Cryopreserved preparation

Abstract

The present disclosure provides cryopreservation formulations, cryopreservation media, and methods of cryopreserving mammalian cells or tissues. The cryopreservation preparation and the culture medium contain a combination of cryoprotectants 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

[0001] Cross-referencing This application claims priority to U.S. Provisional Application No. 63 / 527,462, filed July 18, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the cryopreservation of cells, particularly mammalian cells and solid tissues. Specifically, this disclosure relates to cryopreservation formulations and culture media, as well as methods for cryopreserving cells. Background Technology

[0003] Preserving mammalian cells and solid tissues in liquid nitrogen is a crucial step in cell culture laboratories, enabling the establishment of a valuable sample resource bank for subsequent research and applications. The nutrient-rich culture medium used during cryopreservation has a significant impact on cell viability and biological function after sample thawing.

[0004] The freezing process (also known as cryopreservation) typically uses cell culture media that can be used to grow mammalian cells to be stored (cryopreserved) and cryoprotectants that maintain the function and viability of the cells during freezing.

[0005] The current "gold standard" for cryopreservation requires the use of dimethyl sulfoxide (DMSO) as a cryoprotectant. While DMSO is the gold standard, studies have also shown that it may be harmful to cryopreserved cells. For example, Galvao et al. "demonstrated that DMSO induced retinal apoptosis in vivo at low concentrations (5 μl DMSO intravitreal injection in rats, with stock solution concentrations of 1, 2, 4, and 8% v / v)." FASEB J. 28(3):1317-30 (2014), Abstract. Similarly, a 2020 review by Awan et al. compiled data showing many disadvantages of using DMSO and highlighting the need for DMSO alternatives. Awan et al., Regenerative Medicine , 15(3):1463-1491(2020).

[0006] Given the drawbacks of using DMSO, DMSO-free cryopreservation media have been developed for cell cryopreservation. One example is XT Thive® cryopreservation, which uses N-substituted biomimetic amino acid polymers (peptides) and peptide-peptide hybrids as alternatives to DMSO. However, peptides are generally synthetic molecules with the potential to bind to / target proteins, and therefore require extensive evaluation for each application.

[0007] What is needed are cryopreservation solutions and media that can overcome the potential drawbacks of using DMSO and currently available DMSO-free media, and can be used for the cryopreservation of cells and large tissues. Summary of the Invention

[0008] This disclosure provides cryopreservation formulations, cryopreservation media, and methods for cryopreserving mammalian cells or large tissue masses. The cryopreservation formulations and media contain combinations of cryoprotectants 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 some embodiments, the cryopreservation formulations and media do not contain dimethyl sulfoxide. In other embodiments, the cryopreservation formulations and media do not contain additional cryoprotectants.

[0009] One embodiment of this disclosure is a cryopreservation formulation for cryopreserving mammalian cells, comprising a combination of cryoprotectants 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 cell culture media to produce a cryopreservation medium.

[0010] In some embodiments, the cryopreservation formulation comprises a combination of cryoprotectants selected from the group consisting of: (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 / mL ascorbic acid; (c) 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.

[0011] The cryopreservation formulation may also contain serum (e.g., FBS), for example, in an amount of about 40% to about 75%. Furthermore, the cryopreservation formulation may contain glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and / or L-serine. In some embodiments, the formulation contains about 0.1% to about 2.5% of glycine, about 0.1% to about 2.5% of L-alanine, about 0.1% to about 2.5% of L-asparagine, about 0.1% to about 2.5% of L-aspartic acid, about 0.1% to about 2.5% of L-glutamic acid, about 0.1% to about 2.5% of L-proline, and / or about 0.1% to about 2.5% of L-serine.

[0012] Another aspect of this disclosure is a cryopreservation medium for cryopreserving mammalian cells, comprising: sufficient amounts of amino acids, glucose and ions to enable mammalian cell growth; and a combination of cryoprotectants 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 cryopreservation medium comprises a combination of cryoprotectants selected from the group consisting of: (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 another embodiment, the cryopreservation medium comprises serum (e.g., FBS), for example, about 40% to about 75% FBS. In another embodiment, the cryopreservation medium comprises glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and / or L-serine.

[0014] This disclosure also provides methods for generating cryopreservation culture media and methods for cryopreserving mammalian cells and / or tissues. Furthermore, this disclosure includes kits containing cryopreservation formulations or culture media.

[0015] Other features and advantages of the invention will become apparent from the following detailed description and examples. Attached Figure Description

[0016] The foregoing overview and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. The drawings illustrate embodiments of the invention for illustrative purposes. However, it should be understood that the invention is not limited to the explicit arrangements, examples, and tools shown.

[0017] Figure 1A and Figure 1B A schematic diagram of the frozen culture medium formulation using the present disclosure is shown.

[0018] Figure 2A-2D Results of freezing placental tissue using the cryopreservation formulation of this disclosure are shown compared to commercially available cryopreservation culture media. Figure 2A and 2B The results of the formulation disclosed herein are shown. Figure 2C and 2DThe results of cells cryopreserved in 10% DMSO / 40% FBS / 50% EGM-2-XAP are shown.

[0019] Figure 3A and Figure 3B Results of cryopreservation of sensitive cell lines (elephant endometrial cells) using the cryopreservation formulation of this disclosure (identified as Xo-Chill (XC)) are shown compared to conventional cryopreservation culture medium formulations.

[0020] Figures 4A-4D Results of cryopreservation of bovine embryonic stem cells using the cryopreservation formulation of this disclosure are shown compared to commercially available mFreSR medium. Figures 4A-4C Photographs of cells cryopreserved in XC+bESCM, XC+KSR, and mFreSR are shown. The photos were taken 48 hours after inoculation (cryopreservation). Figure 4D The cell viability and normalized survival percentage after cryopreservation are shown.

[0021] Figures 5A-5D Results of cryopreservation of human organoids using the cryopreservation formulation of this disclosure are shown compared to commercially available Crystal CS10 medium. Figure 5A Images of human organoids pre-incubated with the XC cryogenic medium and CryoStor CS10 (CS) disclosed herein are shown. Figure 5B Images of human organoids incubated with the XC cryogenic medium and CryoStor CS10 disclosed herein are shown. Figure 5C The image shows the cells after thawing and culturing in organoid maintenance medium (OMM) for 24 hours. Figure 5D Cell contractility (organoid beating %) of thawed cells cryopreserved in XC+OMM and CS10 is shown. Detailed Implementation

[0022] This disclosure provides cryopreservation media, formulations, and methods for cryopreserving mammalian cells. This disclosure is based on the finding that cryoprotection with DMSO and FBS can be achieved using a combination of the following cryoprotectants: at least glycerol and trehalose; or glycerol and trehalose, along with ascorbic acid and / or taurine.

[0023] The cryopreservation culture medium and preparation disclosed herein contain at least glycerol and trehalose. The cryopreservation culture medium and preparation may be supplemented with serum, ascorbic acid, and / or taurine.

[0024] In some embodiments, the cryopreservation medium and formulation are DMSO-free, meaning they contain no DMSO whatsoever. In other embodiments, the cryopreservation medium and formulation are free of synthetic molecules (such as peptides) that may bind to / target proteins.

[0025] The cryopreservation formulation is intended to be added to cell culture media. In some embodiments, the cryopreservation medium is a base medium to which the cryopreservation formulation has been added.

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

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

[0028] As used herein, the terms “comprising,” “including,” “containing,” and “characterized in” are interchangeable, inclusive, and open-ended, and do not exclude additional, unreferenced elements or method steps. Any expression of the term “comprising” herein, particularly in the description of the components of a composition or the elements of an apparatus, should be understood to include those compositions and methods that are substantially composed of said components or elements.

[0029] As used herein, the term "composed of" excludes any ingredient, step, or component not specified in the elements of the claims.

[0030] As used herein, when referring to measurable values ​​(such as quantities, durations of time, etc.), the term “about” means including variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, such variations being suitable for performing the disclosed method.

[0031] Before describing certain embodiments in more detail, it should be understood that the invention is not limited to the described embodiments, and of course, these embodiments can be varied. It should also be understood that the terminology used herein is for describing certain embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0032] When a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range (accurate to one-tenth of the lower limit unit unless otherwise explicitly stated in the context), as well as any other specified value or intermediate value within the range, is covered by this invention. The upper and lower limits of these smaller ranges may be independently included within that smaller range and are also covered by this invention, but are limited by any explicitly excluded limits within the range. If the range includes one or two limits, the range excluding one or both of the included limits is also included by this invention.

[0033] Unless otherwise defined, 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 pertains. Although any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, representative illustrative methods and materials are described hereafter.

[0034] As used herein, the term "transformation" or "transformation" refers to the transfer of a nucleic acid fragment into a host cell, such as a host bacterial cell, resulting in genetic stability. A host cell containing the transformed nucleic acid fragment is referred to as a "recombinant," "transgenic," or "transformed" organism.

[0035] As used herein, the term "isolated" means that a biological component (such as a nucleic acid, peptide, or protein) has been substantially isolated, produced, or purified from other biological components of the organism in which the component is naturally present (i.e., other chromosomal and extrachromosomal DNA and RNA, as well as proteins). Therefore, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified using standard purification methods. "Isolated" nucleic acids, peptides, and proteins can form part of a composition, and are considered "isolated" as long as the composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also covers nucleic acids, peptides, and proteins prepared through recombinant expression in host cells, as well as chemically synthesized nucleic acids.

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

[0037] As used in this article, a "promoter" is an example of a transcriptional regulatory sequence, specifically referring to a nucleic acid sequence typically described as located in the proximal region of a gene at the 5' end of the start codon. Transcription of adjacent nucleic acid segments begins in the promoter region. The transcription rate of repressible promoters decreases under the action of repressors, the transcription rate of inducible promoters increases under the action of inducers, while the transcription rate of constitutive promoters, although affected by general metabolic conditions, is not specifically regulated.

[0038] As used herein, the term "gene product" refers to any product encoded by a nucleic acid sequence. Therefore, a gene product can be, for example, a primary transcript, a mature transcript, a processed transcript, or a protein / peptide encoded by a transcript. Accordingly, gene products include mRNA, rRNA, hairpin RNAs (such as microRNAs, shRNAs, siRNAs, tRNAs), and peptides and proteins (such as reporter proteins or therapeutic proteins).

[0039] As used herein, the term "stem cell" refers to a cell capable of self-renewal and differentiation into at least one more differentiated or less developed phenotype. The term "stem cell" includes stem cell lines, induced pluripotent stem cells, non-human embryonic stem cells, pluripotent stem cells, amniotic stem cells, placental stem cells, or adult stem cells. "Induced pluripotent stem cells" refers to cells that, through the introduction of one or more reprogramming factors or genes, are induced from non-pluripotent cells into phenotypes with less differentiation or greater developmental capacity. As used herein, induced stem cells do not need to be pluripotent, but under appropriate conditions possess the ability to differentiate into more than one highly differentiated phenotype. It should be understood that this ability does not exist prior to the introduction of the reprogramming factor. Prior to the introduction of the reprogramming factor, the induced stem cells will express at least one stem cell marker not expressed by the parental cells. In this case, the stem cell marker does not include the factor introduced through reprogramming. Induced pluripotent stem cells or iPS cells, under appropriate conditions, possess the ability to differentiate into cell phenotypes derived from the endoderm, mesoderm, and ectoderm germ layers.

[0040] As used herein, the term "marker" is used to describe the characteristics and / or phenotype of a cell. Markers can be used to screen cells for target characteristics and can vary with specific cell types. A marker is a morphological, structural, functional, or biochemical (enzymatic) characteristic of a specific cell type, or a molecule expressed by that cell type. On one hand, such markers are proteins that may have epitopes recognized by antibodies or other binding molecules available in the art. However, markers can consist of any molecule intracellularly or on the cell surface, 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 nucleocytoplasmic ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to a specific matrix, the ability to take up or exclude specific dyes, the ability to migrate under specific conditions, and the ability to differentiate along a specific lineage. Markers can be detected by any method available to those skilled in the art. A marker can also be the absence of a certain morphological characteristic or protein, lipid, etc. A marker can be a combination of a unique set of properties consisting of the presence / absence of polypeptides and other morphological or structural features. In one implementation, the marker is a cell surface marker.

[0041] The term "exogenous" refers to a substance introduced into a cell through artificial manipulation. As used herein, "exogenous" can refer to nucleic acids (e.g., nucleic acids encoding polypeptides) or polypeptides introduced into a biological system (such as a cell or organism) through methods involving artificial manipulation, where such nucleic acids or polypeptides are not normally present in said biological system. Alternatively, "exogenous" can refer to nucleic acids or polypeptides introduced into a biological system (such as a cell or organism) through methods involving artificial manipulation, where the nucleic acid or polypeptide is present in relatively low amounts in said biological system and there is a desire to increase its content in the cell or organism, for example, to achieve ectopic expression or increase expression levels.

[0042] As used herein, the term "reprogramming gene" or "reprogramming factor" refers to an agent or nucleic acid molecule that can induce a somatic cell reprogramming process to re-express a less differentiated, more stem cell-like phenotype. Reprogramming factors can be nucleic acids, peptides, or small molecules that promote a reprogrammed phenotype when introduced into cells. Non-limiting examples of reprogramming factors include Oct4 (octamer-binding transcription factor 4), SOX2 (sex-determining region Y)-box protein 2, Klf4 (Krüppel-like factor 4), and c-Myc. These are classic or standard combinations of reprogramming factors used to induce pluripotent stem cells, among others. Other factors that can be considered reprogramming factors in the process of reprogramming cells to a lower differentiation or stem cell phenotype include LIN28+Nanog, Esrrb, Pax5 shRNA, C / EBPa, p53 siRNA, UTF1, DNMT shRNA, Wnt3a, SV40 LT(T), hTERT, and small molecule chemical reagents, including but not limited to BIX-01294, BayK8644, RG108, AZA, dexamethasone, VPA, TSA, SAHA, PD0325901+CHIR99021(2i), and A-83-01. In some embodiments, the reprogramming gene or factor is Oct4, Klf4, SOX2, and c-Myc.

[0043] As used herein, the terms “dedifferentiation,” “reversal differentiation,” or “reprogramming” refer to a process that generates a cell type that re-expresses a differentiation phenotype lower than that of its derived cells and / or expresses at least one stem cell marker not expressed prior to the process. For example, terminally differentiated cells can be dedifferentiated into pluripotent cells. Dedifferentiation reverses the lineage of cells from pluripotent to fully differentiated. Typically, reversing a cell's differentiation phenotype requires artificial manipulation of the cell, such as by introducing or expressing exogenous peptide factors. Reprogramming processes are generally not observed under natural conditions, either in vivo or in vitro.

[0044] As used herein, a “reprogrammed cell” refers to a cell that has been exposed to one or more reprogramming factors and expresses a differentiation phenotype lower than that of its derived cell. Reprogrammed cells may also possess self-renewal capacity and express at least one stem cell marker not delivered to the cell as a reprogramming factor. Furthermore, reprogrammed cells will have the ability to differentiate into more differentiated somatic cell types according to the differentiation protocols provided herein or described in the art.

[0045] As used herein, the term "somatic cell" means any cell other than germ cells, cells present in or derived from a pre-implantation embryo, or cells derived from the in vitro proliferation of such cells. In other words, a somatic cell is any cell that forms an organism, excluding germ cells. Every cell type in a mammalian body, except for sperm and eggs and the cells that produce them (gametophytes), is a somatic cell: internal organs, skin, bones, blood, and connective tissue are essentially composed of somatic cells. In some embodiments, a somatic cell is a "non-embryonic somatic cell," meaning a somatic cell that is not present in or derived from an embryo and is not derived from the in vitro proliferation of such cells. In some embodiments, a somatic cell is an "adult cell," meaning a cell present in or derived from an organism other than an embryo or fetus, or a cell derived from the in vitro proliferation of such cells.

[0046] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were expressly and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the reference methods and / or materials relating to those publications. References to any publication are solely for its disclosure prior to the filing date and should not be construed as an admission that the invention is not entitled to a prior art prior to that publication. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification.

[0047] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly specifies otherwise. It should also be noted that the drafting of the claims may exclude any optional elements. Therefore, this statement is intended as a prior basis for the use of such exclusive terms as “alone,” “only,” or “negative” in relation to the statement of a claim element.

[0048] Without departing from the scope or spirit of the invention, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments. Any of the methods described may be implemented in the order of the events stated or in any other logically feasible order.

[0049] Examples of suitable mammalian cells and tissues The cryopreservation formulations and culture media disclosed herein are suitable for the cryopreservation of any mammalian cells or tissues. In some embodiments, the cryopreservation formulations and culture media disclosed herein can be used for the cryopreservation of elephant cells or tissues, including elephant progenitor cells. For example, the elephant cells may be selected from Asian elephant cells (…). Elephas maximus African elephant cells () Loxodonta africana ), African forest elephant cells ( Lofodonta cyclotis ) and Borneo elephant cells ( Elephas maximus bornensis ).

[0050] In particular, the cell culture formulations and culture media of this disclosure are suitable for genetically engineered (e.g., transformed) or reprogrammed mammalian cells or tissues. In some embodiments, the cell culture formulations and culture media of this disclosure are suitable for cryopreservation of stem cells or progenitor cells, including but not limited to cells obtained through dedifferentiation or dedifferentiation, reprogrammed cells, and somatic cells. In other embodiments, the cell culture formulations and culture media of this disclosure are suitable for cryopreservation of tissues obtained from these stem cells or progenitor cells.

[0051] Cryopreservation preparations The cryopreservation formulation disclosed herein contains glycerol and trehalose as cryoprotectants. In some embodiments, the cryopreservation solution contains glycerol, trehalose, ascorbic acid, and / or taurine as cryoprotectants. In alternative embodiments, the cryopreservation solution contains glycerol, trehalose, ascorbic acid, and taurine as cryoprotectants. In some embodiments, the cryopreservation formulation does not contain DMSO and / or any additional cryoprotectants.

[0052] In some embodiments, the formulation further contains glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and / or L-serine. In alternative embodiments, the cryopreserved formulation is supplemented with serum, such as fetal bovine serum.

[0053] In some implementations, the cryopreserved formulations do not contain DMSO, that is, they do not contain any DMSO.

[0054] In one embodiment, the cryopreservation formulation contains glycerol and trehalose. In some embodiments, the cryopreservation formulation contains about 20% to about 60%, or about 30% to about 50%, or about 35% to about 45% glycerol. In other embodiments, 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 trehalose. In other embodiments, the cryopreservation formulation comprises 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.

[0055] In another embodiment, the cryopreservation formulation further contains ascorbic acid and / or taurine. Therefore, in some embodiments, the cryopreservation formulation contains glycerol, trehalose, and ascorbic acid. In other embodiments, the cryopreservation formulation contains glycerol, trehalose, and taurine. In alternative embodiments, the cryopreservation formulation contains glycerol, trehalose, ascorbic acid, and taurine. In some embodiments, the cryopreservation formulation contains ascorbic acid at 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 taurine at 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 3.5 mM. In an alternative embodiment, the cryopreservation formulation contains ascorbic acid at 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, and taurine at 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 3.5 mM.

[0056] In another embodiment, the cryopreservation formulation comprises: (a) about 20% to about 60%, or about 30% to about 50%, or about 35% to about 45% 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 about 1. (c) Trehalose of about 5 mM to about 2.5 mM; (d) Ascorbic acid 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; and (e) Taurine of 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 3.5 mM.

[0057] In alternative embodiments, the cryopreservation formulation further comprises glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and / or L-serine. In some embodiments, the cryopreservation solution comprises glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. In some embodiments, 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 some embodiments, the NEAA cell culture supplement comprises approximately 750 mg / L glycine, approximately 890 mg / L L-alanine, approximately 1320 mg / L L-asparagine, approximately 1330 mg / L aspartic acid, approximately 1470 mg / L L-glutamic acid, and approximately 1150 mg / L L-serine. The content of NEAA may be from about 0.1% to about 2.5%, or from about 0.5% to about 2.0%, or from about 0.8% to about 1.8%.

[0058] In another embodiment, the cryopreservation formulation further comprises serum, such as FBS. In another embodiment, the cryopreservation formulation further comprises about 40% to about 75%, or about 50% to about 70%, or about 55% to about 65% serum (e.g., FBS). In some embodiments, the cryopreservation formulation comprises a combination of serum and a cryoprotectant 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 another embodiment, the cryopreservation formulation comprises glycerol, trehalose, ascorbic acid, taurine, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, and serum.

[0059] In some embodiments, the cryopreservation formulation of this disclosure is prepared and added to a cell culture medium. In some embodiments, the cryopreservation formulation is a 2× base formulation that can be mixed with a suitable cell culture (growth) medium at a ratio of about 1:1.

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

[0061]

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

[0063] Cryopreservation of culture medium Another embodiment of this disclosure relates to a cryopreservation culture medium. The cryopreservation culture medium of this disclosure is a cell culture medium containing (or supplemented with) glycerol and trehalose as cryoprotectants. In some embodiments, the cryopreservation culture medium contains or supplements glycerol, trehalose, and taurine as cryoprotectants. In a further embodiment, the cryopreservation culture medium contains or supplements glycerol, trehalose, and ascorbic acid as cryoprotectants. In yet another embodiment, the cryopreservation culture medium contains or supplements glycerol, trehalose, ascorbic acid, and taurine as cryoprotectants.

[0064] Cryopreservation media contain the essential components necessary for the growth of mammalian cells. In some embodiments, the cryopreservation medium is a basic defined medium. In some embodiments, the cryopreservation medium contains sufficient amounts of amino acids, energy sources (e.g., glucose), and ions to allow for the growth of mammalian cells.

[0065] In some embodiments, the cryopreservation medium contains or is supplemented with a combination of cryoprotectants 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 medium does not contain DMSO and / or additional cryoprotectants.

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

[0067] As used herein, the phrase “cell culture medium supplemented with” or “cell culture medium supplemented with” refers to existing cell culture media or including commercially available media such as DMEM containing glycerol, trehalose, ascorbic acid, taurine, glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and / or L-serine.

[0068] In one embodiment, the cryopreservation medium is a basal cell culture medium supplemented with a combination of cryoprotectants 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 some embodiments, 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 some embodiments, 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, glycine, L-alanine, L-asparagine, L-glutamic acid, L-proline, and L-serine are provided as commercially available non-essential amino acid (NEAA) cell culture supplements. In some embodiments, the NEAA cell culture supplement contains approximately 750 mg / L glycine, approximately 890 mg / L L-alanine, approximately 1320 mg / L L-asparagine, approximately 1330 mg / L aspartic acid, approximately 1470 mg / L L-glutamic acid, and approximately 1150 mg / L L-serine.

[0070] In another embodiment, the cryopreservation medium also contains or is supplemented with serum, such as FBS. In one embodiment, the cryopreservation medium contains or is supplemented with serum and a combination of cryoprotectants 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.

[0071] In some implementations, the cryopreservation media do not contain DMSO, that is, they do not contain any DMSO.

[0072] In some embodiments, the cryopreservation medium is generated by adding a 1:1 ratio of cell culture medium to the cryopreservation formulation described above. Various commercial cell culture media can be used to generate the cryopreservation medium of this disclosure. In one embodiment, the cryopreservation medium is generated by adding the cryopreservation formulation of this disclosure to Dulbecco's Modified Eagle Medium (DMEM). In other embodiments, the cryopreservation medium is generated by adding the cryopreservation formulation of this disclosure to Minimal Essential Medium (MEM), Basal Eagle Medium (BME), Roswell Park Memorial Institute (RPMI) 1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), or Endothelial Growth Medium-2 (EGM-2).

[0073] Examples of cryopreserved culture media are shown in the table below.

[0074]

[0075] The basal cell culture medium used to generate the cryopreservation culture medium of this disclosure can be any cell culture medium, including commercially available media such as well-defined basal media.

[0076] Reagent test kit This disclosure also provides a cryopreservation kit. In one embodiment, the kit comprises the cryopreservation formulation as described above and instructions for use. In other embodiments, the kit comprises the cryopreservation formulation as described above, NEAA cell culture supplement, and instructions for use. In still other embodiments, the kit comprises the cryopreservation formulation as described above, NEAA cell culture supplement, cell culture medium, and instructions for use. In a further embodiment, the kit comprises the cryopreservation formulation as described above, NEAA cell culture supplement, cell culture medium, serum supplement (e.g., FBS), and instructions for use.

[0077] In one embodiment, the kit comprises the cryopreservation medium as described above and instructions for use. In another embodiment, the kit comprises the cryopreservation medium as described above, NEAA cell culture supplement, and instructions for use. In yet another embodiment, the kit comprises the cryopreservation medium as described above, NEAA cell culture supplement, serum supplement (e.g., FBS), and instructions for use.

[0078] Preparation method of cryopreservation culture medium This disclosure also provides a method for producing a cryopreservation culture medium. In one embodiment, the method includes mixing the cryopreservation formulation of this disclosure with a cell culture medium (e.g., a basal culture medium). In some embodiments, the method includes mixing the basal culture medium and the cryopreservation formulation in a ratio of about 1:1.

[0079] In one embodiment, the method includes mixing the cryopreservation formulation of the present disclosure with a cell culture medium containing sufficient amounts of amino acids, energy sources (e.g., glucose) and ions to allow mammalian cell growth.

[0080] Methods of cryopreserving cells or tissues This disclosure also provides a method for cryopreserving mammalian cells or solid tissues. The method for cryopreserving cells includes: gradually freezing the cells in the cryopreservation medium of this disclosure or a medium supplemented with the cryopreservation formulation of this disclosure; and storing the cells / tissue in a suitable environment (e.g., liquid nitrogen). The method may also include rapid thawing of the frozen cells. Figure 1A A schematic diagram of a protocol for cryopreservation of cells / tissues is shown.

[0081] A method for cryopreserving solid tissue includes: gradually freezing the solid in the cryopreservation medium of this disclosure or a medium supplemented with the cryopreservation formulation of this disclosure; and storing the solid in a suitable environment (e.g., liquid nitrogen). The method may also include rapidly thawing the frozen solid tissue. Figure 1B A schematic diagram of a protocol for tissue cryopreservation is shown. The protocol includes washing the tissue sample to be preserved, adding culture medium, and then transferring the tissue sample to a container for cryopreservation. Subsequently, the cryopreservation culture medium or solution of this 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 is ready for long-term storage. In some embodiments, the protocol includes the following steps: 1. Place the tissue in a culture dish and wash twice with physiological saline containing 1% penicillin / streptomycin (optional).

[0082] 2. As soon as possible, use forceps and a scalpel to cut the tissue into small pieces of approximately 5×5 mm in a petri dish. The size of the tissue sections depends on their density. Cell culture medium must be added at this point to prevent the tissue from drying out, and incubate at room temperature for 10 minutes.

[0083] 3. Transfer the tissue block to a cryovial.

[0084] 4. Using a pipette / micropipette, add cryopreservation medium to adequately cover the tissue, with a minimum ratio of 1:1 tissue to cryopreservation medium volume.

[0085] 5. Cover the tube and invert it 8-10 times to thoroughly mix the sample.

[0086] 6. Depending on the size of the tissue at room temperature, equilibrate for 10 to 30 minutes.

[0087] 7. Place the vial at -80°C. The vial can be stored at -80°C for several days, or it can be placed in liquid nitrogen immediately the next day (24 hours) for long-term storage.

[0088] Current cryopreservation protocols employ similar "gradual freezing" and "rapid thawing" principles to minimize the harmful effects of ice crystal formation in cells, which can damage lipid membranes. Traditionally, for cryopreservation media containing DMSO, it is important to minimize the time cells are exposed to the DMSO solution at room temperature, as DMSO increases cell membrane permeability and can impair cell integrity with prolonged exposure. Therefore, DMSO-containing media must be mixed separately from the cells, as exposure to pure DMSO can be highly cytotoxic. The cell preparation formulation disclosed herein is a ready-to-use mixture that can be combined with the growth medium used for a specific population to create a customizable cryopreservation mixture.

[0089] The cell culture media and formulations disclosed herein can be used in this progressive freezing and rapid thawing protocol. In some embodiments, once mixed with a fully frozen culture medium, the cells are stored in an insulated freezer and gradually frozen at -80°C before being transferred for long-term storage in liquid nitrogen.

[0090] In some embodiments, when the cryopreservation formulation and / or culture medium of this disclosure are used for cryopreservation of solid tissues, the method includes an incubation period at room temperature (RT) in the presence of a culture medium (growth medium) and a second incubation at RT with a frozen culture medium.

[0091] Without further description, it is believed that those skilled in the art can use the foregoing description and the following illustrative examples to make and utilize the invention and practice the claimed methods. Therefore, the following working examples specifically point to preferred embodiments of the invention and should not be construed as limiting the remainder of this disclosure in any way.

[0092] Example Example 1: Cells derived from frozen elephant tissue To develop the cell culture media and formulations of this disclosure, a series of experiments were conducted. In these experiments, elephant placental tissue was frozen in the presence of the cryopreservation formulations of this disclosure and 10% DMSO / 40% FBS / 50% EGM-2-XAP or MACS® freezing solutions. The frozen samples were examined by subsequent thawing and amplification monitoring (basic amplification of cell culture or the ability of frozen tissue to develop into cell lines).

[0093] like Figure 2A and Figure 2B As shown, placental tissue preserved using the formulation disclosed herein at the p0 and p2 stages exhibited cell viability. In contrast, cells cryopreserved in 10% DMSO / 40% FBS / 50% EGM-2-XAP showed almost no cell viability. Figure 2C and Figure 2D MACS® cryosol was also tested, but the result was negative (data not shown).

[0094] Other freezing conditions were also tested: 10% DMSO / 90% FBS, flash freezing, and low osmotic pressure. None of these other conditions showed positive cell-derived results. The cryopreservation formulation of this disclosure was compared with commercially available cryopreservation media to understand its ability to cryopreserve sensitive cell lines. Test results are shown in Examples 2 (elephant endometrial cells), 3 (bovine embryonic stem cells), and 4 (human organoids). For the tests in Examples 2-4, [the following methods were used]. Figure 1B The scheme shown is the same as the one described in paragraph

[0079] above.

[0095] Example 2: Comparison of frozen culture medium used for elephant endometrial cells The cryopreservation formulation disclosed herein (Xo-Chill cryopreservation medium) was compared with a formulation containing 10% DMSO / 40% FBS / 50% EGM-2 (see Table 2-1 below). In this test, elephant endometrial cells were frozen and stored at -80°C for one year, and then thawed. The results of this test are as follows: Figure 3A (Xo-Chill) and Figure 3B As shown.

[0096]

[0097] Example 3: Comparison of frozen culture medium used for bovine embryonic stem cells The cryopreservation formulations containing bESCM (“XC+bESCM”) or KSR (“XC+KSR”) disclosed herein were compared with mFreSR™ (Stem Cell Technologies) (see Tables 3-1 and 3-2). Bovine embryonic stem cells were used in this test.

[0098]

[0099]

[0100] Bovine embryonic stem cells (ESCs) p4 were harvested using ReLeSR and frozen in 10% DMSO and 90% serum. Upon thawing, the cells showed a 75% viability (data not shown) and were subsequently seeded into porphyrin-pre-coated wells in bEPSCM medium. Cells were harvested after reaching 80% confluence and cryopreserved at 1×10⁻⁶ cells per medium. 6 Each cell. Figures 4A-4C Photographs of cells cryopreserved in XC+bESCM, XC+KSR, and mFreSR™ are shown. The photos were taken 48 hours after inoculation (cryopreservation). Figure 4D Cell viability and normalized survival percentage after cryopreservation are shown. Tests in this example confirm that the cryopreservation formulation of this disclosure provides similar or improved performance compared to mFreSR™.

[0101] Example 4: Comparison of cryopreservation culture media for human organoids Furthermore, the cryopreservation formulation disclosed herein was compared with CryoStor CS10 (Stem Cell Technologies). Human organoids were used in this test.

[0102] Following the manufacturer's recommendations, the commercially leading cryopreservation medium CryoStor SC10 was used as a reference. XC cryopreservation medium (see Table 4-1 below) was compared to CS10 containing 10% DMSO because DMSO allows for better recovery of spheres / organoids and preservation of their function. Human organoids were grown in organoid maintenance medium (OMM). Samples were then collected and transferred to cryopreservation flasks (…). Figure 5A , Figure 5B The XC frozen medium samples were incubated at room temperature for 10 minutes. Organoids in the XC frozen medium showed equilibrium, while CS10 remained floating. Both groups were frozen at a slow freezing rate. After thawing, the samples were cultured in OMM, and bouncing contractility was monitored after 24 hours. Figure 5C The results of the cell contractility test are as follows: Figure 5D As shown.

[0103]

[0104] Implementation The present invention also provides the following non-limiting embodiments.

[0105] Embodiment 1 is a cryopreservation preparation for cryopreserving mammalian cells or solid mammalian tissues, comprising a combination of cryoprotectants 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 preparation is used for cryopreserving cells. In another embodiment, the preparation is used for cryopreserving solid mammalian tissues.

[0106] Embodiment 2 is a cryopreservation formulation of Embodiment 1, wherein the formulation comprises a combination of cryoprotectants selected from the group consisting of: (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 / mL of anti-inflammatory agents. Ascorbic acid; (c) 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.

[0107] Embodiment 3 is a cryopreservation preparation of Embodiment 1 or 2, wherein the combination of cryoprotectants is glycerol and trehalose.

[0108] Implementation method 4 is a cryopreservation preparation of implementation method 1 or 2, wherein the combination of cryoprotectants is glycerol, trehalose and ascorbic acid.

[0109] Embodiment 5 is a cryopreservation preparation of Embodiment 1 or 2, wherein the combination of cryoprotectants is glycerol, trehalose and taurine.

[0110] Embodiment 6 is a cryopreservation preparation of Embodiment 1 or 2, wherein the combination of cryoprotectants is glycerol, trehalose, ascorbic acid and taurine.

[0111] Embodiment 7 is a cryopreservation preparation of any one of Embodiments 1 to 6, wherein the preparation further comprises serum.

[0112] Embodiment 8 is a cryopreservation preparation of Embodiment 7, wherein the preparation contains about 40% to about 75% serum.

[0113] Embodiment 9 is a cryopreservation preparation of Embodiment 7 or 8, wherein the serum contains FBS.

[0114] Embodiment 10 is a cryopreservation preparation 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.

[0115] Embodiment 11 is a cryopreservation formulation of Embodiment 10, further comprising 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.

[0116] Embodiment 12 is a cryopreservation formulation of Embodiment 10 or 11, wherein the formulation further comprises glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine.

[0117] Embodiment 13 is a cryopreservation formulation of any one of Embodiments 1 to 12, wherein the formulation is free of dimethyl sulfoxide (DMSO).

[0118] Embodiment 14 is a cryopreservation formulation of any one of Embodiments 1 to 13, wherein the formulation does not contain any additional cryoprotectant.

[0119] Embodiment 15 is a cryopreservation culture medium comprising the cryopreservation formulation of any one of Embodiments 1 to 14.

[0120] Implementation 16 is the cryopreservation culture medium of Implementation 15, which contains sufficient amounts of amino acids, energy sources and ions to allow mammalian cell growth.

[0121] Implementation method 17 is the cryopreservation culture medium of implementation method 16, wherein the energy source is glucose.

[0122] Embodiment 18 is a method for producing a cryopreservation culture medium, comprising mixing the cryopreservation preparation of any one of Embodiments 1 to 14 with a cell culture medium.

[0123] Implementation 19 is the method of Implementation 18, wherein the cell culture medium contains sufficient amounts of amino acids, an energy source, and ions to allow mammalian cell growth. Alternatively, the cell culture medium contains sufficient amounts of amino acids, an energy source, and ions to allow mammalian cell growth in solid mammalian tissue.

[0124] Implementation 20 is the method of Implementation 19, wherein the cell culture medium contains sufficient amounts of amino acids, glucose, and ions to allow mammalian cell growth. Alternatively, the cell culture medium contains sufficient amounts of amino acids, glucose, and ions to allow mammalian cell growth in solid mammalian tissue.

[0125] Implementation 21 is a cryopreservation culture medium for cryopreserving mammalian cells, comprising: sufficient amounts of amino acids, glucose and ions to enable mammalian cell growth; and a combination of cryoprotectants 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.

[0126] Implementation 22 is a cryopreservation culture medium of Implementation 21, comprising: sufficient amounts of amino acids, glucose, and ions to allow mammalian cell growth; and a combination of cryoprotectants selected from: (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. Implementation 23 is a cryopreservation culture medium of Implementation 21 or 22, wherein the combination of cryoprotectants is glycerol and trehalose.

[0127] Implementation 24 is a cryopreservation culture medium of Implementation 21 or 22, wherein the combination of cryoprotectants is glycerol, trehalose and ascorbic acid.

[0128] Implementation 25 is a cryopreservation culture medium of Implementation 21 or 22, wherein the combination of cryoprotectants is glycerol, trehalose and taurine.

[0129] Implementation 26 is a cryopreservation culture medium of Implementation 21 or 22, wherein the combination of cryoprotectants is glycerol, trehalose, ascorbic acid and taurine.

[0130] Embodiment 27 is a cryopreservation culture medium of any one of Embodiments 21 to 26, wherein the culture medium further comprises serum.

[0131] Embodiment 28 is the cryopreservation culture medium of Embodiment 27, wherein the culture medium contains about 40% to about 75% serum.

[0132] Implementation 29 is the cryopreservation culture medium of Implementation 26 or 27, wherein the serum contains FBS.

[0133] Embodiment 30 is a cryopreservation culture 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.

[0134] Embodiment 31 is the cryopreservation culture medium of Embodiment 30, which further comprises 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.

[0135] Embodiment 32 is the cryopreservation culture medium of Embodiment 30 or 31, wherein the culture medium further comprises glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine.

[0136] Embodiment 33 is a cryopreservation medium of any one of Embodiments 21 to 32, wherein the cryopreservation medium contains about 25% to about 45% of a basal cell culture medium containing sufficient amounts of amino acids, glucose and ions to allow mammalian cell growth.

[0137] Embodiment 34 is a cryopreservation culture medium of any one of Embodiments 21 to 33, wherein the culture medium does not contain DMSO.

[0138] Embodiment 35 is a cryopreservation culture medium of any one of Embodiments 21 to 33, wherein the culture medium does not contain any additional cryoprotectant.

[0139] Embodiment 36 is a cryopreservation culture medium of any one of Embodiments 21 to 35, wherein mammalian cells are in solid tissue.

[0140] Embodiment 37 is a method for cryopreserving mammalian cells, comprising: freezing mammalian cells in a cryopreservation culture medium of any one of Embodiments 15 to 17 or 21 to 35 or a cell culture medium supplemented with a cryopreservation preparation of any one of Embodiments 1 to 14; and storing the cells in a suitable environment.

[0141] Implementation method 38 is the method of implementation method 37, wherein a suitable environment includes storing cells in liquid nitrogen.

[0142] Embodiment 39 is a method for cryopreserving solid mammalian tissue, comprising: freezing the solid mammalian tissue in a cryopreservation culture medium of any one of Embodiments 15 to 17 or 21 to 36 or a cell culture medium supplemented with a cryopreservation preparation of any one of Embodiments 1 to 14; and storing the solid mammalian tissue in a suitable environment.

[0143] Implementation 40 is the method of Implementation 39, wherein a suitable environment includes storing solid mammalian tissue in liquid nitrogen.

[0144] Implementation 41 is the method of Implementation 39, wherein freezing includes culturing solid mammalian tissue in a growth medium at room temperature, and then culturing the solid mammalian tissue in a cryopreservation medium or a cell culture medium supplemented with a cryopreservation agent.

[0145] Embodiment 42 is a kit comprising the cryopreservation preparation of any one of Embodiments 1 to 14, the cryopreservation culture medium of any one of Embodiments 15 to 17 or 21 to 36, and instructions for use.

[0146] Implementation 43 is a kit of implementation 42, wherein the kit contains the cryopreservation preparation and cell culture medium of any one of implementations 1 to 14.

[0147] Implementation 44 is a kit of Implementation 43, wherein the cell culture medium contains sufficient amounts of amino acids, energy sources and ions to allow mammalian cells to grow.

[0148] Example 45 is the kit of Example 42, wherein the kit contains the cryopreserved culture medium of any one of Examples 15 to 17 or 21 to 35.

[0149] Embodiment 46 is a kit of any one of Embodiments 42 to 45, wherein the kit includes instructions for cryopreservation of mammalian cells.

[0150] While the invention has been described and illustrated herein with reference to various specific materials, procedures, and examples, it should be understood that the invention is not limited to the specific combinations of materials and procedures chosen for this purpose. Those skilled in the art will understand that many variations of these details may be implied. It is intended that the specification and embodiments be considered exemplary only, and the true scope and spirit of the invention are indicated by the following claims. All references, patents, and patent applications cited in this application are incorporated herein by reference in their entirety.

Claims

1. A cryopreservation preparation for cryopreserving mammalian cells or solid mammalian tissues, comprising a combination of cryoprotectants 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 cryopreservation formulation of claim 1, wherein the formulation comprises a combination of cryoprotectants selected from the group consisting of: (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 / mL ascorbic acid; (c) 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.

3. The cryopreservation formulation according to claim 1 or 2, wherein the combination of the cryoprotectants is glycerol and trehalose.

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

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

6. The cryopreservation formulation according to claim 1 or 2, wherein the combination of said cryoprotectants is glycerol, trehalose, ascorbic acid and taurine.

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

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

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

10. The cryopreservation formulation 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. The cryopreservation formulation of claim 10, further comprising 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.

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

14. The cryopreservation formulation according to claim 1 or 13, wherein the formulation does not contain an additional cryoprotectant.

15. A cryopreservation culture medium comprising the cryopreservation formulation as described in claim 1.

16. The cryopreservation culture medium according to claim 15, wherein it contains sufficient amounts of amino acids, energy sources and ions to allow mammalian cell growth.

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

18. A method for producing a cryopreservation culture medium, comprising mixing the cryopreservation formulation as claimed in claim 1 with a cell culture medium.

19. The method of claim 18, wherein the cell culture medium contains sufficient amounts of amino acids, energy sources and ions to allow mammalian cell growth.

20. The method of claim 19, wherein the cell culture medium contains sufficient amounts of amino acids, glucose, and ions to allow mammalian cell growth.

21. A cryopreservation culture medium for cryopreserving mammalian cells, comprising: Sufficient amounts of amino acids, glucose, and ions are required to allow mammalian cells to grow; and Combinations of cryoprotectants selected from the following groups: (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 cryopreservation culture medium according to claim 21, comprising: Sufficient amounts of amino acids, glucose, and ions are required to allow mammalian cells to grow; and Combinations of cryoprotectants selected from the following groups: (a) Approximately 10% to approximately 40% glycerol and approximately 100 mM to approximately 3 M trehalose; (b) Glycerin at about 10% to about 40%, trehalose at about 100 mM to about 3 M and ascorbic acid at about 20 μg / mL to about 250 μg / mL; (c) Approximately 10% to approximately 40% glycerol, approximately 100 mM to approximately 3 M trehalose, and taurine; and (d) Glycerin of about 10% to about 40%, trehalose of about 100 mM to about 3 M, ascorbic acid of about 20 μg / mL to about 250 μg / mL and taurine of about 5 mM to about 30 mM.

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

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

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

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

27. The cryopreservation culture medium according to claim 21 or 22, wherein the culture medium further comprises serum.

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

29. The cryopreservation culture medium according to claim 27, wherein the serum comprises FBS.

30. The cryopreservation culture 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 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.

32. The cryopreservation culture medium according to claim 30, wherein the culture medium further comprises 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 comprises about 25% to about 45% of a basal cell culture medium, wherein the basal cell culture medium contains sufficient amounts of amino acids, glucose and ions to allow mammalian cell growth.

34. The cryopreservation medium according to claim 21 or 22, wherein the medium is free of DMSO.

35. The cryopreservation medium according to claim 21 or 22, wherein the medium does not contain any additional cryoprotectant.

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

37. A method for cryopreserving mammalian cells, comprising: Frozen mammalian cells in the cryopreservation medium as described in claim 15 or in a cell culture medium supplemented with the cryopreservation preparation as described in claim 1; and Store 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 for cryopreserving solid mammalian tissue, comprising: Frozen solid mammalian tissue in the cryopreservation medium of claim 15 or a cell culture medium supplemented with the cryopreservation preparation of claim 1; and Store solid mammalian tissues in a suitable environment.

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

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

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

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

44. The kit of claim 43, wherein the cell culture medium contains sufficient amounts of amino acids, energy sources and ions to allow mammalian cell growth.

45. The kit of claim 42, wherein the kit comprises the cryopreserved culture medium as described in any one of claims 15.

46. ​​The kit of claim 42, wherein the kit includes instructions for cryopreservation of mammalian cells.

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