent advances in Cryobiology,(2004),pp.157-178. 7. W.F.Rall,G.M.Fahy,Ice-free cryopreservation of mouse embryos at -196C, by vitrification.Nature 313,573-575(1985). 8. G.M.Fahy,et al.,Physical and biological aspects of renal vitrification.Organogenesis 5,167-175(2009). 9. E.P.M.A.D.Beljakov,N.A.Rojdestvenskaja,Comparative study of conserved blood stored at a temperature below 0℃ in a liquid supercooling state and in a frozen state.Bibl.Haematol 38,336-338(1969). 10. J.Heyman,Y;Xuan,NB;Renard,Preservation in the supercooled state of one-cell rabbit eggs with reduced cell water content.Cryobiology 25,564-564(1988). 11. R. Monzen, K; Hosoda, T; Nagai, R; Monzen, Koshiro; Hosoda, Toru; Hayashi, Doubun; Imai, Yasushi; Okawa, Yasuhiro; Kohro, Takahide; Uozaki, Hiroshi; Nishiyama, Tomoki; Fukayama, Masashi; Nagai, The use of a supercooling refrigerator improves the preservation of organ grafts. Biochem. Biophys. Res. Commun. 337, 534 - 539 (2005). 12. N. Ishine, B. Rubinsky, C. Y. Lee, A histological analysis of liver injury in freezing storage. Cryobiology 39, 271 - 277 (1999). 13. N. Ishine, B. Rubinsky, C. Y. Lee, Transplantation of mammalian livers following freezing: Vascular damage and functional recovery. Cryobiology 40, 84 - 89 (2000). 14. G. Amir, et al., Improved viability and reduced apoptosis in sub - zero 21 - hour preservation of transplanted rat hearts using anti - freeze proteins. J. Hear. Lung Transplant. 24, 1915 - 1929 (2005). 15. G. Amir, et al., Prolonged 24 - hour subzero preservation of heterotopically transplanted rat hearts using antifreeze proteins derived from arctic fish. Ann. Thorac. Surg. (2004) https: / doi.org / 10.1016 / j.athoracsur.2003.04.004. 16. G. Amir, et al., Subzero nonfreezing cryopreservation of rat hearts using antifreeze protein I and antifreeze protein III. Cryobiology (2004) https: / doi.org / 10.1016 / j.cryobiol.2004.02.009. 17. H. Huang, M. L. Yarmush, O. B. Usta, Long - term deep - supercooling of large - volume water and red cell suspensions via surface sealing with immiscible liquids. Nat. Commun. (2018) https: / doi.org / 10.1038 / s41467 - 018 - 05636 - 0. 18. S. A. Szobota, B. Rubinsky, Analysis of isochoric subcooling. Cryobiology (2006) https: / doi.org / 10.1016 / j.cryobiol.2006.04.001. 19. M. J. Powell - Palm, A. Koh - Bell, B. Rubinsky, Isochoric conditions enhance stability of metastable supercooled water. Appl. Phys. Lett 123702, https: / / doi.org / 10.1063 / 1.5145334(2020). 20. Perez, A. Pedro, “Thermodynamic and heat transfer analysis for isochoric cryopreservation” (2006). 21. B. Rubinsky, P. A. Perez, M. E. Carlson, The thermodynamic principles of isochoric cryopreservation. Cryobiology (2005) https: / doi.org / 10.1016 / j.cryobiol.2004.12.002. 22. A. N. Consiglio, D. Lilley, R. Prasher, B. Rubinsky, M. J. Powell-Palm, Methods to stabilize aqueous supercooling identified by use of an isochoric nucleation detection (INDe) device. Cryobiology (2022) https: / doi.org / 10.1016 / j.cryobiol.2022.03.003. 23. S.-I. Campean, et al., Analysis of the relative supercooling enhancement of two emerging supercooling techniques. AIP Adv. 11, 055125 (2021). 24. M. J. Powell-Palm, et al., Isochoric supercooled preservation and revival of human cardiac microtissues. Commun. Biol. 4 (2021). 25. Y. Zhang, et al., Isochoric vitrification: An experimental study to establish proof of concept. Cryobiology (2018) https: / doi.org / 10.1016 / j.cryobiol.2018.06.005. 26. V. Bhagat, M. Becker, Degradable Adhesives for Surgery and Tissue Engineering. Biomacromolecules 18, 3009 - 3039 (2017). 27. F. Botea, et al., An exploratory study on isochoric supercooling preservation of the pig liver. Biochem. Biophys. Reports 34, 101485 (2023).

Claims

1. A method for reducing the probability of ice nucleation during storage of a biological material in an isovolume system, comprising: (a) placing the biological material in a flexible and impermeable inner container; (b) adding an inner solution having a melting point higher than the desired storage temperature to the inner container; (c) removing bulk gas from the inner container; (d) sealing the inner container; (e) placing the inner container in a rigid and non-insulating outer container to form a space between the outer surface of the inner container and the inner surface of the outer container; (f) filling the space between the outer surface of the inner container and the inner surface of the outer container with an outer solution, wherein the biological material and the inner solution each have an equilibrium melting point, and the outer solution has a melting point lower than the equilibrium melting point of the biological material and lower than the equilibrium melting point of the inner solution; (g) removing bulk gas from the outer container; (h) sealing the outer container; (i) cooling the inner container, the outer container, the biological material, the inner solution, and the outer solution to the desired storage temperature; (j) maintaining the inner container, the outer container, the biological material, the inner solution, and the outer solution at the desired storage temperature for a desired storage period; (k) heating the inner container, the outer container, the biological material, the inner solution, and the outer solution to a temperature higher than the desired storage temperature; (l) opening the outer container and the inner container; (m) removing the biological material from the inner container.

2. A method for reducing the probability of ice nucleation during storage of a biological material in an isovolume system, comprising: (a) placing the biological material in a flexible and impermeable inner container; (b) removing bulk gas from the inner container; (c) sealing the inner container; (d) placing the inner container in a rigid and non-insulating outer container to form a space between the outer surface of the inner container and the inner surface of the outer container; (e) filling the space between the outer surface of the inner container and the inner surface of the outer container with an outer solution, wherein the biological material has a melting point, and the outer solution has a melting point lower than the melting point of the biological material; (f) removing bulk gas from the outer container; (g) a step of sealing the outer container; (h) a step of cooling the inner container, the outer container, the biological substance, and the outer solution to the desired storage temperature; (i) a step of maintaining the inner container, the outer container, the biological substance, and the outer solution at the desired storage temperature for a desired storage period; (j) a step of heating the inner container, the outer container, the biological substance, and the outer solution to a temperature higher than the desired storage temperature; (k) a step of opening the outer container and the inner container; (l) a method comprising a step of removing the biological substance from the inner container.

3. The method according to claim 1 or 2, wherein the desired storage temperature is 0 °C or lower.

4. The desired storage temperature is lower than the equilibrium melting point of the biological substance and lower than the equilibrium melting point of the inner solution, The method according to claim 1, wherein the desired storage temperature is higher than the melting point of the outer solution.

5. The desired storage temperature is lower than the melting point of the biological substance, The method according to claim 2, wherein the desired storage temperature is higher than the melting point of the outer solution.

6. The biological substance and the inner solution each have a glass transition temperature, The outer solution has a glass transition temperature, The desired storage temperature is lower than the glass transition temperature of the biological substance and lower than the glass transition temperature of the inner solution, The desired storage temperature is lower than the glass transition temperature of the outer solution, The method according to claim 1, wherein the glass transition temperature of the outer solution is higher than the glass transition temperature of the biological substance and higher than the glass transition temperature of the inner solution.

7. The biological substance has a glass transition temperature, The outer solution has a glass transition temperature, The desired storage temperature is lower than the glass transition temperature of the biological substance, The desired storage temperature is lower than the glass transition temperature of the outer solution, The method according to claim 2, wherein the glass transition temperature of the outer solution is higher than the glass transition temperature of the biological substance.

8. The method according to claim 1 or 2, wherein the inner container is made of a hydrophobic polymer material.

9. The method according to claim 1, wherein the inner solution is composed of an aqueous solution containing organic molecules at a first concentration.

10. The method according to claim 1, wherein the inner solution is composed of an aqueous solution containing a cryoprotectant at a first concentration.

11. The outer solution is composed of an aqueous solution containing an organic molecule at a second concentration, The method according to claim 9, wherein the second concentration of the organic molecule in the outer solution is higher than the first concentration of the organic molecule in the inner solution.

12. The outer solution is composed of an aqueous solution containing a cryoprotectant at a second concentration, The method according to claim 10, wherein the second concentration of the cryoprotectant in the outer solution is higher than the first concentration of the organic molecule in the inner solution.

13. The method according to claim 1, further comprising the step of perfusing the biological substance together with the inner solution.

14. Both the step of cooling the inner container, the outer container, the biological substance, the inner solution, and the outer solution to the desired storage temperature and the step of heating the inner container, the outer container, the biological substance, the inner solution, and the outer solution to a temperature higher than the desired storage temperature are performed at a rate within the range of 0.01 °C / min to 10 °C / min. The method according to claim 1.

15. Both the step of cooling the inner container, the outer container, the biological substance, the inner solution, and the outer solution to the desired storage temperature and the step of heating the inner container, the outer container, the biological substance, the inner solution, and the outer solution to a temperature higher than the desired storage temperature are performed at a rate within the range of 1 °C / min to 1000 °C / min. The method according to claim 1.

16. Both the step of cooling the inner container, the outer container, the biological substance, and the outer solution to the desired storage temperature and the step of heating the inner container, the outer container, the biological substance, and the outer solution to a temperature higher than the desired storage temperature are performed at a rate within the range of 0.01 °C / min to 10 °C / min. The method according to claim 2.

17. Both the step of cooling the inner container, the outer container, the biological substance, and the outer solution to the desired storage temperature and the step of heating the inner container, the outer container, the biological substance, and the outer solution to a temperature higher than the desired storage temperature are performed at a rate within the range of 1 °C / min to 1000 °C / min. The method according to claim 2.

18. The method according to claim 2, wherein the inner container is made of a flexible material that directly contacts the outer surface of the biological material and does not allow mass transfer.

19. The method according to claim 18, wherein the flexible material is a tissue adhesive.

20. An apparatus for reducing the probability of ice nucleation during the storage of biological materials in an isovolume system, comprising: (a) an outer container that is rigid and non-insulating, an outer container provided with a seal configured to provide an airtight and liquidtight seal; (b) an inner container located within the outer container, an inner container that is flexible but cannot transfer mass; (c) an inner solution within the inner container, an inner solution having an equilibrium melting point above the desired storage temperature below 0 °C; (d) an outer solution within the outer container and outside the inner container, an outer solution composed of a liquid having an equilibrium melting point lower than the desired storage temperature below 0 °C.

21. An apparatus for reducing the probability of ice nucleation during the storage of biological materials in an isovolume system, comprising: (a) an outer container that is rigid and non-insulating, an outer container provided with a seal configured to provide an airtight and liquidtight seal; (b) an inner container located within the outer container, an inner container that is flexible but cannot transfer mass; (c) an inner solution within the inner container, an inner solution having an equilibrium melting point above the desired storage temperature below 0 °C; (d) an outer solution within the outer container and outside the inner container, an outer solution configured to undergo vitrification at the desired storage temperature below 0 °C.

22. An apparatus for reducing the probability of ice nucleation during the storage of biological materials in an isovolume system, comprising: (a) an outer container that is rigid and non-insulating, an outer container provided with a seal configured to provide an airtight and liquidtight seal; (b) at least two inner containers located within the outer container, at least two inner containers that are flexible but cannot transfer mass; (c) an inner solution within each of the inner containers, an inner solution having an equilibrium melting point above the desired storage temperature below 0 °C; (d) an outer solution within the outer container and outside the inner containers, An apparatus comprising an outer solution composed of a liquid having an equilibrium melting point lower than the desired storage temperature below 0°C.

23. An apparatus for reducing the probability of ice nucleation during the storage of biological substances in an isovolumetric system, comprising: (a) an outer container that is rigid and non-insulating, the outer container comprising a seal configured to provide an airtight and liquidtight seal; (b) at least two inner containers positioned within the outer container, the at least two inner containers being flexible but unable to transfer mass; (c) an inner solution within each of the inner containers, the inner solution having an equilibrium melting point above the desired storage temperature below 0°C; (d) an outer solution within the outer container and outside the inner containers, the outer solution being configured to undergo vitrification at the desired storage temperature below 0°C.

24. (e) means for controlling the temperature of the apparatus; (f) means for monitoring the temperature of the outer container; (g) means for monitoring the pressure within the outer container; (h) an external processor configured to communicate with the means for controlling the temperature, the means for monitoring the temperature, and the means for monitoring the pressure. The apparatus according to claim 20, 21, 22, or 23.

25. The apparatus according to claim 20, 21, 22, or 23, wherein each of the inner containers is composed of low-density polyethylene.

26. The apparatus according to claim 20, 21, 22, or 23, wherein the outer container is composed of a transparent rigid material.

27. The apparatus according to claim 20, 21, 22, or 23, further comprising means for protecting the apparatus from vibration.

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