Composition for cryopreserving cells, method for freezing cells, and cell preparation

Abstract

The present disclosure provides a composition for cryopreservation of cells that can protect cells during cell freezing, for example, without the substantial addition of a cell cryoprotectant, such as an organic solvent. SOLUTION: The composition for cryopreservation of cells of the present disclosure contains a sugar or a derivative thereof and / or a surfactant.

Description

[Technical Field]

[0001] The present disclosure relates to compositions for cryopreservation of cells, methods for freezing cells, and cell preparations. [Background technology]

[0002] With the advancement of regenerative medicine, cells isolated from living organisms are cultured on a large scale, and the cultured cells are further processed by gene transfer, etc. Furthermore, attempts are being made to treat diseases by using the cultured or processed cells as cell medicines.

[0003] In the manufacture of cell medicines, cells are cultured and processed in a cell processing facility, and then the processed cells are cryopreserved in order to be transported to a hospital (Non-Patent Documents 1 and 2). In the cryopreservation, the processed cells are dispersed in a cell cryopreservation solution containing a cell cryoprotectant and then frozen. The cells are then transported in the frozen state. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Wang, J., Li, R., “Effects, methods and limits of the cryopreservation on mesenchymal stem cells.”, Stem Cell Res Ther, 2024, 15, Article number:337 [Non-patent document 2] Murray KA, Gibson MI., “Chemical approaches to cryopreservation.”, Nat Rev Chem., 2022, 6(8), p.579-593. Summary of the Invention [Problem to be solved by the invention]

[0005] As the cryopreservation solution, a cryopreservation solution containing dimethyl sulfoxide (DMSO) as the cell cryoprotectant is widely used because of its high cell cryoprotective effect. However, since DMSO is cytotoxic, it is difficult to use a cell medicine containing DMSO. in live It has been reported that toxicity occurs when administered to animals. In addition to DMSO, organic solvents such as propylene glycol, ethylene glycol, glycerol (glycerin), and ethanol have also been reported as cryoprotectants. However, these organic solvent cryoprotectants are also in live Toxicity may occur when administered to

[0006] Therefore, the present disclosure provides, for example, a composition for cryopreserving cells that can protect cells during freezing without substantially adding an organic solvent cell cryoprotectant, a method for freezing cells, and a cell preparation obtained thereby. [Means for solving the problem]

[0007] To achieve the above object, the composition for cryopreservation of cells (hereinafter also referred to as "composition") of the present disclosure contains a sugar or a derivative thereof and / or a surfactant.

[0008] The method for freezing cells of the present disclosure (hereinafter also referred to as "freezing method") comprises a freezing step of freezing cells in the presence of the composition of the present disclosure.

[0009] The cell preparation of the present disclosure comprises cells and a composition of the present disclosure. [Effects of the Invention]

[0010] According to the present disclosure, for example, cells can be protected during freezing without substantially adding an organic solvent cell cryoprotectant. DETAILED DESCRIPTION OF THE INVENTION

[0011] <Definition> In the present disclosure, "sugar" refers to any sugar. The sugar may be, for example, a monosaccharide, a disaccharide, a trisaccharide, or a polysaccharide. In the present disclosure, "sugar derivative" refers to a compound in which a sugar is modified with a modifying group. The sugar derivative is, for example, a glycoside.

[0012] In this disclosure, "reducing sugar" refers to a sugar that has reducing properties and has a free aldehyde group or a free ketone group, and "non-reducing sugar" refers to a sugar that does not exhibit reducing properties and does not have a free aldehyde group or a free ketone group.

[0013] As used herein, "glutathione" refers to a tripeptide composed of glutamic acid, cysteine, and glycine. Glutathione is known to function as an antioxidant in cells and protect cells from reactive oxygen species such as free radicals and peroxides.

[0014] As used herein, "amino acid" refers to an organic compound containing an amino group and a carboxyl group. The amino acid may be an organic compound containing an amino group and a sulfonyl group. The amino acid may be in the D-form, the L-form, or a mixture of both.

[0015] As used herein, "substantially free" means free from or completely free of concentrations above the detection limit. Specifically, a composition without added serum or plasma is serum-free and substantially free from serum or plasma. "Serum-free" means that it does not contain, or does not contain, serum- or plasma-derived components at concentrations above the detection limit. A composition without added animal-derived components is animal-free and substantially free from human and animal-derived components. "Animal-free" means that it does not contain, or does not contain, animal-derived components at concentrations above the detection limit. A composition without added protein is protein-free and does not contain protein. "Protein-free" means that it does not contain, or does not contain, proteins at concentrations above the detection limit. A composition without added organic solvent cellular cryoprotectants is organic solvent-free and substantially free from organic solvent cellular cryoprotectants. "Organic solvent-free" means that it does not contain, or does not contain, the organic solvent cellular cryoprotectants at concentrations above the detection limit. A composition without added albumin is albumin-free and does not contain albumin. The term "albumin-free" means that the albumin is not contained at a concentration above the detection limit or is not contained at all.

[0016] As used herein, the term "organic solvent" refers to an organic compound that is liquid at room temperature and pressure and has the ability to dissolve other substances. The organic compound refers to a compound containing carbon atoms that generates carbon dioxide or carbonizes when burned.

[0017] As used herein, "albumin" refers to a protein known as a plasma component. Examples of human serum albumin include a protein having the amino acid sequence registered in GenBank under accession number AAN17825.1, or a human serum albumin having an amino acid sequence corresponding thereto.

[0018] As used herein, "chemically defined" means that the components contained and their amounts can be chemically defined. Specifically, a "chemically defined composition" refers to a composition in which the compound name or amino acid sequence is clear and the amount thereof can be specified.

[0019] As used herein, "infusion" refers to a liquid preparation, injection, or drip infusion that can be administered intravenously.

[0020] As used herein, the term "medium" refers to a medium used for culturing cells. The medium can be prepared, for example, by adding necessary components to a basal medium.

[0021] As used herein, "isolated" means identified and separated and / or recovered from components of its natural state. The "isolation" can be achieved, for example, by at least one purification step.

[0022] As used herein, "protein" refers to a polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and / or artificial amino acids. The protein is a polymer composed of 10 or more amino acids. As used herein, "peptide" refers to a polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and / or artificial amino acids. The protein is a polymer composed of less than 10 amino acids.

[0023] As used herein, "cell preparation" refers to a composition containing desired cells.

[0024] As used herein, "mesenchymal cells" refer to cells that constitute connective tissue derived from the mesoderm or neural crest and / or cells that have the ability to differentiate into said cells. When the mesenchymal cells have the ability to self-renew, they can also be called mesenchymal stem cells (MSCs).

[0025] As used herein, "immune cells" refers to cells that constitute the immune system and / or cells that have the ability to differentiate into said cells.

[0026] As used herein, "treatment" refers to therapeutic treatment and / or prophylactic treatment. As used herein, "treatment" refers to treating, curing, preventing, suppressing, ameliorating, or improving a disease, pathological condition, or disorder, or halting, suppressing, reducing, or delaying the progression of a disease, pathological condition, or disorder. As used herein, "prevention" refers to reducing the likelihood of developing a disease or pathological condition, or delaying the onset of a disease or pathological condition. The "treatment" may be, for example, treatment of a subject (patient) who develops the target disease, or treatment of an animal model of the target disease.

[0027] As used herein, "subject" means an animal or a cell, tissue, or organ derived from an animal. The subject is used in a sense that includes, in particular, humans. The animals include humans and non-humans. The non-human animals include, for example, mice, rats, rabbits, dogs, cats, and Examples of mammals include sheep, horses, pigs, monkeys, dolphins, and sea lions. "Patient" means a subject receiving prophylactic or therapeutic treatment. For example, in addition to the patients, healthy individuals are included.

[0028] Sequence information for the proteins described herein or the nucleic acids (eg, DNA or RNA) encoding them is available from sources such as Protein Data Bank, UniProt, or Genbank.

[0029] The present disclosure will be described below using examples, but the present disclosure is not limited to the following examples and can be implemented with any modifications. Furthermore, the descriptions in this disclosure are mutually applicable unless otherwise specified. In this specification, the expression "to" is used to mean the numerical or physical value before and after it. In this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B." In the following description, "mol / l" may also be abbreviated as "M."

[0030] <Cell cryopreservation composition> In one aspect, the present disclosure provides a composition for cryopreservation of cells that can protect cells during freezing without the substantial addition of an organic solvent-based cryoprotectant. The composition for cryopreservation of cells of the present disclosure comprises a sugar or a derivative thereof and / or a surfactant.

[0031] As a result of extensive research, the present inventors have found that the sugar or its derivative and / or surfactant functions as a cell cryoprotectant. It is believed that the sugar or sugar derivative, or surfactant, particularly at a certain concentration or higher, inhibits the formation of ice crystals inside and outside the cell, and dehydrates intracellular water due to the difference in osmotic pressure between the inside and outside of the cell, thereby protecting cell membranes and intracellular organelles from freezing damage. However, this assumption does not limit the present disclosure in any way. Therefore, according to the present disclosure, by using the sugar or sugar derivative and / or surfactant as a cell cryoprotectant, cells can be protected during cell freezing, for example, without using an organic solvent cell cryoprotectant.

[0032] The composition of the present disclosure contains, for example, the sugar or its derivative and / or a surfactant as the cell cryoprotectant. The composition of the present disclosure preferably contains the sugar or its derivative, for example, because of its high cell cryoprotective effect. The composition of the present disclosure may use any one of the cell cryoprotectants alone or two or more of them in combination.

[0033] The sugar is not particularly limited, and examples thereof include monosaccharides, disaccharides, trisaccharides, polysaccharides, etc. The sugar is preferably a monosaccharide, disaccharide, or trisaccharide, and more preferably a monosaccharide or disaccharide, for example, because it increases the cell proliferation rate during culture after thawing. One type of sugar may be used alone, or two or more types may be used in combination.

[0034] Examples of the monosaccharides include glucose, galactose, mannose, fructose, xylose, ribose, and arabinose, with glucose being preferred. Examples of the disaccharides include sucrose, maltose, sucralose, lactose, cellobiose, lactobionic acid, lactulose, melibiose, and isomaltose, with sucrose or maltose being preferred, and sucrose being more preferred. Examples of the trisaccharides include raffinose, maltotriose, isomaltotriose, panose, and melezitose, with raffinose being preferred. Examples of the polysaccharides include dextran, starch, hydroxyethyl starch, hyaluronic acid, glycogen, and alginic acid.

[0035] The sugar may be, for example, a non-reducing sugar or a reducing sugar, but is preferably a non-reducing sugar due to its high cell cryoprotective effect. Examples of the non-reducing sugar include sucrose, trehalose, sucralose, maltotriose, isomaltotriose, and panose.

[0036] Examples of the sugar derivative include nucleosides, nucleotides, sugar alcohols, etc. One type of the sugar derivative may be used alone, or two or more types may be used in combination.

[0037] Examples of the nucleoside include ribonucleosides and deoxyribonucleosides. Examples of the ribonucleosides include uridine, adenosine, guanosine, 5-methyluridine, and cytidine, with uridine being preferred due to its high cell cryoprotective effect. Examples of the deoxyribonucleosides include deoxyuridine, deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine, with deoxyuridine being preferred.

[0038] Examples of the nucleotide include ribonucleotides and deoxyribonucleotides. Examples of the ribonucleotide include uridine monophosphate, uridine diphosphate, uridine triphosphate, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, guanosine monophosphate, guanosine diphosphate, guanosine triphosphate, cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, 5-methyluridine monophosphate, 5-methyluridine diphosphate, and 5-methyluridine triphosphate. Examples of the deoxyribonucleotide include deoxyuridine monophosphate, deoxyuridine diphosphate, deoxyuridine triphosphate, deoxyadenosine monophosphate, deoxyadenosine diphosphate, deoxyadenosine triphosphate, deoxyguanosine monophosphate, deoxyguanosine diphosphate, deoxyguanosine triphosphate, deoxycytidine monophosphate, deoxycytidine diphosphate, deoxycytidine triphosphate, thymidine monophosphate, thymidine diphosphate, and thymidine triphosphate.

[0039] Examples of the sugar alcohol include sorbitol and xylitol.

[0040] Examples of the surfactant include polyvinylpyrrolidone (PVP) and modified polyalkylene glycol. The modified polyalkylene glycol is, for example, a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. The polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block is, for example, a polyethylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. The modified polyalkylene glycol is, for example, a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer. The modified polyalkylene glycol is preferably Soluplus. The modified polyalkylene glycol may be, for example, a graft copolymer obtained from (i) N-vinyl caprolactam, (ii) vinyl acetate, and (iii) a polyether. The polyether may be polyethylene glycol. The modified polyalkylene glycol may be, for example, a compound obtained by free radical polymerization of a mixture of the above (i) to (iii). In this case, the composition of the mixture may be, for example, 40 to 60 wt%, 45 to 57 wt%, or 50 to 55 wt% of (i), 15 to 35 wt%, 20 to 30 wt%, or 20 to 25 wt% of (ii), and 10 to 30 wt%, 13 to 25 wt%, or 15 to 20 wt% of (iii). The total of (i) to (iii) may be 100 wt%. The modified polyalkylene glycol may be, for example, a compound represented by the following structural formula (Formula I) or a salt thereof:

[0041] [ka]

[0042] In the above chemical formula, the wt% of l, m, and n are, for example, 40 to 60 wt%, 15 to 35 wt%, and 10 to 30 wt%, respectively. The wt% of l is, for example, 40 to 60 wt%, 45 to 57 wt%, or 50 to 55 wt%. The wt% of m is, for example, 15 to 35 wt%, 20 to 30 wt%, or 20 to 25 wt%. The wt% of n is, for example, 10 to 30 wt%, 13 to 25 wt%, or 15 to 20 wt%. The wt% of l, m, and n may, for example, be 100 wt% in total. The wt% of l, m, and n may be, for example, about 57 wt%, about 30 wt%, and about 13 wt%, respectively.

[0043] In the above chemical formula, the degrees of polymerization of l, m, and n are, for example, 60 to 160, 470 to 1110, and 480 to 1130, respectively. The degree of polymerization of l is, for example, 60 to 160, 70 to 150, 80 to 140, 90 to 130, 100 to 120, or 100 to 110. The degree of polymerization of m is, for example, 470 to 1110, 500 to 1050, 550 to 1000, 600 to 950, 650 to 850, 700 to 800, or 750 to 780. The degree of polymerization of n is, for example, 480 to 1130, 500 to 1050, 550 to 1000, 600 to 950, 650 to 900, 700 to 850, or 750 to 800. The degrees of polymerization of l, m, and n may be average degrees of polymerization. The mass-average molecular weight (Mw) of the modified polyalkylene glycol is, for example, 70,000 to 170,000 g / mol, 80,000 to 160,000 g / mol, 90,000 to 140,000 g / mol, 100,000 to 130,000 g / mol, or 110,000 to 125,000 g / mol. The mass-average molecular weight (Mw) of the modified polyalkylene glycol is preferably about 118,000 g / mol. The modified polyalkylene glycol is preferably Soluplus. Soluplus is a compound represented by the above chemical formula, in which n is 13, m is 30, and l is 57. The l, m, and n of Soluplus may have, for example, the wt% or degree of polymerization described above. The modified polyalkylene glycol may be produced, for example, by the method described in US 2008 / 0293828 A1, US 2010 / 0204425 A1, or US 2018 / 0305636 A1. The components (i) to (iii) may be the components described in these documents.

[0044] The concentration of the sugar or its derivative is, for example, 1 to 90% (w / v), 2 to 80% (w / v), 3 to 70% (w / v), 4 to 60% (w / v), 5 to 50% (w / v), 6 to 40% (w / v), 7 to 30% (w / v), 8 to 25% (w / v), 9 to 20% (w / v), or 10 to 15% (w / v). The concentration of the sugar or its derivative may be, for example, the concentration of one type of compound or the combined concentration of two or more types of compounds.

[0045] When the composition of the present disclosure contains the sugar, the concentration of the sugar is, for example, 1 to 90% (w / v), 2 to 80% (w / v), 3 to 70% (w / v), 4 to 60% (w / v), 5 to 50% (w / v), 6 to 40% (w / v), 7 to 30% (w / v), 5 to 25% (w / v), 7.5 to 20% (w / v), or 10 to 15% (w / v). When the sugar is a monosaccharide, disaccharide, and / or trisaccharide, the concentration of the sugar is, for example, 1 to 90% (w / v), 2 to 80% (w / v), 3 to 70% (w / v), 4 to 60% (w / v), 5 to 50% (w / v), 6 to 40% (w / v), 7 to 30% (w / v), 5 to 25% (w / v), 7.5 to 20% (w / v), or 10 to 15% (w / v). The sugar concentration may be, for example, the concentration of one type of sugar or the combined concentration of two or more types of sugars.

[0046] When the composition of the present disclosure contains the sugar alcohol, the concentration of the sugar alcohol is, for example, 1 to 20% (w / v), 1.5 to 15% (w / v), 2 to 12.5% ​​(w / v), or 2.5 to 10% (w / v). The sugar alcohol concentration may be, for example, the concentration of one type of sugar alcohol or the total concentration of two or more types of sugar alcohols.

[0047] The concentration of the surfactant is, for example, 1 to 20% (w / v), 2.5 to 15% (w / v), 5 to 15% (w / v), or 7.5 to 12.5% ​​(w / v). When the surfactant is PVP or a modified polyalkylene glycol, the concentration of the PVP or modified polyalkylene glycol is, for example, 1 to 20% (w / v), 2.5 to 15% (w / v), 5 to 15% (w / v), or 7.5 to 12.5% ​​(w / v). The concentration of the sugar alcohol may be, for example, the concentration of one type of sugar alcohol or the total concentration of two or more types of sugar alcohols.

[0048] The composition of the present disclosure may contain, for example, an antioxidant (reducing agent). The antioxidant is a substance capable of inhibiting oxidation reactions. By including the antioxidant, the composition of the present disclosure can further improve the cell cryoprotective effect. Examples of the antioxidant include glutathione, lipoic acid (α-lipoic acid, thioctic acid), ascorbic acid (vitamin C) or its derivatives, tocopherol (vitamin E) or its derivatives, acetylcysteine, and chelating agents. Examples of the ascorbic acid derivatives include ascorbic acid 2-glucoside, ascorbic acid 2-phosphate, ascorbyl tetrahexyldecanoate, and 3-O-ethyl ascorbic acid. Examples of the tocopherol derivatives include α-tocopheryl phosphate, tocopherol acetate, and tocopherol succinate. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA), citric acid, gluconic acid, sodium gluconate, phytic acid, etc. One type of the antioxidant may be used alone, or two or more types may be used in combination.

[0049] When the composition of the present disclosure contains the antioxidant, the cell cryoprotective effect can be particularly improved, and therefore, more preferably, the antioxidant includes the lipoic acid and the glutathione.

[0050] The composition of the present disclosure can particularly improve the cell cryoprotective effect, and therefore preferably contains the sugar and the antioxidant, more preferably contains the monosaccharide and / or disaccharide and the antioxidant, and even more preferably contains the disaccharide and the antioxidant.

[0051] When the composition of the present disclosure contains an antioxidant, the concentration of the antioxidant is, for example, 1 nmol / L to 100 mmol / L, 10 nmol / L to 10 mmol / L, 0.1 μmol / L to 10 mmol / L, 1 μmol / L to 1 mmol / L, or 10 to 100 μmol / L. The concentration of the antioxidant may be, for example, the concentration of a single antioxidant or the combined concentration of two or more antioxidants. The concentration of the glutathione is, for example, 10 nmol / L to 10 mmol / L, 100 nmol / L to 1 mmol / L, or 1 μmol / L to 100 μmol / L. The concentration of the lipoic acid is, for example, 1 nmol / L to 100 mmol / L, 10 nmol / L to 10 mmol / L, or 100 nmol / L to 1 mmol / L.

[0052] The composition of the present disclosure may contain, for example, an amino acid. By containing the amino acid, the composition of the present disclosure can further improve the cell cryoprotective effect. Examples of the amino acid include α-amino acids, β-amino acids, and γ-amino acids. Examples of the amino acid include amino acids that constitute proteins. The amino acid may be a sulfur-containing amino acid. One type of amino acid may be used alone, or two or more types may be used in combination. Examples of the amino acid include alanine, taurine, phenylalanine, methionine, proline, cysteine, serine, lysine, arginine, homoserine, aminobutyric acid, aminocaproic acid, tryptophan, and glycine.

[0053] The concentration of the amino acids is, for example, 0.01 to 100 mmol / L, 0.1 to 50 mmol / L, or 0.5 to 10 mmol / L. The concentration of the amino acids may be, for example, the concentration of one type of amino acid or the total concentration of two or more types of amino acids.

[0054] In the composition of the present disclosure, each compound may be, for example, a salt. The salt of each compound is not particularly limited and includes, for example, inorganic salts or organic salts. The salts include, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, etc. The metal salts include, for example, alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, barium salts, etc.), aluminum salts, etc. Salts with organic bases include, for example, salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, etc. Salts with inorganic acids include, for example, salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. The salts with organic acids include, for example, salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, mesylic acid, tosylic acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. The salts with basic amino acids include, for example, salts with arginine, lysine, ornithine, etc. The salts with acidic amino acids include, for example, salts with aspartic acid, glutamic acid, etc. The salts include pharmaceutically acceptable salts. The pharmaceutically acceptable salts include, for example, forms having reasonable benefits for pharmaceutical use.

[0055] The composition of the present disclosure may be, for example, a liquid or a solid. When the composition of the present disclosure is a liquid, the composition of the present disclosure may further include, for example, an aqueous solvent.

[0056] The aqueous solvent is, for example, an aqueous solvent used for cells, and specific examples include water, buffer solutions, infusion solutions, and culture media, with buffer solutions and infusion solutions being preferred. Examples of the buffer solution include Hank's balanced salt solution (HBSS) and phosphate buffer solution. Examples of the infusion solution include isotonic electrolyte infusion solutions such as bicarbonate Ringer's, physiological saline, Ringer's solution, lactate Ringer's solution, and acetate Ringer's solution; hypotonic electrolyte infusion solutions such as Solution No. 1 (initiation solution), Solution No. 2 (dehydration replacement solution), Solution No. 3 (maintenance solution), and Solution No. 4 (postoperative recovery solution); and amino acid infusion solutions. The culture medium can be, for example, a basal medium. Examples of the basal medium include MEM medium (Gibco Invitrogen and others), MEMα (Gibco Invitrogen and others), DMEM medium (Gibco Invitrogen and others), IMDM medium (Wako Pure Chemical and others), RPMI1640 medium (Gibco Invitrogen and others), Ham F-12 medium (Gibco and others), RD medium, and mixtures thereof.

[0057] The composition of the present disclosure has, for example, a cell viability after a cell freeze-thaw test of 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. The composition of the present disclosure has, for example, a cell viability after a cell freeze-thaw test of 60 to 100%, 65 to 99%, 70 to 99%, 75 to 98%, 80 to 97%, 85 to 96%, or 90 to 95%. The cell freeze-thaw test can be carried out in the same manner as in Example 1 described below. Specifically, 1×10 5 ~2×10 6Adipose-derived stromal cells (AD-MSCs) from 1000 cells are suspended in 0.5 mL of the target cell cryopreservation solution, and the resulting cell suspension is quickly frozen in a deep freezer (-80°C). The frozen AD-MSCs are thawed by placing the tube in a water bath at approximately 25°C from the day after the start of freezing. After thawing, the AD-MSCs are washed with HBSS(+) and recovered from the tube. After centrifugation, the supernatant is removed and the AD-MSCs are suspended in culture medium. Trypan blue is mixed with the resulting cell suspension, and a portion of the resulting mixture is added to a slide and the number of viable cells is counted using the cell counter. The viability of the cells is then calculated based on the total cell number (N a ) to the number of viable cells after freezing (N b ) proportion (N b / N a × 100(%)).

[0058] The composition of the present disclosure is preferably substantially free of, for example, organic solvent cellular cryoprotectants. Examples of the organic solvent cellular cryoprotectants include dimethyl sulfoxide, propylene glycol, ethylene glycol, glycerol (glycerin), ethanol, etc. The composition of the present disclosure is preferably substantially free of dimethyl sulfoxide, propylene glycol, ethylene glycol, glycerol (glycerin), and ethanol.

[0059] The composition of the present disclosure is preferably substantially free of, for example, animal-derived components. Examples of the animal-derived components include proteins, lipids, peptides, hormones, amino acids, vitamins, etc. More preferably, the composition of the present disclosure is substantially free of, for example, animal-derived proteins, lipids, peptides, hormones, amino acids, and / or vitamins, preferably proteins, lipids, peptides, hormones, amino acids, and vitamins derived from animal sources.

[0060] The compositions of the present disclosure are preferably substantially free of, for example, serum and / or plasma. More preferably, the compositions of the present disclosure are free of, for example, serum and plasma.

[0061] The compositions of the present disclosure are, for example, preferably chemically defined compositions.

[0062] In the composition of the present disclosure, the target cells can be any cells. Examples of such cells include primary cultured cells cultured in vitro, cells isolated from living organisms, and cultured cell lines. Specific examples of such cells include mesenchymal cells and immune cells. Mesenchymal cells are typically found in blood vessels, inside and around organs such as the liver or pancreas, fat, bone marrow, or umbilical cord. Mesenchymal stem cells are a type of multipotent stem cell and are capable of differentiating into adipocytes, osteocytes, chondrocytes, muscle cells, hepatocytes, tendon cells, and / or nerve cells. Depending on the tissue from which they are collected, mesenchymal stem cells are also referred to as adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, and umbilical cord-derived mesenchymal stem cells. Examples of such immune cells include T cells, B cells, NK cells, NKT cells, monocytes, macrophages, and dendritic cells. In addition to these, the cells may be, for example, skin cells, keratinocytes, skeletal muscle cells, cardiac muscle cells, lung cells, mesenteric cells, adipocytes, stem cells, hepatocytes, epithelial cells (epithelial-like cells), Kupffer cells, fibroblasts, neurons, cardiac muscle cells, muscle cells, chondrocytes, pancreatic acinar cells, islets of Langerhans, osteocytes, myoblasts, satellite cells, endothelial cells, preadipocytes, bile duct epithelial cells, progenitor cells, pluripotent stem cells, adult stem cells, germ cells, blood cells, nerve cells, corneal epithelial cells, corneal endothelial cells, osteocytes, chondrocytes, insulin-producing cells, and the like.

[0063] The cell may be, for example, a tissue or organ composed of multiple cells. Examples of the tissue include epithelial tissues such as skin and intestinal epithelium, muscle tissues such as connective tissue, skeletal muscle and cardiac muscle, and nervous tissues such as brain, spinal cord and nerves.

[0064] The cells may be, for example, the cell-derived components, such as exosomes (EVs, extracellular vesicles), microvesicles, and apoptotic bodies.

[0065] The composition of the present disclosure can be prepared, for example, by mixing the components when the composition is solid. For example, the composition of the present disclosure can be prepared, for example, by mixing the components and the aqueous solvent when the composition is liquid.

[0066] The composition of the present disclosure can be suitably used for cryopreserving the cells, for example, without the use of an organic solvent cryoprotectant.

[0067] <Cell freezing method> In another aspect, the present disclosure provides a method for freezing cells, which can protect cells during freezing without substantially adding an organic solvent-based cell cryoprotectant. The method for freezing cells of the present disclosure includes a freezing step of freezing cells in the presence of the composition for cryopreservation of cells of the present disclosure.

[0068] In the following description, unless otherwise specified, the description of the concentration of each component in the composition of the present disclosure can be used as the description of the concentration of each component in the freezing step.

[0069] The freezing method of the present disclosure may include, for example, a step of contacting the cells with a composition of the present disclosure prior to the freezing step. The contact can be achieved, for example, by mixing the cells with the composition of the present disclosure or suspending the cells in the composition of the present disclosure. The concentration of the cells in the resulting cryopreservation solution containing the cells can be, for example, 1 x 10 2 ~1×10 11 cells / mL, 1×10 3 ~1×10 10 cells / mL, 1×10 4 ~1×10 9 cells / mL.

[0070] Next, in the freezing step, for example, the cryopreservation solution containing the cells is frozen. The freezing can be performed, for example, by a conventional freezing method used for freezing cells. The freezing may be, for example, rapid freezing or slow freezing. Furthermore, the freezing may be performed by continuously or discontinuously cooling the cryopreservation solution. Specific examples of the freezing method include storing the cryopreservation solution containing the cells in a tube and placing it in a freezer at −20°C, −40°C, or −80°C, or by placing the tube in the gas or liquid phase of liquid nitrogen, or by placing it in a freezer such as a proton freezer. In the freezing step, the cooling rate is, for example, −0.1 to −30°C / min, −0.2 to −25°C / min, −0.3 to −20°C / min, −0.4 to −15°C / min, −0.5 to −10°C / min, or −1 to −5°C / min. In the freezing step, the final temperature range during freezing is, for example, -20 to -196°C, -40 to -150°C, or -70 to -90°C.

[0071] The freezing method of the present disclosure may further include, for example, a preservation step of preserving the frozen cells in a frozen state. The preservation can be carried out, for example, by placing a cryopreservation solution containing the cells in a freezer; the vapor phase of liquid nitrogen; or the like. The storage temperature in the preservation step is, for example, −20 to 196°C, −40 to −150°C, or −70 to −90°C. The storage period in the preservation step can be any period, for example, 1 day to 5 years, 10 days to 3 years, or 1 to 2 years, as specific examples.

[0072] The freezing method of the present disclosure may further include, for example, a thawing step of thawing the cryopreserved cells. The thawing step can be carried out, for example, by warming the cryopreservation solution containing the cells. The warming can be carried out, for example, by thawing at 20 to 40°C or 30 to 37°C.

[0073] The freezing methods of the present disclosure are carried out, for example, in vitro or in vivo.

[0074] The freezing method of the present disclosure allows for suitable cryopreservation of cells, for example, without using an organic solvent cryoprotectant.

[0075] <Cell preparation> In another aspect, the present disclosure provides a cell preparation that is substantially free of the organic solvent cell cryoprotectant. The cell preparation of the present disclosure comprises cells and the cell cryopreservation composition of the present disclosure.

[0076] The description of the concentration of each component in the composition of the present disclosure can be used, for example, as a description of the concentration of each component in the cell preparation of the present disclosure.

[0077] The state of the cell preparation of the present disclosure may be, for example, a post-freezing state, i.e., a frozen state or solid, or a pre-freezing or post-freeze-thaw state, i.e., a liquid. In the latter case, the cell preparation of the present disclosure is, for example, the cells present in the composition of the present disclosure. The state of the cell preparation of the present disclosure is preferably a frozen state or solid.

[0078] The cell preparations of the present disclosure are obtained, for example, by the cell freezing methods of the present disclosure.

[0079] The cell preparation of the present disclosure is substantially free of, for example, the organic solvent-based cell cryoprotectant, thereby suppressing the toxicity caused by the organic solvent, and therefore is suitable for use as, for example, a cell medicine.

[0080] <Treatment method> In another aspect, the present disclosure provides a treatment method in which the toxicity of the organic solvent as a cellular cryoprotectant is reduced. The treatment method of the present disclosure includes administering to a subject the cell preparation of the present disclosure.

[0081] In the present disclosure, the subject is, for example, a patient with a disease that can be treated with cells containing the cell preparation. The treatment method of the present disclosure is carried out, for example, in vitro or in vivo.

[0082] The cell preparation of the present disclosure can protect cells during freezing without substantially containing the organic solvent-based cellular cryoprotectant, and therefore, according to the treatment method of the present disclosure, the amount of the organic solvent-based cellular cryoprotectant administered to the subject can be reduced, thereby reducing the toxicity of the organic solvent-based cellular cryoprotectant. [Example]

[0083] Next, examples of the present disclosure will be described. However, the present disclosure is not limited by the following examples. Commercially available reagents were used according to their protocols unless otherwise specified. In the following examples, "mol / l" may be abbreviated as "M."

[0084] [reagent] In the following examples, the following reagents were used unless otherwise specified.

[0085] [Table 1]

[0086] [solvent] In each of the following examples, the compositions of the solvents used in the preservative solutions were as shown in Tables 2 and 3 below.

[0087] [Table 2]

[0088] [Table 3]

[0089] [Cell preparation] The cells used in the following examples were prepared as follows.

[0090] (1) Autologous preparation of adipose-derived stromal cells (adipose-derived mesenchymal stem cells (AD-MSCs)) Adipose-derived stromal cells were prepared from fresh adipose tissue (via StemExpress). After washing the adipose tissue with HBSS(+), a collagenase solution (Solaris Bio, Inc.) was added in an amount equal to the adipose tissue weight, and the adipose tissue was treated with collagenase at 37°C for 3.5 hours. The resulting treatment solution was then centrifuged (800g x 5 minutes). The supernatant containing oil was discarded. The precipitate was washed with the resulting HBSS(+) and then centrifuged twice. The red blood cells were then lysed using RBC Lysis Solution (Solaris Bio, Inc.). The lysed solution was then centrifuged (800g x 5 minutes). The supernatant was then discarded. The resulting precipitate (tissue fragments) was suspended in medium (M101-AF-500, manufactured by Solaris Bio, Inc.). The tissue fragments were then filtered through a cell strainer. The obtained filtrate was used as a cell suspension containing SVF (stromal vascular fraction, stromal vascular cell group), and the solvent was replaced with a cell cryopreservation solution (Solaris Bio Co., Ltd., C101-AF-100). The cell suspension was then cryopreserved at -80°C. The cryopreserved SVF was washed with HBSS. The SVF was then suspended in culture medium. The suspension was then seeded into a T-25 flask (Sumitomo Bakelite Co., Ltd., MS-23050). The seeded cells were recovered and cryopreserved at -80°C. The cryopreserved cells were expanded in culture medium using a T-150 flask (Sumitomo Bakelite Co., Ltd., MS-23600). After the expansion culture, the AD-MSCs were recovered when the cells reached a subconfluent state.

[0091] (2) Preparation from adipose-derived stromal cells (commercially available AD-MSCs) Adipose-derived stromal cells (PT-5006, manufactured by Lonza) were expanded in a T-150 flask (MS-23600, manufactured by Sumitomo Bakelite Co., Ltd.) After the expansion, the AD-MSCs were collected when they reached a subconfluent state.

[0092] (3) Preparation of umbilical cord-derived mesenchymal cells (UC-MSCs) The UC-MSCs were collected in the same manner as in (2), except that umbilical cord-derived stromal cells (LIFELINE, FC-0020) were used instead of the commercially available AD-MSCs.

[0093] (4) Preparation of THP-1 cells The THP-1 cells were collected in the same manner as in (2) above, except that THP-1 cells (ATCC, TIB-202) were used instead of the commercially available AD-MSCs, RPMI-1640 + 10% FBS (Gibco, 72400047 and A31604-01) was used instead of the AD-MSC medium, and a T-25 non-adherent flask (Sumitomo Bakelite Co., Ltd., MS-2305R) was used instead of a T-150 flask.

[0094] (5) Preparation of Jurkat cells The Jurkat cells were collected in the same manner as in (4), except that Jurkat cells (manufactured by KAC Corporation, EC88042803-G0) were used instead of the THP-1 cells.

[0095] [Example 1] We confirmed that the use of a cell cryopreservation solution containing sucrose can improve cell viability during cryopreservation.

[0096] (Freezing test) Autologous AD-MSCs were seeded into a T-150 flask supplemented with medium and cultured for 3 to 4 days. After the AD-MSCs were grown to subconfluence, a detachment agent (D101-AF-500, manufactured by Solaris Bio Inc.) was added to the T-150 flask and incubated at 37°C for 5 minutes. The AD-MSCs were then detached, centrifuged (400 xg, 5 minutes), and the supernatant was removed. The recovered AD-MSCs were suspended in HBSS(+) to prepare a cell suspension. Trypan blue (15250-061, manufactured by Gibco) was added to the cell suspension, and the number of viable cells was counted using a cell counter (Countess3, manufactured by Thermofisher Scientific). Furthermore, the AD-MSCs were diluted to 1 x 10 5~2×10 6 The AD-MSCs were then dispensed into tubes to form 1000 cells. After dispensing, the AD-MSCs were centrifuged (400 × g, 5 minutes) and the supernatant was removed. Then, 0.5 mL of each cell cryopreservation solution listed in Table 4 below was added to each tube and suspended, and the resulting cell suspension was quickly frozen in a deep freezer (−80°C). DMSO is commonly used as a cell cryoprotectant and serves as a positive control. HBSS+ alone serves as a negative control, lacking a cell cryoprotectant.

[0097] [Table 4]

[0098] The frozen AD-MSCs were thawed by placing the tube in a water bath at room temperature (approximately 25°C) from the day after the start of freezing. After thawing, the AD-MSCs were washed with HBSS(+), recovered from the tube, and centrifuged (400 x g, 5 minutes). After separation, the supernatant was removed, and the AD-MSCs were suspended in the medium. Trypan blue was mixed with the obtained cell suspension, and a portion of the obtained mixture was added to a slide, and the number of viable cells was counted using the cell counter. The total number of cells after cryopreservation (N b ) and the number of live cells after cryopreservation (N a ) and survival rate (N a / N b × 100(%)) were calculated. The results are shown in Table 5 below.

[0099] [Table 5]

[0100] Table 5 shows the results of the survival rate of AD-MSCs after freezing and thawing. As shown in Table 5, the survival rate of the cells after freezing and thawing was significantly reduced in the negative control (HBSS+ only). In contrast, the survival rate of the cells was higher in the case of 10% sucrose and 10% DMSO compared to HBSS+ only. This indicates that the use of sucrose can improve the survival rate of cells when frozen, that is, sucrose acts as a cell cryoprotectant.

[0101] [Example 2] We confirmed that cell survival rates during cryopreservation can be improved by using cell cryopreservation solutions containing different concentrations of sucrose.

[0102] The survival rate of AD-MSCs after freezing and thawing was calculated in the same manner as in Example 1, except that the cell cryopreservation solution in Table 6 below was used instead of the cell cryopreservation solution in Table 4 above. The results are shown in Table 7 below.

[0103] [Table 6]

[0104] [Table 7]

[0105] Table 7 shows the results of the survival rate of AD-MSCs after freezing and thawing. As shown in Table 7, the survival rate of cells after freezing and thawing was improved at all sucrose concentrations. In particular, by increasing the sucrose concentration to 5% or more, the survival rate was about 70%, which was a high survival rate. Generally, a cell cryopreservation solution with a survival rate of more than 70% after freezing and thawing is considered to be excellent. Therefore, it was found that the survival rate of cells after freezing and thawing can be significantly improved by increasing the sucrose concentration to 5% or more.

[0106] [Example 3] We confirmed that the survival rate of cells during cryopreservation can be improved by using a cell cryopreservation solution containing an antioxidant (reducing agent).

[0107] The survival rates of AD-MSCs after freezing and thawing were calculated in the same manner as in Example 1, except that the cell cryopreservation solutions shown in Tables 8 to 10 below were used instead of the cell cryopreservation solutions shown in Table 4 above. Table 12 below shows the survival rates (N c ) as the standard, the survival rate (N d ) for the specific survival rate (N d / N c × 100(%)) was calculated. Furthermore, in Table 13 below, the number of viable cells (N E ) and the number of viable cells (N f ) and the specific viable cell number (N f / N E × 100(%)) were calculated. The results are shown in Tables 11 to 13 below.

[0108] [Table 8]

[0109] [Table 9]

[0110] [Table 10]

[0111] [Table 11]

[0112] [Table 12]

[0113] [Table 13]

[0114] Table 11 shows the results for AD-MSC viability. Table 12 shows the results for AD-MSC specific viability. Table 13 shows the results for AD-MSC specific viable cell count. As shown in Table 11, all antioxidants improved cell viability after freezing and thawing. In particular, a high viability of approximately 86% was achieved by setting the glutathione concentration at 32.5%. This indicates that the use of glutathione as an antioxidant can further improve post-thaw viability. Furthermore, as shown in Table 12, the addition of glutathione increased the specific cell viability after freezing and thawing compared with the absence of glutathione. This indicates that adding glutathione at a concentration of 1 μM or higher can improve cell viability during cell freezing. Furthermore, as shown in Table 13, the addition of glutathione and alpha-lipoic acid as antioxidants increased the specific cell count after freezing and thawing compared with the absence of glutathione alone. In other words, the use of glutathione and alpha-lipoic acid as antioxidants can improve cell survival rates after freezing and thawing, and it was found that glutathione and alpha-lipoic acid act as antioxidants in cell cryoprotectants.

[0115] [Example 4] We confirmed that the viability of cells during cryopreservation can be improved by using a cell cryopreservation solution containing sugars, sugar alcohols, nucleotides, PVP, or Soluplus.

[0116] The survival rates of AD-MSCs after freezing and thawing were calculated in the same manner as in Example 1, except that the cell cryopreservation solutions shown in Tables 14 to 16 below were used instead of the cell cryopreservation solutions shown in Table 4. The results are shown in Tables 17 to 19 below.

[0117] [Table 14]

[0118] [Table 15]

[0119] [Table 16]

[0120] [Table 17]

[0121] [Table 18]

[0122] [Table 19]

[0123] Tables 17 to 19 show the results of the survival rate of AD-MSCs after freezing and thawing. As shown in Tables 17 to 19, the addition of not only sucrose but also other disaccharides, monosaccharides, trisaccharides, polysaccharides, sugar alcohols, nucleosides, PVP, and Soluplus improved the survival rate of cells after freezing and thawing. In other words, it was found that disaccharides, monosaccharides, trisaccharides, polysaccharides, sugar alcohols, nucleosides, PVP, and Soluplus act as cell cryoprotectants.

[0124] [Example 5] We confirmed that the viability of cells during cryopreservation can be improved by using solvents other than HBSS as the cell cryopreservation medium.

[0125] The survival rate of AD-MSCs after freezing and thawing was calculated in the same manner as in Example 1, except that the cell cryopreservation solution shown in Table 20 below was used instead of the cell cryopreservation solution shown in Table 4. The results are shown in Tables 21 and 22 below.

[0126] [Table 20]

[0127] [Table 21]

[0128] [Table 22]

[0129] Tables 21 and 22 show the results of the survival rate of AD-MSCs after freezing and thawing. As shown in Tables 21 and 22, the survival rate of cells after freezing and thawing was improved not only with HBSS+ but also with other solvents. In other words, it was found that the survival rate of cells during cryopreservation can be improved when using not only HBSS+ but also other solvents in cell cryopreservation solutions containing sucrose and glutathione.

[0130] [Example 6] We confirmed that the viability of cells during cryopreservation can be improved using various freezing methods in a cell cryopreservation solution containing sucrose and glutathione.

[0131] The survival rate of AD-MSCs after freezing and thawing was calculated in the same manner as in Example 1, except that the cell cryopreservation solution in Table 23 below was used instead of the cell cryopreservation solution in Table 4 above, and the freezing method was changed to rapid freezing in a deep freezer (-80°C) using the freezing method in Table 21 below. The results are shown in Table 24 below.

[0132] [Table 23]

[0133] [Table 24]

[0134] Table 24 shows the results of the survival rate of AD-MSCs after freezing and thawing. As shown in Table 24, the survival rate of cells after freezing and thawing was improved regardless of the freezing method. In other words, it was found that the survival rate of cells during cryopreservation can be improved using various freezing methods in a cell cryopreservation solution containing sucrose and glutathione.

[0135] [Example 7] We confirmed that a cell cryopreservation solution containing sucrose and amino acids can improve cell viability during cryopreservation.

[0136] The survival rate of AD-MSCs after freezing and thawing was calculated in the same manner as in Example 1, except that the cell cryopreservation solution shown in Table 25 below was used instead of the cell cryopreservation solution shown in Table 4. The results are shown in Table 26 below.

[0137] [Table 25]

[0138] (Culture test) Next, for samples 5 to 8, the cells were frozen and thawed, and the number of viable cells was counted when further cultured. After freezing and thawing, the cells were washed. Next, the cell suspension suspended in the medium was seeded on a 6-well plate (Sumitomo Bakelite Co., Ltd., MS-80060) to which 2 mL of medium had been added in advance. The density of the AD-MSCs was 5 × 10 3 ~7.5×10 3 cells / cm 2The cells were then prepared so that the concentration of the detachment agent was 500 μL and cultured for 3 days. Thereafter, the number of viable cells of the AD-MSCs was counted. To count the number of cells, first, 500 μL of the detachment agent was added to the 6-well plate and incubated at 37°C for 5 minutes. Next, the mixture containing the detachment agent was stirred well, and trypan blue was added. Furthermore, a portion of the mixture was added to a slide, and the number of viable cells after culturing the AD-MSCs for 3 days was counted using a cell counter. Then, the number of viable cells (Ni) without addition of amino acids (Sample 4) and the number of viable cells (N) at each alanine addition concentration (Samples 5 to 7) were calculated. j ) and the specific survival rate (N j / N i × 100(%)) were calculated. The results are shown in Table 25 below.

[0139] [Table 26]

[0140] [Table 27]

[0141] Table 26 shows the results of the survival rate of AD-MSCs after freezing and thawing. Table 27 shows the specific viable cell count of AD-MSCs when further cultured after freezing and thawing. As shown in Table 26, the addition of alanine and taurine improved the survival rate of cells after freezing and thawing. Furthermore, as shown in Table 27, the survival rate of cells after freezing and thawing improved concentration-dependently by increasing the alanine concentration to 0.05 mM or higher. In other words, it was found that the addition of amino acids to a sucrose-containing cell cryopreservation solution can improve the survival rate of cells after freezing and thawing.

[0142] [Example 8] We confirmed that a cell cryopreservation solution containing sucrose and glutathione can improve the survival rate of cells during cryopreservation using various cells.

[0143] The survival rates of AD-MSCs after freezing and thawing were calculated in the same manner as in Example 1, except that the cell cryopreservation solution in Table 28 below was used instead of the cell cryopreservation solution in Table 4 above, and adipose-derived stromal cells (commercially available AD-MSCs), umbilical cord-derived mesenchymal cells (UC-MSCs), THP-1 cells, and Jurkat cells were used instead of autologous AD-MSCs. The results are shown in Table 29 below.

[0144] [Table 28]

[0145] [Table 29]

[0146] Table 29 shows the results of the survival rate of AD-MSCs after freezing and thawing in various cell types. As shown in Table 29, the survival rate of cells after freezing and thawing was improved in all cell types. In other words, it was found that the cell cryopreservation solution containing sucrose and glutathione can improve the survival rate of cells after freezing and thawing in various cell types.

[0147] Although the present disclosure has been described above with reference to embodiments and examples, the present disclosure is not limited to the above embodiments and examples. Various modifications that can be understood by a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0148] The patents, patent applications, and publications cited herein are incorporated by reference into this specification in their entirety as if the contents were specifically set forth herein.

[0149] <Additional Notes> Some or all of the above-described embodiments and examples can be described as, but are not limited to, the following supplementary notes. <Cell cryopreservation composition> (Appendix 1) A composition for cryopreserving cells, comprising a sugar or a derivative thereof and / or a surfactant. (Appendix 2) 2. The composition of cells of claim 1, wherein the sugar is a monosaccharide, a disaccharide, a trisaccharide, and / or a polysaccharide. (Appendix 3) 3. The composition of claim 2, wherein the disaccharide is selected from the group consisting of sucrose, maltose, sucralose, lactose, cellobiose, lactobionic acid, lactulose, melibiose, and isomaltose. (Appendix 4) 4. The composition of claim 2 or 3, wherein the disaccharide comprises sucrose. (Appendix 5) 5. The composition of any of claims 2 to 4, wherein the monosaccharide is selected from the group consisting of glucose, galactose, mannose, fructose, xylose, ribose, and arabinose. (Appendix 6) 6. The composition of any of Appendices 2 to 5, wherein the trisaccharide is selected from the group consisting of raffinose, maltotriose, isomaltotriose, panose, and melezitose. (Appendix 7) 7. The composition of any of claims 2 to 6, wherein the polysaccharide is selected from the group consisting of dextran, starch, hydroxyethyl starch, hyaluronic acid, glycogen, and alginic acid. (Appendix 8) 8. The composition of any of claims 1 to 7, wherein the sugar is a non-reducing sugar and / or a reducing sugar. (Appendix 9) 9. The composition of any one of claims 1 to 8, wherein the sugar derivative is selected from the group consisting of a nucleoside, a nucleotide, and a sugar alcohol. (Appendix 10) 10. The composition of claim 9, wherein the nucleoside comprises uridine and / or deoxyuridine. (Appendix 11) 11. The composition of claim 9 or 10, wherein the sugar alcohol is selected from the group consisting of sorbitol and xylitol. (Appendix 12) 12. The composition of any one of claims 1 to 11, wherein the surfactant comprises polyvinylpyrrolidone (PVP) and / or a modified polyalkylene glycol. (Appendix 13) 13. The composition of claim 12, wherein the modified polyalkylene glycol comprises Soluplus. (Appendix 14) comprising the sugar, 14. The composition according to any one of claims 1 to 13, wherein the sugar has a concentration of 5 to 25% (w / v). (Appendix 15) the sugar is a monosaccharide, disaccharide, and / or trisaccharide; 15. The composition according to any one of claims 1 to 14, wherein the sugar has a concentration of 5 to 25% (w / v). (Appendix 16) the sugar derivative is a sugar alcohol, 16. The composition according to any one of claims 1 to 15, wherein the sugar alcohol has a concentration of 1 to 20% (w / v). (Appendix 17) 17. The composition according to any one of claims 1 to 16, wherein the concentration of the surfactant is 1 to 20% (w / v). (Appendix 18) 18. The composition of any of Appendices 1 to 17, further comprising an antioxidant. (Appendix 19) 19. The composition of claim 18, wherein the antioxidant comprises an antioxidant selected from the group consisting of glutathione, lipoic acid (alpha lipoic acid, thioctic acid), ascorbic acid (vitamin C) or a derivative thereof, tocopherol (vitamin E) or a derivative thereof, acetylcysteine, and a chelating agent. (Appendix 20) 20. The composition of claim 19, wherein the ascorbic acid derivative is selected from the group consisting of ascorbic acid 2-glucoside, ascorbic acid 2-phosphate, ascorbyl tetrahexyldecanoate, and 3-O-ethyl ascorbic acid. (Appendix 21) 21. The composition of claim 19 or 20, wherein the tocopherol derivative is selected from the group consisting of α-tocopheryl phosphate, tocopherol acetate, and tocopherol succinate. (Appendix 22) 22. The composition of any of claims 19 to 21, wherein the antioxidant comprises lipoic acid and glutathione. (Appendix 23) 23. The composition according to any one of claims 18 to 22, wherein the concentration of the antioxidant is 1 nmol / L to 100 mmol / L. (Appendix 24) 24. The composition of any of Appendices 1 to 23, further comprising an amino acid. (Appendix 25) 25. The composition of claim 24, wherein the amino acid is selected from the group consisting of alanine, taurine, phenylalanine, methionine, proline, cysteine, serine, lysine, arginine, homoserine, aminobutyric acid, aminocaproic acid, tryptophan, and glycine. (Appendix 26) 26. The composition according to claim 24 or 25, wherein the concentration of the amino acid is 0.01 to 100 mmol / L. (Appendix 27) 27. The composition of any one of claims 1 to 26, further comprising an aqueous solvent. (Appendix 28) 28. The composition of claim 27, wherein the aqueous solvent is selected from the group consisting of water, a buffer solution, an infusion solution, and a culture medium. (Appendix 29) 29. The composition of claim 28, wherein the buffer is selected from the group consisting of Hank's Balanced Salt Solution (HBSS), phosphate buffer, Dulbecco's phosphate buffered saline, Hepes buffered saline, and amino acid buffer. (Appendix 30) 30. The composition of any one of Appendices 1 to 29, wherein the cell viability after a cell freeze-thaw test is 60% or more. (Appendix 31) 31. The composition of any of claims 1 to 30, wherein the composition is substantially free of organic solvent cellular cryoprotectants. (Appendix 32) 32. The composition of claim 31, wherein the organic solvent cellular cryoprotectant is selected from the group consisting of dimethyl sulfoxide, propylene glycol, ethylene glycol, glycerol, and ethanol. (Appendix 33) 33. The composition of any of claims 1 to 32, wherein the composition is substantially free of animal-derived components. (Appendix 34) 34. The composition of claim 33, wherein the animal-derived component is a protein, lipid, peptide, hormone, amino acid, and / or vitamin obtained from an animal source. (Appendix 35) 35. The composition of any of claims 1 to 34, wherein the composition is substantially free of serum and / or plasma. (Appendix 36) 36. The composition of any of Appendices 1 to 35, which is a chemically defined composition. <Cell freezing method> (Appendix 37) A method for freezing cells, comprising a freezing step of freezing cells in the presence of a composition described in any one of appendices 1 to 36. (Appendix 38) 38. The freezing method according to claim 37, further comprising a preservation step of preserving the frozen cells in a frozen state. (Appendix 39) The freezing method according to claim 37 or 38, wherein the cells are primary cultured cells cultured in vitro and / or cells isolated from a living organism. (Appendix 40) 40. The freezing method of any one of appendix 37 to 39, wherein the cells are selected from the group consisting of mesenchymal cells, immune cells, fibroblasts, epithelial-like cells, pluripotent stem cells, adult stem cells, germ cells, muscle cells, blood cells, nerve cells, hepatocytes, adipocytes, corneal epithelial cells, corneal endothelial cells, bone cells, chondrocytes, and insulin-producing cells. (Appendix 41) 41. The freezing method of any one of claims 37 to 40, which is carried out in vitro or in vivo. <Cell preparation> (Appendix 42) A cell preparation comprising cells and a composition according to any one of appendices 1 to 36. (Appendix 43) 43. The cell preparation of claim 42, which is frozen or solid. (Appendix 44) 44. The cell preparation of claim 42 or 43, wherein the cells are selected from the group consisting of mesenchymal cells, immune cells, fibroblasts, epithelial-like cells, pluripotent stem cells, adult stem cells, germ cells, muscle cells, blood cells, nerve cells, hepatocytes, adipocytes, corneal epithelial cells, corneal endothelial cells, bone cells, chondrocytes, and insulin-producing cells. (Appendix 45) 45. A cell preparation according to any one of appendices 42 to 44, obtained by the freezing method of any one of appendices 37 to 41. <Treatment method> (Appendix 46) A method of treatment comprising administering to a subject a cell preparation according to any one of claims 42 to 45. (Appendix 47) 47. The method of claim 46, wherein the subject is a patient with a disease that can be treated with the cells contained in the cell preparation. [Industrial Applicability]

[0150] As described above, according to the present disclosure, cells can be protected during freezing without the substantial addition of, for example, an organic solvent cell cryoprotectant. Therefore, the present disclosure can be said to be extremely useful in, for example, the fields of cell medicine and regenerative medicine.

Claims

1. A composition comprising sucrose and glutathione, the concentration of the sucrose is 5 to 20% (w / v); A composition for cryopreservation of cells, wherein the concentration of glutathione is 1 to 2000 μmol / L.

2. The composition of claim 1 , comprising a lipoic acid.

3. The composition according to claim 1 or 2, further comprising an aqueous solvent.

4. The composition of claim 3 , wherein the aqueous solvent is selected from the group consisting of water, a buffer solution, an infusion solution, and a culture medium.

5. 3. The composition according to claim 1, wherein the cell viability after a cell freeze-thaw test is 60% or more.

6. 3. The composition of claim 1 or 2, which is substantially free of organic solvent cellular cryoprotectants.

7. 7. The composition of claim 6, wherein the organic solvent cellular cryoprotectant is selected from the group consisting of dimethyl sulfoxide, propylene glycol, ethylene glycol, glycerol, and ethanol.

8. A method for freezing cells, comprising a freezing step of freezing cells in the presence of the composition according to claim 1 or 2.

9. A cell preparation comprising cells and the composition of claim 1 or 2.

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