WO2017187287A1

WO2017187287A1 - Medium for biopreservation of cells and other biological materials at cryogenic and hypothermic conditions and uses thereof

Info

Publication number: WO2017187287A1
Application number: PCT/IB2017/052099
Authority: WO
Inventors: Yiling Lu; Galdys MONTENEGRO; Jennifer Nicole FELIX; Annette RUFF; Jacquelyn COVARRUBIAS; Danielle EDWARDS
Original assignee: Smith & Nephew, Inc.
Priority date: 2016-04-25
Filing date: 2017-04-11
Publication date: 2017-11-02

Abstract

Biopreservation mediums containing a pan-oxygenase inhibitor, a sugar alcohol, an amino alcohol, an ionic metal halide, and/or a biguanide, useful for all phases of biopreservation including cryopreservation, thawing and hypothermic (cold storage) of biological materials such as cells, cell preparations, biological tissues, and/or organs, plus their uses are disclosed. All phases of biopreservation can be conducted with one biopreservation medium.

Description

DESCRIPTION

MEDIUM FOR BIOPRESERVATION OF CELLS AND OTHER BIOLOGICAL MATERIALS AT CRYOGENIC AND HYPOTHERMIC CONDITIONS AND USES

THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent

Application Serial No. 62/327,066, filed April 25, 2016, hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION A. Field of the Invention

[0002] The invention generally relates to biopreservation of biological materials. In particular, the invention relates to the cryopreservation, thawing, and/or hypothermic preservation (cold storage) of cells, cell preparations, biological tissues, and/or organs.

B. Description of Relevant Art [0003] Biopreservation involves the preservation of biological materials including cells, cell preparations, biological tissues, and organs at cryogenic temperatures and/or hypothermic temperatures (cold storage). Generally, biological materials such as cells and cell preparations are preserved at cryogenic temperatures for long periods of time (e.g., up to 1 to 2 years), thawed, and then used immediately in uses such as tissue regeneration, wound healing, and cellular therapy.

[0004] For example, the cell preparation DERMAGRAFT^® must be stored continuously at minus 65°C to minus 85°C in order to ensure cell viability, according to "DERMAGRAFT Directions for Use", Organogenesis Inc., 2014, herein incorporated by reference. This requirement presents challenges in shipping the cell preparation to the end user's facility from the manufacturer, and in subsequent storage of the cell preparation at the end user's facility. Special shipping services must be employed and expensive cryogenic freezers or liquid nitrogen containers must be on site at the end user's facility, such as hospitals, clinics, nursing facilities, or doctor's offices, in order to maintain the cell preparation at cryogenic temperatures. Once thawed, cell preparations are typically rinsed to remove any hazardous materials. By way of example, DERMAGRAFT^® must be immediately rinsed three times with saline solution to remove the hazardous cryoprotectant solution containing dimethyl sulfoxide (DMSO) from the cell preparation. After rinsing, cell preparations are typically used within 30 minutes to ensure the delivery of living cells to the patient's wound; otherwise, the preparations should be discarded. These requirements are restrictive and problematic for the end user, because if the directions are not followed properly, the cells in the cell preparation will die and the preparation will not be efficacious.

[0005] Additional problems that are associated with the biopreservation of cells and cell preparations include the use of hazardous materials such as DMSO. DMSO is commonly used as a cryoprotectant, but must be removed before the cells or cell preparation is used on humans because DMSO is irritating to the skin and mucous membranes. [0006] Hypothermic (cold storage) media solutions such as HYPOTHERMOSOL®

FRS available from BioLife Solutions, and PRIME-XV™ available from Irvine Scientific, are commercially available for the transport and short term storage of biological materials; however, these solutions are not suitable for cryopreservation. Thus, if a manufacturer of a cell preparation were to thaw the cell preparation prior to shipping so that hypothermic temperatures could be maintained during shipping and storage of the preparation at the end user's facilities, the manufacturer would have to remove the cryoprotectant medium from the cell preparation and add the hypothermic medium solution to the cell preparation before shipping. This process would present the opportunity for contamination of the preparation and potential loss of cell viability, plus this would entail additional handling costs at the manufacturer. An additional problem with hypothermic media solutions is that generally, the reculturability of cells (the ability of cells to regrow in culture) after storage at hypothermic conditions in hypothermic media solutions is poor.

SUMMARY OF THE INVENTION

[0007] One or more aspects of the present invention provides a solution to the aforementioned limitations and deficiencies in the art relating to biopreservation of biological materials. In one instance, the present invention provides for a biopreservation medium comprising a basal medium and at least one of a pan-oxygenase inhibitor, a sugar alcohol, an amino alcohol, an ionic metal halide, and/or a biguanide, or combinations thereof. This biopreservation medium can be used for each phase of a continuous biopreservation process, i.e., cryopreserving, thawing, and hypothermic storing of biological materials such as cells, cell preparations, biological tissues and/or organs carried out in succession. The biopreservation medium can provide for good viable cell recovery and cell reculturability of the cells subsequent to the biopreservation process. This eliminates the need to change the medium or open the vial/container between phases or change the medium after thawing, and thus facilitates the shipment and short term storage of biological materials plus reduces the chances for contamination. The biological materials can remain under hypothermic conditions for a period of time to allow for shipping and storing the biological materials at the end user's facility, such as a hospital, clinic, nursing facility, or doctor's office. It also eliminates the need for the end user to have a cryogenic freezer or liquid nitrogen on site. The end user only needs a refrigerator for storage of the thawed biological materials at hypothermic temperatures. Thus, in certain non-limiting instances, the end user can even be a patient at home administering self-treatment, where the biological material could be stored in a home refrigerator.

[0008] Additionally, the biopreservation medium of the invention does not necessarily need the inclusion of DMSO for cryopreservation. Thus, the biopreservation medium may include DMSO in some instances and may exclude DMSO in other instances. In one non-limiting aspect, the biopreservation medium of the invention has demonstrated an improvement of cell viability when used for cell preservation during continuous cryopreservation, thawing, and hypothermic storage conditions over commercially available conventional hypothermic storage mediums such as HYPOTHERMOSOL®.

[0009] Another advantage of the present invention is that only one medium is needed for all the phases of biopreservation, i.e. cryopreservation, thawing, and hypothermic storage, which is more economical than using a different medium for each phase. The biopreservation medium of the invention can be animal-origin free (AOF). The biopreservation medium of the invention can be ASA-free, i.e., antibiotic/antimycotic-free, serum-free, and animal-origin-free. [0010] Additionally, the biopreservation medium of the invention has been shown to improve the reculturability of cells after cryogenic storage followed by hypothermic conditions versus commercial hypothermic storage mediums such as HYPOTHERMOSOL. This improvement of reculturability of cells stored cryogenically followed by hypothermic conditions is particularly significant as reculturability of cells is generally poor with commercially sourced storage media.

[0011] In one aspect of the invention, there is disclosed a biopreservation medium compositions comprising a basal medium and at least one of a pan-oxygenase inhibitor, a sugar alcohol, an amino alcohol, an ionic metal halide, and/or a biguanide, or combinations thereof. In another aspect of the invention, there is disclosed a biopreservation medium comprising a basal medium and at least one pan-oxygenase inhibitor. In another aspect of the invention, there is disclosed a biopreservation medium comprising: (a) a basal medium and at least one pan-oxygenase inhibitor; and (b) at least one of a sugar alcohol, an amino alcohol, an ionic metal halide, and/or a biguanide; or combinations thereof.

[0012] In some embodiments, the pan-oxygenase inhibitor is IOX1 (5-carboxy-8- hydroxyquinoline) .

[0013] In some embodiments, the sugar alcohol is sorbitol or mannitol, or a combination thereof. In other embodiments, the sugar alcohol is sorbitol.

[0014] In some embodiments the amino alcohol is ethanolamine.

[0015] In some embodiments, the ionic metal halide salt is at least one of sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or combinations thereof. In other embodiments, the ionic metal halide salt is sodium chloride plus at least one of potassium chloride, calcium chloride, or magnesium chloride, or a combination thereof. In other embodiments, the ionic metal halide salt is potassium chloride, plus at least one of sodium chloride, calcium chloride, or magnesium chloride, or combinations thereof. In other embodiments, the ionic metal halide salt is sodium chloride. In other embodiments, the ionic metal halide salt is potassium chloride. In still other embodiments, the ionic metal halide is sodium chloride and potassium chloride.

[0016] In some embodiments, the biguanide is metformin, or the hydrochloride salt of metformin, i.e., metformin HC1.

[0017] In some embodiments, the biopreservation medium composition further comprises glycerol and/or other cryoprotectants. [0018] In one aspect of the invention, there is disclosed a method for a continuous process of cryopreserving, thawing, and subsequent hypothermic storing a cell preparation or a plurality of cells, the method comprising: a. combining the cell preparation or a plurality of cells with a biopreservation medium composition comprising a basal medium and at least one of a pan- oxygenase inhibitor, a sugar alcohol, an amino alcohol, an ionic metal halide, or a biguanide, or combinations thereof, to form a mixture;

b. cryopreserving the mixture;

c. thawing the mixture; and

d. storing the thawed mixture at hypothermic conditions.

[0019] In some embodiments, the biopreservation medium composition is not changed between steps a and b. In further embodiments, the biopreservation medium is not changed between steps c and d.

[0020] In some embodiments, the mixture is stored at cryogenic temperatures for at least 1 week prior to thawing. In some embodiments, the mixture is stored at hypothermic conditions for at least 1 week after thawing.

[0021] In some embodiments, the biopreservation medium comprises a basal medium and at least one pan-oxygenase inhibitor. In other embodiments, the biopreservation medium comprises (a) a basal medium and at least one pan-oxygenase inhibitor and (b) at least one of a sugar alcohol, an amino alcohol, an ionic metal halide, and/or a biguanide, or combinations thereof.

[0022] The terms "biopreservation", "biopreserving", "biopreserve", and

"biopreserved" relate to either one of, two of, or all three of the processes of cryopreservation, thawing, and/or hypothermic storage. A "continuous process" of biopreservation includes instances where all three of cryopreservation, thawing, and hypothermic storage are being performed.

[0023] The terms "cryogenic conditions", "cryogenic temperatures", or

"cryopreservation temperatures" mean temperatures at minus 20°C to minus 196°C.

[0024] The terms "hypothermic conditions", "hypothermic temperatures", "refrigeration temperatures", "refrigerated conditions" or "cold storage" mean temperatures at 0°C to 10 °C or 2°C to 8°C.

[0025] The term "about" or "approximately" are defined as being close to as understood by one of ordinary skill in the art, and in one non-limiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%. [0026] For purposes of this application, a number value with one or more decimal places can be rounded to the nearest whole number using standard rounding guidelines, i.e. round up if the number being rounded is 5, 6, 7, 8, or 9, and round down if the number being rounded is 0, 1, 2, 3, or 4. For example, 3.7 can be rounded to 4. [0027] Also disclosed in the context of the present invention are embodiments 1 to

34. Embodiment 1 is a method for a continuous process of cryopreserving, thawing, and subsequent hypothermic storing a cell preparation or a plurality of cells, the method comprising: (a) combining the cell preparation or a plurality of cells with a biopreservation medium composition comprising a basal medium and at least one pan-oxygenase inhibitor to form a mixture; (b) cryopreserving the mixture; (c) thawing the mixture; and (d) storing the thawed mixture at hypothermic conditions. Embodiment 2 is the method of embodiment 1, wherein the composition further comprises a sugar alcohol, an amino alcohol, an ionic metal halide, or a biguanide, or combinations thereof. Embodiment 3 is the method of embodiment 2, wherein the sugar alcohol is mannitol or sorbitol, or a combination thereof. Embodiment 4 is the method of embodiment 2, wherein the sugar alcohol is sorbitol. Embodiment 5 is the method of embodiment 2, wherein the amino alcohol is ethanolamine. Embodiment 6 is the method of embodiment 2, wherein the ionic metal halide is sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or a combination thereof. Embodiment 7 is the method of embodiment 2, wherein the ionic metal halide is sodium chloride. Embodiment 8 is the method of embodiment 2, wherein the ionic metal halide is potassium chloride. Embodiment 9 is the method of embodiment 2, wherein the biguanide is metformin HC1. Embodiment 10 is the method of any one of embodiments 1 to 9, wherein the biopreservation medium composition is not changed between steps a and b and between steps c and d. Embodiment 11 is the method of any of embodiments 1 to 10, wherein the mixture is stored at cryogenic temperatures for at least 1 week prior to thawing and at hypothermic conditions for at least 1 week after thawing. Embodiment 12 is the method of any of embodiments 1 to 11, wherein the pan-oxygenase inhibitor is IOX1 (5-carboxy-8- hydroxyquinoline). Embodiment 13 is the method of embodiment 12, wherein the IOX1 is at a concentration of at least 0.01% w/v. Embodiment 14 is the method of any of embodiments 1 to 13, wherein the plurality of cells comprises fibroblasts or keratinocytes, or combinations thereof. Embodiment 15 is the method of embodiment 14, wherein the fibroblasts or keratinocytes, or combinations thereof, are mitotically inactivated. Embodiment 16 is the method of any of embodiments 1 to 13, wherein the plurality of cells comprises stem cells. Embodiment 17 is the method of any of embodiments 1 to 13, wherein the cell preparation comprises a carrier and fibroblasts or keratinocytes, or combinations thereof. Embodiment 18 is the method of embodiment 17, wherein the fibroblasts or keratinocytes, or combinations thereof, are mitotically inactivated. Embodiment 19 is the method of any of embodiments 1 to 13, wherein the cell preparation comprises a carrier and stem cells. Embodiment 20 is the method of any of embodiments 1 to 19, wherein the percentage of viable cells in the mixture after hypothermic storage is at least 25% of the initial loading number of cells in the mixture prior to cryopreserving the mixture. Embodiment 21 is the method of any of embodiments 1 to 19, wherein the percentage of viable cells in the mixture after hypothermic storage is at least 55% of the initial loading number of cells in the mixture prior to cryopreserving the mixture. Embodiment 22 is the method of any of embodiments 1 to 19, wherein the percentage of viable cells in the mixture after hypothermic storage is at least 65% of the initial loading number of cells in the mixture prior to cryopreserving the mixture. Embodiment 23 is a biopreservation medium composition useful for cryopreserving, thawing, and hypothermic storing a cell preparation or a plurality of cells, the biopreservation medium comprising a basal medium and at least one pan-oxygenase inhibitors. Embodiment 24 is the composition of embodiment 23, wherein the composition further comprises a sugar alcohol, an amino alcohol, an ionic metal halide, or a biguanide, or combinations thereof. Embodiment 25 is the composition of embodiment 23 or 24, wherein the pan-oxygenase inhibitor is IOX1 (5-carboxy-8-hydroxyquinoline). Embodiment 26 is the composition of embodiment 25, wherein the IOX1 is at a concentration of at least 0.01% w/v. Embodiment

27 is the composition of embodiment 24, wherein the sugar alcohol is sorbitol. Embodiment

28 is the composition of embodiment 24, wherein the amino alcohol is ethanolamine. Embodiment 29 is the composition of embodiment 24, wherein the ionic metal halide is sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or combinations thereof. Embodiment 30 is the composition of embodiment 24, wherein the ionic metal halide is sodium chloride. Embodiment 31 is the composition of embodiment 24, wherein the ionic metal halide is potassium chloride. Embodiment 32 is the composition of embodiment 24, wherein the wherein the biguanide is metformin HC1. Embodiment 33 is a method for cryopreserving a cell preparation or a plurality of cells, the method comprising combining the cell preparation or a plurality of cells with a biopreservation medium composition comprising a basal medium and at least one pan-oxygenase inhibitor to form a mixture and cryopreserving the mixture. Embodiment 34 is the method of embodiment 33 further comprising thawing the mixture and storing the thawed mixture at hypothermic conditions.

[0028] The words "comprising" (and any form of comprising, such as "comprise" and

"comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0029] The use of the word "a" or "an" when used in conjunction with the terms

"comprising", "having", "including", or "containing" (or any variations of these words) may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0030] The compositions and methods for their use can "comprise," "consist essentially of," or "consist of any of the ingredients or steps disclosed throughout the specification. [0031] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0032] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1: Graph of re-check of cell counts at time 0 after initial loading of cells.

[0034] FIG. 2: Graph of cell counts after two-week storage at hypothermic conditions.

[0035] FIG. 3: Graph of cell counts after 1-week at cryogenic conditions followed by

1-week at hypothermic conditions. [0036] FIG. 4: Images of re-cultured cells after 2- weeks at hypothermic conditions. [0037] FIG. 5: Images of re-cultured cells after 1-week at cryogenic conditions followed by 1-week at hypothermic conditions.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention provides for a biopreservation medium composition useful for biopreservation of cells, cell preparations, biological tissues and/or organs at both cryogenic and hypothermic conditions.

A. Biopreservation Medium Compositions

[0039] In one aspect of the invention, there is disclosed a biopreservation medium that is suitable for the continuous biopreservation process comprising a cryopreservation phase, a thawing phase, and a hypothermic preservation phase (cold storage) of biological materials such as cells, cell preparations, biological tissues and/or organs. In some embodiments, the biopreservation medium composition of the present invention comprises a basal medium and at least one of a pan-oxygenase inhibitor, a sugar alcohol, an amino alcohol, an ionic metal halide, and/or a biguanide, or combinations thereof. In other embodiments, the biopreservation medium of the present invention comprises a basal medium and at least one pan-oxygenase inhibitor. In still other embodiments, the biopreservation medium of the present invention comprises (a) a basal medium and at least one pan-oxygenase inhibitor and (b) at least one of a sugar alcohol, an amino alcohol, an ionic metal halide, and/or a biguanide, or combinations thereof. The advantage of the biopreservation medium of the present invention is that it can be used for all phases of biopreservation including cryopreservation, thawing, and hypothermic preservation carried out in succession without the need to change the medium between phases.

[0040] In some embodiments, the pan-oxygenase inhibitor is IOX1 (5-carboxy-8- hydroxyquinoline) . [0041] In some embodiments, the sugar alcohol is sorbitol or mannitol, or a combination thereof. In other embodiments, the sugar alcohol is sorbitol.

[0042] In some embodiments the amino alcohol is ethanolamine.

[0043] In some embodiments, the ionic metal halide salt is at least one of sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or combinations thereof. In other embodiments, the ionic metal halide salt is sodium chloride plus at least one of potassium chloride, calcium chloride, or magnesium chloride, or combinations thereof. In other embodiments, the ionic metal halide salt is potassium chloride, plus at least one of sodium chloride, calcium chloride, or magnesium chloride, or combinations thereof. In other embodiments, the ionic metal halide salt is sodium chloride. In other embodiments, the ionic metal halide salt is potassium chloride. In still other embodiments, the ionic metal halide is sodium chloride and potassium chloride.

[0044] In some embodiments, the biguanide is metformin, or the hydrochloride salt of metformin, i.e., metformin HC1. [0045] In some embodiments, the biopreservation medium composition further comprises glycerol and/or other cryoprotectants.

[0046] Various cryoprotectants known in the art may be used in the biopreservation medium of the present invention. Glycerol, also known as glycerin, is a suitable cryoprotectant for use in the present invention. For use in an animal-origin free (AOF) biopreservation medium, a non-animal source of glycerol is preferred, such as a synthetic or vegetable source of glycerol both of which are widely available commercially. In some embodiments, the biopreservation medium comprises glycerol. In some embodiments, the biopreservation medium does not contain DMSO. In various embodiments, the concentration of the cryoprotectant in the biopreservation medium of the present invention can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25% v/v or any percentage derivable therein of the total composition weight.

[0047] The biopreservation medium of the present invention can be serum free and/or

AOF, i.e., substantially free from animal derived components. The biopreservation medium of the present invention can be antibiotic and/or antimycotic- free, i.e., substantially free from antibiotic and/or antimycotic agents. The biopreservation medium of the present invention can be ASA-free, i.e., substantially free from antibiotic/antimycotic agents, serums, and animal derived components.

[0048] The biopreservation medium of the present invention is suitable for use in each phase of biopreservation separately and independent of the other phases. For example, the biopreservation medium of the present invention can be used solely as a cryopreservation medium, or solely as a thawing medium, or solely as a hypothermic (cold storage) medium. In some embodiments, the biopreservation medium is used solely as a cryopreservation medium. In some embodiments, the biopreservation medium is used solely as a thawing medium. In some embodiments, the biopreservation medium is used solely as a hypothermic (cold storage) medium. In other embodiments, the biopreservation medium is used as the cryopreservation medium, the thawing medium, and the hypothermic (cold storage) medium.

[0049] The biopreservation medium of the present invention is suitable for the continuous biopreservation of cells, cell preparations, biological tissues or organs for long term storage at cryogenic conditions followed by short term storage at hypothermic conditions. In some embodiments, the storage time at cryogenic conditions is at least 1 week, or at least 1 month, or at least 2 months, or at least 3 months, or at least 6 months, or at least 1 year, or at least 2 years. In some embodiments, the storage time at hypothermic conditions is at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least 5 weeks, or at least 6 weeks.

[0050] The biopreservation medium of the present invention can be made by blending the pan-oxygenase inhibitor, a sugar alcohol, an amino alcohol, an ionic metal halide, and/or a biguanide with a basal medium until a homogenous mixture is obtained using mixing and blending techniques and equipment known in the art. Non-limiting examples of suitable mixing and blending equipment include lab mixers, lab shakers, lab stirrers, Lightnin' propeller-type mixers, Silverson rotor/stator homogenizers, and MorehouseCowles dissolvers. Use of aseptic techniques are desirable when producing the medium.

Pan-oxygenase Inhibitors

[0051] The biopreservation medium of the present invention can comprise one or more pan-oxygenase inhibitors, such as IOX1 (5-carboxy-8-hydroxyquinoline). Pan- oxygenase inhibitors, including 2-oxoglutarate (20G) oxygenase inhibitors, are agents that can induce expression of hypoxia-inducible factors (HIFs) in cells. It is known that cells stored at refrigerated (hypothermic) temperatures are still taking oxygen and are metabolically active. It is possible that hypothermically stored cells need to have a mechanism to fight oxygen shortage and be pro-active on hypoxia stress. Cells exposed to pan-oxygenase inhibitors are known to have induced HIFs and are protected under hypoxic conditions. For example, IOX1 is known to induce the HIF-Ια subunit of HIF-1. It was discovered that agents that induce expression of hypoxia-inducible factors (HIFs), such as the pan-oxygenase inhibitors IOX1, have a positive impact on cell survival in the hypothermic environment, and showed improvements, when combined with a basal medium, in the viability and re-culturability of cells after storage in cryogenic followed by hypothermic conditions versus the basal medium alone.

[0052] IOX1 (5-carboxy-8-hydroxyquinoline), is a broad spectrum, cell permeable inhibitor of 2-oxyglutarate (20G) oxygenase commercially available from SIGMA- ALDRICH. IOX1 has the formula as shown in Formula I:

Formula I

[0053] Derivatives of IOX1, including ester derivatives of IOX1 are also suitable pan- oxygenase inhibitors. Suitable ester derivatives of IOX1 are disclosed in "A Cell-Permeable Ester Derivative of the JmjC Histone Demethylase Inhibitor IOX1", Schiller et al., ChemMedChem 2014, 9, 566-571 herein incorporated by reference.

[0054] Other suitable pan-oxygenase inhibitors include, but are not limited to IOX2

(N- [[ 1 ,2-Dihydro-4-hydroxy-2-oxo- 1 -(phenylmethyl)-3 -quinolinyl]carbonyl] -glycine) and DMOG (dimethyloxalylglycine).

[0055] In some embodiments, the pan-oxygenase inhibitor is IOX1. The concentration of the IOX1 in the composition can be from 0.002% w/v (0.1% v/v of ImM solution of IOX1) to 0.01% w/v (0.5% v/v of a 1 mM solution of IOX1); or from 0.002% w/v to 0.02% w/v; or from 0.01% w/v to 0.02% w/v; or 0.002% w/v; or 0.003% w/v; or 0.004% w/v; or 0.005% w/v; or 0.006% w/v; or 0.007% w/v; or 0.008% w/v; or 0.009% w/v; or 0.01% w/v; or 0.011% w/v; or 0.012% w/v; or 0.013% w/v; or 0.014% w/v; or 0.015% w/v; or 0.016% w/v; or 0.017% w/v; or 0.018% w/v; or 0.019% w/v; or 0.02% w/v; or at least 0.002% w/v; or at least 0.01% w/v.

Sugar Alcohols [0056] Sugar alcohols, also called polyhydric alcohols, have the general formula as shown in Formula II and differ in chain length. Formula II HOCH₂(CHOH)_nCH₂OH

[0057] Suitable sugar alcohols include, but are not limited to sorbitol and mannitol.

In some embodiments, the sugar alcohol is sorbitol and/or mannitol, or combinations thereof. The mannitol can be D-mannitol. In other embodiments, the sugar alcohol is sorbitol. Concentrations of the total sugar alcohols in the compositions can generally be from about 0.05% to about 5% w/v. The concentration of mannitol can be from 0.1% w/v to 0.5% w/v; or can be 0.2% w/v; or can be at least 0.2% w/v. The concentration of sorbitol can be from 0.1%w/v to 0.5% w/v; or can be 0.2% w/v; or can be at least 0.2% w/v.

Amino Alcohols [0058] Amino alcohols are compounds that contain both amine and alcohol functional group. Suitable amino alcohols include, but are not limited to ethanolamine, diethanolamine, and triethanolamine. Concentrations of amino alcohols can generally be from about 0.0005% to about 0.1% w/v. In some embodiments, the amino alcohol is ethanolamine. The concentration of ethanolamine can be 0.001% w/v (0.2% v/v of a 100 mM solution of ethanolamine); or at least 0.001% w/v.

Ionic Metal Halides

[0059] Ionic metal halides are ionic compounds between metals and halogens.

Suitable ionic metal halides include, but are not limited to sodium chloride, potassium chloride, calcium chloride and its various hydrates such as calcium chloride dihydrate, magnesium chloride and its various hydrates such as magnesium chloride hexahydrate. In some embodiments, the ionic metal halide salt is at least one of sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or a combination thereof. In other embodiments, the ionic metal halide salt is sodium chloride plus at least one of potassium chloride, calcium chloride, or magnesium chloride, or combinations thereof. In other embodiments, the ionic metal halide salt is potassium chloride, plus at least one of sodium chloride, calcium chloride, or magnesium chloride, or combinations thereof. In other embodiments, the ionic metal halide salt is sodium chloride. In other embodiments, the ionic metal halide is potassium chloride. In still other embodiments, the ionic metal halide is sodium chloride and potassium chloride. Concentrations of the total ionic metal halides in the compositions can generally be from about 0.0005% to about 5% w/v. The concertation of sodium chloride can be from 0.05% w/v to 0.2% w/v, or from 0.06% w/v (1% v/v of a 1M NaCl solution) to 0.12% w/v (2% v/v of a IM NaCl solution); or can be 0.12% w/v; or can be at least 0.12% w/v. The concentration of potassium chloride can be from 0.03 to 0.2% w/v, or from 0.04% w/v (0.5% v/v of a IM KCl solution) to 0.19% w/v (2.5% v/v of a IM KCl solution); or can be 0.19% w/v; or can be at least 0.19% w/v. The concentration of calcium chloride dihydrate can be from 0.0005 to 0.001% w/v, or can be 0.0008% w/v (0.05% v/v of a lOmM calcium chloride dihydrate), or can be at least 0.0008% w/v. The concentration of magnesium chloride hexahydrate can be from 0.01 to 0.05% w/v, or can be 0.01% w/v (0.25% v/v of a 200mM solution of magnesium chloride hexahydrate), or can be at least 0.01% w/v. Biguanides

[0060] Biguanides have the general formula as shown in Formula III

Formula III HN(C(NH)NH₂)₂

[0061] Suitable biguanides include but are not limited to metformin (N,N- dimethylimidodicarbonimidic diamide) and its hydrochloride salt, metformin HC1. In some embodiments, the biguanide is metformin HC1. The concentration of metformin HC1 can be 0.003% w/v (0.1% v/v of 200mM solution of metformin HC1).

Basal Medium

[0062] Any basal medium compatible with the cells being preserved can be used in the present invention. The basal medium can be AOF and/or ASA-free. The basal medium can be obtained through commercial means from a manufacture or it can be manufactured in- situ. Components of the basal medium can be selected from various compounds known in the art for use in such mediums.

[0063] Suitable components of the basal medium can be selected from various compounds commercially available and generally known in the art for use in such mediums, such as vitamins, nutrients, amino acids, and/or organic salts. Non-limiting examples are glycerol (glycerin), dextran-40, sucrose, trehalose, lactobionic acid, D-mannitol, D-(-)- glucose, potassium chloride, potassium sulfate, tripotassium phosphate, 2-phospho-L- ascorbic acid trisodium salt, GIBCO® MEM amino acid solution, ROCK inhibitor Y-27632, potassium acetate, human albumin, dimethyl a-ketoglutarate, potassium carbonate, HEPES, calcium chloride dehydrate, magnesium chloride hexahydrate, glycine, L-alanine, L-arginine hydrochloride, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L- histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L- phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, choline chloride, D-pantothenic acid, folic acid, myo-inositol, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B 12, D-biotin, ammonium metavanadate, ammonium molybdate, calcium chloride, cupric sulfate, ferric sulfate, magnesium chloride, manganese sulfate, nickelous chloride, potassium chloride, potassium hydroxide, sodium bicarbonate, sodium chloride, sodium hydroxide, sodium meta silicate, sodium phosphate dibasic, sodium selenite, tin chloride, zinc sulfate, adenine HC1, D-glucose (dextrose), DL- alpha-lipoic acid, ethanolamine, O-phosphorylethanolamine, phenol red (optional), putrescine 2HC1, sodium pyruvate, thymidine, or water, or any combination thereof.

[0064] Various cryoprotectants known in the art, such as glycerol, may be used in the present invention. DMSO is widely used as a cryoprotectant of cells, but it is toxic (irritating) to the skin and mucous membranes; thus, DMSO is not typically considered a suitable cryoprotectant for use in biopreserving biological materials that would be used in direct contact with body tissues or used for organ transplants. Glycerol, also known as glycerin, is a suitable cryoprotectant for use in the present invention. For use in an AOF biopreservation medium, a non-animal source of glycerol is preferred, such as a synthetic or vegetable source of glycerol both of which are widely available commercially. In some embodiments, the biopreservation medium of the invention does not include DMSO.

[0065] Concentrations of the components of the basal medium can be based on the desired functionality of each component.

[0066] An exemplary basal medium suitable for inclusion in the biopreservation medium of the invention is shown in Table 1 below.

Table 1

Trehalose (50%) 0.40

Lactobionic acid (25%) 2.00

D-Mannitol (20%) 1.00

D-(-)-Glucose (250 niM) 1.00

Potassium chloride (1000 mM) 0.50

Potassium sulfate (200 mM) 0.05

Tripotassium phosphate (100 mM) 0.10

2-phospho-L-ascorbic acid trisodium salt (250 mM) 2.00

GIBCO® MEM Amino Acid Solution (50X) (no glutamine) 0.20

ROCK inhibitor Y-27632 (0.5 mM) 1.00

Potassium acetate (20 mM) 1.00

Human Albumin (2 mg/mL) 1.00

Dimethyl a-ketoglutarate (250 mM) 1.00

Potassium carbonate (1500 mM) 1.00

HEPES (pH 7.6) (1000 mM) 2.00

Calcium chloride dehydrate (10 mM) 0.05

Magnesium chloride hexahydrate (200 mM) 0.25

KOH (0.5 M) (as necessary for pH adjustment) qs to pH

Water for Injection qs ad 100%

B. Biopreservation Methods

[0067] In one aspect of the invention the biopreservation method of the invention relates to the cryopreservation, thawing, and/or hypothermic preservation (cold storage) of cells, cell preparations, biological tissues and organs, carried out in succession using the biopreservation medium composition of the invention without changing the biopreservation medium composition at any time throughout the biopreservation method. In another aspect of the invention, the biopreservation method of the invention relates to the cryopreservation of cells, cell preparations, biological tissues and organs using the biopreservation medium composition of the invention. In another aspect of the invention, the biopreservation method of the invention relates to the thawing of cells, cell preparations, biological tissues and organs from cryogenic conditions using the biopreservation medium composition of the invention. In another aspect of the invention, the biopreservation method of the invention relates to the hypothermic preservation (cold storage) of cells, cell preparations, biological tissues and organs using the biopreservation medium composition of the invention. [0068] In one aspect of the invention, there is disclosed a method for a continuous process of cryopreserving, thawing, and subsequent hypothermic storing a cell preparation or a plurality of cells, the method comprising: a. combining the cell preparation or a plurality of cells with a biopreservation medium composition comprising a basal medium, and at least one of a pan- oxygenase inhibitor, a sugar alcohol, an amino alcohol, an ionic metal halide, or a biguanide; or a combination thereof to form a mixture;

b. cryopreserving the mixture;

c. thawing the mixture; and

d. storing the thawed mixture at hypothermic conditions. [0069] In some embodiments, the biopreservation medium composition is not changed between steps a and b. In other embodiments, the biopreservation medium is not changed between steps c and d.

[0070] In some embodiments, the mixture is stored at cryogenic temperatures for at least 1 week prior to thawing. In some embodiments, the mixture is stored at hypothermic conditions for at least 1 week after thawing.

[0071] In some embodiments, the biopreservation medium comprises a basal medium and at least one pan-oxygenase inhibitor. In other embodiments, the biopreservation medium comprises (a) a basal medium and at least one pan-oxygenase inhibitor and (b) at least one of a sugar alcohol, an amino alcohol, an ionic metal halide, and/or a biguanide, or combinations thereof.

[0072] In some embodiments, the pan-oxygenase inhibitor is IOX1 (5-carboxy-8- hydroxyquinoline) .

[0073] In some embodiments, the sugar alcohol is sorbitol or mannitol, or a combination thereof. In other embodiments, the sugar alcohol is sorbitol. [0074] In some embodiments the amino alcohol is ethanolamine. [0075] In some embodiments, the ionic metal halide salt is sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or combinations thereof.

[0076] In some embodiments, the biguanide is metformin or the hydrochloride salt of metformin. [0077] In some embodiments, the ionic metal halide salt is at least one of sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or combinations thereof. In other embodiments, the ionic metal halide salt is sodium chloride plus at least one of potassium chloride, calcium chloride, or magnesium chloride, or combinations thereof. In other embodiments, the ionic metal halide salt is potassium chloride, plus at least one of sodium chloride, calcium chloride, or magnesium chloride, or combinations thereof. In other embodiments, the ionic metal halide salt is sodium chloride. In other embodiments, the ionic metal halide salt is potassium chloride. In still other embodiments, the ionic metal halide is sodium chloride and potassium chloride.

[0078] In some embodiments, the plurality of cells comprises fibroblasts or keratinocytes, or a combination thereof. In other embodiments the plurality of cells comprises fibroblasts or keratinocytes, or a combination thereof; wherein the fibroblasts or keratinocytes, or a combination thereof, are mitotically inactivated. In still other embodiments, the plurality of cells comprises stem cells.

[0079] In some embodiments, the cell preparation comprises a carrier; and fibroblasts or keratinocytes, or combinations thereof. In other embodiments, the cell preparation comprises a carrier; and fibroblasts or keratinocytes, or combinations thereof, wherein the fibroblasts or keratinocytes, or combinations thereof, are mitotically inactivated. In still other embodiments, the cell preparation comprises a carrier and stem cells.

[0080] In some embodiments, the percentage of viable cells from the plurality of cells or the cell preparation remaining after biopreservation (viable cell recovery) is at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75% of the initial loading number of cells prior to biopreservation. Cryopreservation

[0081] Cryopreservation techniques for cryopreserving cells, cell preparations, biological tissues and organs are known in the art. Generally, the cryopreservation temperatures are between minus 20°C to minus 196°C. Cryogenic freezers are commonly used for cryopreservation and are commercially available. The use of liquid nitrogen in storage tanks is another known technique for cryopreservation.

Thawing

[0082] Techniques for thawing biological materials from cryogenic conditions are known in the art. Care must be exercised during thawing to prevent damage or death to cells and/or tissues. Generally, water baths or electric heating devices are used for thawing cryopreserved biological materials. Other non-limiting examples of thawing devices include the THAWSTAR™ Automated Cryogenic Vial Thawing System available from BioCision LLC and the BARKEY® Plasmatherm available from Barkey GmbH & Co.

Hypothermic storage (Cold Storage) [0083] Techniques for hypothermic storage (cold storage) of biological materials are known in the art and include the transport and storage of the materials. Hypothermic conditions include temperatures at 0°C to 10 °C or 2°C to 8°C. Ordinary household or commercial grade refrigerators can be used for hypothermic storage. Care must be exercised during the transport of biological materials to prevent exposure of the materials to temperatures above hypothermic conditions. Biological materials can be shipped via refrigerated trucks or rail cars. Insulated shipping containers with cold packs can be used for shipment by mail or express delivery services. Ice chests with cold packs or ice can also be employed for transport in automobiles.

C. Cells and Cell Preparations Cell Types

[0084] The biopreservation medium of the present invention is suitable for the biopreservation of a variety of cell types including animal (mammalian and non-mammalian) and plant cells. The biopreservation medium of the present invention is suitable for the biopreservation of one cell, a plurality of cells which can be of one cell type or of mixed cell types, a population of cells of one cell type, or a population of mixed cells.

[0085] Non-limiting examples of mammalian animal cell types include fibroblasts, keratinocytes, dermal cells, melanocytes, hair cells, outer root sheath cells, epithelial cells, corneal epithelial cells, progenitor cells, stromal cells, lymphocytes including plasma cells, B cells, T cells, cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells, myeloid cells, granulocytes, basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes, hypersegmented neutrophils, monocytes, macrophages, reticulocytes, platelets, mast cells, thrombocytes, megakaryocytes, dendritic cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, oxyphil cells, adrenal cells, chromaffin cells, pineal cells, pinealocytes, glial cells, glioblasts, astrocytes, oligodendrocytes, microglial cells, magnocellular neurosecretory cells, stellate cells, boettcher cells, pituitary cells, gonadotropes, corticotropes, thyrotropes, somatotrope, lactotrophs, pneumocyte, type I pneumocytes, type II pneumocytes, Clara cells; goblet cells, alveolar macrophages, myocardiocytes, pericytes, gastric cells, gastric chief cells, parietal cells, goblet cells, paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells, enteroendocrine cells, enterochromaffin cells, APUD cell, liver cells, hepatocytes, Kupffer cells, bone cells, osteoblasts, osteocytes, osteoclast, odontoblasts, cementoblasts, ameloblasts, cartilage cells, chondroblasts, chondrocytes, trichocytes, nevus cells, muscle cells, neurons, nerve cells, neuroglia cells, myocytes, myoblasts, myotubes, adipocytes, tendon cells, podocytes, juxtaglomerular cells, intraglomerular mesangial cells, extraglomerular mesangial cells, kidney cells, macula densa cells, spermatozoa, Sertoli cells, leydig cells, oocytes, caco-2 cells, blood-derived stem cells, umbilical cord blood cells, mononuclear cells, differentiated cells, differentiated fibroblasts, mitotically active, mitotically inactive cells, irradiated cells, autologous cells, allogeneic cells, xenogeneic, isograft cells, allograft cells, xenograft cells, genetically engineered cells, immortalized cells, stem cells including mammalian and non- mammalian stem cells, or any combination thereof.

[0086] Non limiting examples of stem cells include adult stem cells, fetal stem cells, embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, totipotent stem cells, oligopotent stem cells, unipotent stem cells, mesenchymal stem cells, amniotic stem cells, umbilical cord blood stem cells, bone marrow stem cells, hematopoietic stem cells, endothelial stem cells, adipose stem cells, dental pulp stem cells, cancer stem cells, or any combination thereof.

[0087] In some embodiments, the biological material is a plurality of cells. In other embodiments, the plurality of cells comprises fibroblasts and/or keratinocytes. In other embodiments the plurality of cells comprises fibroblasts and/or keratinocytes, wherein the fibroblasts and/or keratinocytes are mitotically inactivated. In other embodiments, the plurality of cells comprises stem cells.

Cell Preparations

[0088] The biopreservation medium of the present invention is suitable for the biopreservation of cell preparations. Cell preparations are known in the art and are useful for a variety of medical and clinical uses including, but not limited to, tissue regeneration, wound healing, and cellular therapy. Cell preparations include, but are not limited to, cells in or on a carrier, substrate or matrix, a suspension of cells in a culture medium or carrier, skin equivalents, grafts, and/or skin substitutes. An example of a cell preparation is DERMAGRAFT®, available from Organogenesis, Inc. Other examples of cell preparations are the cell preparations disclosed in US patents US 6673603, US 7144729, US 7449333, US 7700351, US 7879605, US 8137965, US 8323638, and US 8679475 all of which are herein incorporated by reference.

[0089] In some embodiments, the biological material is a cell preparation. In other embodiments, the cell preparation comprises a carrier, fibroblasts, and/or keratinocytes. In other embodiments, the cell preparation comprises a carrier, fibroblasts, and/or keratinocytes, wherein the fibroblasts and/or keratinocytes are mitotically inactivated. In other embodiments, the cell preparation comprises a carrier and stem cells.

Cell Count and Cell Viability Determination [0090] Counts of cells and viable cells biopreserved in the biopreservation medium of the present invention may be determined with instruments and methods known in the art. A suitable instrument for determining viable cell counts is the Vi-CELL® XR Cell Viability Analyzer available from Beckman Coulter, Inc. The viable cell recovery after biopreservation can be expressed as a percentage of viable cells of the initial loading number of cells prior to biopreservation. D. Biological Tissues and Organs

[0091] The biopreservation medium of the present invention is suitable for the biopreservation of a variety of biological tissues and organs including animal (mammalian and non-mammalian) tissues and organs. Non-limiting examples of mammalian animal tissues include chondral tissue, cartilage, tendons, ligaments, vertebral discs, osteochondral tissue, islet tissue, osteogenic tissue, neural tissue, skin, mucous membranes, bone tissue, bone marrow, adipose tissue, muscle tissue, blood, corneas, lens, ocular tissue, meniscus, hair follicles, striated muscle, smooth muscle, cardiac muscle, connective tissue, blood vessels, functional spine units, or muscle-tendon grafts. Non-limiting examples of organs include heart, liver, or kidney.

EXAMPLES

[0092] The following examples are included to demonstrate certain non-limiting aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the applicants to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - Exemplary Formulas

[0093] The basal medium formula known as the "Core" formula was prepared and is shown in Table 2 below.

Table 2

D-(-)-Glucose (250 niM) 1.00

Potassium chloride (1000 mM) 0.50^B

Potassium sulfate (200 mM) 0.05

Tripotassium phosphate (100 mM) 0.10

2-phospho-L-ascorbic acid trisodium salt (250 mM) 2.00

GIBCO® MEM Amino Acid Solution (50X) (no glutamine) 0.20

ROCK inhibitor Y-27632 (0.5 mM) 1.00

Potassium acetate (20 mM) 1.00

Human Albumin (2 mg/mL) 1.00

Dimethyl a-ketoglutarate (250 mM) 1.00

Potassium carbonate (1500 mM) 1.00

HEPES (pH 7.6) (1000 mM) 2.00

Calcium chloride dihydrate (10 mM) 0.05

Magnesium chloride hexahydrate (200 mM) 0.25°

KOH (0.5 M) (pH adjustment) qs to pH

Water for Injection qs ad 100%

Equivalent to 0.20% w/v D-mannitol (100%)

Equivalent to 0.04% w/v potassium chloride

Equivalent to 0.0008% w/v calcium chloride dihydrate

^DEquivalent to 0.01% w/v magnesium chloride hexahydrate [0094] The pH of the "Core" formula was 7.66. The osmolality of the "Core" formula was 800.

[0095] The biopreservation medium formulas shown in Tables 3 and 4 below were prepared using the basal medium "Core" formula from Table 2. These formulas include the "Core" formula plus the listed ingredients. The biopreservation medium formula shown in Table 5 is a complete formula that includes a basal medium.

Table 3

D-(-)

Fructose 0.3 - - - - - - - - - -

(70%)

D-Sorbitol

- 0.2 - - - - - - - - - (100%)

Spermidine

- - 0.1 0.2 - - - - - - - (100 mM)

Potassium

Propionate - - - - 0.1 0.2 - - - - - (100 mM)

Sodium

Butyrate - - - - - - 0.1 0.2 - - - (100 mM)

Sodium

Lactate - - - - - - - - 0.1 0.2 - (100 mM)

Ethanolamine

- - - - - - - - - - 0.1 (100 mM)

q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad

Core

100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%

Table 4

Metformin

Hydrochloride - - - - - - - - - 0.1^L 0.2^M (200 mM)

q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad q.s.ad

Core

100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%

Equivalent to 0.001% w/v ethanolamine

FEquivalent to 0.002% w/v IOX1

GEquivalent to 0.01% w/v IOX1

Equivalent to 0.06% w/v sodium chloride

^IEquivalent to 0.12% w/v sodium chloride

^JTotal KCl (1000 mM) in Core+31 is 1.5% v/v equivalent to 0.12% w/v potassium chloride

^Total KCl (1000 mM) in Core+31x2 is 2.5% v/v equivalent to 0.19% w/v potassium chloride

^LEquivalent to 0.003% w/v metformin HC1

Equivalent to 0.007% w/v metformin HC1

Table 5 - Lot HBW7-18

Potassium chloride (1000 mM) 2.00^p

Sodium Chloride (1000 mM) 1.00^R

KOH (0.5 M) (pH adjustment) qs to pH

Water for Injection qs ad 100%

Equivalent to 0.1% w/v D-mannitol

pEquivalent to 1.15% w/v potassium chloride

Equivalent to 0.06% w/v sodium chloride

Example 2 -Cell Recovery in Biopreservation Medium Studies

[0096] Human keratinocytes were added to HYPOTHERMOSOL (HTS) and each of the formulas listed in Tables 3, 4, and 5 at an initial loading number of lxlO⁶ cells/mL. After the cells were loaded, the cell count of total and viable cells was re-checked at time 0 using a Vi-CELL XR cell viability analyzer and the results reported in FIG. 1. [0097] One set of samples was stored in a refrigerator at 2-8°C (hypothermic conditions) for 2 weeks. Another set of samples was stored in a freezer at minus 80°C (cryogenic conditions) for 1 week, thawed for 2 minutes at 37°C, and then stored in a refrigerator at 2-8°C (hypothermic conditions) for 1 week. After storage, the cell count of total and viable cells was then determined for each sample using a Vi-CELL XR cell viability analyzer and the results reported in FIG. 2 (hypothermic conditions) and FIG. 3 (cryogenic plus hypothermic conditions). The total cell counts and the viable cell counts are reported in each figure.

[0098] Table 6 reports the % viable cell recovery for samples stored at 1-week cryogenic plus 1-week hypothermic conditions expressed as a percentage of viable cells of the initial loading number of cells.

Table 6 - Viable cell recovery after 1-week Cryo+ 1-week Hypothermic

Core+7 lxlO⁶ 6.00x10^s 42.2%

Core+27x2 lxlO⁶ 6.00x10^s 42.8

Core+28x5 lxlO⁶ 6.60x10^s 66%

Core+30x2 lxlO⁶ 6.90x10^s 50.3%

Core+31x2 lxlO⁶ 7.20x10^s 53.5%

Core+32 lxlO⁶ 6.10x10^s 49.1

[0099] As can be seen in the results in Table 6 and FIG. 3, the viable cell counts of formulas Core+7, Core+27x2, Core+28x5, Core+30x2, Core+31x2, and Core+32 after storage at 1-week cryogenic plus 1-week hypothermic conditions are higher than the viable cell counts of the basal medium formula alone and HTS under the same storage conditions.

Example 3 - Re-culture of Cells Post Cryogenic and Hypothermic Storage

[00100] The set of samples of cells stored for 2-weeks at hypothermic conditions and the set of samples of cells stored for 1-week at cryogenic conditions followed by 1-week at hypothermic storage conditions from Example 2 were re-cultured after storage and assessed for growth qualitatively. Aliquots (50 μί) of cells in suspension were seeded in duplicate in 6-well plates. Whenever any condition reached confluence, the culture was stopped for all of the test conditions from the same storage condition. Cells were fixed with 10% Formalin and stained with Acid Fuchsin. Images of the stained plates are shown in FIG. 4 (after two-week hypothermic conditions) and in FIG. 5 (after 1-week cryogenic conditions plus 1-week hypothermic conditions). As can be seen by the images in FIG. 5, the reculturability of cells in formulas Core+7, Core+27x2, Core+28x5, Core+30x2, Core+31x2, and Core+32 was significantly better than HTS, which showed virtually no cell growth.

Claims

1. A method for a continuous process of cryopreserving, thawing, and subsequent hypothermic storing a cell preparation or a plurality of cells, the method comprising: a. combining the cell preparation or a plurality of cells with a biopreservation medium composition comprising a basal medium and at least one pan-oxygenase inhibitor to form a mixture; b. cryopreserving the mixture; c. thawing the mixture; and d. storing the thawed mixture at hypothermic conditions.

2. The method of claim 1, wherein the composition further comprises a sugar alcohol, an amino alcohol, an ionic metal halide, or a biguanide, or a combination thereof.

3. The method of claim 2, wherein the sugar alcohol is mannitol or sorbitol, or a combination thereof.

4. The method of claim 2, wherein the amino alcohol is ethanolamine.

5. The method of claim 2, wherein the ionic metal halide is sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or a combination thereof.

6. The method of claim 2, wherein the biguanide is metformin HC1.

7. The method of any one of claims 1 to 6, wherein the biopreservation medium composition is not changed between steps a and b and between steps c and d.

8. The method of any one of claims 1 to 7, wherein the mixture is stored at cryogenic temperatures for at least 1 week prior to thawing and at hypothermic conditions for at least 1 week after thawing.

9. The method of any of claims 1 to 8, wherein the pan-oxygenase inhibitor is IOX1 (5- carboxy- 8-hydroxyquinoline) .

10. The method of claim 9, wherein the IOX1 is at a concentration of 0.01% w/v - 0.02% w/v.

11. The method of any of claims 1 to 10, wherein the percentage of viable cells in the mixture after hypothermic storage is at least 25% of the initial loading number of cells in the mixture prior to cryopreserving the mixture.

12. A biopreservation medium composition useful for cryopreserving, thawing, and hypothermic storing a cell preparation or a plurality of cells, the biopreservation medium comprising a basal medium and at least one pan-oxygenase inhibitor.

13. The composition of claim 12, wherein the composition further comprises a sugar alcohol, an amino alcohol, an ionic metal halide, or a biguanide, or a combination thereof.

14. The composition of claim 12 or 13, wherein the pan-oxygenase inhibitor is IOX1 (5- carboxy- 8-hydroxyquinoline) .

15. The composition of claim 14, wherein the IOX1 is at a concentration of 0.01% w/v - 0.02% w/v.

16. The composition of claim 13, wherein the sugar alcohol is mannitol or sorbitol, or a combination thereof.

17. The composition of claim 13, wherein the amino alcohol is ethanolamine.

18. The composition of claim 13, wherein the ionic metal halide is sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or a combination thereof.

19. The composition of claim 13, wherein the wherein the biguanide is metformin HC1.

20. A method for cryopreserving a cell preparation or a plurality of cells, the method comprising combining the cell preparation or a plurality of cells with a biopreservation medium composition comprising a basal medium and at least one pan-oxygenase inhibitor to form a mixture and cryopreserving the mixture.

21. The method of claim 20, further comprising thawing the mixture and storing the thawed mixture at hypothermic conditions.