Method and apparatus for reducing excess pressure in an isochoric system

Abstract

A method for reducing overpressure in an isochoric system, comprising: providing a rigid, sealable master container; disposing a primary subsystem containing biological material within the master container; disposing a secondary subsystem within the master container; removing bulk vapor from the master container; sealing the master container; cooling the master container to a desired storage temperature below 0 degrees Celsius; maintaining the master container at the storage temperature for a storage period; warming the master container to a temperature above the equilibrium melting point of the primary subsystem; opening the master container; and removing the biological material from the master container. The secondary subsystem is a water-immiscible liquid having a positive thermal expansion coefficient greater in absolute magnitude than the thermal expansion coefficient of water at temperatures below 0 degrees Celsius. An apparatus for performing the foregoing method steps.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. § 119(e), this application claims the benefit of U.S. Patent No. 6,339,0688, filed July 20, 2022, the contents of which are incorporated herein by reference. [Background technology]

[0002] Background of the Invention 1. Field of the Invention The present invention relates generally to methods and systems for preserving biological material, and more particularly to methods and apparatus for preventing pressure-based damage to biological material stored in an isochoric system at temperatures below zero degrees Celsius by reducing excessive positive or negative pressures caused by thermal volumetric changes in the contents of the system.

[0003] 2. Description of Related Technology There has been growing interest in the use of isochoric (constant volume) systems for the cryopreservation of biological materials [1]. These systems exploit the unique thermodynamic effects of isochoric conditions to prevent undesired freezing of aqueous solutions containing biological contents and damage the contents, while maintaining the contents at a low enough temperature to halt metabolism and extend the period during which they can be preserved. Among isochoric preservation techniques, isochoric supercooling [2], [3] and isochoric vitrification [4] do not involve a first-order phase change. That is, isochoric supercooling and isochoric vitrification involve maintaining the contained aqueous contents (including the aqueous contents within any contained biological material) in a single homogeneous phase. During isochoric supercooling, which generally proceeds in the temperature range of 0°C to -40°C, these aqueous contents are liquid. During isochoric vitrification, which generally proceeds in the temperature range of -80°C to -273°C, the aqueous contents are initially liquid and then vitrified [5].

[0004] All materials expand or contract when their temperature changes; that is, all materials have a finite, non-zero coefficient of thermal expansion (although selected materials may have a zero coefficient of thermal expansion at a singular value, at the temperature where the thermal expansion value changes from positive to negative). Thus, when confined within a rigid, constant-volume, isochoric system, a change in temperature will either increase (if the internal contents tend to significantly expand) or decrease (if the internal contents tend to significantly contract) the pressure experienced within the system. In an isochoric system, the resulting pressure is a function of temperature only, and the relationship between temperature and pressure is an invariant function of the thermovolumetric properties of the contained contents.

[0005] The term "thermal volume" as applied to a property or change of a material describes how the material changes volume in response to changes in temperature and pressure. The specific properties that govern the relationship between temperature and pressure in an isochoric system are the coefficient of thermal expansion, or the degree to which a material changes volume with temperature at a given pressure, and the compressibility, or the degree to which a material changes volume with pressure at a given temperature. The absolute change in volume of a material is also proportional to its initial volume. It is important to note that any pressure that appears in a single-phase isochoric system results from the thermal volume change of the material present, as opposed to the expansion or contraction of additional material (such as ice) produced by a phase change, as is often encountered during conventional isochoric freezing [6], [7].

[0006] For biological specimens, prolonged exposure to pressures greater or less than those experienced under normal homeostatic conditions can prove harmful [8], [9]. Therefore, during isochoric cryopreservation protocols, it is crucial to influence the relationship between temperature and pressure experienced within the system to ensure that the pressure is maintained at the desired value at the desired storage temperature. In other words, it is desirable to reduce excess pressure that may be generated by thermal volumetric changes of the internal contents, regardless of whether the pressure is positive or negative.

[0007] The temperature-pressure relationship in systems where a two-phase liquid-ice configuration exists (commonly referred to as "isochoric freezing") has been well characterized and exploited in many previous devices. [6] However, methods to influence the pressure-temperature relationship and reduce overpressure in single-phase supercooled or vitrified isochoric systems have not previously been reported.

[0008] Various different aspects of isochoric cryopreservation have been taught in the prior art. Rubinsky and Szobota teach a method of using isochoric conditions to stabilize isochoric supercooling against unintended homogeneous ice nucleation (U.S. Patent Application Publication No. 20070042337). Powell-Palm and Rubinsky teach a method of using isochoric conditions to stabilize isochoric supercooling against unintended heterogeneous ice nucleation, and a method of using a device to monitor ice nucleation from a supercooling system using a pressure transducer (International Patent Application No. PCT / US21 / 12863). Rubinsky et al. teach how multi-stage temperature cycling can be used to achieve vitrification in an isochoric system, and how monitoring pressure for the large increase associated with ice formation can be used to verify successful vitrification (U.S. Patent Application Publication No. 20200178518). All of these disclosures relate to the many beneficial aspects of isochoric subcooling and isochoric vitrification, but ignore the potentially harmful overpressure (positive or negative) that can be generated by simple thermal volumetric changes of the contents within the isochoric chamber (as opposed to a phase change), and none provide a means to reduce this overpressure. Summary of the Invention

[0009] C. Objectives of the Invention The present invention relates to methods and apparatus for reducing excessive positive or negative pressure during isochoric storage. The present invention relates to the preservation of biological material by isochoric vitrification and isochoric supercooling. More specifically, the present invention provides methods and apparatus for reducing excessive positive or negative pressure on biological material in an isochoric system by storing the biological material in a supercooled state at a temperature below the equilibrium melting point of the biological material and the solution in which it is maintained. The present invention also provides methods and apparatus for reducing excessive positive or negative pressure on biological material in an isochoric system by storing the biological material in a vitrified or partially vitrified state at a temperature below the glass transition temperature of the biological material and the solution in which it is maintained. In both cases, the present invention reduces excessive positive or negative pressure on biological material by reducing the net thermal volume change of the different elements stored in the isochoric container at the desired storage temperature.

[0010] Brief Summary of the Invention The present invention is a method for reducing overpressure in an isochoric system, the method comprising the steps of providing a rigid, sealable master container; disposing a primary subsystem comprised of biological material within the master container; disposing a secondary subsystem within the master container, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius and the secondary subsystem is a water-immiscible liquid and has a positive coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; removing the bulk gas phase from the master container; sealing the master container; cooling the master container to a desired storage temperature below 0 degrees Celsius; maintaining the master container at the desired storage temperature for a desired storage period, wherein the primary subsystem has an equilibrium melting point; warming the master container to a temperature above the equilibrium melting point of the primary subsystem; opening the master container; and removing the biological material from the master container.

[0011] In an alternative embodiment, the present invention is a method for reducing overpressure in an isochoric system, the method comprising the steps of providing a rigid, sealable master container; disposing a primary subsystem comprised of biological material within the master container; disposing a secondary subsystem within the master container, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, and the secondary subsystem is a water-immiscible liquid and has a negative coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; removing bulk gas phase from the master container; sealing the master container; cooling the master container to a desired storage temperature below 0 degrees Celsius; maintaining the master container at the desired storage temperature for a desired storage period, wherein the primary subsystem has an equilibrium melting point; warming the master container to a temperature above the equilibrium melting point of the primary subsystem; opening the master container; and removing the biological material from the master container.

[0012] In an alternative embodiment, the present invention is a method for reducing overpressure in an isochoric system, the method comprising the steps of providing a rigid, sealable master container; disposing a primary subsystem comprised of biological material within the master container; disposing a secondary subsystem within the master container, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius and the secondary subsystem is a water-immiscible liquid and has a positive coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; removing bulk gas phase from the master container; sealing the master container; cooling the master container to a desired storage temperature below 0 degrees Celsius; maintaining the master container at the desired storage temperature for a desired storage period, wherein the primary subsystem has an equilibrium melting point; warming the master container to a temperature above the equilibrium melting point of the primary subsystem; opening the master container; and removing the biological material from the master container.

[0013] In an alternative embodiment, the present invention is a method for reducing overpressure in an isochoric system, the method comprising the steps of providing a rigid, sealable master container; disposing a primary subsystem comprised of biological material within the master container; disposing a secondary subsystem within the master container, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, and the secondary subsystem is a water-immiscible liquid and has a negative coefficient of thermal expansion that is less in absolute magnitude than the thermal expansion coefficient of water; removing the bulk gas phase from the master container; sealing the master container; cooling the master container to a desired storage temperature below 0 degrees Celsius; maintaining the master container at the desired storage temperature for a desired storage period, wherein the primary subsystem has an equilibrium melting point; warming the master container to a temperature above the equilibrium melting point of the primary subsystem; opening the master container; and removing the biological material from the master container.

[0014] In an alternative embodiment, the present invention is a method for reducing overpressure in an isochoric system, the method comprising the steps of providing a rigid, sealable master container; disposing a primary subsystem comprised of biological material within the master container; disposing a secondary subsystem within the master container, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius and the secondary subsystem is a water-immiscible solid and has a positive coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; removing the bulk gas phase from the master container; sealing the master container; cooling the master container to a desired storage temperature below 0 degrees Celsius; maintaining the master container at the desired storage temperature for a desired storage period, wherein the primary subsystem has an equilibrium melting point; warming the master container to a temperature above the equilibrium melting point of the primary subsystem; opening the master container; and removing the biological material from the master container.

[0015] In an alternative embodiment, the present invention is a method for reducing overpressure in an isochoric system, the method comprising the steps of providing a rigid, sealable master container; disposing a primary subsystem comprised of biological material within the master container; disposing a secondary subsystem within the master container, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, and the secondary subsystem is a water-immiscible solid and has a negative coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; removing the bulk gas phase from the master container; sealing the master container; cooling the master container to a desired storage temperature below 0 degrees Celsius; maintaining the master container at the desired storage temperature for a desired storage period, wherein the primary subsystem has an equilibrium melting point; warming the master container to a temperature above the equilibrium melting point of the primary subsystem; opening the master container; and removing the biological material from the master container.

[0016] In an alternative embodiment, the present invention is a method for reducing overpressure in an isochoric system, the method comprising the steps of providing a rigid, sealable master container; disposing a primary subsystem comprised of biological material within the master container; disposing a secondary subsystem within the master container, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius and the secondary subsystem is a water-immiscible solid and has a positive coefficient of thermal expansion that is less in absolute magnitude than the thermal expansion coefficient of water; removing the bulk gas phase from the master container; sealing the master container; cooling the master container to a desired storage temperature below 0 degrees Celsius; maintaining the master container at the desired storage temperature for a desired storage period, wherein the primary subsystem has an equilibrium melting point; warming the master container to a temperature above the equilibrium melting point of the primary subsystem; opening the master container; and removing the biological material from the master container.

[0017] In an alternative embodiment, the present invention is a method for reducing overpressure in an isochoric system, the method comprising the steps of providing a rigid, sealable master container; disposing a primary subsystem comprised of biological material within the master container; disposing a secondary subsystem within the master container, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, and the secondary subsystem is a water-immiscible solid and has a negative coefficient of thermal expansion that is less in absolute magnitude than the thermal expansion coefficient of water; removing the bulk gas phase from the master container; sealing the master container; cooling the master container to a desired storage temperature below 0 degrees Celsius; maintaining the master container at the desired storage temperature for a desired storage period, wherein the primary subsystem has an equilibrium melting point; warming the master container to a temperature above the equilibrium melting point of the primary subsystem; opening the master container; and removing the biological material from the master container.

[0018] In an alternative embodiment, the present invention is a method for reducing overpressure in an isochoric system, the method comprising the steps of providing a rigid, sealable master container; disposing a primary subsystem comprised of biological material within the master container; disposing a secondary subsystem within the master container, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, the secondary subsystem being a water-miscible liquid having a positive coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water, the liquid being separated from the primary subsystem by a mass-impermeable barrier; removing a bulk gas phase from the master container; sealing the master container; cooling the master container to a desired storage temperature below 0 degrees Celsius; maintaining the master container at the desired storage temperature for a desired storage period, wherein the primary subsystem has an equilibrium melting point; warming the master container to a temperature above the equilibrium melting point of the primary subsystem; opening the master container; and removing the biological material from the master container.

[0019] In an alternative embodiment, the present invention is a method for reducing overpressure in an isochoric system, the method comprising the steps of providing a rigid, sealable master container; disposing a primary subsystem comprised of biological material within the master container; disposing a secondary subsystem within the master container, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, the secondary subsystem being a water-immiscible liquid having a negative coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water, the liquid being separated from the primary subsystem by a mass-impermeable barrier; removing a bulk gas phase from the master container; sealing the master container; cooling the master container to a desired storage temperature below 0 degrees Celsius; maintaining the master container at the desired storage temperature for a desired storage period, wherein the primary subsystem has an equilibrium melting point; warming the master container to a temperature above the equilibrium melting point of the primary subsystem; opening the master container; and removing the biological material from the master container.

[0020] In an alternative embodiment, the present invention is a method for reducing overpressure in an isochoric system, the method comprising the steps of providing a rigid, sealable master container; disposing a primary subsystem comprised of biological material within the master container; disposing a secondary subsystem within the master container, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, the secondary subsystem being a water-immiscible liquid having a positive coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water, the liquid being separated from the primary subsystem by a mass-impermeable barrier; removing a bulk gas phase from the master container; sealing the master container; cooling the master container to a desired storage temperature below 0 degrees Celsius; maintaining the master container at the desired storage temperature for a desired storage period, wherein the primary subsystem has an equilibrium melting point; warming the master container to a temperature above the equilibrium melting point of the primary subsystem; opening the master container; and removing the biological material from the master container.

[0021] In an alternative embodiment, the present invention is a method for reducing overpressure in an isochoric system, the method comprising the steps of providing a rigid, sealable master container; disposing a primary subsystem comprised of biological material within the master container; disposing a secondary subsystem within the master container, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, the secondary subsystem being a water-immiscible liquid having a negative coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water, the liquid being separated from the primary subsystem by a mass-impermeable barrier; removing a bulk gas phase from the master container; sealing the master container; cooling the master container to a desired storage temperature below 0 degrees Celsius; maintaining the master container at the desired storage temperature for a desired storage period, wherein the primary subsystem has an equilibrium melting point; warming the master container to a temperature above the equilibrium melting point of the primary subsystem; opening the master container; and removing the biological material from the master container.

[0022] In a preferred embodiment, the secondary subsystem is comprised of the group consisting of mineral oil, vegetable oil, silicone oil, and perfluorocarbon. In an alternative embodiment, the secondary subsystem is comprised of pure water.

[0023] In a preferred embodiment, the invention further comprises providing a mechanical element configured to increase or decrease the volume of the master vessel, hi a preferred embodiment, the master vessel is constructed from a material having a thermal expansion coefficient higher than that of grade 5 titanium.

[0024] In one embodiment, the invention further comprises combining at least one primary subsystem with two or more secondary subsystems in the same master vessel. In another embodiment, the invention further comprises combining two or more primary subsystems with at least one secondary subsystem in the same master vessel. In yet another embodiment, the invention further comprises combining two or more primary subsystems with two or more secondary subsystems in the same master vessel.

[0025] The present invention is also an apparatus for reducing excess pressure in an isochoric system, the apparatus comprising: a rigid, sealable master vessel having a volume and wherein any bulk gas phase within the master vessel comprises less than 5 percent of the volume of the master vessel; a primary subsystem comprised of biological material contained within the master vessel; a secondary subsystem contained within the master vessel, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, and the secondary subsystem is a water-immiscible liquid and has a positive coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; means for monitoring and controlling the temperature of the master vessel; and means external to the master vessel for monitoring the pressure within the master vessel.

[0026] In an alternative embodiment, the present invention is an apparatus for reducing excess pressure in an isochoric system, the apparatus comprising: a rigid, sealable master vessel having a volume and wherein any bulk gas phase within the master vessel comprises less than 5 percent of the volume of the master vessel; a primary subsystem comprised of biological material contained within the master vessel; a secondary subsystem contained within the master vessel, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, the secondary subsystem being a water-immiscible liquid and having a negative coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; means for monitoring and controlling the temperature of the master vessel; and means external to the master vessel for monitoring the pressure within the master vessel.

[0027] In an alternative embodiment, the present invention is an apparatus for reducing excess pressure in an isochoric system, the apparatus comprising: a rigid, sealable master vessel having a volume and wherein any bulk gas phase within the master vessel comprises less than 5 percent of the volume of the master vessel; a primary subsystem comprised of biological material contained within the master vessel; a secondary subsystem contained within the master vessel, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius and the secondary subsystem is a water-immiscible liquid and has a positive coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; means for monitoring and controlling the temperature of the master vessel; and means external to the master vessel for monitoring the pressure within the master vessel.

[0028] In an alternative embodiment, the present invention is an apparatus for reducing excess pressure in an isochoric system, the apparatus comprising: a rigid, sealable master vessel having a volume and wherein any bulk gas phase within the master vessel comprises less than 5 percent of the volume of the master vessel; a primary subsystem comprised of biological material contained within the master vessel; a secondary subsystem contained within the master vessel, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, the secondary subsystem being a water-immiscible liquid and having a negative coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; means for monitoring and controlling the temperature of the master vessel; and means external to the master vessel for monitoring the pressure within the master vessel.

[0029] In an alternative embodiment, the present invention is an apparatus for reducing excess pressure in an isochoric system, the apparatus comprising: a rigid, sealable master vessel having a volume and wherein any bulk gas phase within the master vessel comprises less than 5 percent of the volume of the master vessel; a primary subsystem comprised of biological material contained within the master vessel; a secondary subsystem contained within the master vessel, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius and the secondary subsystem is a water-immiscible solid and has a positive coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; means for monitoring and controlling the temperature of the master vessel; and means external to the master vessel for monitoring the pressure within the master vessel.

[0030] In an alternative embodiment, the present invention is an apparatus for reducing excess pressure in an isochoric system, the apparatus comprising: a rigid, sealable master vessel having a volume and any bulk gas phase within the master vessel comprising less than 5 percent of the volume of the master vessel; a primary subsystem comprised of biological material contained within the master vessel; a secondary subsystem contained within the master vessel, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, and the secondary subsystem is a water-immiscible solid and has a negative coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; means for monitoring and controlling the temperature of the master vessel; and means external to the master vessel for monitoring the pressure within the master vessel.

[0031] In an alternative embodiment, the present invention is an apparatus for reducing excess pressure in an isochoric system, the apparatus comprising: a rigid, sealable master vessel having a volume and wherein any bulk gas phase within the master vessel comprises less than 5 percent of the volume of the master vessel; a primary subsystem comprised of biological material contained within the master vessel; a secondary subsystem contained within the master vessel, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius and the secondary subsystem is a water-immiscible solid and has a positive coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; means for monitoring and controlling the temperature of the master vessel; and means external to the master vessel for monitoring the pressure within the master vessel.

[0032] In an alternative embodiment, the present invention is an apparatus for reducing excess pressure in an isochoric system, the apparatus comprising: a rigid, sealable master vessel having a volume and wherein any bulk gas phase within the master vessel comprises less than 5 percent of the volume of the master vessel; a primary subsystem comprised of biological material contained within the master vessel; a secondary subsystem contained within the master vessel, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, and the secondary subsystem is a water-immiscible solid and has a negative coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; means for monitoring and controlling the temperature of the master vessel; and means external to the master vessel for monitoring the pressure within the master vessel.

[0033] In an alternative embodiment, the present invention is an apparatus for reducing overpressure in an isochoric system, the apparatus comprising: a rigid, sealable master vessel having a volume and any bulk gas phase within the master vessel comprising less than 5 percent of the volume of the master vessel; a primary subsystem comprised of biological material contained within the master vessel; a secondary subsystem contained within the master vessel, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, the secondary subsystem being a water-miscible liquid having a positive coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water, the liquid being separated from the primary subsystem by a mass-impermeable barrier; means for monitoring and controlling the temperature of the master vessel; and means external to the master vessel for monitoring the pressure within the master vessel.

[0034] In an alternative embodiment, the present invention is an apparatus for reducing overpressure in an isochoric system, the apparatus comprising: a rigid, sealable master vessel having a volume and any bulk gas phase within the master vessel comprising less than 5 percent of the volume of the master vessel; a primary subsystem comprised of biological material contained within the master vessel; a secondary subsystem contained within the master vessel, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, the secondary subsystem being a water-miscible liquid having a negative coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water, the liquid being separated from the primary subsystem by a mass-impermeable barrier; means for monitoring and controlling the temperature of the master vessel; and means external to the master vessel for monitoring the pressure within the master vessel.

[0035] In an alternative embodiment, the present invention is an apparatus for reducing overpressure in an isochoric system, the apparatus comprising: a rigid, sealable master vessel having a volume and any bulk gas phase within the master vessel comprising less than 5 percent of the volume of the master vessel; a primary subsystem comprised of biological material contained within the master vessel; a secondary subsystem contained within the master vessel, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, the secondary subsystem being a water-miscible liquid having a positive coefficient of thermal expansion that is less in absolute magnitude than the thermal expansion coefficient of water, the liquid being separated from the primary subsystem by a mass-impermeable barrier; means for monitoring and controlling the temperature of the master vessel; and means external to the master vessel for monitoring the pressure within the master vessel.

[0036] In an alternative embodiment, the present invention is an apparatus for reducing overpressure in an isochoric system, the apparatus comprising: a rigid, sealable master vessel having a volume and any bulk gas phase within the master vessel comprising less than 5 percent of the volume of the master vessel; a primary subsystem comprised of biological material contained within the master vessel; a secondary subsystem contained within the master vessel, wherein water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius, the secondary subsystem being a water-miscible liquid having a negative coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water, the liquid being separated from the primary subsystem by a mass-impermeable barrier; means for monitoring and controlling the temperature of the master vessel; and means external to the master vessel for monitoring the pressure within the master vessel. [Brief explanation of the drawings]

[0037] [Figure 1] 1 is a flow chart illustrating the steps of a preferred embodiment of the method of the present invention. [Figure 2] This is a continuation of Figure 1. [Figure 3]1 is a schematic cross-sectional view showing the core components of a preferred embodiment of the device of the present invention. [Figure 4] 1 is a graphical plot showing how different ratios of oil to water can change the pressure-temperature relationship of an isochoric system. DETAILED DESCRIPTION OF THE INVENTION

[0038] Detailed Description of the Invention A. Overview Traditional preservation by isochoric vitrification or isochoric supercooling involves placing the biological material and surrounding aqueous solution in a rigid container that does not transmit 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 isochoric freezing[8,10], isochoric supercooling

[11] , and isochoric vitrification[4], and the pressure reading within the system for a given temperature indicates whether (and, if so, to what extent) ice nucleation has occurred.

[0039] In current methods and devices for preservation by isochoric supercooling or isochoric vitrification, the pressures to which biological materials are exposed during cooling, warming, and storage are determined by the thermovolumetric properties (i.e., coefficients of thermal expansion and compressibility) of the solution containing the biological material, and these pressures may prove excessive and harmful to the biological material.

[0040] The present invention aims to reduce the excess positive or negative pressure to which biological material is exposed during isochoric storage by adding one or more subsystems to the internal contents of an isochoric chamber, the thermal volumetric properties of which compensate for those of the biological material itself and / or the aqueous material in which it is stored, resulting in a net reduction in the thermal volumetric change of the system at the desired storage temperature, and therefore a reduction in the excess pressure generated.

[0041] Specifically, when the biological material and / or the solution in which it is contained, referred to as the "primary subsystem," expands at the desired storage temperature, creating a positive overpressure, an additional subsystem, referred to as the "secondary subsystem," contracts at the desired storage temperature to compensate for the primary subsystem and reduce the final positive pressure to which the biological material is exposed. Similarly, when the primary subsystem contracts at the desired storage temperature, the secondary subsystem expands, reducing the final negative pressure to which the biological material is exposed. The secondary subsystem does not contain the biological material and is composed of a material that is immiscible with the aqueous solution or includes a mass-impermeable barrier, thereby not significantly altering the chemical composition of the primary subsystem.

[0042] The present invention reduces the excessive positive or negative pressure to which biological material is exposed during isochoric storage by reducing the net thermal volume change within the isochoric container at a given desired storage temperature.

[0043] B. Detailed explanation of the diagram FIG. 1 is a flowchart illustrating the initial steps of a preferred embodiment of the method of the present invention. First, a rigid, sealable master container is provided (101). Preferably, this container is constructed of an anodized aluminum alloy, titanium alloy, stainless steel alloy, or another metallic material with high strength and corrosion resistance. Next, a primary subsystem, optionally comprised of a biological material in an aqueous solution having an equilibrium melting point higher than the desired storage temperature, is placed within the master container (102). Next, a secondary subsystem is placed within the master container. This secondary subsystem has the following properties: it does not substantially change the chemical composition of the primary subsystem; it does not contain biological material; and it has thermovolumetric properties that compensate for the thermovolumetric properties of the primary subsystem to reduce excess positive or negative pressure within the master container at the desired storage temperature (103). Preferably, this secondary subsystem is constructed of a water-immiscible liquid and has a positive thermal expansion coefficient greater in absolute magnitude than the negative thermal expansion coefficient of water at temperatures below 0 degrees Celsius. Next, all or most of the bulk gas phase is removed from the master container (104). The master container is then sealed (105).

[0044] Referring to Figure 2, the master container is cooled to a desired sub-zero degree Celsius storage temperature (201). The master container is then maintained at this desired storage temperature for a desired storage period (202). The master container is then warmed to a temperature above the equilibrium melting point of the primary subsystem (203). Finally, the master container is opened and the biological material is removed (204).

[0045] 3 is a schematic cross-sectional view showing the core components of a preferred embodiment of the device of the present invention. The device comprises a master container 301 having a seal 302 that is rigid and capable of providing an airtight and liquid-tight seal, a primary subsystem comprised of biological material 303 optionally housed in an aqueous solution 304 having an equilibrium melting point above a desired sub-zero degree Celsius storage temperature, and a secondary subsystem 305 comprised of a liquid or solid that does not substantially alter the chemical composition of the biological material, is itself free of biological material, and whose thermal volumetric properties compensate for those of the biological material 303 and / or its housing aqueous solution 304, thereby minimizing excessive positive or negative pressure at the desired temperature.

[0046] In a preferred embodiment, the aqueous solution 304 in the primary subsystem is composed of a dilute saline solution typically used in organ or tissue preservation, and the secondary subsystem 305 is composed of mineral oil, a perfluorocarbon, or another water-immiscible liquid. Similar to pure water, diluted saline expands as it cools below 0°C, creating excess positive pressure in a closed isochoric system, such as that provided by the master vessel 301. Mineral oil, perfluorocarbons, and nearly all other water-immiscible liquids contract at these same temperatures, thereby compensating for the expansion of the saline solution and reducing excess pressure to the benefit of the biological material 303 stored therein.

[0047] In an alternative embodiment, the aqueous solution 304 in the primary subsystem is composed of an aqueous solution containing a high concentration of cryoprotective additives (e.g., dimethyl sulfoxide, ethylene glycol, glycerol, propylene glycol, etc.). The aqueous solution 304 contracts significantly upon cooling, creating excessive negative pressure within the master vessel 301. The secondary subsystem 305 may be composed of pure water or a dilute solution contained within a mass-impermeable barrier. Pure water and its dilute solutions expand upon cooling in the temperature range below 0°C, thereby compensating for the contracting aqueous solution 304 in the primary subsystem and reducing the excessive negative pressure. The mass-impermeable barrier prevents chemical interactions between the secondary subsystem 305, on the one hand, and the aqueous solution 304 and biological material 303 in the primary subsystem, on the other hand.

[0048] The apparatus may also optionally include external means 306 for providing temperature control and cooling / warming to the system, 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 307 for monitoring the temperature of the system, such as a thermocouple, resistor, or thermometer; means 308 for monitoring the pressure within the outer vessel, such as a digital pressure transducer, a pressure gauge, a pressure-sensitive optical port, or a strain gauge; and a control system 309, such as a computer or microprocessor, in communication with the temperature and / or pressure measurement means and the temperature control and cooling / warming means.

[0049] The biological material 303 to be preserved is often contained in an aqueous solution 304, with water acting as the solvent. In some embodiments of the present invention, this solution 304 is composed of water with or without the addition of organic molecules or chemical cryoprotectants. These additives may determine the temperature range to which the system can be supercooled without ice nucleation or may increase the stability of supercooling at a given storage temperature. They may also increase the glass transition temperature of the solution to increase the ease of vitrification, decrease the melting or freezing point of the solution, and / or minimize toxicity to the biological material 303. These additives may also affect the thermovolumetric properties of water, reducing or increasing the extent to which the primary subsystem expands or contracts at a given temperature and / or reducing or increasing the pressure generated from said expansion or contraction. 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.

[0050] By way of example and not limitation, biological material 303 may be comprised of human or non-human cells, organic molecules, multicellular constructs, tissues, organs, complete organisms and / or food, including, but not limited to, stem cells, blood, bone marrow, blood vessels, pancreatic islets, reproductive tissue, skin, etc.; may be comprised of heart, liver, kidney, lung, pancreas, spleen, etc.; may be comprised of eye, all or part of a limb, finger or toe, brain, spinal column, dorsal ganglion, nervous tissue, etc.; may be comprised of artificial tissue such as 3D microtissue constructs, liver-on-chip constructs, lung-on-chip constructs, heart-on-chip constructs, etc.; may be comprised of complete organisms such as zebrafish, corals, nematodes, or other marine or terrestrial animals; and / or may be comprised of food products such as cherries, berries, potatoes, tomatoes, fish, beef, etc.

[0051] The biological material 303 may be perfused with or in the aqueous solution 304 prior to storage. The biological material may also undergo some form of conditioning prior to storage, including, but not limited to, normothermic or hypothermic machine perfusion, passive or active perfusion with fluid, or immersion in any type of fluid.

[0052] In some embodiments, multiple separate and / or different primary subsystems, each consisting of a biological material 303 and, optionally, an aqueous solution 304 containing the biological material, can be added to the master vessel 301. Multiple separate and / or different secondary subsystems 305 can also be added, all of which may have different thermal volumetric properties. The volume-averaged thermal volumetric properties of the primary subsystems are compensated for by the volume-averaged thermal volumetric properties of the secondary subsystems.

[0053] For example, in one embodiment, a master container 301 can contain both a human heart 303 contained in a dilute saline solution 304 of one composition and a human brain 303 contained in a dilute solution 304 of another composition. Both dilute aqueous solutions 304 expand upon cooling, creating undesirable excess positive pressure. The total excess pressure created is a function of the combined net expansion of the two primary subsystems. Two secondary subsystems, one composed of mineral oil and the other of perfluorocarbon, may be added to the master container 301, both of which contract upon cooling, thereby compensating for the expansion of the primary subsystem and reducing the excess positive pressure. The total excess pressure reduction is a function of the combined net contraction of the two secondary subsystems 305. The final pressure within the master container 301 at the desired storage temperature is a function of the net thermal volume change of all subsystems, primary and secondary combined.

[0054] The secondary subsystem 305 may be constructed of liquid materials that are generally immiscible with water, including hydrocarbons such as mineral oil, vegetable oil, or silicone oil; perfluorocarbons such as perfluorodecalin, perfluorotributylamine, perfluorooctyl bromide, or any other immiscible liquid. The secondary subsystem 305 may also be constructed of liquid materials that are miscible with water but are protected from mixing with the biological material and / or its aqueous housing solution by a mass-impermeable membrane or container. The secondary subsystem 305 may also be constructed of solids such as rubber or plastic. Polymeric materials, particularly ABS plastic, nylon, and the like, are known to contract significantly with temperature and can be used to compensate for the expansion of the primary subsystem.

[0055] The master container 301 and all contents therein may be stored for any amount of time at one or more temperatures between 0°C and -273°C 302, and may be cooled (201) and / or warmed (203) at any rate. In some embodiments, when the biological material 303 to be stored is a human organ, an isochoric supercooling approach may be used, where the desired storage temperature may be in the range of 0°C to -20°C to ensure avoidance of nucleation from supercooled conditions, and the desired cooling (201) and warming (203) rates may be between 0.01°C / min and 10°C / min to avoid damage from excessively fast temperature changes. In other embodiments, when the biological material 303 to be stored is reproductive material such as sperm, oocytes, or embryos, or living organisms such as corals, an isochoric vitrification technique may be used, where the desired storage temperature may be in the range of −80° C. to −196° C. to facilitate the glass transition process, and the desired cooling and warming rates may be in the range of 1° C. / min to 1000° C. / min to ensure the avoidance of ice nucleation during the vitrification process.

[0056] 3, the master vessel 301 is cooled by a cooling and / or warming system 306 external to the outer vessel, although internal cooling and / or warming systems can also be used, examples of which include internal heat exchanger tubes or internal phase change materials. In all cases, the cooling and / or warming system 306 that regulates the temperature of the outer vessel 301 can be active (i.e., requiring the input of thermodynamic work), as in the case of a refrigerator or circulating bath, or passive (i.e., proceeding on its own), as in the case of a phase change material such as ice or a eutectic salt.

[0057] The master vessel 301 may be equipped with instruments to measure or infer the pressure within 308, such as a pressure transducer, pressure gauge, pressure-sensitive optical port, or strain gauge. This instrumentation can be used to monitor the pressure at continuous or discrete points during the cooling 201, storage 202, or warming 203 process. This pressure can be used to verify the success of the reduction of excess pressure; if the pressure is higher or lower than expected or desired, a control system 309 in communication with the temperature control means 306 and the pressure measurement device 308 can implement a temperature change based on such readings from the pressure measurement device. For example, if the biological material 303 within the device is a human heart intended for transplantation and the heart is stored 207 at a temperature of −4° C., if the pressure reading is higher than atmospheric pressure (0.1 MPa), the control system 309 will issue a command to the temperature control device 306 to warm the system in increments of less than 1° C. in order to reduce this excess pressure while maintaining a tolerable supercooled storage temperature. The control system 309 can also be used to change or adjust the temperature of the system in response to any changes in measured or inferred pressure in the system, since in an isochoric system, temperature and pressure are coupled.

[0058] In one embodiment, the master vessel 301 may also be equipped with a piston, threaded rod, or another mechanical element configured to increase or decrease the volume of the master vessel, which may provide an additional means for varying the pressure within the master vessel and may be used in combination with one or more secondary subsystems 305 to more precisely regulate the pressure during storage.

[0059] Master vessel 301 may be equipped with additional means to protect its internal contents from vibration, including sleeves, coatings, mounts, or other external features made of vibration-damping materials such as neoprene or other rubber, springs or other mechanical features for vibration damping, and / or combinations thereof. Vibrations that may be encountered during flight, ground transport, or general use can cause undesirable fluctuations in pressure in a subcooled isochoric system.

[0060] Undesired or uncontrolled changes in temperature can adversely affect the stored biological material 303 and can create undesired fluctuations in pressure due to the temperature-pressure coupling inherent in isochoric systems. Therefore, the master container 301 may be equipped with additional means to protect the stored supercooled or vitrified biological material 303 from undesired temperature changes, including, but not limited to, thermal insulating sheaths, sleeves, or coatings; surrounding phase change materials; vacuum insulating panels, materials, or chambers; and / or other insulating means.

[0061] The master container 301, the primary subsystem, comprised of the biological material 203 and, optionally, the aqueous solution 304 in which it is contained, and the secondary subsystem 305 may each comprise or be comprised of any volume, with a wide range of volumes being desirable depending on the biological material 303 to be stored or the overpressure to be reduced. For example, to preserve mesenchymal stem cells by isovolumetric vitrification, the master container 301 may contain a volume ranging from 1 microliter to 10 mL. In contrast, to preserve human liver by isovolumetric supercooling, the master container may contain a volume ranging from 1 L to 20 L. For bulk agricultural applications, particularly those intended to preserve food during transport, master containers 201 on the 20-1000 L scale may also be desirable.

[0062] The master container 301 may also be fabricated in whole or in part from 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 201, storage 202, or warming 203 of the system, including, but not limited to, the behavior of the stored biologic or any phase transitions that may occur. In some embodiments, the fully or partially transparent master container 301 is integrated into a microscope platform, thereby allowing microscopic examination of the contents contained therein. The container may also be geometrically configured on the millimeter or micron length scale for these purposes.

[0063] While the master container 301 is generally assumed to be substantially independent of the thermal volumetric contraction / expansion processes that dictate its internal pressure, it may be specifically configured to have a substantial thermal volumetric effect. To this end, the container may be constructed from a material with a high coefficient of thermal expansion relative to stainless steel or titanium, such as ABS plastic, or from a shape selected to maximize contraction or expansion effects in a given direction, such as a cylinder, whose contraction / expansion acts primarily radially. This may be particularly preferable when the primary subsystem, consisting of the biological material 303 optionally stored in the aqueous solution 304, is highly contractile (as may be the case when the aqueous solution 304 is highly concentrated). In this case, the additional contraction of the master container 301 itself around the primary subsystem can complement the reduction provided by the secondary subsystem 305 to help reduce excess negative pressure.

[0064] Figure 4 is a graphical plot 400 showing how, using the known thermovolumetric properties of the primary and secondary subsystems and applying the simple principle of volume conservation, the overpressure generated at a given temperature can be calculated for different ratios of the volumes of the two subsystems. Such calculations allow for the rational design of isochoric protocols to maximize overpressure reduction. In this figure, the primary subsystem is approximated as having the properties of water (a reasonable approximation for most biological materials 303 and dilute aqueous solutions 304), and the secondary subsystem is approximated as having the properties of mineral oil. The figure legend gives the resulting pressure as a function of temperature for different volume percentages of the secondary subsystem. C. Example

[0065] To test the present invention, a device was fabricated according to the general design of Figure 3 and tested in the preservation of biological material by isochoric supercooling. In this example, the preserved biological model 303 was a rodent heart, which was stored in two supercooling configurations for 24 hours at -4°C without ice nucleation (202).

[0066] The first configuration involves a single-phase isochoric system without the excess pressure drop provided by the present invention, and the second configuration involves an isochoric system with a secondary subsystem 305 comprised of a fixed volume of mineral oil. The first configuration without pressure control resulted in a hydrostatic pressure increase of 61 bar at -4°C due to uncompensated expansion of the aqueous storage solution 304 (University of Wisconsin Solution, a clinical standard, approximately 300 mM dilute aqueous organ preservation fluid).

[0067] The second configuration, in accordance with the methods and apparatus disclosed herein, incorporates a secondary subsystem 305 constructed from mineral oil. Mineral oil is immiscible with aqueous solutions, thereby not altering the chemical composition of the biological material 303 or its aqueous solution 304. Mineral oil also contracts as temperatures drop below 0 degrees Celsius, thereby counteracting the expansion of the University of Wisconsin solution and the heart itself. Based on baseline estimates plotted at 400 in FIG. 4, the secondary subsystem 305 was selected to occupy 21% of the initial volume of the master vessel 301, thereby providing the greatest reduction in overpressure. At the desired storage temperature of -4°C, the pressure was 1 bar, identical to standard atmospheric conditions.

[0068] After 24 hours of storage at -4°C (202), rewarming (203), and removal of rodent hearts from the chamber (204), the hearts were evaluated in a Langendorff perfusion device using various functional metrics, including heart rate, left ventricular developed pressure (LVDP), rate-pressure product (RPP), and contraction / relaxation rates. Reduced heart rate, LVDP, RPP, and contraction / relaxation rates were measured for hearts stored in a conventional isovolumic system without pressure regulation. Meanwhile, hearts stored in an isovolumic system with pressure control exhibited normal cardiac function. References [1] S. Giwa et al., “Organ and tissue preservation has the potential to revolutionize medicine,” Nat. Biotechnol., vol. 35, no. 6, pp. 530-542, 2017, doi:10.1038 / nbt.3889. [2] M.J. Powell-Palm, A. Koh-Bell, and B. Rubinsky, “Isovolumetric conditions enhance the stability of metastable supercooled water,” Appl. Phys. Lett., vol. 116, no. 12, 2020, doi:10.1063 / 1.5145334. [3] A. N. Consiglio, D. Lilley, R. Prasher, B. Rubinsky, and M. J. Powell-Palm, “Methods for stabilizing aqueous supercooling identified by use of an isochoric nucleation detection (INDe) device,” Cryobiology, 2022, doi:https: / / doi.org / 10.1016 / j.cryobiol.2022.03.003. [4] Y. Zhang et al., “Isovolumetric Vitrification: Experimental Study to Establish Proof of Concept,” Cryobiology, 2018, doi:10.1016 / j.cryobiol.2018.06.005. [5] G.M.Fahy, D.R.MacFarlane, C.A.Angell, and H.T.Meryman, “Vitrification as an Approach to Cryopreservation,” Cryobiology, vol. 21, no. 4, pp. 407-426, Aug. 1984, doi:10.1016 / 0011-2240(84)90079-8. [6] B. Rubinsky, PAPerez, and MECarlson, “Thermodynamic principles of isovolume cryopreservation,” Cryobiology, 2005, doi:10.1016 / j.cryobiol.2004.12.002. [7] B. Chang et al., “On the pressure dependence of salt-water eutectic,” Cell Rep. Phys. Sci., p. 100856, 2022, doi:https: / / doi.org / 10.1016 / j.xcrp.2022.100856. [8] L.Wan et al., “Preservation of rat hearts under subzero isovolumic conditions (-8°C, 78MPa),” Biochem.Biophys.Res.Commun., vol.496, no.3, pp.852-857, Feb.2018, doi:10.1016 / j.bbrc.2018.01.140. [9] L. Wan, M.J. Powell-Palm, M.G. Clemens, and B. Rubinsky, “Time-dependent effects of pressure during preservation of rat hearts at subzero temperatures in an isochoric system,” Cryoletters, vol. 40, no. 1, pp. 64-70, Jan. 2019.

[10] M.J. Powell-Palm, Y. Zhang, J. Aruda, and B. Rubinsky, “Isovolumetric conditions enable pancreatic islet preservation at subzero temperatures without the use of osmotic cryoprotectants,” Cryobiology, vol. 86, pp. 130-133, February 2019, doi:10.1016 / j.cryobiol.2019.01.003.

[11] “Preservation and regeneration of human cardiac microtissues by isovolumetric supercooling | Communication Biology.” https: / / www.nature.com / articles / s42003-021-02650-9 (accessed July 11, 2023).

Claims

1. 1. A method for reducing overpressure in an isochoric system, comprising: (a) providing a rigid, sealable master container; (b) placing a primary subsystem containing biological material into said master container; (c) placing a secondary subsystem within the master vessel; Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem being a water-immiscible liquid and having a positive coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; (d) removing bulk vapor from the master vessel; (e) sealing the master container; (f) cooling the master container to a desired sub-zero degree Celsius storage temperature; (g) maintaining said master container at a desired storage temperature for a desired storage period; (h) the primary subsystem has an equilibrium melting point, and warming the master container to a temperature greater than the equilibrium melting point of the primary subsystem; (i) opening the master container; (j) removing said biological material from said master container.

2. 1. A method for reducing overpressure in an isochoric system, comprising: (a) providing a rigid, sealable master container; (b) placing a primary subsystem containing biological material into said master container; (c) placing a secondary subsystem within the master vessel; Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; a secondary subsystem that is a water-immiscible liquid and has a negative coefficient of thermal expansion that is greater in absolute magnitude than the coefficient of thermal expansion of water; (d) removing bulk vapor from the master vessel; (e) sealing the master container; (f) cooling the master container to a desired sub-zero degree Celsius storage temperature; (g) maintaining said master container at a desired storage temperature for a desired storage period; (h) the primary subsystem has an equilibrium melting point, and warming the master container to a temperature greater than the equilibrium melting point of the primary subsystem; (i) opening the master container; (j) removing said biological material from said master container.

3. 1. A method for reducing overpressure in an isochoric system, comprising: (a) providing a rigid, sealable master container; (b) placing a primary subsystem containing biological material into said master container; (c) placing a secondary subsystem within the master vessel; Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem being a water-immiscible liquid and having a positive coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; (d) removing bulk vapor from the master vessel; (e) sealing the master container; (f) cooling the master container to a desired sub-zero degree Celsius storage temperature; (g) maintaining said master container at a desired storage temperature for a desired storage period; (h) the primary subsystem has an equilibrium melting point, and warming the master container to a temperature greater than the equilibrium melting point of the primary subsystem; (i) opening the master container; (j) removing said biological material from said master container.

4. 1. A method for reducing overpressure in an isochoric system, comprising: (a) providing a rigid, sealable master container; (b) placing a primary subsystem containing biological material into said master container; (c) placing a secondary subsystem within the master vessel; Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem being a water-immiscible liquid and having a negative coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; (d) removing bulk vapor from the master vessel; (e) sealing the master container; (f) cooling the master container to a desired sub-zero degree Celsius storage temperature; (g) maintaining said master container at a desired storage temperature for a desired storage period; (h) the primary subsystem has an equilibrium melting point, and warming the master container to a temperature greater than the equilibrium melting point of the primary subsystem; (i) opening the master container; (j) removing said biological material from said master container.

5. 1. A method for reducing overpressure in an isochoric system, comprising: (a) providing a rigid, sealable master container; (b) placing a primary subsystem containing biological material into said master container; (c) placing a secondary subsystem within the master vessel; Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem being a water-immiscible solid and having a positive coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; (d) removing bulk vapor from the master vessel; (e) sealing the master container; (f) cooling the master container to a desired sub-zero degree Celsius storage temperature; (g) maintaining said master container at a desired storage temperature for a desired storage period; (h) the primary subsystem has an equilibrium melting point, and warming the master container to a temperature greater than the equilibrium melting point of the primary subsystem; (i) opening the master container; (j) removing said biological material from said master container.

6. 1. A method for reducing overpressure in an isochoric system, comprising: (a) providing a rigid, sealable master container; (b) placing a primary subsystem containing biological material into said master container; (c) placing a secondary subsystem within the master vessel; Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem being a water-immiscible solid and having a negative coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; (d) removing bulk vapor from the master vessel; (e) sealing the master container; (f) cooling the master container to a desired sub-zero degree Celsius storage temperature; (g) maintaining said master container at a desired storage temperature for a desired storage period; (h) the primary subsystem has an equilibrium melting point, and warming the master container to a temperature greater than the equilibrium melting point of the primary subsystem; (i) opening the master container; (j) removing said biological material from said master container.

7. 1. A method for reducing overpressure in an isochoric system, comprising: (a) providing a rigid, sealable master container; (b) placing a primary subsystem containing biological material into said master container; (c) placing a secondary subsystem within the master vessel; Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem being a water-immiscible solid and having a positive coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; (d) removing bulk vapor from the master vessel; (e) sealing the master container; (f) cooling the master container to a desired sub-zero degree Celsius storage temperature; (g) maintaining said master container at a desired storage temperature for a desired storage period; (h) the primary subsystem has an equilibrium melting point, and warming the master container to a temperature greater than the equilibrium melting point of the primary subsystem; (i) opening the master container; (j) removing said biological material from said master container.

8. 1. A method for reducing overpressure in an isochoric system, comprising: (a) providing a rigid, sealable master container; (b) placing a primary subsystem containing biological material into said master container; (c) placing a secondary subsystem within the master vessel; Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem being a water-immiscible solid and having a negative coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; (d) removing bulk vapor from the master vessel; (e) sealing the master container; (f) cooling the master container to a desired sub-zero degree Celsius storage temperature; (g) maintaining said master container at a desired storage temperature for a desired storage period; (h) the primary subsystem has an equilibrium melting point, and warming the master container to a temperature greater than the equilibrium melting point of the primary subsystem; (i) opening the master container; (j) removing said biological material from said master container.

9. 1. A method for reducing overpressure in an isochoric system, comprising: (a) providing a rigid, sealable master container; (b) placing a primary subsystem containing biological material into said master container; (c) placing a secondary subsystem within the master vessel; Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem is a water-miscible liquid and has a positive coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; the liquid being separated from the primary subsystem by a mass impermeable barrier; (d) removing bulk vapor from the master vessel; (e) sealing the master container; (f) cooling the master container to a desired sub-zero degree Celsius storage temperature; (g) maintaining said master container at a desired storage temperature for a desired storage period; (h) the primary subsystem has an equilibrium melting point, and warming the master container to a temperature greater than the equilibrium melting point of the primary subsystem; (i) opening the master container; (j) removing said biological material from said master container.

10. 1. A method for reducing overpressure in an isochoric system, comprising: (a) providing a rigid, sealable master container; (b) placing a primary subsystem containing biological material into said master container; (c) placing a secondary subsystem within the master vessel; Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem being a water-immiscible liquid and having a negative coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; the liquid being separated from the primary subsystem by a mass impermeable barrier; (d) removing bulk vapor from the master vessel; (e) sealing the master container; (f) cooling the master container to a desired sub-zero degree Celsius storage temperature; (g) maintaining said master container at a desired storage temperature for a desired storage period; (h) the primary subsystem has an equilibrium melting point, and warming the master container to a temperature greater than the equilibrium melting point of the primary subsystem; (i) opening the master container; (j) removing said biological material from said master container.

11. 1. A method for reducing overpressure in an isochoric system, comprising: (a) providing a rigid, sealable master container; (b) placing a primary subsystem containing biological material into said master container; (c) placing a secondary subsystem within the master vessel; Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem is a water-immiscible liquid and has a positive coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; the liquid being separated from the primary subsystem by a mass impermeable barrier; (d) removing bulk vapor from the master vessel; (e) sealing the master container; (f) cooling the master container to a desired sub-zero degree Celsius storage temperature; (g) maintaining said master container at a desired storage temperature for a desired storage period; (h) the primary subsystem has an equilibrium melting point, and warming the master container to a temperature greater than the equilibrium melting point of the primary subsystem; (i) opening the master container; (j) removing said biological material from said master container.

12. 1. A method for reducing overpressure in an isochoric system, comprising: (a) providing a rigid, sealable master container; (b) placing a primary subsystem containing biological material into said master container; (c) placing a secondary subsystem within the master vessel; Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem being a water-immiscible liquid and having a negative coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; the liquid being separated from the primary subsystem by a mass impermeable barrier; (d) removing bulk vapor from the master vessel; (e) sealing the master container; (f) cooling the master container to a desired sub-zero degree Celsius storage temperature; (g) maintaining said master container at a desired storage temperature for a desired storage period; (h) the primary subsystem has an equilibrium melting point, and warming the master container to a temperature greater than the equilibrium melting point of the primary subsystem; (i) opening the master container; (j) removing said biological material from said master container.

13. 13. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, wherein the secondary subsystem is comprised of the group consisting of mineral oil, vegetable oil, silicone oil, and perfluorocarbon.

14. 13. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, wherein the secondary subsystem comprises purified water.

15. 13. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, further comprising the step of: (k) providing a mechanical element configured to increase or decrease the volume of the master container.

16. 13. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, wherein the master container comprises a material having a coefficient of thermal expansion higher than that of grade 5 titanium.

17. 13. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, further comprising the step of: (k) combining at least one primary subsystem with two or more secondary subsystems in the same master vessel.

18. (k) combining two or more primary subsystems and at least one secondary subsystem in the same master vessel.

19. 13. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, further comprising the step of: (k) combining two or more primary subsystems and two or more secondary subsystems in the same master vessel.

20. 1. An apparatus for reducing excess pressure in an isochoric system, comprising: (a) a rigid, sealable master container; the master reservoir having a volume; a master vessel, wherein any bulk vapor phase within said master vessel comprises less than 5 percent of the volume of said master vessel; (b) a primary subsystem containing biological material contained within said master container; (c) a secondary subsystem contained within the master vessel, Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; a secondary subsystem that is a water-immiscible liquid and has a positive coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; (d) means for monitoring and controlling the temperature of said master vessel; (e) means external to said master vessel for monitoring the pressure within said master vessel.

21. 1. An apparatus for reducing excess pressure in an isochoric system, comprising: (a) a rigid, sealable master container; the master reservoir having a volume; a master vessel, wherein any bulk vapor phase within said master vessel comprises less than 5 percent of the volume of said master vessel; (b) a primary subsystem containing biological material contained within said master container; (c) a secondary subsystem contained within the master vessel, Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; a secondary subsystem that is a water-immiscible liquid and has a negative coefficient of thermal expansion that is greater in absolute magnitude than the coefficient of thermal expansion of water; (d) means for monitoring and controlling the temperature of said master vessel; (e) means external to said master vessel for monitoring the pressure within said master vessel.

22. 1. An apparatus for reducing excess pressure in an isochoric system, comprising: (a) a rigid, sealable master container; the master reservoir having a volume; a master vessel, wherein any bulk vapor phase within said master vessel comprises less than 5 percent of the volume of said master vessel; (b) a primary subsystem containing biological material contained within said master container; (c) a secondary subsystem contained within the master vessel, Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; a secondary subsystem, the secondary subsystem being a water-immiscible liquid and having a positive coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; (d) means for monitoring and controlling the temperature of said master vessel; (e) means external to said master vessel for monitoring the pressure within said master vessel.

23. 1. An apparatus for reducing excess pressure in an isochoric system, comprising: (a) a rigid, sealable master container; the master reservoir having a volume; a master vessel, wherein any bulk vapor phase within said master vessel comprises less than 5 percent of the volume of said master vessel; (b) a primary subsystem containing biological material contained within said master container; (c) a secondary subsystem contained within the master vessel, Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; a secondary subsystem, the secondary subsystem being a water-immiscible liquid and having a negative coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; (d) means for monitoring and controlling the temperature of said master vessel; (e) means external to said master vessel for monitoring the pressure within said master vessel.

24. 1. An apparatus for reducing excess pressure in an isochoric system, comprising: (a) a rigid, sealable master container; the master reservoir having a volume; a master vessel, wherein any bulk vapor phase within said master vessel comprises less than 5 percent of the volume of said master vessel; (b) a primary subsystem containing biological material contained within said master container; (c) a secondary subsystem contained within the master vessel, Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; a secondary subsystem that is a water-immiscible solid and has a positive coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; (d) means for monitoring and controlling the temperature of said master vessel; (e) means external to said master vessel for monitoring the pressure within said master vessel.

25. 1. An apparatus for reducing excess pressure in an isochoric system, comprising: (a) a rigid, sealable master container; the master reservoir having a volume; a master vessel, wherein any bulk vapor phase within said master vessel comprises less than 5 percent of the volume of said master vessel; (b) a primary subsystem containing biological material contained within said master container; (c) a secondary subsystem contained within the master vessel, Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; a secondary subsystem that is a water-immiscible solid and has a negative coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; (d) means for monitoring and controlling the temperature of said master vessel; (e) means external to said master vessel for monitoring the pressure within said master vessel.

26. 1. An apparatus for reducing excess pressure in an isochoric system, comprising: (a) a rigid, sealable master container; the master reservoir having a volume; a master vessel, wherein any bulk vapor phase within said master vessel comprises less than 5 percent of the volume of said master vessel; (b) a primary subsystem containing biological material contained within said master container; (c) a secondary subsystem contained within the master vessel, Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; a secondary subsystem that is a water-immiscible solid and has a positive coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; (d) means for monitoring and controlling the temperature of said master vessel; (e) means external to said master vessel for monitoring the pressure within said master vessel.

27. 1. An apparatus for reducing excess pressure in an isochoric system, comprising: (a) a rigid, sealable master container; the master reservoir having a volume; a master vessel, wherein any bulk vapor phase within said master vessel comprises less than 5 percent of the volume of said master vessel; (b) a primary subsystem containing biological material contained within said master container; (c) a secondary subsystem contained within the master vessel, Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; a secondary subsystem that is a water-immiscible solid and has a negative coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; (d) means for monitoring and controlling the temperature of said master vessel; (e) means external to said master vessel for monitoring the pressure within said master vessel.

28. 1. An apparatus for reducing excess pressure in an isochoric system, comprising: (a) a rigid, sealable master container; the master reservoir having a volume; a master vessel, wherein any bulk vapor phase within said master vessel comprises less than 5 percent of the volume of said master vessel; (b) a primary subsystem containing biological material contained within said master container; (c) a secondary subsystem contained within the master vessel, Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem is a water-miscible liquid and has a positive coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; a secondary subsystem, the liquid being separated from the primary subsystem by a mass-impermeable barrier; (d) means for monitoring and controlling the temperature of said master vessel; (e) means external to said master vessel for monitoring the pressure within said master vessel.

29. 1. An apparatus for reducing excess pressure in an isochoric system, comprising: (a) a rigid, sealable master container; the master reservoir having a volume; a master vessel, wherein any bulk vapor phase within said master vessel comprises less than 5 percent of the volume of said master vessel; (b) a primary subsystem containing biological material contained within said master container; (c) a secondary subsystem contained within the master vessel, Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem is a water-miscible liquid and has a negative coefficient of thermal expansion greater in absolute magnitude than the coefficient of thermal expansion of water; a secondary subsystem, the liquid being separated from the primary subsystem by a mass-impermeable barrier; (d) means for monitoring and controlling the temperature of said master vessel; (e) means external to said master vessel for monitoring the pressure within said master vessel.

30. 1. An apparatus for reducing excess pressure in an isochoric system, comprising: (a) a rigid, sealable master container; the master reservoir having a volume; a master vessel, wherein any bulk vapor phase within said master vessel comprises less than 5 percent of the volume of said master vessel; (b) a primary subsystem containing biological material contained within said master container; (c) a secondary subsystem contained within the master vessel, Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; a secondary subsystem that is a water-miscible liquid and has a positive coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; a secondary subsystem, the liquid being separated from the primary subsystem by a mass-impermeable barrier; (d) means for monitoring and controlling the temperature of said master vessel; (e) means external to said master vessel for monitoring the pressure within said master vessel.

31. 1. An apparatus for reducing excess pressure in an isochoric system, comprising: (a) a rigid, sealable master container; the master reservoir having a volume; a master vessel, wherein any bulk vapor phase within said master vessel comprises less than 5 percent of the volume of said master vessel; (b) a primary subsystem containing biological material contained within said master container; (c) a secondary subsystem contained within the master vessel, Water has a negative coefficient of thermal expansion at temperatures below 0 degrees Celsius; the secondary subsystem is a water-miscible liquid and has a negative coefficient of thermal expansion that is less in absolute magnitude than the coefficient of thermal expansion of water; a secondary subsystem, the liquid being separated from the primary subsystem by a mass-impermeable barrier; (d) means for monitoring and controlling the temperature of said master vessel; (e) means external to said master vessel for monitoring the pressure within said master vessel.

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