WO2025019671A1 - Cryopreservation formulations - Google Patents

Abstract

The disclosure provides for cryopreservation formulations, cryopreservation media and method of cryopreserving mammalian cells or tissues. The cryopreservation formulations and media contain a combination of cryoprotective agents selected from the group consisting of; (a) glycerol and trehalose; (b) glycerol, trehalose, and ascorbic acid; (c) glycerol, trehalose, and taurine: and (d) glycerol, trehalose, ascorbic acid, and taurine. In certain embodiments, the cryopreservation formulations and media are free of dimethyl sulfoxide.

Description

CRYOPRESERVATION FORMULATIONS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/527,462, filed July 18, 2023, the disclosure of which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] This disclosure relates to cryopreservation of cells, in particular mammalian cells and solid tissues. Specifically, this disclosure relates to cryopreservation formulations and media, as well as methods of cry opreserving cells.

BACKGROUND OF THE INVENTION

[0003] The preservation of mammalian cells and solid tissues in liquid nitrogen storage is an essential step in cell culture labs as it provides the ability to bank precious samples for use in downstream studies/applications. The nutrient rich media utilized during this freezing process has a substantial impact on both the overall survival and performance of cells post sample thawing.

[0004] The freezing process (also known as cry opreservation) typically uses a cell culture medium in which the mammalian cells to be stored (cryopreserved) can grow and a cryoprotectant agent that preserves the cells’ function and viability during freezing.

[0005] The current “gold standard’ for cry opreservation requires use of dimethyl sulfoxide (DMSO) as the cryoprotectant agent. While DMSO is the gold standard, research has also shown it may be detrimental to the cryopreserved cells. For example, Galvao et al. “demonstrated that DMSO induces retinal apoptosis in vivo at low concentrations (5 pl intravitreally dosed DMSO in rat from a stock concentration of 1, 2, 4, and 8% v/v).” Galvao et al., FASEB J. 28(3): 1317-30 (2014) at Abstract. Similarly, a 2020 review by Awan et al. compiled data showing many of the downsides of using DMSO and highlights the need for DMSO alternatives. Awan c/ al. Regenerative Medicine, 15(3): 1463-1491 (2020).

[0006] In view of the downsides of using DMSO, DMSO-free cry opreservation media have been developed for cryopreservation of cells. One example is the XT-Thive® cry opreservation which uses N-substituted biomimetic amino acid polymers (peptoids) and peptoid-peptide hybrids as substitute for DMSO. However, generally speaking, peptoids are synthetic molecules with the potential to bind/target proteins, thus, they need extensive evaluation for each application.

[0007] What is needed are cry opreservation solutions and media that overcome the potential downsides of using DMSO and the currently available DMSO-free media and that are useful for cryopreservation of cells and bulk tissues.

SUMMARY OF THE INVENTION

[0008] The disclosure provides for cryopreservation formulations, cryopreservation media, and methods of cryopreserving mammalian cells or bulk tissues. The cryopreservation formulations and media contain a combination of cryoprotective agents selected from the group consisting of: (a) glycerol and trehalose; (b) glycerol, trehalose, and ascorbic acid; (c) glycerol, trehalose, and taurine; and (d) glycerol, trehalose, ascorbic acid, and taurine. In certain embodiments, the cry opreservation formulations and media are free of dimethyl sulfoxide. In other embodiments, the cryopreservation formulations and media do not contain an additional cryoprotective agent.

[0009] One embodiment of the disclosure is cryopreservation formulation for cry opreserving mammalian cells containing a combination of cryoprotective agents selected from the group consisting of: (a) glycerol and trehalose; (b) glycerol, trehalose, and ascorbic acid; (c) glycerol, trehalose, and taurine; and (d) glycerol, trehalose, ascorbic acid, and taurine. These formulations can be added to a cell culture medium to generate a cryopreservation medium. [0010] In certain embodiments, the cry opreservation formulations contain a combination of cryoprotective agents selected from the group consisting of: (a) from about 20% to about 60% of glycerol and from about 200 mM to about 3 M or from about 200 mM to about 600 mM of trehalose; (b) from about 20% to about 60% of glycerol, from about 200 mM to about 3 M or from about 200 mM to about 600 mM of trehalose, and from about 100 pg/mL to about 250 pg/mL of ascorbic acid; (c) from about 20% to about 60% of glycerol, from about 200 mM to about 3 M or from about 200 mM to about 600 mM of trehalose, and from about 10 mM to about 40 mM of taurine; and (d) from about 20% to about 60% of glycerol, from about 200 mM to about 3 M or from about 200 mM to about 600 mM of trehalose, from about 100 pg/mL to about 250 pg/mL of ascorbic acid, and from about 10 mM to about 40 mM of taurine.

[0011] The cryopreservation formulation can also contain serum (e.g., FBS), for example, in amounts from about 40% to about 75%. In addition, the cry opreservation formulation can contain glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and/or L-serine. In certain embodiments, the formulations contain from about 0.1% to about 2.5% of glycine, from about 0.1% to about 2.5% of L-alanine, from about 0.1% to about 2.5% of L-asparagine, from about 0.1% to about 2.5% of L-aspartic acid, from about 0. 1% to about 2.5% of L-glutamic acid, from about 0.1% to about 2.5% of L-proline, and/or from about 0.1% to about 2.5% of L-serine.

[0012] Another aspect of the disclosure is a cry opreservation medium for cry opreserving mammalian cells containing: a sufficient amount of amino acids, glucose, and ions to allow mammalian cells to grow; and a combination of cryoprotective agents selected from the group consisting of: (a) glycerol and trehalose; (b) glycerol, trehalose, and ascorbic acid; (c) glycerol, trehalose, and taurine; and (d) glycerol, trehalose, ascorbic acid, and taurine.

[0013] In one embodiment, the cry opreservation medium contains a combination of cryoprotective agents selected from the group consisting of: (a) from about 10% to about 40% of glycerol and from about 100 mM to about 3 M of trehalose; (b) from about 10% to about 40% of glycerol, from about 100 mM to about 3 M of trehalose, and from about 20 pg/mL to about 250 pg/mL of ascorbic acid; (c) from about 10% to about 40% of glycerol, from about 100 mM to about 3 M of trehalose, and taurine; and (d) from about 10% to about 40% of glycerol, from about 100 mM to about 3 M of trehalose, from about 20 pg/mL to about 250 pg/mL of ascorbic acid, and from about 5 mM to about 30 mM of taurine. In another embodiment, the cryopreservation media contain serum (e.g., FBS), such as, for example, from about 40% to about 75% of FBS. In a further embodiment, the cryopreservation medium contains glycine, L-alanine, L-asparagine, L-aspartic acid, L- glutamic acid, L-proline, and/or L-serine.

[0014] The disclosure also provides for methods of generating the cry' opreservation media and method of cry opreserving mammalian cells and/or tissues. In addition, the disclosure includes kit containing the cry opreservation formulations or media.

[0015] Other features and advantages of the invention will be apparent from the detailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The foregoing summary⁷, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended figures. For illustrating the invention, the figures demonstrate embodiments of the present invention. It should be understood, however, that the invention is not limited to the precise arrangements, examples, and instrumentalities shown.

[0017] FIG. 1A and FIG. IB show schematics of using the cry o-media formulations of the disclosure.

[0018] FIGS. 2A-2D show the results of cryopreserving placental issue frozen using the cry opreservation formulations of the disclosure when compared to commercially available cry opreservation media. FIGS. 2A and 2B show the results for the formulations for the disclosure. FIGS. 2C and 2D show the results for cells cryopreserved in 10% DMSO/ 40% FBS/50% EGM-2-XAP.

[0019] FIG. 3A and FIG 3B show the results of cry opreserving sensitive cell lines (Elephant endometrial cells) using a cry opreservation formulation of the disclosure (identified as Xo- Chill (XC)) when compared to a traditional cry opreservation media formulation.

[0020] FIGS. 4A-4D show the results of cryopreserving bovine embryonic stem cells using cry opreservation formulations of the disclosure when compared to the commercially available mFreSR media. FIGS. 4A-4C show pictures of cells cryopreserved in XC + bESCM, XC + KSR. and mFreSR, respectively. The picture was taken 48 hours after seeding (cryopreservation). FIG. 4D shows the percentage cell viability and normalized viability after cryopreservation.

[0021] FIGS. 5A-5D show the results of cryopreserving human organoids using a cry opreservation formulation of the disclosure when compared to the commercially available Crystor CS 10 media. FIG. 5 A show-s a picture of human organoids pre-incubation with XC cryomedia of the disclosure and CryoStor CS10 (CS). FIG. 5B shows a picture of human organoids post-incubation with XC cryomedia of the disclosure and CryoStor CS10. FIG. 5C shows a picture of the cells after thawing and culture in organoid maintenance medium (OMM) for twenty-four hours. FIG. 5D show's the cell contractility' (beating organoids %) for cells after thawing that were cryopreserved in XC+ OMM and CS10.

DETAILED DESCRIPTION

[0022] The disclosure provides cryopreservation media, formulations, and methods of cry opreserving mammalian cells. This disclosure is based on the discovery it is possible to achieve the cryoprotective effect of DMSO and FBS using combination of the follow ing cryoprotective agents: at least glycerol and trehalose; or glycerol and trehalose, as well as ascorbic acid, and/or taurine.

[0023] The cry opreservation media and formulations of the disclosure contain at least glycerol and trehalose. The cryopreservation media and formulations may be supplemented with serum, ascorbic acid, and/or taurine.

[0024] In certain embodiments, the cryopreservation media and formulations are free of DMSO, i.e., the cry opreservation media and formulations do not contain any DMSO. In other embodiments, the cry opreservation media and formulations do not contain synthetic molecules (such as e.g., peptoids) with the potential to bind/target proteins.

[0025] The cryopreservation formulations are designed to be added to cell culture media. In certain embodiments, the cry opreservation media are basal media to which the cryopreservation formulation has been added.

[0026] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into subsections that describe or illustrate certain features, embodiments, or applications of the present invention.

Definitions

[0027] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0028] As used herein, the terms “comprising,” “including.” “containing” and “characterized by” are exchangeable, inclusive, open-ended and do not exclude additional, unrecited elements or method steps. Any recitation herein of the term “comprising,” particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements.

[0029] As used herein, the term “consisting of’ excludes any element, step, or ingredient not specified in the claim element.

[0030] As used herein, the term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ± 20% or ± 10%, more preferably ± 5%, even more preferably ± 1%, and still more preferably ± 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. [0031] Before certain embodiments are described in greater detail, it is to be understood that this invention is not limited to certain embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing certain embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0032] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods, and materials are now described.

[0034] As used herein, the term "transform’⁷ or "transformation” refers to the transfer of a nucleic acid fragment into a host cell, such as a host bacterial cell, resulting in genetically stable inheritance. Host cells comprising the transformed nucleic acid fragment are referred to as “recombinant” or “transgenic” or “transformed” organisms.

[0035] As used herein, the term “isolated” means a biological component (such as a nucleic acid, peptide, or protein) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins.

Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. “Isolated” nucleic acids, peptides and proteins can be part of a composition and still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0036] As used herein, “gene” refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.

[0037] As used herein, a “promoter” is an example of a transcriptional regulatory sequence and is specifically a nucleic acid sequence generally described as the proximal region of a gene located 5' to the start codon. The transcription of an adjacent nucleic acid segment is initiated at the promoter region. A repressible promoter's rate of transcription decreases in response to a repressing agent. An inducible promoter's rate of transcription increases in response to an inducing agent. A constitutive promoter's rate of transcription is not specifically regulated, though it can vary under the influence of general metabolic conditions.

[0038] The term “gene product,” as used herein, refers to any product encoded by a nucleic acid sequence. Accordingly, a gene product may. for example, be a primary transcript, a mature transcript, a processed transcript, or a protein or peptide encoded by a transcript. Examples for gene products, accordingly, include mRNAs, rRNAs, hairpin RNAs (e.g., microRNAs, shRNAs, siRNAs, tRNAs), and peptides and proteins, for example, reporter proteins or therapeutic proteins.

[0039] As used herein, the term “stem cell” refers to a cell that can self-renew and differentiate to at least one more differentiated or less developmentally capable phenotype. The term “stem cell” encompasses stem cell lines, induced stem cells, non-human embryonic stem cells, pluripotent stem cells, multipotent stem cells, amniotic stem cells, placental stem cells, or adult stem cells. An “induced stem cell” is one derived from a non-pluripotent cell induced to a less differentiated or more developmentally capable phenotype by introduction of one or more reprogramming factors or genes. As the term is used herein, an induced stem cell need not be pluripotent, but has the capacity to differentiate, under appropriate conditions, to more than one more-highly-differentiated phenotype. It should be understood that the capacity was not present prior to the introduction of reprogramming factors. An induced stem cell will express at least one stem cell marker not expressed by the parent cell prior to introduction of reprogramming factors. In this context, a stem cell marker is exclusive of a factor introduced by reprogramming. An induced pluripotent stem cell, or iPS cell, has the induced capacity to differentiate, under appropriate conditions, to a cell phenotype derived from each of the endoderm, mesoderm, and ectoderm germ layers.

[0040] The term “marker” as used herein is used to describe a characteristic and/or phenotype of a cell. Markers can be used for selection of cells comprising characteristics of interest and can vary with specific cells. Markers are characteristics, whether morphological, structural, functional, or biochemical (enzymatic) characteristics of the cell of a particular cell ty pe, or molecules expressed by the cell ty pe. In one aspect, such markers are proteins. Such proteins can possess an epitope for antibodies or other binding molecules available in the art. However, a marker can consist of any molecule found in or on a cell, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological characteristics or traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to particular substrates, ability to incorporate or exclude particular dyes, ability to migrate under particular conditions, and the ability to differentiate along particular lineages. Markers can be detected by any method available to one of skill in the art. Markers can also be the absence of a morphological characteristic or absence of proteins, lipids etc. Markers can be a combination of a panel of unique characteristics of the presence and/or absence of polypeptides and other morphological or structural characteristics. In one embodiment, the marker is a cell surface marker.

[0041] The term “exogenous” refers to a substance present in a cell that was introduced by the hand of man. The term “exogenous” when used herein can refer to a nucleic acid (e.g. , a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively lower amounts and in which one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels.

[0042] As used herein, the term “reprogramming genes” or “reprogramming factors” refers to agents or nucleic acid molecules that can induce the reprogramming process in a somatic cell to re-express a less-differentiated, more stem-cell like phenot pe. The reprogramming factor can be a nucleic acid, a polypeptide, or a small molecule that promotes a reprogrammed phenotype when introduced to a cell. Non-limiting examples of reprogramming factors include: Oct4 (Octamer binding transcription factor-4), SOX2 (Sex determining region Y)- box 2, Klf4 (Kruppel Like Factor-4), and c-Myc. These are the so-called “classical” or “standard” set of reprogramming factors used to derive, for example, induced pluripotent stem cells. Additional factors that can be considered reprogramming factors when introduced in the process of reprogramming cells to a less differentiated or stem cell phenotype include LIN28 + Nanog, Esrrb, Pax5 shRNA, C/EBPa, p53 siRNA, UTF1, DNMT shRNA, Wnt3a, SV40 LT(T), hTERT, small molecule chemical agents including, but not limited to BIX- 01294, BayK8644, RG108, AZA, dexamethasone, VP A, TSA, SAHA, PD0325901 + CHIR99021(2i) and A-83-01. In some embodiments, the reprogramming genes or factors are Oct4, Klf4, SOX2, and c-Myc. [0043] As used herein, the terms ‘‘dedifferentiation'’ or “retrodifferentiation’' or “reprogramming” refer to a process that generates a cell that re-expresses a less differentiated phenotype than the cell from which it is derived and/or expresses at least one stem cell marker not expressed prior to that process. For example, a terminally differentiated cell can be dedifferentiated to a multipotent cell. That is, dedifferentiation shifts a cell backward along the differentiation spectrum of totipotent cells to fully differentiated cells. Typically, reversal of the differentiation phenotype of a cell requires artificial manipulation of the cell, for example, by introducing or expressing exogenous polypeptide factors. Reprogramming is not typically observed under native conditions in vivo or in vitro.

[0044] As used herein, a “reprogrammed cell” is a cell that has been contacted with one or more reprogramming factors and expresses a less differentiated phenotype than the cell from which it was derived. The reprogrammed cell can also have the capacity to self-renew and will express at least one stem cell marker that was not delivered to the cell as a reprogramming factor. Furthermore, the reprogrammed cell will have the capacity to differentiate into a more-differentiated somatic cell type following differentiation protocols provided herein or described in the art.

[0045] As used herein, the term “somatic cell” refers to any cell other than a germ cell, a cell present in or obtained from a pre-implantation embryo, or a cell resulting from proliferation of such a cell in vitro. Stated another way, a somatic cell refers to any cells forming the body of an organism, excluding germ cells. Every cell type in the mammalian body-apart from the sperm and ova and the cells from which they are made (gametocytes)-is a somatic cell: internal organs, skin, bones, blood, and connective tissue are all substantially made up of somatic cells. In some embodiments the somatic cell is a “non-embryonic somatic cell,” by which is meant a somatic cell that is not present in or obtained from an embryo and does not result from proliferation of such a cell in vitro. In some embodiments the somatic cell is an “adult somatic cell,” by which is meant a cell that is present in or obtained from an organism other than an embry o or a fetus or results from proliferation of such a cell in vitro.

[0046] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. [0047] It is noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0048] Each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

Examples of suitable mammalian cells and tissues

[0049] The cry opreservation formulations and media of the disclosure are suitable for cry opreservation of any mammalian cell or tissue. In certain embodiments, the cry opreservation formulations and media of the disclosure can be used to cry opreserve elephant cells or tissues, including elephant progenitor cells. The elephant cell can, for example, be selected from an Asian elephant cell (Elephas maximus). an African elephant cell (Loxodonta africana), an African forest elephant cell (Loxodonta cyclotis), and a Bornean elephant cell (Elephas maximus borneensis).

[0050] In particular, the cell culture formulations and media of the disclosure are suitable for mammalian cells or tissues that have been genetically engineered (e. , transformed) or reprogrammed. In certain embodiments, the cell culture formulations and media of the disclosure are suitable for cry opreserving stem cells or progenitor cells, including but not limited to cells obtained by dedifferentiation or retrodifferentiation, reprogrammed cells, and somatic cells. In other embodiments, the cell culture formulations and media of the disclosure are suitable for cry opreserving tissues obtained from such stem cells or progenitor cells.

Cryo reservation Formulations

[0051] The cryopreservation formulations of the disclosure contain glycerol and trehalose as cryoprotective agents. In certain embodiments, the cryopreservation solutions contain glycerol, trehalose, ascorbic acid, and/or taurine as cryoprotective agents. In alternate embodiments, the cryopreservation solutions contain glycerol, trehalose, ascorbic acid, and taurine as cryoprotective agents. In certain embodiments, the cryopreservation formulations do not contain DMSO and/or any additional cryoprotective agent.

[0052] In certain embodiments, the formulations also contain glycine, L-alanine, L- asparagine. L-aspartic acid, L-glutamic acid, L-proline, and/or L-serine. In alternate embodiments, cryopreservation formulations are supplemented with serum, such as e.g., fetal bovine serum.

[0053] In certain embodiments, the cry opreservation formulations are free of DMSO, i.e., they do not contain any DMSO.

[0054] In one embodiment, the cry opreservation formulation contains glycerol and trehalose. In certain embodiments, the cryopreservation formulation contains from about 20% to about 60%, alternatively from about 30% to about 50%, alternatively from about 35% to about 45% of glycerol. In other embodiments, the cryopreservation formulation contains from about 200 mM to about 3 mM, alternatively from about 300 mM to about 3 mM, alternatively from about 200 mM to about 600 mM, alternatively from about 300 mM to about 500 mM, alternatively from about 350 mM to about 450 mM, alternatively from about 1 mM to about 3 mM, alternatively from about 1.5 mM to about 2.5 mM of trehalose. In other embodiments, the cryopreservation formulation contains from about 20% to about 60%. alternatively from about 30% to about 50%, alternatively from about 35% to about 45% of glycerol and from about 200 mM to about 3 mM, alternatively from about 300 mM to about 3 mM, alternatively from about 200 mM to about 600 mM, alternatively from about 300 mM to about 500 mM, alternatively from about 350 mM to about 450 mM, alternatively from about 1 mM to about 3 mM, alternatively from about 1.5 mM to about 2.5 mM of trehalose.

[0055] In another embodiment, the cryopreservation formulation also contains ascorbic acid and/or taurine. Accordingly, in certain embodiments, the cry opreservation formulation contains glycerol, trehalose, and ascorbic acid. In other embodiments, the cry opreservation formulation contains glycerol, trehalose, and taurine. In alternate embodiments, the cry opreservation formulation contains glycerol, trehalose, ascorbic acid, and taurine. In certain embodiments, the cry opreservation formulation contains from about 100 pg/mL to about 250 pg/mL, alternatively from about 100 pg/mL to about 200 pg/mL, alternatively from about 120 pg/mL to about 180 pg/mL of ascorbic acid. In other embodiments, the cry opreservation formulation contains from about 10 mM to about 40 mM. alternatively from about 15 mM to about 35 mM, alternatively from about 20 mM to about 35 mM, alternatively from about 25 mM to about 35 mM of taurine. In alternate embodiments, the cry opreservation formulation contains from about 100 pg/mL to about 250 pg/mL. alternatively from about 100 pg/mL to about 200 pg/mL, alternatively from about 120 pg/mL to about 180 pg/mL of ascorbic acid and from about 10 mM to about 40 mM, alternatively from about 15 mM to about 35 mM, alternatively from about 20 mM to about 35 mM, alternatively from about 25 mM to about 35 mM of taurine.

[0056] In an alternate embodiment, the cry opreservation formulation contains: (a) from about 20% to about 60%, alternatively from about 30% to about 50%, alternatively from about 35% to about 45% of glycerol; (b) from about 200 mM to about 3 mM, alternatively from about 300 mM to about 3 mM, alternatively from about 200 mM to about 600 mM, alternatively from about 300 mM to about 500 mM, alternatively from about 350 mM to about 450 mM, alternatively from about 1 mM to about 3 mM, alternatively from about 1.5 mM to about 2.5 mM of trehalose; (c) from about 100 pg/mL to about 250 pg/mL, alternatively from about 100 pg/mL to about 200 pg/mL, alternatively from about 120 pg/mL to about 180 pg/mL of ascorbic acid; and (d) from about 10 mM to about 40 mM, alternatively from about 15 mM to about 35 mM, alternatively from about 20 mM to about 35 mM, alternatively from about 25 mM to about 35 mM of taurine.

[0057] In alternate embodiments, the cryopreservation formulation also contains glycine, L- alanine, L-asparagine, L-aspartic acid. L-glutamic acid, L-proline, and/or L-serine. In certain embodiments, the cryopreservation solution contains glycine. L-alanine, L-asparagine. L- aspartic acid, L-glutamic acid, L-proline, and L-serine. In some embodiments, the glycine, L-alanine, L-asparagine, L-glutamic acid, L-proline, and L-serine are provided as a commercially available Non-Essential Amino Acid (NEAA) cell culture supplement. In certain embodiments, NEAA cell culture supplement comprises about 750 mg/L of glycine, about 890 mg/L of L-alanine, about 1320 mg/L of L-asparagine, about 1330 mg/L of aspartic acid, about 1470 mg/L of L-glutamic acid, about 1150 mg/L of L-Serine. The NEAA may be present from about 0. 1% to about 2.5%, alternatively from about 0.5% to about 2.0%, alternatively from about 0.8% to about 1.8%.

[0058] In yet an alternate embodiment, the cry opreservation formulation further contains serum, e.g., FBS. In an alternate embodiment, the cry opreservation formulation further contains from about 40% to about 75%, alternatively from about 50% to about 70%, alternatively from about 55% to about 65%of serum (e.g., FBS). In certain embodiments, the cry opreservation formulation contains serum and a combination of cryoprotective agents selected from the group consisting of: (a) glycerol and trehalose, (b) glycerol, trehalose, and ascorbic acid, (c) glycerol, trehalose, and taurine, and (d) glycerol, trehalose, ascorbic acid, and taurine. In an alternate embodiment, the cryopreservation formulation contains glycerol, trehalose, ascorbic acid, taurine, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, and serum.

[0059] In certain embodiments, the cry opreservation formulations of the disclosure are formulated to be added to cell culture media. In certain embodiments, the cryopreservation formulation are 2 X base formulations that can be mixed approximately 1 : 1 with a suitable cell culture (growth) medium.

[0060] Examples of cry opreservation formulations are shown in the table below.

[0061] The cryopreservation formulations described above can be added to existing cell culture media, including commercially available media to generate cry opreservation media.

Cryopreservation media

[0062] Another embodiment of the disclosure is directed to cry opreservation media. The cry opreservation media of the disclosure are cell culture media containing (or supplemented with) glycerol and trehalose as cryoprotective agents. In certain embodiments, the cryopreservation media contain or are supplemented with glycerol, trehalose, and taurine as cryoprotective agents. In further embodiments, the cryopreservation media contain or are supplemented with glycerol, trehalose, and ascorbic acid as cryoprotective agents. In additional embodiments, the cryopreservation media contain or are supplemented with glycerol, trehalose, ascorbic acid, and taurine as cry oprotective agents.

[0063] The cryopreservation media contain the necessary ingredient for allowing growth of mammalian cells. In some embodiments, the cryopreservation media are basal defined media. In certain embodiments, the cryopreservation media contain a sufficient amount of amino acids, an energy' source (e.g., glucose), and ions to allow' mammalian cells to grow. [0064] In certain embodiments, the cryopreservation media contain or are supplemented a combination of cryoprotective agents selected from the group consisting of: (a) glycerol and trehalose; (b) glycerol, trehalose, and ascorbic acid; (c) glycerol, trehalose, and taurine; and (d) glycerol, trehalose, ascorbic acid, and taurine. In specific embodiments, the cry opreservation media do not contain DMSO and/or additional cryoprotective agents. [0065] In certain embodiments, the cry opreservation media also contain glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and/or L-serine. In further embodiments, cryopreservation media are supplemented with serum, such as e.g., fetal bovine serum.

[0066] As used herein, the phrases “cell culture medium supplemented with” or “cell culture media supplemented with” refer to existing cell culture media or, including commercially available media, such as e.g., DMEM, to which glycerol, trehalose, ascorbic acid, taurine, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and/or L-serine. [0067] In one embodiment, the cryopreservation medium is a basic cell culture medium that has been supplemented with a combination of cryoprotective agents selected from the group consisting of: (a) glycerol and trehalose; (b) glycerol, trehalose, and ascorbic acid; (c) glycerol, trehalose, and taurine; and (d) glycerol, trehalose, ascorbic acid, and taurine. In certain embodiments, cry opreservation medium contains from about 25% to about 45%, alternatively from about 30% to about 45%. alternatively from about 30% to about 40% of a basic cell culture medium.

[0068] In an alternate embodiment, the cryopreservation medium also contains or is supplemented with glycine, L-alanine. L-asparagine, L-aspartic acid, L-glutamic acid, L- proline, and/or L-serine. In certain embodiments, the cry opreservation medium contains or is supplemented with glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L- proline, and L-serine. In some embodiments, the glycine, L-alanine, L-asparagine, L- glutamic acid, L-proline, and L-serine are provided as a commercially available Non- Essential Amino Acid (NEAA) cell culture supplement. In certain embodiments, NEAA cell culture supplement comprises about 750 mg/L of glycine, about 890 mg/L of L-alanine, about 1320 mg/L of L-asparagine, about 1330 mg/L of aspartic acid, about 1470 mg/L of L- glutamic acid, about 1150 mg/L of L-Serine.

[0069] In yet an alternate embodiment, the cry opreservation medium further contains or is supplemented with serum, e.g., FBS. In one embodiment, the cryopreservation medium contains or is supplemented serum and a combination of cryoprotective agents selected from the group consisting of: (a) glycerol and trehalose, (b) glycerol, trehalose, and ascorbic acid, (c) glycerol, trehalose, and taurine, and (d) glycerol, trehalose, ascorbic acid, and taurine. [0070] In certain embodiments, the cry opreservation media are free of DMSO. i.e.. they do not contain any DMSO. [0071] In certain embodiments, the cry opreservation media are generated by adding a 1 : 1 ratio of a cell culture medium to a cryopreservation formulation described above. A variety of commercially cell culture media may be used to generate the cry opreservation media of the disclosure. In one embodiment, the cryopreservation medium is generated by adding the cry opreservation formulation of the disclosure to Dulbecco’s Modified Eagle Medium (DMEM). In other embodiments, the cryopreservation medium is generated by adding the cry opreservation formulation of the disclosure to Minimum Essential Medium (MEM), Basal Medium Eagle (BME), Roswell Park Memorial Institute (RPMI) 1640 Medium, Iscove’s Modified Dulbecco’s Media (IMDM) or Endothelial Cell Growth Medium-2 (EGM-2).

[0072] Examples of cryopreservation media are shown in the table below.

[0073] The basic cell culture medium used to generate the cry opreservation media of the disclosure can be any cell culture medium including commercially available media such a defined basal media. Kits

[0074] The disclosure also provides for cryopreservation kits. In one embodiment, the kits contain a cry opreservation formulation as described above and instructions for use. In other embodiments, the kits contain a cry opreservation formulation as described above, aNEAA cell culture supplement, and instructions for use. In other embodiments, the kits contain a cry opreservation formulation as described above, a NEAA cell culture supplement, a cell culture medium, and instructions for use. In further embodiments, the kits contain a cry opreservation formulation as described above, aNEAA cell culture supplement, a cell culture medium, a serum supplement (e.g, FBS), and instructions for use.

[0075] In one embodiment, the kits contain a cryopreservation medium as described above and instructions for use. In another embodiment, the kits contain a cry opreservation medium as described above, a NEAA cell culture supplement, and instructions for use. In yet another embodiment, the kits contain a cryopreservation medium as described above. aNEAA cell culture supplement, a serum supplement (e.g, FBS), and instructions for use.

Methods of seneratins cryopreservation media

[0076] The disclosure also provides for methods of generating the cryopreservation media. In one embodiment, the methods include mixing a cryopreservation formulation of the disclosure and a cell culture medium (e.g, a basal medium). In certain embodiments, the method includes mixing approximately a 1 : 1 ratio of the basal medium and the cryopreservation formulation.

[0077] In one embodiment, the method includes mixing a cryopreservation formulation of the disclosure and a cell culture medium containing a sufficient amount of amino acids, an energy source (e.g., glucose), and ions to allow mammalian cells to grow.

Methods of cryopreservins cells or tissues

[0078] The disclosure also provides method of cryopreserving mammalian cells or solid tissues. The method of cryopreserving cells includes: gradually freezing the cells in a cryopreservation medium of the disclosure or a culture medium supplemented with a cryopreservation formulation of the disclosure; and storing the cells/tissues in a suitable environment (e.g., in liquid nitrogen). The method can also include rapid thaw of the frozen cells. FIG. 1 A shows a schematic of a protocol for cryopreservation of cells/tissues.

[0079] The method of cry opreserving solid tissue includes: gradually freezing the solid in a cryopreservation medium of the disclosure or a culture medium supplemented with a cry opreservation formulation of the disclosure; and storing the solid in a suitable environment (e.g, in liquid nitrogen). The method can also include rapid thaw of the frozen solid tissue. FIG. IB shows a schematic of a protocol for cryopreservation of tissues. The protocol includes washing the tissue sample to be preserved, adding culture medium, and then transferring the tissue sample to a container for cry opreservation. Subsequently, the cry opreservation medium or solution of the disclosure is added to the sample and allowed to equilibrate under appropriate conditions (e.g., room temperature). The sample is then cooled to about -80°C. Within 24 hours after cooling at -80°C, the sample is ready for long term storage. In certain embodiments, the protocol includes the following steps:

1. Place a tissue in a petri dish and wash twice with the saline solution containing 1% anti/anti (1% pen/strep, optional).

2. As soon as possible, with the help of forceps and scalpel in a petri dish, cut the tissue into small pieces, approximately 5x5 mm. The tissue section's size would depend on its density. Cell culture medium must be added at this point to prevent tissues from drying out and incubate for 10 minutes at room temperature.

3. Transfer the pieces of tissue to the cryovial.

4. With a transfer pipette/micropipette, add cryopreservation medium, enough to cover the tissues in a minimum ratio of 1 : 1 tissue: cryomedia volume.

5. Close the lid and thoroughly mix samples by inverting the tube 8-10 times.

6. Equilibrate for 10 up to 30 minutes, depending on the size of the tissue at room temperature.

7. Place the vials at -80°C. Vials can be stored at -80°C for several days or placed in LN2 as soon as the following day (24 hours) for long term storage.

[0080] The most commonly applied cryopreservation protocols currently employ the similar philosophy of ‘‘Gradual Freeze” & “Rapid Thaw” to minimize the deleterious effects of ice cry stal formation in cells that could compromise the lipid membrane. Traditionally for cry opreservation mediums containing DMSO. it is important to minimize the time that cells are exposed to the DMSO solution while at room temperature since the DMSO increases the permeability of the cell membrane and can itself lead to compromises of integrity with extended exposure. For this reason, mediums containing DMSO must be mixed separately from cell since exposure to neat DMSO can be highly cytotoxic. The cell formulation formulations of the disclosure are a ready to use mix that can be coupled with the grow th medium a given group is utilizing for a customizable cryo-mix.

[0081] The cell culture media and formulations of the disclosure may be used in such a Gradual Freeze” & “Rapid Thaw” protocol. In certain embodiments, once mixed with complete freezing media, the cells are stored in an insulated cryo-chamber and placed at -80C to gradually freeze before transfer for liquid nitrogen storage for long-term.

[0082] In some embodiments, when the cry opreservation formulation and/or media of the disclosure are used to cry opreserve solid tissues, the method includes a room temperature (RT) incubation period in the presence of a culture medium (growth medium) and a second incubation with the freezing media at RT.

[0083] Without further description, it is believed that one of ordinary skill in the art can. using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples, therefore, specifically point out the preferred embodiments of the present invention and are not to be construed as limiting in any way the remainder of the disclosure.

Examples

Example 1: Cell derivation from elephant frozen tissue

[0084] To develop the cell culture media and formulation of the disclosure, a series of experiments were conducted. In this experiment, placental tissue from elephants was frozen in the presence of a cryopreservation formulation of the disclosure and 10% DMSO/ 40% FBS/50% EGM-2-XAP or MACS® Freezing Solution. Examination of samples that underw ent freezing was conducted by subsequent thawing and monitoring for expansion (either of basic expansion for cell culture or at ability to develop cell lines in the case of frozen tissues).

[0085] As shown in FIG. 2A and FIG. 2B, placental tissue preserved using the formulations of the disclosure at pO and p2 show survival of cells. In contrast, cells cryopreserved in 10% DMSO/ 40% FBS/50% EGM-2-XAP showed little survival (FIG. 2C and FIG. 2D).

MACS® Freezing Solution was also tested but with negative results (data not shown). [0086] Other freezing conditions were also tested: 10% DMSO/90% FBS, flash freeze and low' osmolarity. None of these other conditions showed positive results for cell derivation. The cry opreservation formulations of the disclosure were compared to commercially available cryopreservation media for their ability to cryopreserve sensitive cell lines. The result of the testing is show n in Example 2 (elephant endometrial cells), Example 3 (bovine embryonic stem cells), and Example 4 (human organoids). For the testing in Examples 2-4, the protocol shown in FIG. IB and described in paragraph [0079] above was used.

Example 2: Comparison of cryomedia for elephant endometrial cells

[0087] A cry opreservation formulation of the disclosure (Xo-Chill freezing media) was compared to a formulation containing 10% DMSO/ 40% FBS/50% EGM-2 (see Table 2-1 below ). For this testing, elephant endometrial cells were frozen and were kept in -80 °C for a year before thawing. The results of this testing are shown in FIG.3 A (Xo-Chill) and FIG. 3 B, respectively.

Example 3: Comparison of cryomedia for bovine embryonic stem cells

[0088] Cry opreservation formulations of the disclosure containing bESCM (“XC + bESCM”) or KSR (“ ‘XC+KSR’ ) were compared to mFreSR™ (Stem Cell Technologies) (see Tables 3-1 and 3-2). For this testing, bovine embryonic stem cells were used.

[0089] Bovine ESCs p4 were harvested with ReLeSR and frozen in 10% DMSO and 90% serum. After thawing, cells showed a viability of 75% (data not shown) and were seeded in vitronectin precoated well in bEPSCM. After samples reached 80% confluence, cells were harvested, and 1 x 10⁶ cells were cryobanked for each medium condition. FIGS. 4A-4C show pictures of cells cryopreserved in XC + bESCM, XC + KSR, and mFreSR™. respectively. The picture was taken 48 hours after seeding (cry opreservation). FIG. 4D shows the percentage cell viability and normalized viability after cry opreservation. The testing in this example confirms that the cry opreservation formulation of the disclosure offers similar or improved performance compared to mFreSR™. Example 4: Comparison of cryomedia for human organoids

[0090] In addition, a cry opreservation formulation of the disclosure was compared to CryoStor CS10 (Stem Cell Technologies). For this testing, human organoids were used. [0091] The commercial leading cryomedia CryoStor SCIO was used as reference, following manufacturer’s recommendation. Since better recovery and function maintenance of spheroids/organoids has been reported using DMSO, XC cryomedia (see Table 4-1 below) was compared with CS10 which has 10% DMSO. Human organoids were grown in organoid maintenance medium (OMM). Then, samples were collected and transferred to a cryovial (FIG. 5A, FIG. 5B). XC cryomedia samples were incubated for 10 minutes at room temperature. Organoids in XC cryomedia showed to equilibrate, while CS10 kept floating. Both groups were frozen following a slow-freezing rate. After thawing, samples were cultured with OMM and 24 hours later, monitored for beating contractility (FIG. 5C). The results of the cell contractility testing are shown in FIG. 5D.

EMBODIMENTS

[0092] The invention provides also the following non-limiting embodiments.

[0093] Embodiment 1 is a cryopreservation formulation for cryopreserving mammalian cells or mammalian solid tissues comprising a combination of cry ©protective agents selected from the group consisting of: (a) glycerol and trehalose: (b) glycerol, trehalose, and ascorbic acid; (c) glycerol, trehalose, and taurine; and (d) glycerol, trehalose, ascorbic acid, and taurine. In one embodiment, the formulation is for cryopreservmg cells. In another embodiment, the formulation is for cryopreserving mammalian solid tissues.

[0094] Embodiment 2 is the cry opreservation formulation of embodiment 1, wherein the formulation comprises a combination of cryoprotective agents selected from the group consisting of: (a) from about 20% to about 60% of glycerol and from about 200 mM to about 3 M or from about 200 mM to about 600 mM of trehalose; (b) from about 20% to about 60% of glycerol, from about 200 mM to about 3 M or from about 200 mM to about 600 mM of trehalose, and from about 100 pg/mL to about 250 pg/mL of ascorbic acid; (c) from about 20% to about 60% of glycerol, from about 200 mM to about 3 M or from about 200 mM to about 600 mM of trehalose, and from about 10 mM to about 40 mM of taurine: and (d) from about 20% to about 60% of glycerol, from about 200 mM to about 3 M or from about 200 mM to about 600 mM of trehalose, from about 100 pg/mL to about 250 pg/mL of ascorbic acid, and from about 10 mM to about 40 mM of taurine.

[0095] Embodiment 3 is the cryopreservation formulation of embodiments 1 or 2, wherein the combination of cry ©protective agents is glycerol and trehalose.

[0096] Embodiment 4 is the cryopreservation formulation of embodiments 1 or 2, wherein the combination of cryoprotective agents is glycerol, trehalose, and ascorbic acid.

[0097] Embodiment 5 is the cryopreservation formulation of embodiments 1 or 2, wherein the combination of cryoprotective agents is glycerol, trehalose, and taurine.

[0098] Embodiment 6 is the cryopreservation formulation of embodiments 1 or 2, wherein the combination of cryoprotective agents is glycerol, trehalose, ascorbic acid, and taurine. [0099] Embodiment 7 is the cryopreservation formulation of any one of embodiments 1 to 6, wherein the formulation further comprises serum.

[0100] Embodiment 8 is the cry opreservation formulation of embodiment 7, wherein the formulation comprises from about 40% to about 75% of serum.

[0101] Embodiment 9 is the cryopreservation formulation of embodiments 7 or 8, wherein the serum comprises FBS. [0102] Embodiment 10 is the cry opreservation formulation of any one of embodiments 1 to 9, further comprising glycine, L-alanine, L-asparagine, L-aspartic acid. L-glutamic acid, L- proline, and/or L-serine.

[0103] Embodiment 11 is the cry opreservation formulation of embodiment 10, further comprising from about 0.1% to about 2.5% of glycine, from about 0.1% to about 2.5% of L- alanine, from about 0. 1% to about 2.5% of L-asparagine, from about 0.1% to about 2.5% of L-aspartic acid, from about 0.1% to about 2.5% of L-glutamic acid, from about 0.1% to about 2.5% of L-proline, and/or from about 0.1% to about 2.5% of L-serine.

[0104] Embodiment 12 is the cryopreservation formulation of embodiments 10 or 11, wherein the formulation further comprises glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine.

[0105] Embodiment 13 is the cry opreservation formulation of any one of embodiments 1 to

12, wherein the formulation is free of dimethyl sulfoxide (DMSO).

[0106] Embodiment 14 is the cry opreservation formulation of any one of embodiments 1 to

13, wherein the formulation does not comprise an additional cryoprotective agent.

[0107] Embodiment 15 is a cryopreservation medium comprising the cry opreservation formulation of any one of embodiments 1 to 14.

[0108] Embodiment 16 is the cryopreservation medium of embodiment 15 comprising a sufficient amount of amino acids, an energy source, and ions to allow mammalian cells to grow.

[0109] Embodiment 17 is the cry opreservation medium of embodiment 16, wherein the energy source is glucose.

[0110] Embodiment 18 is a method of generating a cry opreservation medium comprising mixing a cry opreservation formulation of any one of embodiments 1 to 14 and a cell culture medium.

[OHl] Embodiment 19 is the method of embodiment 18, wherein the cell culture medium comprises a sufficient amount of amino acids, an energy' source, and ions to allow mammalian cells to grow. Alternatively, the cell culture medium comprises a sufficient amount of amino acids, an energy source, and ions to allow mammalian cells in a solid mammalian tissue to grow.

[0112] Embodiment 20 is the method of embodiment 19, wherein the cell culture medium comprises a sufficient amount of amino acids, glucose, and ions to allow mammalian cells to grow. Alternatively, the cell culture medium comprises a sufficient amount of amino acids, glucose, and ions to allow mammalian cells in a solid mammalian tissue to grow. [0113] Embodiment 21 is a cry opreservation medium for cry opreserving mammalian cells comprising: a sufficient amount of amino acids, glucose, and ions to allow mammalian cells to grow; and a combination of cryoprotective agents selected from the group consisting of: (a) glycerol and trehalose; (b) glycerol, trehalose, and ascorbic acid; (c) glycerol, trehalose, and taurine; and (d) glycerol, trehalose, ascorbic acid, and taurine.

[0114] Embodiment 22 is the cry opreservation medium of embodiment 21 comprising: a sufficient amount of amino acids, glucose, and ions to allow mammalian cells to grow; and a combination of cryoprotecti ve agents selected from the group consisting of: (a) from about 10% to about 40% of glycerol and from about 100 mM to about 3 M of trehalose; (b) from about 10% to about 40% of glycerol, from about 100 mM to about 3 M of trehalose, and from about 20 pg/mL to about 250 pg/mL of ascorbic acid; (c) from about 10% to about 40% of glycerol, from about 100 mM to about 3 M of trehalose, and taurine; and (d) from about 10% to about 40% of glycerol, from about 100 mM to about 3 M of trehalose, from about 20 pg/mL to about 250 pg/mL of ascorbic acid, and from about 5 mM to about 30 mM of taurine [0115] Embodiment 23 is the cry opreservation medium of embodiments 21 or 22, wherein the combination of cryoprotective agents is glycerol and trehalose.

[0116] Embodiment 24 is the cryopreservation medium of embodiments 21 or 22, wherein the combination of cry oprotective agents is glycerol, trehalose, and ascorbic acid.

[0117] Embodiment 25 is the cryopreservation medium of embodiments 21 or 22, wherein the combination of cryoprotective agents is glycerol, trehalose, and taurine.

[0118] Embodiment 26 is the cry opreservation medium of embodiments 21 or 22, wherein the combination of cry oprotective agents is glycerol, trehalose, ascorbic acid, and taurine. [0119] Embodiment 27 is the cry opreservation medium of any one of embodiments 21 to 26, wherein the medium further comprises serum.

[0120] Embodiment 28 is the cry opreservation medium of embodiment 27, wherein the medium comprises from about 40% to about 75% of serum.

[0121] Embodiment 29 is the cry' opreservation medium of embodiments 26 or 27, wherein the serum comprises FBS.

[0122] Embodiment 30 is the cryopreservation medium of any one of embodiments 21 to 29, further comprising glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L- proline, and/or L-serine.

[0123] Embodiment 31 is the cry opreservation medium of embodiment 30. further comprising from about 0.1% to about 2% of glycine, from about 0.1% to about 2% of L- alanine, from about 0.1% to about 2% of L-asparagine, from about 0.1% to about 2% of L- aspartic acid, from about 0. 1% to about 2% of L-glutamic acid, from about 0. 1% to about 2% of L-proline, and/or from about 0. 1% to about 2% of L-serine.

[0124] Embodiment 32 is the cry opreservation medium of embodiments 30 or 31, wherein the medium further comprises glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine.

[0125] Embodiment 33 is the cryopreservation medium of any one of embodiments 21 to 32, wherein the cryopreservation medium comprises from about 25% to about 45% of a basic cell culture medium comprising the sufficient amount of amino acids, glucose, and ions to allow mammalian cells to grow.

[0126] Embodiment 34 is the cry opreservation medium of any one of embodiments 21 to 33, wherein the medium is free of DMSO.

[0127] Embodiment 35 is the cry opreservation medium of any one of embodiments 21 to 33, wherein the medium does not comprise an additional cryoprotective agent.

[0128] Embodiment 36 is the cry opreservation medium of any one of embodiments 21 to 35, wherein the mammalian cells are in a solid tissue.

[0129] Embodiment 37 is a method of cryopreserving mammalian cells comprising: freezing mammalian cells in a cry opreservation medium of any one of embodiments 15 to 17 or 21 to 35 or a cell culture medium supplemented with a cryopreservation formulation of any one of embodiments 1 to 14; and storing the cells in a suitable environment.

[0130] Embodiment 38 is the method of embodiment 37, wherein the suitable environment comprises storing the cells in liquid nitrogen.

[0131] Embodiment 39 is a method of cryopreserving solid mammalian tissue comprising: freezing solid mammalian tissue in a cry opreservation medium of any one of embodiments 15 to 17 or 21 to 36 or a cell culture medium supplemented with a cryopreservation formulation of any one of embodiments 1 to 14; and storing the solid mammalian tissue in a suitable environment.

[0132] Embodiment 40 is the method of embodiment 39, wherein the suitable environment comprises storing the solid mammalian tissue in liquid nitrogen.

[0133] Embodiment 41 is the method of embodiment 39, wherein the freezing comprises culturing the solid mammalian tissue at room temperature in a growth medium and then culturing the solid mammalian tissue with a cryopreservation medium or the cell culture medium supplemented with a cry opreservation formulation. [0134] Embodiment 42 is a kit comprising the cryopreservation formulation of any one of embodiments 1 to 14, the cry opreservation medium of any one of embodiments 15 to 17 or 21 to 36 and instructions for use.

[0135] Embodiment 43 is the kit of embodiment 42, wherein the kit comprises the cry opreservation formulation of any one of embodiments 1 to 14 and a cell culture medium.

[0136] Embodiment 44 is the kit of embodiment 43, wherein the cell culture medium comprises a sufficient amount of amino acids, an energy source, and ions to allow mammalian cells to grow.

[0137] Embodiment 45 is the kit of embodiment 42, wherein the kit comprises the cry opreservation medium of any one of embodiments 15 to 17 or 21 to 35.

[0138] Embodiment 46 is the kit of any one of embodiments 42 to 45. wherein the kit comprises instructions for cry opreserving mammalian cells.

[0139] While the invention has been described and illustrated herein by references to various specific materials, procedures, and examples, it is understood that the invention is not restricted to the particular combinations of material and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplar}', only, with the true scope and spirit of the invention being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.

Claims

CLAIMS What is claimed is:

1. A cry opreservation formulation for cry opreserving mammalian cells or solid mammalian tissue comprising a combination of cryoprotective agents selected from the group consisting of: (a) glycerol and trehalose; (b) glycerol, trehalose, and ascorbic acid; (c) glycerol, trehalose, and taurine; and (d) glycerol, trehalose, ascorbic acid, and taurine.

2. The cry opreservation formulation of claim 1, wherein the formulation comprises a combination of cryoprotective agents selected from the group consisting of:

(a) from about 20% to about 60% of glycerol and from about 200 mM to about 3 M or from about 200 mM to about 600 mM of trehalose;

(b) from about 20% to about 60% of glycerol, from about 200 mM to about 3 M or from about 200 mM to about 600 mM of trehalose, and from about 100 pg/mL to about 250 pg/mL of ascorbic acid;

(c) from about 20% to about 60% of glycerol, from about 200 mM to about 3 M or from about 200 mM to about 600 mM of trehalose, and from about 10 mM to about 40 mM of taurine; and

(d) from about 20% to about 60% of glycerol, from about 200 mM to about 3 M or from about 200 mM to about 600 mM of trehalose, from about 100 pg/mL to about 250 pg/mL of ascorbic acid, and from about 10 mM to about 40 mM of taurine.

3. The cryopresen ation formulation of claims 1 or 2, wherein the combination of cryoprotective agents is glycerol and trehalose.

4. The cryopreservation formulation of claims 1 or 2, wherein the combination of cryoprotective agents is glycerol, trehalose, and ascorbic acid.

5. The cryopreservation formulation of claims 1 or 2, wherein the combination of cryoprotective agents is glycerol, trehalose, and taurine.

6. The cryopresen ation formulation of claims 1 or 2, wherein the combination of cryoprotective agents is glycerol, trehalose, ascorbic acid, and taurine.

7. The cryopreservation formulation of claim 1, wherein the formulation further comprises serum.

8. The cryopreservation formulation of claim 7, wherein the formulation comprises from about 40% to about 75% of serum.

9. The cryopreservation formulation of claim 7, wherein the serum comprises FBS.

10. The cry opreservation formulation of claims 1 or 7, further comprising glycine, L- alanine, L-asparagine, L-aspartic acid. L-glutamic acid, L-proline, and/or L-serine.

11. The cry opreservation formulation of claim 10, further comprising from about 0.1 % to about 2.5% of glycine, from about 0.1% to about 2.5% of L-alanine, from about 0.1% to about 2.5% of L-asparagine, from about 0.1% to about 2.5% of L-aspartic acid, from about 0.1% to about 2.5% of L-glutamic acid, from about 0. 1% to about 2.5% of L-proline, and/or from about 0.1% to about 2.5% of L-serine.

12. The cry opreservation formulation of claims 10, wherein the formulation further comprises glycine. L-alanine, L-asparagine. L-aspartic acid, L-glutamic acid. L-proline, and L-serine.

13. The cryopreservation formulation of claim 1, wherein the formulation is free of dimethyl sulfoxide (DMSO).

14. The cry opreservation formulation of claims 1 or 13, wherein the formulation does not comprise an additional cryoprotective agent.

15. A cry opreservation medium comprising the cry opreservation formulation of claim 1.

16. The cry opreservation medium of claim 15 comprising a sufficient amount of amino acids, an energy source, and ions to allow mammalian cells to grow.

17. The cry opreservation medium of claim 16, wherein the energy source is glucose.

18. A method of generating a cryopreservation medium comprising mixing a cry opreservation formulation of claim 1 and a cell culture medium.

19. The method of claim 18, wherein the cell culture medium comprises a sufficient amount of amino acids, an energy source, and ions to allow mammalian cells to grow.

20. The method of claim 19, wherein the cell culture medium comprises a sufficient amount of amino acids, glucose, and ions to allow mammalian cells to grow.

21. A cry opreservation medium for cry opreserving mammalian cells comprising: a sufficient amount of amino acids, glucose, and ions to allow mammalian cells to grow; and a combination of cryoprotective agents selected from the group consisting of: (a) glycerol and trehalose; (b) glycerol, trehalose, and ascorbic acid; (c) glycerol, trehalose, and taurine; and (d) glycerol, trehalose, ascorbic acid, and taurine.

22. The cry opreservation medium of claim 21 comprising: a sufficient amount of amino acids, glucose, and ions to allow mammalian cells to grow; and a combination of cryoprotective agents selected from the group consisting of:

(a) from about 10% to about 40% of glycerol and from about 100 mM to about 3 M of trehalose;

(b) from about 10% to about 40% of glycerol, from about 100 mM to about 3 M of trehalose, and from about 20 pg/mL to about 250 pg/mL of ascorbic acid;

(c) from about 10% to about 40% of glycerol, from about 100 mM to about 3 M of trehalose, and taurine; and

(d) from about 10% to about 40% of glycerol, from about 100 mM to about 3 M of trehalose, from about 20 pg/mL to about 250 pg/mL of ascorbic acid, and from about 5 mM to about 30 mM of taurine.

23. The cry opreservation medium of claims 21 or 22, wherein the combination of cryoprotective agents is glycerol and trehalose.

24. The cryopreservation medium of claims 21 or 22, wherein the combination of cryoprotective agents is glycerol, trehalose, and ascorbic acid.

25. The cry opreservation medium of claims 21 or 22, wherein the combination of cryoprotective agents is glycerol, trehalose, and taurine.

26. The cry opreservation medium of claims 21 or 22, wherein the combination of cry opro tech ve agents is glycerol, trehalose, ascorbic acid, and taurine.

27. The cryopreservation medium of claims 21 or 22, wherein the medium further comprises serum.

28. The cryopreservation medium of claim 27, wherein the medium comprises from about 40% to about 75% of serum.

29. The cryopreservation medium of claim 27, wherein the serum comprises FBS.

30. The cry opreservation medium of claims 21 or 22, further comprising glycine, L- alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and/or L-serine.

31. The cry opreservation medium of claim 30, further comprising from about 0.1% to about 2% of glycine, from about 0.1 % to about 2% of L-alanine, from about 0. 1 % to about 2% of L-asparagine, from about 0.1% to about 2% of L-aspartic acid, from about 0.1% to about 2% of L-glutamic acid, from about 0. 1% to about 2% of L-proline, and/or from about 0. 1% to about 2% of L-serine.

32. The cryopreservation medium of claim 30, wherein the medium further comprises glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline. and L-serine.

33. The cry opreservation medium of claims 21 or 22, wherein the cry opreservation medium comprises from about 25% to about 45% of a basic cell culture medium comprising the sufficient amount of amino acids, glucose, and ions to allow mammalian cells to grow.

34. The cry opreservation medium of claims 21 or 22, wherein the medium is free of DMSO.

35. The cry opreservation medium of claims 21 or 22, wherein the medium does not comprise an additional cryoprotective agent.

36. The cry opreservation medium of claims 21 or 22, wherein the mammalian cells are in a solid tissue.

37. A method of cryopreserving mammalian cells comprising: freezing mammalian cells in a cry opreservation medium of claim 15 or a cell culture medium supplemented with a cry opreservation formulation of claim 1; and storing the cells in a suitable environment.

38. The method of claim 37, wherein the suitable environment comprises storing the cells in liquid nitrogen.

39. A method of cry opreserving solid mammalian tissue comprising: freezing solid mammalian tissue in a cryopreservation medium of claim 15 or a cell culture medium supplemented with a cry opreservation formulation of claim 1; and storing the solid mammalian tissue in a suitable environment.

40. The method of claim 39, wherein the suitable environment comprises storing the solid mammalian tissue in liquid nitrogen.

41. The method of claim 39, wherein the freezing comprises culturing the solid mammalian tissue at room temperature in a growth medium and then culturing the solid mammalian tissue with a cry opreservation medium or the cell culture medium supplemented with a cry opreservation formulation.

42. A kit comprising the cry opreservation formulation of claim 1, or the cry opreservation medium of claim 15, and instructions for use.

43. The kit of claim 42, wherein the kit comprises the cryopreservation formulation of claim 1 and a cell culture medium.

44. The kit of claim 43, wherein the cell culture medium comprises a sufficient amount of amino acids, an energy source, and ions to allow mammalian cells to grow.

45. The kit of claim 42, wherein the kit comprises the cry opreservation medium of anyone of claim 15.

46. The kit of claim 42, wherein the kit comprises instructions for cryopreserving mammalian cells.