Cryopreservation preparations

Abstract

This disclosure provides cryopreservation formulations, cryopreservation media, and methods for cryopreserving mammalian cells or tissues. The cryopreservation formulations and cryopreservation media contain a combination of cryoprotective agents selected from the group consisting of (a) to (d) below: (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 cryopreservation media do not contain dimethyl sulfoxide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Application No. 63 / 527462 filed on July 18, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] The present disclosure relates to cryopreservation of cells, in particular to cryopreservation of mammalian cells and solid tissues. Specifically, the present disclosure relates to cryopreservation preparations (cryopreservation compositions), cryopreservation media, and methods for cryopreserving cells. Background Art

[0003] Storage of mammalian cells and solid tissues in liquid nitrogen is an essential step in cell culture laboratories, as it enables preservation of valuable samples for use in downstream research or applications. The nutrient-rich medium used in the freezing process has a great impact on the overall viability and function of cells after sample thawing.

[0004] In the freezing step (also referred to as cryopreservation), a cell culture medium that allows proliferation of mammalian cells to be stored (cryopreserved) and a cryoprotectant that maintains cell function and viability during freezing are generally used.

[0005] Currently, the "gold standard" for cryopreservation requires the use of dimethyl sulfoxide (DMSO) as a cryoprotective agent. However, while DMSO is the gold standard, studies have shown that it can have harmful effects on cryopreserved cells. For example, Galvao et al. reported demonstrating that "DMSO induces retinal apoptosis in vivo even at low concentrations (5 μL of DMSO from 1%, 2%, 4%, and 8% (v / v) stock solutions administered intravitreally to rats)" (Non-Patent Literature 1). Similarly, a 2020 review by Awan et al. summarized data showing numerous drawbacks of using DMSO and emphasized the need for alternatives to DMSO (Non-Patent Literature 2).

[0006] Given the drawbacks of using DMSO, cryopreservation media that do not contain DMSO have been developed for cell cryopreservation. One example is the XT-Thive® cryopreservation method, which uses N-substituted biomimetic amino acid polymers (peptoids) and peptoid-peptide hybrids as substitutes for DMSO. However, since peptoids are generally synthetic molecules that can bind to or target proteins, they require extensive evaluation depending on their application.

[0007] Therefore, there is a need for cryopreservation solutions and culture media that overcome the potential drawbacks of using DMSO, as well as the shortcomings of currently available DMSO-free media, and are useful for the cryopreservation of cells and solid tissues. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Galvao et al.,FASEB J.28(3):1317-30(2014)at Abstract [Non-Patent Document 2] Awan et al.Regenerative Medicine,15(3):1463-1491(2020) [Overview of the Initiative]

[0009] This disclosure provides cryopreservation formulations, cryopreservation media, and methods for cryopreserving mammalian cells or solid tissues. The cryopreservation formulations and cryopreservation media contain a combination of cryoprotective agents selected from the group consisting of (a) to (d) below: (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 cryopreservation media do not contain dimethyl sulfoxide (DMSO). In other embodiments, the cryopreservation formulations and cryopreservation media do not contain additional cryoprotective agents.

[0010] In one embodiment, the present disclosure provides a cryopreservation formulation for cryopreserving mammalian cells, comprising a combination of cryoprotective agents selected from the group consisting of (a) to (d) below: (a) glycerol and trehalose; (b) glycerol, trehalose and ascorbic acid; (c) glycerol, trehalose and taurine; and (d) glycerol, trehalose, ascorbic acid and taurine. These cryopreservation formulations can be added to cell culture media to produce cryopreservation media.

[0011] In a particular embodiment, the cryopreservation formulation contains a combination of cryoprotective agents selected from the group consisting of (a) to (d) below: (a) about 20% to about 60% glycerol and about 200 mM to about 3 M or about 200 mM to about 600 mM trehalose; (b) about 20% to about 60% glycerol, about 200 mM to about 3 M or about 200 mM to about 600 mM trehalose and about 100 μg / mL to about 250 μg (c) ascorbic acid in g / mL; (d) about 20% to about 60% glycerol, about 200 mM to about 3 M or about 200 mM to about 600 mM trehalose, and about 10 mM to about 40 mM taurine; and (d) about 20% to about 60% glycerol, about 200 mM to about 3 M or about 200 mM to about 600 mM trehalose, about 100 μg / mL to about 250 μg / mL ascorbic acid, and about 10 mM to about 40 mM taurine.

[0012] In certain embodiments, the cryopreservation preparation may contain serum (e.g., fetal bovine serum) in an amount of about 40% to about 75%. Furthermore, the cryopreservation preparation may contain glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and / or L-serine. In certain embodiments, the cryopreservation preparation contains about 0.1% to about 2.5% glycine, about 0.1% to about 2.5% L-alanine, about 0.1% to about 2.5% L-asparagine, about 0.1% to about 2.5% L-aspartic acid, about 0.1% to about 2.5% L-glutamic acid, about 0.1% to about 2.5% L-proline and / or about 0.1% to about 2.5% L-serine.

[0013] In certain embodiments, the Disclosure provides a cryopreservation medium for cryopreserving mammalian cells, comprising a combination of amino acids, glucose and ions in sufficient quantities to enable the proliferation of mammalian cells, and a cryoprotective agent selected from the group consisting of (a) to (d) below: (a) glycerol and trehalose; (b) glycerol, trehalose and ascorbic acid; (c) glycerol, trehalose and taurine; and (d) glycerol, trehalose, ascorbic acid and taurine.

[0014] In a particular embodiment, the cryopreservation medium contains a combination of cryoprotectants selected from the group consisting of (a) to (d) below: (a) about 10% to about 40% glycerol and about 100 mM to about 3 M trehalose; (b) about 10% to about 40% glycerol, about 100 mM to about 3 M trehalose and about 20 μg / mL to about 250 μg / mL ascorbic acid; (c) about 10% to about 40% glycerol, about 100 mM to about 3 M trehalose and taurine; and (d) about 10% to about 40% glycerol, about 100 mM to about 3 M trehalose, about 20 μg / mL to about 250 μg / mL ascorbic acid and about 5 mM to about 30 mM taurine. In certain embodiments, the cryopreservation medium contains serum (e.g., fetal cow serum) in the medium, for example, containing about 40% to about 75% of the total medium as FBS. In certain embodiments, the cryopreservation medium contains glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and / or L-serine.

[0015] This disclosure also provides methods for producing cryopreservation media, and methods for cryopreserving mammalian cells and / or tissues. Furthermore, this disclosure includes kits comprising cryopreservation formulations or cryopreservation media.

[0016] Other features and advantages of the present invention will become apparent from the following detailed description and examples. [Brief explanation of the drawing]

[0017] The above summary and the detailed description of the present invention below can be better understood by reading them in conjunction with the accompanying drawings. Embodiments of the present invention are shown in the drawings to illustrate the invention. However, it should be understood that the present invention is not limited to the specific configurations, examples, and means shown.

[0018] [Figure 1] Figures 1A and 1B schematically illustrate the use of the cryopreservation culture medium and cryopreservation preparation of this disclosure.

[0019] [Figure 2] Figures 2A to 2D show the results of cryopreserving placental tissue using the cryopreservation preparation of this disclosure compared to that using commercially available cryopreservation media. Figures 2A and 2B show the results using the cryopreservation preparation of this disclosure. Figures 2C and 2D show the results for cells cryopreserved in 10% DMSO / 40% FBS / 50% EGM-2-XAP.

[0020] [Figure 3] Figures 3A and 3B show the results of cryopreserving a highly sensitive cell line (elephant endometrial cells) using the cryopreservation preparation of this disclosure (identified as Xo-Chill(XC)) compared with the results using a conventional cryopreservation medium preparation.

[0021] Figures 4A to 4D show the results of cryopreserving bovine embryonic stem cells using the cryopreservation formulations of this disclosure, compared to the results using commercially available mFreSR medium. Figures 4A to 4C show images of cells cryopreserved in XC+bESCM, XC+KSR, and mFreSR, respectively. These images were taken 48 hours after seeding (cryopreservation). Figure 4D shows the cell viability and normalized viability after cryopreservation.

[0022] Figures 5A to 5D show the results of comparing the cryopreservation of human organoids using the cryopreservation preparation of the present disclosure with the case of using a commercially available CryoStor CS10 medium. Figure 5A shows images of human organoids before incubation with the XC cryopreservation medium of the present disclosure and CryoStor CS10 (CS). Figure 5B shows images of human organoids after incubation with the XC cryopreservation medium of the present disclosure and CryoStor CS10. Figure 5C shows images of cells after thawing and culturing for 24 hours in organoid maintenance medium (OMM). Figure 5D shows the post-thaw cell contractility (percentage of beating organoids) for cells cryopreserved in XC+OMM and CS10. MODE FOR CARRYING OUT THE INVENTION

[0023] The present disclosure provides a cryopreservation medium, a cryopreservation preparation, and a method for cryopreserving mammalian cells. The present disclosure is based on the finding that the cryoprotective effect achieved by DMSO and FBS can be obtained by using a combination of cryoprotective agents comprising at least glycerol and trehalose, or glycerol and trehalose plus ascorbic acid and / or taurine.

[0024] The cryopreservation medium and cryopreservation preparation of the present disclosure contain at least glycerol and trehalose. The cryopreservation medium and cryopreservation preparation may further contain serum, ascorbic acid and / or taurine.

[0025] In a specific embodiment, the cryopreservation medium and the cryopreservation preparation do not contain DMSO. That is, the cryopreservation medium and the cryopreservation preparation do not contain any DMSO. In a specific embodiment, the cryopreservation medium and the cryopreservation preparation do not contain synthetic molecules that have the potential to bind to or target proteins (e.g., peptoids).

[0026] The cryopreservation preparation is designed to be added to a cell culture medium. In a particular embodiment, the cryopreservation medium is a basal medium to which the cryopreservation preparation has been added.

[0027] For the purpose of clarifying this disclosure and not limiting the present invention, a detailed description of the present invention is divided into several subsections that describe or illustrate specific features, embodiments, or uses of the present invention.

[0028] [Definition] Furthermore, it should be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to limit the invention.

[0029] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” and “characterized by” are mutually interchangeable, inclusive and open in meaning, and do not exclude any additional elements or methods not described herein. Furthermore, any mention of the term “comprising” herein, particularly in reference to components of a composition or apparatus, is understood to include compositions and methods essentially composed of the described components or elements, as well as compositions and methods composed of such components or elements.

[0030] As used herein, the term "consisting of" means excluding any elements, processes, or components not specified in the claim element.

[0031] As used herein, when the term "about" is used in reference to a measurable value such as a quantity or time period, it shall include a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the stated value, to the extent that it is appropriate for carrying out the disclosed method.

[0032] Before describing specific embodiments in more detail below, it should be understood that the present invention is not limited to such specific embodiments, and these can naturally be modified in various ways. Furthermore, it should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be restrictive, and the scope of the present invention is limited only by the appended claims.

[0033] Where a numerical range is specified, unless the context clearly indicates otherwise, each intermediate value up to one-tenth of the lower limit of that range, i.e., any value located between the upper and lower limits of that range, as well as any other value explicitly or implicitly indicated within that range, are understood to be included in the present invention. The upper and lower limits of these smaller ranges may be independently included within those smaller ranges and are also included in the present invention unless specifically excluded in the numerical range. Furthermore, if a specified numerical range includes one or both limits, the range excluding one or both of those included limits is also included in the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which the present invention pertains. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention; however, representative exemplary methods and materials are described below.

[0035] As used herein, the terms “transform” or “transformation” refer to the introduction of a nucleic acid fragment into a host cell, such as a host bacterial cell, resulting in genetically stable inheritance. A host cell containing a transformed nucleic acid fragment is called a “recombinant,” “transgenic,” or “transformed” organism.

[0036] As used herein, the term “isolated” means that a biological component (e.g., nucleic acid, peptide, or protein) is substantially separated from, produced separately from, or purified from other biological components of an organism in which it is naturally present (i.e., other chromosomes and extrachromosomal DNA and RNA, as well as proteins). Thus, “isolated” nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. “Isolated” nucleic acids, peptides, and proteins are still isolated if the composition is not part of the natural environment of the nucleic acid, peptide, or protein, even if it is part of a composition. The term also includes nucleic acids, peptides, and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids.

[0037] As used herein, “gene” means a nucleic acid containing an open reading frame that encodes a polypeptide, including both exon sequences and (if applicable) intron sequences.

[0038] As used herein, “promoter” is an example of a transcriptional regulatory sequence, specifically a nucleic acid sequence commonly described as the proximal region of a gene located 5' to the start codon. Transcription of adjacent nucleic acid segments is initiated at the promoter region. The transcription rate of a repressive promoter decreases in response to an inhibitor. The transcription rate of an inducible promoter increases in response to an inducer. The transcription rate of a constitutive promoter is not particularly regulated, but may change under the influence of general metabolic conditions.

[0039] The term "gene product," as used herein, refers to any product encoded by a nucleic acid sequence. Therefore, a gene product may be, for example, a primary transcript, a mature transcript, a processed transcript, or a protein or peptide encoded by a transcript. Examples of gene products include mRNA, rRNA, hairpin RNA (e.g., microRNA, shRNA, siRNA, tRNA), and peptides and proteins, such as reporter proteins or therapeutic proteins.

[0040] As used herein, the term “stem cell” means a cell that is capable of self-renewal and can differentiate into at least one more differentiated phenotype or a less developmentally potent 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. “Induced stem cells” are derived from non-pluripotent cells that have been induced into a less differentiated phenotype or a more developmentally potent phenotype by the introduction of one or more reprogramming factors or genes. As used herein, induced stem cells do not need to be pluripotent, but have the ability to differentiate into one or more more highly differentiated phenotypes under appropriate conditions. It should be understood that this ability was not present before the introduction of the reprogramming factor. Induced stem cells express at least one stem cell marker that was not expressed by the parental cell before the introduction of the reprogramming factor. In this context, the stem cell marker excludes factors introduced by reprogramming. Induced pluripotent stem cells, or iPS cells, have the ability to differentiate into cellular phenotypes derived from each of the germ layers: endoderm, mesoderm, and ectoderm, under appropriate conditions.

[0041] As used herein, the term “marker” is used to describe the characteristics and / or phenotype of a cell. Markers can be used to select cells containing desired characteristics and are varied by specific cells. A marker is a morphological, structural, functional, or biochemical (enzymatic) feature unique to a particular cell type, or a molecule expressed by that cell type. In one embodiment, such a marker is a protein. Such a protein may possess an epitope to antibodies or other binding molecules available in the art. However, a marker can consist of any molecule found inside or on a cell, including but not limited to proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological features or traits include, but are not limited to, shape, size, and nucleus-to-cytoplasmic ratio. Examples of functional features or traits include, but are not limited to, the ability to adhere to a particular substrate, the ability to take up or reject a particular dye, the ability to migrate under specific conditions, and the ability to differentiate along a particular lineage. Markers can be detected by any method available to those skilled in the art. Markers may also be a lack of morphological features, or a lack of proteins, lipids, etc. The marker may be a combination of a set of unique features relating to the presence and / or absence of polypeptides, as well as other morphological or structural characteristics. In one embodiment, the marker is a cell surface marker.

[0042] The term “exogenous” refers to a substance present in a cell that has been introduced by artificial means. As used herein, “exogenous” can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide introduced by artificial means into a biological system, such as a cell or organism, where it is not normally found. Alternatively, “exogenous” may refer to a nucleic acid or polypeptide introduced by artificial means into a biological system, such as a cell or organism, where it is desirable to increase the amount of nucleic acid or polypeptide in the cell or organism, for example, to produce ectopic expression or a higher level.

[0043] In this specification, the terms “reprogramming gene” or “reprogramming factor” refer to drugs or nucleic acid molecules that induce a reprogramming process in somatic cells, thereby re-expressing a less differentiated, more stem cell-like phenotype. Reprogramming factors may be nucleic acids, polypeptides, or small molecules that, when introduced into cells, promote the reprogrammed phenotype. Non-exclusive 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, for example, to induce induced pluripotent stem cells. Additional factors that may be introduced as reprogramming factors in the process of reprogramming cells into a poorly differentiated or stem cell phenotype include, but are not limited to, small molecule chemicals such as LIN28+Nanog, Esrrb, Pax5 shRNA, C / EBPa, p53 siRNA, UTF1, DNMT shRNA, Wnt3a, SV40 LT(T), hTERT, BIX-01294, BayK8644, RG108, AZA, dexamethasone, VPA, TSA, SAHA, PD0325901+CHIR99021(2i), and A-83-01. In some embodiments, the reprogramming genes or factors are Oct4, Klf4, SOX2, and c-Myc.

[0044] As used herein, the terms “dedifferentiation,” “reverse differentiation,” or “reprogramming” refer to the process of generating cells that re-express a less differentiated phenotype than the cells from which they originate, and / or express at least one stem cell marker that was not expressed prior to the process. For example, terminally differentiated cells can be dedifferentiated into pluripotent cells. That is, dedifferentiation shifts cells backward along the differentiation spectrum from totipotent to fully differentiated cells. Typically, reversing the differentiation phenotype of a cell requires artificial manipulation of the cell, for example, by introducing or expressing exogenous polypeptide factors. Reprogramming is typically not observed under in vivo or in vitro natural conditions.

[0045] As used herein, “reprogrammed cells” are cells that have been exposed to one or more reprogramming factors and express a less differentiated phenotype than the cells from which they originated. Reprogrammed cells also possess the ability to self-replicate and express at least one stem cell marker that was not delivered to the cells as a reprogramming factor. Furthermore, reprogrammed cells have the ability to differentiate into more differentiated somatic cell types according to differentiation protocols described herein or known in the art.

[0046] As used herein, the term “somatic cell” means any cell other than germ cells, cells present in or obtained from a preimplantation embryo, or cells resulting from the in vitro proliferation of such cells. In other words, somatic cells refer to all cells that make up the body of an organism, excluding germ cells. Except for sperm and eggs, and the cells that form them (germ cells), all cell types in the mammalian body are somatic cells. Internal organs, skin, bone, blood, and connective tissue are all substantially composed of somatic cells. In some embodiments, somatic cells are “non-embryonic somatic cells,” meaning somatic cells that are not present in or obtained from an embryo, and do not result from the in vitro proliferation of such cells. In some embodiments, somatic cells are “adult somatic cells,” meaning cells present in or obtained from an organism other than an embryo or fetus, or cells resulting from the in vitro proliferation of such cells.

[0047] All publications and patents cited herein are invoked by reference in the same way that each publication or patent is individually and expressly indicated as being invoked by reference, and are invoked by reference herein for the purpose of disclosing and explaining the methods and / or materials cited in such publications. Any citation of a publication is for the sole purpose of indicating its disclosure prior to the filing date and should not be construed as acknowledging that the present invention does not have the right to retroactively apply to the date of such publication based on prior art. Furthermore, the stated publication date may differ from the actual publication date, and such publication date should be independently verified as necessary.

[0048] Note that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple subjects unless the context clearly indicates otherwise. Also note that claims may be constructed to exclude any element. Therefore, this description is intended to serve as an antecedent basis when using exclusive terms such as "solely," "only," or "negative limitation" in relation to the description of elements of a claim.

[0049] Each individual embodiment described and illustrated herein has its own independent components and features, which can be readily separated from or combined with the components and features of any other embodiment without departing from the scope or spirit of the invention. Furthermore, any method described may be carried out in the order of steps described, or in any other logically possible order.

[0050] Examples of suitable mammalian cells and tissues The cryopreservation formulations and culture media of this disclosure are suitable for the cryopreservation of any mammalian cells or tissues. In certain embodiments, the cryopreservation formulations and culture media of this disclosure can be used for the cryopreservation of elephant cells or tissues, including elephant progenitor cells. The elephant cells may be selected from, for example, Asian elephant cells (Elephas maximus), African elephant cells (Loxodonta africana), African forest elephant cells (Loxodonta cyclotis), and Bornean elephant cells (Elephas maximus borneensis).

[0051] In particular, the cryopreservation formulations and culture media of the present disclosure are suitable for the cryopreservation of genetically modified (e.g., transformed) or reprogrammed mammalian cells or tissues. In certain embodiments, the cryopreservation formulations and culture media of the present disclosure are suitable for the cryopreservation of stem cells or progenitor cells, including but not limited to cells obtained by dedifferentiation or dedifferentiation, reprogrammed cells, and somatic cells. In certain embodiments, the cryopreservation formulations and culture media of the present disclosure are suitable for the cryopreservation of tissues obtained from such stem cells or progenitor cells.

[0052] Cryopreservation preparations The cryopreservation formulations relating to this disclosure contain glycerol and trehalose as cryoprotective agents. In certain embodiments, the cryopreservation solution contains glycerol, trehalose, ascorbic acid and / or taurine as cryoprotective agents. In other embodiments, the cryopreservation solution contains glycerol, trehalose, ascorbic acid and taurine as cryoprotective agents. In certain embodiments, the cryopreservation formulation does not contain dimethyl sulfoxide (DMSO) and / or additional cryoprotective agents.

[0053] In certain embodiments, the formulation further contains glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and / or L-serine. In other embodiments, the cryopreservation formulation is supplemented with serum, such as fetal cow serum.

[0054] In certain embodiments, the cryopreservation preparation does not contain DMSO, that is, it does not contain any DMSO.

[0055] In one embodiment, the cryopreservation formulation contains glycerol and trehalose. In a particular embodiment, the cryopreservation formulation contains about 20% to about 60%, or about 30% to about 50%, or about 35% to about 45% of glycerol. In another embodiment, the cryopreservation formulation contains about 200 mM to about 3 mM, or about 300 mM to about 3 mM, or about 200 mM to about 600 mM, or about 300 mM to about 500 mM, or about 350 mM to about 450 mM, or about 1 mM to about 3 mM, or about 1.5 mM to about 2.5 mM of trehalose. In yet another embodiment, the cryopreservation formulation contains about 20% to about 60%, or about 30% to about 50%, or about 35% to about 45% of glycerol, and about 200 mM to about 3 mM, or about 300 mM to about 3 mM, or about 200 mM to about 600 mM, or about 300 mM to about 500 mM, or about 350 mM to about 450 mM, or about 1 mM to about 3 mM, or about 1.5 mM to about 2.5 mM of trehalose.

[0056] In another embodiment, the cryopreservation preparation further contains ascorbic acid and / or taurine. Therefore, in a particular embodiment, the cryopreservation preparation contains glycerol, trehalose, and ascorbic acid. In another embodiment, the cryopreservation preparation contains glycerol, trehalose, and taurine. In yet another embodiment, the cryopreservation preparation contains glycerol, trehalose, ascorbic acid, and taurine. In a particular embodiment, the cryopreservation preparation contains ascorbic acid in an amount of about 100 μg / mL to about 250 μg / mL, or about 100 μg / mL to about 200 μg / mL, or about 120 μg / mL to about 180 μg / mL. In other embodiments, the cryopreservation formulation contains about 10 mM to about 40 mM, or about 15 mM to about 35 mM, or about 20 mM to about 35 mM, or about 25 mM to about 35 mM of taurine. In yet another embodiment, the cryopreservation formulation contains about 100 μg / mL to about 250 μg / mL, or about 100 μg / mL to about 200 μg / mL, or about 120 μg / mL to about 180 μg / mL of ascorbic acid, and about 10 mM to about 40 mM, or about 15 mM to about 35 mM, or about 20 mM to about 35 mM, or about 25 mM to about 35 mM of taurine.

[0057] In yet another embodiment, the cryopreservation formulation contains (a) about 20% to about 60%, or about 30% to about 50%, or about 35% to about 45% of glycerol, (b) about 200 mM to about 3 mM, or about 300 mM to about 3 mM, or about 200 mM to about 600 mM, or about 300 mM to about 500 mM, or about 350 mM to about 450 mM, or about 1 mM to about 3 mM, or It contains (c) approximately 1.5 mM to approximately 2.5 mM trehalose, (d) approximately 100 μg / mL to approximately 250 μg / mL, or approximately 100 μg / mL to approximately 200 μg / mL, or approximately 120 μg / mL to approximately 180 μg / mL ascorbic acid, and (d) approximately 10 mM to approximately 40 mM, or approximately 15 mM to approximately 35 mM, or approximately 20 mM to approximately 35 mM, or approximately 25 mM to approximately 35 mM taurine.

[0058] In yet another embodiment, the cryopreservation preparation further contains glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and / or L-serine. In a particular embodiment, 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 supplied as commercially available non-essential amino acid (NEAA) cell culture supplements. In certain embodiments, the NEAA cell culture supplement contains approximately 750 mg / L of glycine, approximately 890 mg / L of L-alanine, approximately 1320 mg / L of L-asparagine, approximately 1330 mg / L of aspartic acid, approximately 1470 mg / L of L-glutamic acid, and approximately 1150 mg / L of L-serine. The NEAA may be contained in a range of approximately 0.1% to approximately 2.5%, or approximately 0.5% to approximately 2.0%, or approximately 0.8% to approximately 1.8%.

[0059] In yet another embodiment, the cryopreservation preparation further contains serum, such as FBS. In yet another embodiment, the cryopreservation preparation further contains about 40% to about 75%, or about 50% to about 70%, or about 55% to about 65% of serum (e.g., FBS). In a particular embodiment, the cryopreservation preparation contains serum and a combination of cryoprotective agents selected from the group consisting of (a) to (d) below: (a) glycerol and trehalose, (b) glycerol, trehalose and ascorbic acid, (c) glycerol, trehalose and taurine, and (d) glycerol, trehalose, ascorbic acid and taurine. In another embodiment, the cryopreservation preparation contains glycerol, trehalose, ascorbic acid, taurine, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, and serum.

[0060] In certain embodiments, the cryopreservation formulation according to the Disclosure is formulated to be added to a cell culture medium. In certain embodiments, the cryopreservation formulation is a 2× basic formulation that can be mixed with a suitable cell culture (growth) medium in approximately a 1:1 ratio.

[0061] Examples of cryopreservation preparations are shown in the table below. TIFF2026529521000001.tif210170TIFF2026529521000002.tif143170

[0062] The cryopreservation preparations described above can be added to existing cell culture media, including commercially available culture media, to produce cryopreservation media.

[0063] Cryopreservation culture medium Another embodiment of the present disclosure relates to a cryopreservation medium. The cryopreservation medium according to the present disclosure is a cell culture medium containing (or supplemented with) glycerol and trehalose as cryoprotective agents. In a particular embodiment, the cryopreservation medium contains, or is supplemented with, glycerol, trehalose, and taurine as cryoprotective agents. In yet another embodiment, the cryopreservation medium contains, or is supplemented with, glycerol, trehalose, and ascorbic acid as cryoprotective agents. In yet another embodiment, the cryopreservation medium contains, or is supplemented with, glycerol, trehalose, ascorbic acid, and taurine as cryoprotective agents.

[0064] The cryopreservation medium contains components necessary to enable the proliferation of mammalian cells. In some embodiments, the cryopreservation medium is a basal definition medium. In certain embodiments, the cryopreservation medium contains sufficient amounts of amino acids, energy sources (e.g., glucose), and ions to enable the proliferation of mammalian cells.

[0065] In certain embodiments, the cryopreservation medium contains, or is added to, a combination of cryoprotective agents selected from the group consisting of (a) to (d) below: (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 medium does not contain DMSO and / or additional cryoprotective agents.

[0066] In certain embodiments, the cryopreservation medium further contains glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and / or L-serine. In yet another embodiment, the cryopreservation medium is supplemented with serum, such as fetal cow serum.

[0067] As used herein, the phrase "cell culture medium supplemented with" or "cell culture media supplemented with" refers to existing cell culture media, including commercially available media such as 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 have been added.

[0068] In one embodiment, the cryopreservation medium is a basal cell culture medium to which a combination of cryoprotective agents selected from the group consisting of (a) to (d) below is added: (a) glycerol and trehalose; (b) glycerol, trehalose and ascorbic acid; (c) glycerol, trehalose and taurine; and (d) glycerol, trehalose, ascorbic acid and taurine. In a particular embodiment, the cryopreservation medium contains about 25% to about 45%, or about 30% to about 45%, or about 30% to about 40% of the basal cell culture medium.

[0069] In another embodiment, the cryopreservation medium further contains, or is supplemented with, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and / or L-serine. In a particular embodiment, the cryopreservation 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 supplied as commercially available non-essential amino acid (NEAA) cell culture supplements. In certain embodiments, the NEAA cell culture supplement comprises approximately 750 mg / L of glycine, approximately 890 mg / L of L-alanine, approximately 1320 mg / L of L-asparagine, approximately 1330 mg / L of aspartic acid, approximately 1470 mg / L of L-glutamic acid, and approximately 1150 mg / L of L-serine.

[0070] In yet another embodiment, the cryopreservation medium further contains or is added to serum, such as FBS. In one embodiment, the cryopreservation medium contains or is added to serum and contains or is added to a combination of cryoprotective agents selected from the group consisting of (a) to (d) below: (a) glycerol and trehalose, (b) glycerol, trehalose and ascorbic acid, (c) glycerol, trehalose and taurine, and (d) glycerol, trehalose, ascorbic acid and taurine.

[0071] In certain embodiments, the cryopreservation medium does not contain DMSO, that is, it does not contain any DMSO.

[0072] In certain embodiments, the cryopreservation medium is prepared by adding the cryopreservation preparation described above to a cell culture medium in a 1:1 ratio. The cryopreservation medium of this disclosure may be prepared using various commercially available cell culture media. In one embodiment, the cryopreservation medium is prepared by adding the cryopreservation preparation of this disclosure to Dulbecco's modified Eagle medium (DMEM). In another embodiment, the cryopreservation medium is prepared by adding the cryopreservation preparation of this disclosure to minimal essential medium (MEM), basal medium Eagle (BME), Roswell Park Memorial Laboratory (RPMI) 1640 medium, Iscove modified Dulbecco's medium (IMDM), or endothelial cell growth medium-2 (EGM-2).

[0073] Examples of culture media for cryopreservation are shown in the table below. TIFF2026529521000003.tif231170TIFF2026529521000004.tif103170

[0074] The basal cell culture medium used to produce the cryopreservation medium of this disclosure may be any cell culture medium, including commercially available media such as basal definition medium.

[0075] kit This disclosure also provides cryopreservation kits. In one embodiment, the kit includes the cryopreservation formulation and instructions for use described above. In another embodiment, the kit includes the cryopreservation formulation, NEAA cell culture supplement, and instructions for use described above. In yet another embodiment, the kit includes the cryopreservation formulation, NEAA cell culture supplement, cell culture medium, and instructions for use described above. In yet another embodiment, the kit includes the cryopreservation formulation, NEAA cell culture supplement, cell culture medium, serum supplement (e.g., FBS), and instructions for use described above.

[0076] In one embodiment, the kit includes the cryopreservation medium and instructions for use described above. In another embodiment, the kit includes the cryopreservation medium, NEAA cell culture supplement, and instructions for use described above. In yet another embodiment, the kit includes the cryopreservation medium, NEAA cell culture supplement, serum supplement (e.g., FBS), and instructions for use described above.

[0077] Method for manufacturing culture media for cryopreservation This disclosure also provides a method for producing cryopreservation media. In one embodiment, the method includes mixing the cryopreservation preparation according to this disclosure with a cell culture medium (e.g., a basal cell culture medium). In a particular embodiment, the method includes mixing the basal cell culture medium and the cryopreservation preparation in approximately a 1:1 ratio.

[0078] In one embodiment, the method includes the step of mixing the cryopreservation formulation according to the Disclosure with a cell culture medium containing an amount of amino acids, an energy source (e.g., glucose), and ions sufficient to enable the proliferation of mammalian cells.

[0079] Methods for cryopreserving cells or tissues This disclosure also provides a method for cryopreserving mammalian cells or solid tissues. The method for cryopreserving cells comprises the steps of: freezing the cells stepwise in a cell culture medium supplemented with the cryopreservation medium or cryopreservation formulation according to this disclosure; and storing the cells or tissues under appropriate conditions (e.g., in liquid nitrogen). The method may further include the step of rapidly thawing the frozen cells. Figure 1A shows a schematic diagram of a protocol for cryopreserving cells or tissues.

[0080] A method for cryopreserving solid tissue comprises the steps of: freezing the solid tissue in stages in a culture medium to which the cryopreservation medium or cryopreservation formulation according to the Disclosure has been added; and storing the solid tissue under appropriate conditions (e.g., in liquid nitrogen). The method may further include the step of rapidly thawing the frozen solid tissue. Figure 1B shows a schematic diagram of a protocol for cryopreserving tissue. The protocol includes the steps of: washing the tissue sample to be preserved; adding a culture medium; and then transferring the tissue sample to a cryopreservation container. Subsequently, the cryopreservation medium or solution according to the Disclosure is added to the sample and equilibrated under appropriate conditions (e.g., room temperature). The sample is then cooled to approximately -80°C. Within 24 hours of cooling to -80°C, the sample can be used for long-term storage. In a particular embodiment, the protocol includes the following steps. 1. Place the tissue in a Petri dish and wash twice with saline solution containing a 1% antibiotic mixture (1% penicillin / streptomycin, optional). 2. As quickly as possible, use tweezers and a scalpel to cut the tissue into small pieces of approximately 5 x 5 mm in a petri dish. The size of the tissue pieces can be adjusted according to their density. At this stage, add cell culture medium to prevent the tissue from drying out and incubate at room temperature for 10 minutes. 3. Transfer the tissue sample to a cryovial. 4. Using a transfer pipette or micropipette, add enough cryopreservation medium to completely cover the tissue, such that the tissue:cryopreservation medium volume ratio is at least 1:1. 5. Close the lid and thoroughly mix the sample by inverting the tube 8 to 10 times. 6. Allow to equilibrate at room temperature for 10-30 minutes, depending on the size of the tissue. 7. Place the vial at -80°C. The vial can be stored at -80°C for several days, or it can be placed in LN2 the following day (24 hours later) for long-term storage.

[0081] The most commonly used cryopreservation protocols today employ a similar approach of "gradual freezing" and "rapid thawing" to minimize the harmful effects of intracellular ice crystal formation, which can compromise the integrity of lipid membranes. Conventionally, when using cryopreservation media containing DMSO, it is important to minimize the time cells are exposed to DMSO solution at room temperature, as DMSO increases cell membrane permeability and can compromise membrane integrity with prolonged exposure. For this reason, media containing DMSO must be mixed separately from cells, as exposure to undiluted DMSO can be highly cytotoxic. In contrast, the cell formulations described herein are ready-to-use mixtures that can be combined with the growth media used by each research group to prepare cryopreservation mixtures that can be adjusted according to the application.

[0082] The cell culture media and formulations relating to this disclosure can be used in the "Gradual Freeze" and "Rapid Thaw" protocols described above. In certain embodiments, after mixing with a complete cryopreservation medium, the cells are stored in an insulated cryo-chamber, placed at -80°C for gradual freezing, and then transferred to liquid nitrogen for long-term storage.

[0083] In some embodiments, when cryopreserving solid tissue using the cryopreservation formulation and / or cryopreservation medium according to the present disclosure, the incubation step includes an incubation step at room temperature (RT) in the presence of a culture medium (growth medium), followed by a second incubation step at room temperature (RT) using the cryopreservation medium.

[0084] Without further detailed explanation, it is expected that those skilled in the art will be able to manufacture and use the present invention and carry out the claimed methods based on the above description and the following exemplary embodiments. Accordingly, the following embodiments illustrate preferred embodiments of the present invention and should not be construed as limiting the remaining parts of this disclosure in any way. [Examples]

[0085] Example 1: Acquisition of cells from cryopreserved elephant tissue A series of experiments were conducted to develop the cell culture medium and formulations related to this disclosure. In these experiments, elephant placental tissue was cryopreserved in the presence of the cryopreservation formulations related to this disclosure, as well as in the presence of 10% DMSO / 40% FBS / 50% EGM-2-XAP or MACS® cryopreservation solution. The cryopreserved samples were examined by monitoring subsequent thawing and proliferation (basic proliferation in cell culture, or, in the case of cryopreserved tissue, the ability to establish cell lines).

[0086] As shown in Figures 2A and 2B, cell viability was observed in placental tissue preserved at p0 and p2 using the formulations of this disclosure. In contrast, cell viability was almost nonexistent in cells cryopreserved in 10% DMSO / 40% FBS / 50% EGM-2-XAP (Figures 2C and 2D). Tests were also conducted on MACS® cryopreservation solution, but the results were negative (data not shown).

[0087] Other freezing conditions were also tested. Specifically, these included 10% DMSO / 90% FBS, flash freeze, and low osmotic pressure conditions. However, no favorable results were observed for cell acquisition under any of these other conditions. The cryopreservation formulation according to this disclosure was compared with commercially available cryopreservation media in terms of its ability to cryopreservate highly sensitive cell lines. The test results are shown in Example 2 (elephant endometrial cells), Example 3 (bovine embryonic stem cells), and Example 4 (human organoids). The tests in Examples 2 to 4 are shown in Figure 1B and followed the protocol described in paragraph

[0079] above.

[0088] Example 2: Comparison of cryopreservation media for elephant endometrial cells The cryopreservation preparation described herein (Xo-Chill cryopreservation medium) was compared with a preparation containing 10% DMSO / 40% FBS / 50% EGM-2 (see Table 2-1 below). In this study, elephant endometrial cells were frozen and stored at -80°C for one year before thawing. The results of this study are shown in Figure 3A (Xo-Chill) and Figure 3B, respectively. [Table 2-1] Formulations with identifier = Xo-Chill (XC) TIFF2026529521000005.tif74166

[0089] Example 3: Comparison of cryopreservation media for bovine embryonic stem cells The cryopreservation formulations of this disclosure containing bESCM ("XC+bESCM") or the cryopreservation formulations of this disclosure containing KSR ("XC+KSR") were compared with mFreSR (trademark: STEMCELL Technologies) (see Tables 3-1 and 3-2 below). Bovine embryonic stem cells were used in this study. [Table 3-1] Formulations with identifier = XC+bESCM TIFF2026529521000006.tif132166 [Table 3-2] Formulation with identifier = XC+KSR TIFF2026529521000007.tif76166

[0090] Bovine embryonic stem cells (Bovine ESCs) p4 were harvested using ReLeSR and frozen in 10% DMSO and 90% serum. After thawing, the cells showed a viability of 75% (data not shown) and were seeded in wells pre-coated with vitronectin in bEPSCM. After the samples reached 80% confluence, the cells were harvested and 1 × 10⁶ cells were collected for each medium condition. 6 The cells were cryopreserved. Figures 4A to 4C show images of cells cryopreserved in XC+bESCM, XC+KSR, and mFreSR®, respectively. These images were taken 48 hours after seeding (cryopreservation). Figure 4D shows the percentage of cell viability and the normalized viability after cryopreservation. From the test results in this example, it was confirmed that the cryopreservation formulation according to this disclosure has performance equivalent to or better than mFreSR®.

[0091] Example 4: Comparison of cryopreservation media for human organoids Furthermore, the cryopreservation formulation described herein was compared with CryoStor CS10 (Stem Cell Technologies). Human organoids were used in this study.

[0092] CryoStor CS10, a major commercially available cryopreservation medium, was used as a reference example according to the manufacturer's recommendations. Since it has been reported that spheroid / organoid recovery and functional maintenance are improved by using DMSO, CS10 containing 10% DMSO was compared with the XC cryopreservation medium of this disclosure (see Table 4-1 below). Human organoids were cultured in organoid maintenance medium (OMM). The samples were then collected and transferred to cryovials (see Figures 5A and 5B). Samples in the XC cryopreservation medium were incubated at room temperature for 10 minutes. Organoids in the XC cryopreservation medium showed equilibrium, while organoids in CS10 remained suspended. Both groups were frozen according to a slow freezing rate. After thawing, the samples were cultured in OMM, and pulsatile contractility was monitored after 24 hours (see Figure 5C). The results of the cell contractility test are shown in Figure 5D. [Table 4-1] Formulations with identifier = XC TIFF2026529521000008.tif120170 Embodiment

[0093] The present invention further provides the following non-limiting embodiments.

[0094] Embodiment 1 is a cryopreservation formulation for cryopreserving mammalian cells or solid mammalian tissues, comprising a combination of cryoprotective agents selected from the group consisting of (a) to (d) below: (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 cryopreserving cells. In another embodiment, the formulation is for cryopreserving solid mammalian tissues.

[0095] Embodiment 2 is a cryopreservation formulation according to Embodiment 1, wherein the formulation contains a combination of cryoprotective agents selected from the group consisting of (a) to (d) below: (a) about 20% to about 60% glycerol and about 200 mM to about 3 M or about 200 mM to about 600 mM trehalose; (b) about 20% to about 60% glycerol, about 200 mM to about 3 M or about 200 mM to about 600 mM trehalose and about 100 μg / mL (c) Approximately 250 μg / mL of ascorbic acid; (d) Approximately 20% to 60% of glycerol, approximately 200 mM to 3 M or approximately 200 mM to 600 mM of trehalose, and approximately 10 mM to 40 mM of taurine; and (d) Approximately 20% to 60% of glycerol, approximately 200 mM to 3 M or approximately 200 mM to 600 mM of trehalose, approximately 100 μg / mL to 250 μg / mL of ascorbic acid, and approximately 10 mM to 40 mM of taurine.

[0096] Embodiment 3 is a cryopreservation formulation according to Embodiment 1 or 2, wherein the combination of cryoprotective agents is glycerol and trehalose.

[0097] Embodiment 4 is a cryopreservation formulation according to Embodiment 1 or 2, wherein the combination of cryoprotective agents is glycerol, trehalose, and ascorbic acid.

[0098] Embodiment 5 is a cryopreservation formulation according to Embodiment 1 or 2, wherein the combination of cryoprotective agents is glycerol, trehalose, and taurine.

[0099] Embodiment 6 is a cryopreservation formulation according to Embodiment 1 or 2, wherein the combination of cryoprotective agents is glycerol, trehalose, ascorbic acid, and taurine.

[0100] Embodiment 7 is a cryopreservation preparation according to any one of Embodiments 1 to 6, further containing serum.

[0101] Embodiment 8 is a cryopreservation preparation according to Embodiment 7, wherein the preparation contains approximately 40% to approximately 75% serum.

[0102] Embodiment 9 is a cryopreservation preparation according to Embodiment 7 or 8, wherein the serum contains FBS.

[0103] Embodiment 10 is a cryopreservation preparation according to any one of Embodiments 1 to 9, further containing glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and / or L-serine.

[0104] Embodiment 11 is a cryopreservation preparation according to Embodiment 10, further containing about 0.1% to about 2.5% glycine, about 0.1% to about 2.5% L-alanine, about 0.1% to about 2.5% L-asparagine, about 0.1% to about 2.5% L-aspartic acid, about 0.1% to about 2.5% L-glutamic acid, about 0.1% to about 2.5% L-proline and / or about 0.1% to about 2.5% L-serine.

[0105] Embodiment 12 is a cryopreservation preparation according to Embodiment 10 or 11, further containing glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine.

[0106] Embodiment 13 is a cryopreservation preparation according to any one of Embodiments 1 to 12, and does not contain dimethyl sulfoxide (DMSO).

[0107] Embodiment 14 is a cryopreservation preparation according to any one of Embodiments 1 to 13, and does not contain an additional cryoprotective agent.

[0108] Embodiment 15 is a cryopreservation medium containing the cryopreservation preparation described in any one of Embodiments 1 to 14.

[0109] Embodiment 16 is a cryopreservation medium according to Embodiment 15, which contains sufficient amounts of amino acids, energy sources, and ions to enable the proliferation of mammalian cells.

[0110] Embodiment 17 is a cryopreservation medium according to Embodiment 16, wherein the energy source is glucose.

[0111] Embodiment 18 is a method for producing a cryopreservation medium, comprising the step of mixing a cryopreservation preparation described in any one of Embodiments 1 to 14 with a cell culture medium.

[0112] Embodiment 19 is the method according to Embodiment 18, wherein the cell culture medium contains an amount of amino acids, an energy source, and ions sufficient to enable the proliferation of mammalian cells. Alternatively, the cell culture medium contains an amount of amino acids, an energy source, and ions sufficient to enable the proliferation of mammalian cells in solid mammalian tissue.

[0113] Embodiment 20 is the method according to Embodiment 19, wherein the cell culture medium contains an amount of amino acids, glucose, and ions sufficient to enable the proliferation of mammalian cells. Alternatively, the cell culture medium contains an amount of amino acids, glucose, and ions sufficient to enable the proliferation of mammalian cells in solid mammalian tissue.

[0114] Embodiment 21 is a cryopreservation medium for cryopreserving mammalian cells, comprising a combination of amino acids, glucose and ions in sufficient quantities to enable the proliferation of mammalian cells, and a cryoprotective agent selected from the group consisting of (a) to (d) below: (a) glycerol and trehalose; (b) glycerol, trehalose and ascorbic acid; (c) glycerol, trehalose and taurine; and (d) glycerol, trehalose, ascorbic acid and taurine.

[0115] Embodiment 22 is a cryopreservation medium according to Embodiment 21, comprising a combination of amino acids, glucose and ions in sufficient quantities to enable the proliferation of mammalian cells, and a cryoprotectant selected from the group consisting of (a) to (d) below: (a) about 10% to about 40% glycerol and about 100 mM to about 3 M trehalose; (b) about 10% to about 40% glycerol and about 100 mM (c) M to approximately 3M trehalose, and approximately 20 μg / mL to approximately 250 μg / mL ascorbic acid; (d) approximately 10% to approximately 40% glycerol, approximately 100 mM to approximately 3M trehalose, and taurine; and (d) approximately 10% to approximately 40% glycerol, approximately 100 mM to approximately 3M trehalose, approximately 20 μg / mL to approximately 250 μg / mL ascorbic acid, and approximately 5 mM to approximately 30 mM taurine.

[0116] Embodiment 23 is a culture medium for cryopreservation according to Embodiment 21 or 22, wherein the combination of cryoprotective agents is glycerol and trehalose.

[0117] Embodiment 24 is a cryopreservation medium according to Embodiment 21 or 22, wherein the combination of cryoprotective agents is glycerol, trehalose, and ascorbic acid.

[0118] Embodiment 25 is a cryopreservation medium according to Embodiment 21 or 22, wherein the combination of cryoprotective agents is glycerol, trehalose, and taurine.

[0119] Embodiment 26 is a cryopreservation medium according to Embodiment 21 or 22, wherein the combination of cryoprotective agents is glycerol, trehalose, ascorbic acid, and taurine.

[0120] Embodiment 27 is a cryopreservation medium according to any one of Embodiments 21 to 26, wherein the medium further contains serum.

[0121] Embodiment 28 is a cryopreservation medium described in Embodiment 27, wherein the medium contains approximately 40% to approximately 75% serum.

[0122] Embodiment 29 is a cryopreservation medium according to either Embodiment 26 or 27, wherein the serum contains FBS.

[0123] Embodiment 30 is a cryopreservation medium according to 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.

[0124] Embodiment 31 is a cryopreservation medium according to Embodiment 30, further comprising about 0.1% to about 2% glycine, about 0.1% to about 2% L-alanine, about 0.1% to about 2% L-asparagine, about 0.1% to about 2% L-aspartic acid, about 0.1% to about 2% L-glutamic acid, about 0.1% to about 2% L-proline and / or about 0.1% to about 2% L-serine.

[0125] Embodiment 32 is a cryopreservation medium according to Embodiment 30 or 31, further comprising glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine.

[0126] Embodiment 33 is a cryopreservation medium according to any one of Embodiments 21 to 32, wherein the cryopreservation medium contains about 25% to about 45% of a basal cell culture medium containing an amount of amino acids, glucose and ions sufficient to enable the proliferation of mammalian cells.

[0127] Embodiment 34 is a cryopreservation medium according to any one of Embodiments 21 to 33, wherein the medium does not contain DMSO.

[0128] Embodiment 35 is a cryopreservation medium according to any one of Embodiments 21 to 33, wherein the medium does not contain an additional cryoprotectant.

[0129] Embodiment 36 is a cryopreservation medium according to any one of Embodiments 21 to 35, wherein the mammalian cells are present in a solid tissue.

[0130] Embodiment 37 is a method for cryopreserving mammalian cells, comprising the steps of freezing mammalian cells in a cell culture medium to which a cryopreservation medium described in any one of Embodiments 15-17 or 21-35, or a cryopreservation preparation described in any one of Embodiments 1-14, is added, and storing the cells in an appropriate environment.

[0131] Embodiment 38 is the method according to Embodiment 37, wherein the suitable environment includes preserving the cells in liquid nitrogen.

[0132] Embodiment 39 is a method for cryopreserving mammalian solid tissue, comprising the steps of: freezing the mammalian solid tissue in a cell culture medium to which the cryopreservation medium described in any one of Embodiments 15-17 or 21-36, or the cryopreservation preparation described in any one of Embodiments 1-14, is added; and preserving the mammalian solid tissue in a suitable environment.

[0133] Embodiment 40 is the method according to Embodiment 39, wherein the suitable environment includes preserving the solid tissue of the mammal in liquid nitrogen.

[0134] Embodiment 41 is the method according to Embodiment 39, wherein the freezing step includes the step of culturing the mammalian solid tissue in a growth medium at room temperature, and the step of culturing the mammalian solid tissue together with a cell culture medium to which a cryopreservation medium or cryopreservation preparation has been added.

[0135] Embodiment 42 is a kit comprising a cryopreservation preparation described in any one of Embodiments 1 to 14, a cryopreservation culture medium described in any one of Embodiments 15 to 17 or 21 to 36, and instructions for use.

[0136] Embodiment 43 is the kit described in Embodiment 42, wherein the kit includes the cryopreservation preparation and cell culture medium described in any one of Embodiments 1 to 14.

[0137] Embodiment 44 is the kit described in Embodiment 43, wherein the cell culture medium contains an amount of amino acids, an energy source, and ions sufficient to enable the proliferation of mammalian cells.

[0138] Embodiment 45 is a kit as described in Embodiment 42, wherein the kit includes a cryopreservation medium as described in any one of Embodiments 15-17 or 21-35.

[0139] Embodiment 46 is a kit according to any one of Embodiments 42 to 45, wherein the kit includes instructions for cryopreserving mammalian cells.

[0140] While the present invention is described and illustrated herein with reference to various specific materials, procedures, and examples, it should be understood that the invention is not limited to any specific combination of materials and procedures selected for its purpose. As will be apparent to those skilled in the art, numerous variations may be implied to these details. This specification and the examples should be understood to be illustrative only, and the true scope and spirit of the invention are shown by the following claims. All documents, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A cryopreservation preparation for cryopreserving mammalian cells or solid mammalian tissues, A cryopreservation preparation containing a combination of cryoprotective agents selected from the group consisting of (a) to (d) below: (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 cryopreservation preparation according to claim 1, wherein the cryopreservation preparation contains a combination of cryoprotective agents selected from the group consisting of (a) to (d) below: (a) Approximately 20% to 60% glycerol, and approximately 200 mM to 3 M or approximately 200 mM to 600 mM trehalose; (b) Approximately 20% to 60% glycerol, approximately 200 mM to 3 M or approximately 200 mM to 600 mM trehalose, and approximately 100 μg / mL to 250 μg / mL ascorbic acid; (c) Approximately 20% to 60% glycerol, approximately 200 mM to 3 M or approximately 200 mM to 600 mM trehalose, and approximately 10 mM to 40 mM taurine; and (d) Approximately 20% to 60% glycerol, approximately 200 mM to 3 M or approximately 200 mM to 600 mM trehalose, approximately 100 μg / mL to 250 μg / mL ascorbic acid, and approximately 10 mM to 40 mM taurine.

3. The cryopreservation preparation according to claim 1 or 2, wherein the combination of cryoprotective agents is glycerol and trehalose.

4. The cryopreservation preparation according to claim 1 or 2, wherein the combination of cryoprotective agents is glycerol, trehalose, and ascorbic acid.

5. The cryopreservation preparation according to claim 1 or 2, wherein the combination of cryoprotective agents is glycerol, trehalose, and taurine.

6. The cryopreservation preparation according to claim 1 or 2, wherein the combination of cryoprotective agents is glycerol, trehalose, ascorbic acid, and taurine.

7. The cryopreservation preparation according to claim 1, further containing serum.

8. The cryopreservation preparation according to claim 7, wherein the preparation contains about 40% to about 75% of the serum.

9. The cryopreservation preparation according to claim 7, wherein the serum comprises FBS (fetal cow serum).

10. The cryopreservation preparation according to claim 1 or 7, further comprising glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and / or L-serine.

11. Furthermore, the cryopreservation preparation according to claim 10 contains approximately 0.1% to approximately 2.5% glycine, approximately 0.1% to approximately 2.5% L-alanine, approximately 0.1% to approximately 2.5% L-asparagine, approximately 0.1% to approximately 2.5% L-aspartic acid, approximately 0.1% to approximately 2.5% L-glutamic acid, approximately 0.1% to approximately 2.5% L-proline and / or approximately 0.1% to approximately 2.5% L-serine.

12. The cryopreservation preparation according to claim 10, further containing glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine.

13. A cryopreservation preparation according to claim 1, which does not contain dimethyl sulfoxide (DMSO).

14. A cryopreservation preparation according to claim 1 or 13, which does not contain an additional cryoprotectant.

15. A culture medium for cryopreservation containing the cryopreservation preparation described in claim 1.

16. A cryopreservation medium according to claim 15, comprising sufficient amounts of amino acids, energy sources, and ions to enable the proliferation of mammalian cells.

17. The cryopreservation medium according to claim 16, wherein the energy source is glucose.

18. A method for producing a cryopreservation medium, comprising the step of mixing the cryopreservation preparation described in claim 1 with a cell culture medium.

19. The method according to claim 18, wherein the cell culture medium contains an amount of amino acids, an energy source, and ions sufficient to enable the proliferation of mammalian cells.

20. The method according to claim 19, wherein the cell culture medium contains an amount of amino acids, glucose, and ions sufficient to enable the proliferation of mammalian cells.

21. A cryopreservation medium for cryopreserving mammalian cells, Sufficient amounts of amino acids, glucose and ions to enable the proliferation of mammalian cells, and A culture medium for cryopreservation containing a combination of cryoprotective agents selected from the group consisting of (a) to (d) below: (a) Glycerol and trehalose; (b) Glycerol, trehalose, and ascorbic acid; (c) Glycerol, trehalose and taurine; and (d) Glycerol, trehalose, ascorbic acid, and taurine.

22. Sufficient amounts of amino acids, glucose and ions to enable the proliferation of mammalian cells, and A cryopreservation medium according to claim 21, comprising a combination of cryoprotectants selected from the group consisting of (a) to (d) below: (a) Approximately 10% to 40% glycerol, and approximately 100 mM to 3 M trehalose; (b) Approximately 10% to 40% glycerol, approximately 100 mM to 3 M trehalose, and approximately 20 μg / mL to 250 μg / mL ascorbic acid; (c) Approximately 10% to 40% glycerol, approximately 100 mM to 3 M trehalose, and taurine; and (d) Approximately 10% to 40% glycerol, approximately 100 mM to 3 M trehalose, approximately 20 μg / mL to 250 μg / mL ascorbic acid, and approximately 5 mM to 30 mM taurine.

23. The cryopreservation medium according to claim 21 or 22, wherein the combination of cryoprotective agents is glycerol and trehalose.

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

25. The cryopreservation medium according to claim 21 or 22, wherein the combination of cryoprotective agents is glycerol, trehalose, and taurine.

26. The cryopreservation medium according to claim 21 or 22, wherein the combination of cryoprotective agents is glycerol, trehalose, ascorbic acid, and taurine.

27. The cryopreservation medium according to claim 21 or 22, further containing serum.

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

29. The cryopreservation medium according to claim 27, wherein the serum comprises FBS (fetal cow serum).

30. The cryopreservation medium according to claim 21 or 22, further comprising glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and / or L-serine.

31. The cryopreservation medium according to claim 30, further containing approximately 0.1% to approximately 2% glycine, approximately 0.1% to approximately 2% L-alanine, approximately 0.1% to approximately 2% L-asparagine, approximately 0.1% to approximately 2% L-aspartic acid, approximately 0.1% to approximately 2% L-glutamic acid, approximately 0.1% to approximately 2% L-proline and / or approximately 0.1% to approximately 2% L-serine.

32. The cryopreservation medium according to claim 30, further containing glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine.

33. The cryopreservation medium according to claim 21 or 22, wherein the cryopreservation medium contains about 25% to about 45% of a basal cell culture medium containing an amount of amino acids, glucose and ions sufficient to enable the proliferation of mammalian cells.

34. A culture medium for cryopreservation according to claim 21 or 22, which does not contain dimethyl sulfoxide (DMSO).

35. A culture medium for cryopreservation according to claim 21 or 22, which does not contain additional cryoprotective agents.

36. The cryopreservation medium according to claim 21 or 22, wherein the mammalian cells are present in a solid tissue.

37. A method for cryopreserving mammalian cells, A step of freezing mammalian cells in a cell culture medium to which the cryopreservation medium described in claim 15 or the cryopreservation preparation described in claim 1 has been added, A step of storing the aforementioned cells in an appropriate environment. Methods that include...

38. The method according to claim 37, wherein the appropriate environment includes preserving the cells in liquid nitrogen.

39. A method for cryopreserving solid mammalian tissue, A step of freezing the solid tissue of the mammal in a cell culture medium to which the cryopreservation medium described in claim 15 or the cryopreservation preparation described in claim 1 has been added, A step of preserving the solid tissue of the aforementioned mammal under appropriate conditions. Methods that include...

40. The method according to claim 39, wherein the appropriate environment includes preserving the solid tissue of the mammal in liquid nitrogen.

41. The method according to claim 39, wherein the freezing step comprises the steps of culturing the solid tissue of the mammal in a growth medium at room temperature, and thereafter culturing the solid tissue of the mammal together with a cryopreservation medium or a cell culture medium to which the cryopreservation preparation has been added.

42. A kit comprising the cryopreservation preparation according to claim 1, or the cryopreservation culture medium according to claim 15, and instructions for use.

43. The kit according to claim 42, comprising the cryopreservation preparation and cell culture medium according to claim 1.

44. The kit according to claim 43, wherein the cell culture medium contains an amount of amino acids, an energy source, and ions sufficient to enable the proliferation of mammalian cells.

45. The kit according to claim 42, comprising the cryopreservation medium described in claim 15.

46. The kit according to claim 42, comprising instructions for cryopreserving mammalian cells.

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