Compositions for cryopreservation of cells, methods for freezing cells, and cell preparations

Abstract

This disclosure provides a cell cryopreservation composition that can protect cells during cell freezing, for example, without substantially adding organic solvent cell cryoprotective agents. [Solution] The cell cryopreservation composition of this disclosure comprises a sugar or a derivative thereof and / or a surfactant.

Description

[Technical Field]

[0001] This disclosure relates to compositions for cryopreservation of cells, methods for cryopreserving cells, and cell preparations. [Background technology]

[0002] With advances in regenerative medicine, cells isolated from living organisms are being cultured in large quantities, and further processing, such as gene introduction, is being performed on these cultured cells. In addition, these cultured or processed cells are being used as cell therapies to treat diseases.

[0003] In the manufacture of the aforementioned cell-based pharmaceutical product, after culturing and processing the cells at a cell processing facility, the processed cells are cryopreserved for transport to the hospital (Non-Patent Documents 1 and 2). In this cryopreservation process, the processed cells are dispersed in a cell cryopreservation solution containing a cell cryoprotective agent and then frozen. The cells are then transported in their 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. [Overview of the project] [Problems that the invention aims to solve]

[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 has cytotoxicity, cell medicaments containing DMSO are in live reported to exhibit toxicity when administered to. In addition to DMSO, cell cryoprotectants that are organic solvents such as propylene glycol, ethylene glycol, glycerol (glycerin), and ethanol have been reported. However, regarding these organic solvent cell cryoprotectants, in live toxicity may also develop when administered to.

[0006] Accordingly, the present disclosure provides, for example, a cryopreservation composition for cells that can protect cells during cryopreservation without substantially adding the aforementioned organic solvent cell cryoprotectant, a method for cryopreserving cells, and a cell preparation obtained thereby. [Means for Solving the Problem]

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

[0008] The method for cryopreserving cells of the present disclosure (hereinafter also referred to as "cryopreservation method") comprises a cryopreservation 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 the composition of the present disclosure. [Effect of the Invention]

[0010] According to the present disclosure, for example, cells can be protected during cryopreservation without substantially adding an organic solvent cell cryoprotectant. [Mode for Carrying Out the Invention]

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

[0012] In the present disclosure, "reducing sugar" is a sugar having reducing properties, and refers to a sugar having a free aldehyde group or a free ketone group. Further, "non-reducing sugar" is a sugar that does not exhibit reducing properties, and refers to a sugar having no free aldehyde group or free ketone group.

[0013] As used herein, "glutathione" is a tripeptide composed of glutamic acid-cysteine-glycine. It is known that glutathione functions as an antioxidant in cells and protects cells from reactive oxygen species such as free radicals and peroxides.

[0014] As used herein, "amino acid" means 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 D-form, L-form, or a mixture of both.

[0015] In this specification, “substantially free” means not containing or completely not containing any substance at concentrations above the detection limit. Specifically, a serum-free composition is serum-free and substantially free of serum or plasma. The term “serum-free” means not containing or not containing any serum or plasma-derived components at concentrations above the detection limit. A composition free of animal-derived components is animal-free and substantially free of human and animal-derived components. The term “animal-free” means not containing or not containing any animal-derived components at concentrations above the detection limit. A protein-free composition is protein-free and does not contain any protein. The term “protein-free” means not containing or not containing any protein at concentrations above the detection limit. A composition free of organic solvent cryoprotective agents is organic solvent-free and substantially free of organic solvent cryoprotective agents. The term “organic solvent-free” means not containing or not containing any organic solvent cryoprotective agents at concentrations above the detection limit. A composition free of albumin is albumin-free and does not contain albumin. The term "albumin-free" means that the product does not contain albumin at a concentration above the detection limit, or does not contain albumin at all.

[0016] In this specification, "organic solvent" means an organic compound that is liquid at room temperature and pressure and has the property of dissolving other substances. The organic compound means a compound containing carbon atoms that produces carbon dioxide or carbonizes when burned.

[0017] In this specification, "albumin" refers to a protein known as a component of plasma. For example, human serum albumin may be a protein having the amino acid sequence registered in GenBank under registration number AAN17825.1, or human serum albumin having the corresponding amino acid sequence.

[0018] In this specification, "chemically defined" means that the components and their quantities can be chemically defined. Specifically, a "chemically defined composition" means a composition in which the compound names or amino acid sequences are clearly defined and their quantities can also be specified.

[0019] In this specification, “infusion” means a liquid preparation, injection, or drip preparation that can be administered intravenously.

[0020] In this specification, “culture medium” means a culture medium used for culturing cells. The culture medium can be prepared, for example, by adding the necessary components to a basal culture medium.

[0021] In this specification, “isolated” means identified and separated, and / or recovered from a component in its natural state. Such “isolation” can be carried out, for example, by obtaining at least one purification step.

[0022] In this specification, “protein” means 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. In this specification, “peptide” means 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 fewer than 10 amino acids.

[0023] In this specification, “cell preparation” means a composition containing the desired cells.

[0024] In this specification, “mesenchymal cells” means cells that constitute connective tissue derived from the mesoderm or neural crest and / or cells that have the ability to differentiate into such cells. If the mesenchymal cells have the ability to self-renew, they may also be called mesenchymal stem cells (MSCs).

[0025] In this specification, “immune cells” means cells that constitute the immune system and / or cells that have the ability to differentiate into such cells.

[0026] In this specification, “treatment” means therapeutic treatment and / or preventive treatment. In this specification, “treatment” means the treatment, cure, prevention, suppression, remission, improvement of a disease, condition, or disorder, or the cessation, suppression, reduction, or delay of the progression of a disease, condition, or disorder. In this specification, “prevention” means a reduction in the likelihood of developing a disease or condition, or a delay in the development of a disease or condition. The “treatment” may be, for example, treatment of a subject (patient) who develops the disease in question, or treatment of a model animal of the disease in question.

[0027] In this specification, “subject” means an animal or a cell, tissue, or organ of animal origin. The term "subject" is used in a sense that includes humans. The animals include humans and non-humans. It means animals. The aforementioned non-human animals include, for example, mice, rats, rabbits, dogs, cats, and cows. Examples of mammals include horses, pigs, monkeys, dolphins, and sea lions. "Patient" means the person receiving preventive or therapeutic treatment. The aforementioned person is, for example, In addition to the aforementioned patients, healthy individuals are also included.

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

[0029] The following explanation of this disclosure includes examples, but this disclosure is not limited to these examples and can be modified as needed. Furthermore, unless otherwise specified, each explanation in this disclosure is interchangeable with one another. In this specification, the expression "~" includes the numerical or physical values ​​before and after it. Also, in this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B." In the following explanation, "mol / l" may also be abbreviated as "M."

[0030] <Composition for cryopreservation of cells> In one embodiment, the Disclosure provides a cell cryopreservation composition capable of protecting cells during cell freezing without substantially adding the organic solvent cell cryoprotectant. The cell cryopreservation composition of the Disclosure comprises a sugar or a derivative thereof and / or a surfactant.

[0031] As a result of diligent research, the present inventors have found that the sugar or its derivative and / or surfactant functions as a cell cryoprotective agent. This is presumed to be because the sugar or sugar derivative or surfactant, in particular when present at a certain concentration or higher, inhibits the formation of ice crystals inside and outside the cell, and dehydrates intracellular water due to the osmotic pressure difference inside and outside the cell, thereby protecting the cell membrane and intracellular organelles from freezing damage. However, this presumption 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 cryoprotective agent, cells can be protected during cell freezing without using, for example, the organic solvent cell cryoprotective agent.

[0032] The compositions of the present disclosure include, for example, the sugar or its derivative and / or surfactant as the cell cryoprotective agent. The compositions of the present disclosure preferably include the sugar or its derivative because of its high cell cryoprotective effect. The compositions of the present disclosure may use any one of the cell cryoprotective agents alone or two or more in combination.

[0033] The sugar is not particularly limited and can be a monosaccharide, disaccharide, trisaccharide, polysaccharide, etc. The sugar is preferably a monosaccharide, disaccharide, or trisaccharide, and more preferably a monosaccharide or disaccharide, because it allows for a high cell proliferation rate in culture after thawing. The sugar may be used alone or in combination of two or more types.

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

[0035] The aforementioned sugar may be a non-reducing sugar or a reducing sugar, but it is preferably a non-reducing sugar because it has a high cell cryopreservation effect. Examples of the aforementioned non-reducing sugars include sucrose, trehalose, sucralose, maltotriose, isomalttriose, and panose.

[0036] Examples of the sugar derivatives include nucleosides, nucleotides, and sugar alcohols. One type of 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 ribonucleoside include uridine, adenosine, guanosine, 5-methyluridine, and cytidine, and uridine is preferred because it has a high cell cryoprotective effect. Examples of the deoxyribonucleoside include deoxyuridine, deoxyadenosine, deoxyguanosine, thymidine, and deoxycytidine, and deoxyuridine is preferred.

[0038] Examples of the nucleotides include ribonucleotides and deoxyribonucleotides. Examples of the ribonucleotides 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, 5-methyluridine triphosphate, and the like. Examples of the aforementioned deoxyribonucleotides 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, thymidine triphosphate, and the like.

[0039] Examples of the aforementioned sugar alcohols 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 polyvinylcaprolactam block and polyvinyl acetate block. The polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block is, for example, polyethylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block. The modified polyalkylene glycol is, for example, a polyvinylcaprolactam-polyvinylacetate-polyethylene glycol graft copolymer. The modified polyalkylene glycol is preferably Soluplus. The modified polyalkylene glycol may also be, for example, a graft copolymer obtained from (i) N-vinylcaprolactam, (ii) vinyl acetate, and (iii) polyether. The polyether may be polyethylene glycol. The modified polyalkylene glycol may, for example, be a compound obtained by free radical polymerization of the mixture of (i) to (iii) above. In this case, the composition of the mixture may be, for example, (i) 40-60 wt%, 45-57 wt%, or 50-55 wt%, (ii) 15-35 wt%, 20-30 wt%, or 20-25 wt%, and (iii) 10-30 wt%, 13-25 wt%, or 15-20 wt%. The total of (i) to (iii) may be 100 wt%. The modified polyalkylene glycol may, for example, be 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, 40-60 wt%, 15-35 wt%, and 10-30 wt%, respectively. The wt% of l is, for example, 40-60 wt%, 45-57 wt%, or 50-55 wt%. The wt% of m is, for example, 15-35 wt%, 20-30 wt%, or 20-25 wt%. The wt% of n is, for example, 10-30 wt%, 13-25 wt%, or 15-20 wt%. The wt% of l, m, and n are, for example, 100 wt% in total. The wt% of l, m, and n may also be, for example, about 57, about 30, and about 13 wt%, respectively.

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

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

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

[0046] If the composition of the present disclosure contains the sugar alcohol, the concentration of the sugar alcohol is, for example, 1-20% (w / v), 1.5-15% (w / v), 2-12.5% ​​(w / v), or 2.5-10% (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.

[0047] The concentration of the surfactant is, for example, 1-20% (w / v), 2.5-15% (w / v), 5-15% (w / v), or 7.5-12.5% ​​(w / v). If the surfactant is PVP or modified polyalkylene glycol, the concentration of the PVP or modified polyalkylene glycol is, for example, 1-20% (w / v), 2.5-15% (w / v), 5-15% (w / v), or 7.5-12.5% ​​(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 compositions of this disclosure may, for example, contain antioxidants (reducing agents). The antioxidant is a substance capable of inhibiting oxidation reactions. By including the antioxidant, the compositions of this disclosure can further improve the cell cryoprotective effect. Examples of the antioxidants include glutathione, lipoic acids (alpha-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, tetrahexyldecyl ascorbate, and 3-O-ethyl ascorbic acid. Examples of the tocopherol derivatives include alpha-tocopheryl phosphate, tocopherol acetate, and tocopherol succinate. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA), citric acid, gluconic acid, sodium gluconate, and phytic acid. The antioxidant may be used alone or in combination of two or more types.

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

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

[0051] If the composition of the present disclosure contains the 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 one type of antioxidant, or the total concentration of two or more types of antioxidants. The concentration of 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 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 compositions of this disclosure may, for example, contain amino acids. By including the amino acids, the cell cryoprotective effect of the compositions of this disclosure can be further improved. Examples of the amino acids include α-amino acids, β-amino acids, γ-amino acids, etc. The amino acids are, for example, amino acids that make up proteins. The amino acids may also be sulfur-containing amino acids. One type of amino acid may be used alone, or two or more types may be used in combination. Examples of the amino acids include alanine, taurine, phenylalanine, methionine, proline, cysteine, serine, lysine, arginine, homoserine, aminobutyric acid, aminocaproic acid, tryptophan, and glycine.

[0053] The concentration of the amino acid 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 acid 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 compositions of this disclosure, each compound may be, for example, a salt. The salts of each compound are not particularly limited and include, 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 the 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 the basic amino acids include, for example, salts with arginine, lysine, ornithine, etc. The salts with the acidic amino acids include, for example, salts with aspartic acid, glutamic acid, etc. The salts include pharmaceutically acceptable salts. "Pharmaceutically acceptable" includes, for example, forms that have reasonable benefit for pharmaceutical use.

[0055] The compositions of the present disclosure may be, for example, liquids or solids. If the composition of the present disclosure is a liquid, it 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, intravenous fluids, and culture media, preferably buffer solutions and intravenous fluids. Examples of buffer solutions include Hanks equilibrium salt solution (HBSS) and phosphate buffer. Examples of intravenous fluids include isotonic electrolyte solutions such as Ringer's bicarbonate solution, physiological saline, Ringer's solution, Ringer's lactate solution, and Ringer's acetate solution; hypotonic electrolyte solutions such as Solution No. 1 (initial solution), Solution No. 2 (dehydration replacement solution), Solution No. 3 (maintenance solution), and Solution No. 4 (postoperative recovery solution); and amino acid solutions. For the culture media, for example, basal culture media can be used. Examples of the aforementioned basal media include MEM medium (Gibco Invitrogen, etc.), MEMα (Gibco Invitrogen, etc.), DMEM medium (Gibco Invitrogen, etc.), IMDM medium (Wako Pure Chemical, etc.), RPMI1640 medium (Gibco Invitrogen, etc.), Ham F-12 medium (Gibco, etc.), RD medium, or mixtures thereof.

[0057] The compositions of this disclosure, for example, have a cell viability 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 after a cell freeze-thaw test. The compositions of this disclosure, for example, have a cell viability of 60-100%, 65-99%, 70-99%, 75-98%, 80-97%, 85-96%, or 90-95% after a cell freeze-thaw test. 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 6Adding 0.5 mL of the target cell cryopreservation solution to adipocyte-derived stromal cells (AD-MSCs) of cells, the cells are suspended, and the resulting cell suspension is rapidly frozen in a deep freezer (-80°C). After freezing, the AD-MSCs are thawed by placing the tubes in a water bath at approximately 25°C on or after the day following the start of freezing. After thawing, the AD-MSCs are washed with HBSS(+), collected from the tubes, centrifuged, 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 in the cell counter is counted. The viability of the cells is then calculated by multiplying the total number of cells (N) after cell freezing. a Number of viable cells (N) in cell freezes relative to ) b ) proportion (N b / N a It can be calculated as ×100(%).

[0058] The compositions of this disclosure preferably substantially contain, for example, organic solvent cryoprotective agents. Examples of such organic solvent cryoprotective agents include dimethyl sulfoxide, propylene glycol, ethylene glycol, glycerol (glycerin), and ethanol. The compositions of this disclosure preferably substantially contain dimethyl sulfoxide, propylene glycol, ethylene glycol, glycerol (glycerin), and ethanol.

[0059] The compositions of this disclosure preferably substantially contain, for example, animal-derived components. Examples of such animal-derived components include proteins, lipids, peptides, hormones, amino acids, and vitamins. The compositions of this disclosure preferably substantially contain, for example, animal-derived proteins, lipids, peptides, hormones, amino acids, and / or vitamins, and more preferably, animal-derived proteins, lipids, peptides, hormones, amino acids, and vitamins.

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

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

[0062] In the compositions of this 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; in fat; in bone marrow or umbilical cord; etc. Mesenchymal stem cells are a type of pluripotent stem cell and refer to cells that have the ability to differentiate into adipocytes, osteocytes, chondrocytes, muscle cells, hepatocytes, tendinocytes, and / or nerve cells. Depending on the tissue from which they are collected, these mesenchymal stem cells are also called, for example, 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 aforementioned cells may also include, for example, skin cells, keratinocytes, skeletal muscle cells, cardiomyocytes, 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, etc.

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

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

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

[0066] The composition of the present disclosure can be suitably used for cryopreservation of the cells, for example, even without using an organic solvent-based cryoprotectant.

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

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

[0069] The freezing method of the present disclosure may, for example, include a step of bringing the composition of the present disclosure into contact with the cells prior to the freezing step. The contact can be prepared, 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 cell concentration in the obtained cryopreservation solution containing cells is, for example, 1×10 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 carried out, for example, by a normal freezing method used for freezing cells. The freezing may be, for example, rapid freezing or slow freezing. Furthermore, the freezing may be carried out, for example, by continuously cooling the cryopreservation solution or by discontinuous cooling. Specifically, the freezing may be carried out, for example, by 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 phase or liquid phase of liquid nitrogen and freezing, or by placing it in a freezer such as a proton freezer and freezing. 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 process, 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 this disclosure may further include, for example, a preservation step of storing the frozen cells in a frozen state. This preservation can be carried out, for example, by placing the cryopreservation solution containing the cells in a freezer; in the gas phase of liquid nitrogen; etc. The preservation temperature in the preservation step is, for example, -20 to 196°C, -40 to -150°C, or -70 to -90°C. The preservation 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.

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

[0073] The freezing method described herein can be performed, for example, in vitro or in vivo.

[0074] The freezing method of this disclosure allows for suitable cryopreservation of cells, for example, without the use of the aforementioned organic solvent cryoprotective agent.

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

[0076] The descriptions of the concentrations of each component in the compositions of this disclosure can be used, for example, to describe the concentrations of each component in the cell preparations of this disclosure.

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

[0078] The cell preparations of this disclosure can be obtained, for example, by the cell freezing method of this disclosure.

[0079] The cell preparations of this disclosure, for example, are substantially free of the cell cryoprotective agents of the organic solvent, and therefore the toxicity caused by the organic solvent can be suppressed. For this reason, the cell preparations of this disclosure can be suitably used, for example, as cell therapies.

[0080] <Treatment Method> In another embodiment, the Disclosure provides a treatment method in which the toxicity of the organic solvent cell cryoprotectant is reduced. The treatment method of the Disclosure includes an administration step of administering the cell preparation of the Disclosure to a subject.

[0081] In this 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 this disclosure is carried out, for example, in vitro or in vivo.

[0082] The cell preparations of this disclosure can protect cells during cell freezing, for example, without substantially containing the organic solvent cryoprotective agent. Therefore, the treatment method of this disclosure can reduce the amount of the organic solvent cryoprotective agent administered to the subject, thereby reducing the toxicity caused by the organic solvent cryoprotective agent. [Examples]

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

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

[0085] [Table 1]

[0086] [solvent] In each of the following examples, the solvent used for the preservation solution had the composition described in Tables 2 and 3 below.

[0087] [Table 2]

[0088] [Table 3]

[0089] [Cell preparation] The cells used in each of the following examples were prepared as described below.

[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(+), an equal volume of collagenase solution (Solaris Bio Co., Ltd.) was added to the adipose tissue, and the tissue was treated with collagenase at 37°C for 3.5 hours. Next, the resulting solution was centrifuged (800g x 5 minutes). The supernatant containing oil was discarded. The precipitate was washed with the obtained HBSS(+) and then centrifuged twice more. Subsequently, the precipitate was treated with RBC Lysis Solution (Solaris Bio Co., Ltd.) to lyse the red blood cells. Next, the lysed solution was centrifuged (800g x 5 minutes). The supernatant was then discarded. The resulting precipitate (tissue fragments) was suspended in culture medium (Solaris Bio Co., Ltd., M101-AF-500). The tissue fragments were then filtered through a cell strainer. The obtained filtrate was prepared as a cell suspension containing SVF (stromal vascular fraction), and the solvent was replaced with cell cryopreservation solution (Solaris Bio Co., Ltd., C101-AF-100). The suspension was then cryopreserved at -80°C. The cryopreserved SVF was washed with HBSS. The SVF was then suspended in culture medium. Next, the suspension was seeded in a T-25 flask (Sumitomo Bakelite Co., Ltd., MS-23050). The seeded cells were harvested and cryopreserved at -80°C. The cryopreserved cells were then cultured in culture medium using a T-150 flask (Sumitomo Bakelite Co., Ltd., MS-23600). After the culture, when the cells reached a subconfluent state, the AD-MSCs were harvested.

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

[0092] (3) Preparation of umbilical cord-derived mesenchymal cells (UC-MSCs) The UC-MSCs were recovered 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 recovered in the same manner as in (2) above, except that THP-1 cells (manufactured by ATCC, TIB-202) were used instead of the commercially available AD-MSCs, RPMI-1640 + 10% FBS (manufactured by Gibco, 72400047 and A31604-01) were used instead of the AD-MSC medium, and a T-25 non-adhesive flask (manufactured by Sumitomo Bakelite Co., Ltd., MS-2305R) was used instead of a T-150 flask.

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

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

[0096] (Freezing and preservation test) The in-house prepared AD-MSCs were seeded in T-150 flasks with culture medium and cultured for 3-4 days. After growing the AD-MSCs to subconfluence, a detaching agent (D101-AF-500, Solaris Bio Co., Ltd.) was added to the T-150 flasks and incubated at 37°C for 5 minutes. Next, the AD-MSCs were 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 (Gibco, 15250-061) was added to the cell suspension, and the number of viable cells was counted using a cell counter (Thermofisher Scientific, Countess3). Furthermore, the AD-MSCs were divided into 1 × 10⁶ cells. 5~2×10 6 The cells were dispensed into tubes. 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 (Table 4 below) was added to each tube to suspend the cells, and the resulting cell suspensions were rapidly frozen in a deep freezer (-80°C). DMSO is commonly used as a cell cryoprotective agent and is a positive control. HBSS+ is a negative control that does not contain a cell cryoprotective agent.

[0097] [Table 4]

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

[0099] [Table 5]

[0100] Table 5 shows the results of the survival rate of AD-MSCs after freeze-thawing. As shown in Table 5, the survival rate of the cells after freeze-thawing was significantly reduced in the negative control (HBSS+ only). In contrast, the survival rate of the cells was higher in the 10% sucrose and 10% DMSO groups compared to HBSS+ only. In other words, it was found that the survival rate of cells during cell freezing can be improved by using sucrose, meaning that sucrose acts as a cell cryoprotective agent.

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

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

[0103] [Table 6]

[0104] [Table 7]

[0105] Table 7 shows the results of the cell viability after freeze-thawing of AD-MSCs. As shown in Table 7, the cell viability after freeze-thawing improved at all sucrose concentrations. In particular, a sucrose concentration of 5% or higher resulted in a viability of approximately 70%, which is a high viability rate. Generally, a cell cryopreservation solution is considered excellent if the viability after freeze-thawing exceeds 70%. Therefore, it was found that increasing the sucrose concentration to 5% or higher can significantly improve the cell viability after freeze-thawing.

[0106] [Example 3] We confirmed that the cell survival rate during cryopreservation can be improved by using a cell cryopreservation solution containing antioxidants (reducing agents).

[0107] Except for using the cell cryopreservation solutions listed in Tables 8-10 below instead of the cell cryopreservation solution listed in Table 4 above, the survival rate of AD-MSCs after freezing and thawing was calculated in the same manner as in Example 1. In Table 12 below, the survival rate (N) of samples without glutathione addition (Sample 8) is shown. c Based on this, the survival rate (N) at each glutathione addition concentration (samples 9-14) was determined. d Regarding the relative survival rate (N d / N c The number of viable cells (N) was calculated as ×100(%). Furthermore, in Table 13 below, the number of viable cells (N) was calculated for the sample without alpha-lipoic acid (Sample 15). E ) and the number of viable cells (N) at the alpha-lipoic acid addition concentration (Sample 16). f Based on this, the relative number of viable cells (N f / N E The percentage (×100(%)) was calculated. These results are shown in Tables 11-13 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 the survival rate of AD-MSCs. Table 12 shows the results for the specific survival rate of AD-MSCs. Table 13 shows the results for the specific number of viable cells of AD-MSCs. As shown in Table 11, the survival rate of cells after freeze-thawing improved with all antioxidants. In particular, a glutathione concentration of 32.5% resulted in a survival rate of approximately 86%, which is a high survival rate. In other words, it was found that using glutathione as an antioxidant can further improve the survival rate after thawing. Also, as shown in Table 12, the specific survival rate of cells after freeze-thawing increased with the addition of glutathione compared to the case without glutathione addition. In other words, it was found that the survival rate of cells during cell freezing can be improved by adding glutathione to a concentration of 1 μM or higher. Furthermore, as shown in Table 13, the specific number of cells after freeze-thawing increased when glutathione and alpha-lipoic acid (lipoic acid) were added as antioxidants compared to glutathione alone. In other words, it was found that using glutathione and alpha-lipoic acid as antioxidants can improve the viability of cells after freezing and thawing, and that glutathione and alpha-lipoic acid act as antioxidants in cell cryoprotection agents.

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

[0116] Except for using the cell cryopreservation solutions shown in Tables 14-16 below instead of the cell cryopreservation solutions shown in Table 4 above, the survival rate of AD-MSCs after freezing and thawing was calculated in the same manner as in Example 1. These results are shown in Tables 17-19 below.

[0117] [Table 14]

[0118] [Table 15]

[0119] [Table 16]

[0120] [Table 17]

[0121] [Table 18]

[0122] [Table 19]

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

[0124] [Example 5] We confirmed that using a solvent other than HBSS in the cell cryopreservation solution can improve the cell viability during cryopreservation.

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

[0126] [Table 20]

[0127] [Table 21]

[0128] [Table 22]

[0129] Tables 21 and 22 above show the results of the survival rate of AD-MSCs after freeze-thawing. As shown in Tables 21 and 22 above, the survival rate of cells after freeze-thawing 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 not only with HBSS+ but also with other solvents in cell cryopreservation solutions containing sucrose and glutathione.

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

[0131] Except for using the cell cryopreservation solution shown in Table 23 below instead of the cell cryopreservation solution shown in Table 4 above, and using the freezing method shown in Table 21 below instead of rapid freezing in a deep freezer (-80°C) as the freezing method, the survival rate of AD-MSCs after freezing and thawing was calculated in the same manner as in Example 1. These 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 cell survival rate after freezing and thawing improved regardless of the freezing method. In other words, it was found that the cell survival rate 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 the viability of cells during cryopreservation.

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

[0137] [Table 25]

[0138] (Culture test) Next, for samples 5-8, the number of viable cells was counted after freezing and thawing the cells and then culturing them further. After freezing and thawing the cells, they were washed. Next, the cell suspension, suspended in the culture medium, was seeded onto a 6-well plate (Sumitomo Bakelite, MS-80060) to which 2 mL of culture medium had been added beforehand. The density of the AD-MSCs was 5 × 10⁻⁶. 3 ~7.5×10 3 cells / cm 2The mixture was prepared as described above and cultured for 3 days. After that, the number of viable cells of the AD-MSCs was counted. To count the number of cells, first, 500 μL of a release agent was added to the 6-well plate and incubated at 37°C for 5 minutes. Next, the mixture containing the release agent was thoroughly stirred and trypan blue was added. Furthermore, a portion of the mixture was added to a slide and the number of viable cells of the AD-MSCs cultured for 3 days was counted using a cell counter. The number of viable cells (Ni) without amino acid addition (Sample 4) and the number of viable cells (N) at each alanine addition concentration (Samples 5-7) were then recorded. j Based on this, the relative survival rate (N j / N i The result was calculated as ×100(%). These 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 freeze-thawing. Table 27 shows the relative number of viable AD-MSCs when they were further cultured after freeze-thawing. As shown in Table 26, the survival rate of cells after freeze-thawing improved by adding alanine and taurine. Furthermore, as shown in Table 27, the survival rate of cells after freeze-thawing improved in a concentration-dependent manner by increasing the alanine concentration to 0.05 mM or higher. In other words, it was found that the survival rate of cells after freeze-thawing can be improved by adding amino acids to a cell cryopreservation solution containing sucrose.

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

[0143] Except for using the cell cryopreservation solution shown in Table 4 as the cell cryopreservation solution, the cell cryopreservation solutions shown in Table 28 below were used, and the cells used were replaced with adipocyte-derived stromal cells (commercially available AD-MSCs), umbilical cord-derived mesenchymal cells (UC-MSCs), THP-1 cells, and Jurkat cells instead of in-house prepared AD-MSCs, the survival rate of AD-MSCs after freezing and thawing was calculated in the same manner as in Example 1. These results are shown in Table 29 below.

[0144] [Table 28]

[0145] [Table 29]

[0146] Table 29 shows the results of freeze-thaw survival rates of AD-MSCs in various cell tumors. As shown in Table 29, cell survival rates improved in all cell tumors. In other words, it was found that cell cryopreservation solutions containing sucrose and glutathione can improve cell survival rates after freeze-thawing in various cell tumors.

[0147] While 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 to the structure and details of the present disclosure are possible, as can be understood by those skilled in the art within the scope of the present disclosure.

[0148] The patents, patent applications, and documents cited herein are incorporated herein by reference in the same manner as their contents are specifically described herein.

[0149] <Note> Some or all of the above embodiments and examples may be described as follows, but are not limited to the following. <Composition for cryopreservation of cells> (Note 1) A composition for cryopreserving cells, comprising a sugar or its derivative and / or a surfactant. (Note 2) The cell composition according to Appendix 1, wherein the sugar is a monosaccharide, disaccharide, trisaccharide, and / or polysaccharide. (Note 3) The composition described in Appendix 2, wherein the disaccharide is selected from the group consisting of sucrose, maltose, sucralose, lactose, cerbiose, lactobionic acid, lactulose, melibiose, and isomaltose. (Note 4) The composition according to Appendix 2 or 3, wherein the disaccharide includes sucrose. (Note 5) The composition according to any one of the appendices 2 to 4, wherein the monosaccharide is selected from the group consisting of glucose, galactose, mannose, fructose, xylose, ribose, and arabinose. (Note 6) The composition according to any one of the appendices 2 to 5, wherein the trisaccharide is selected from the group consisting of raffinose, maltotriose, isomalttriose, panose, and melegitose. (Note 7) The polysaccharide is selected from the group consisting of dextran, starch, hydroxyethyl starch, hyaluronic acid, glycogen, and alginic acid, as described in any of the descriptions in Appendix 2 to 6. (Note 8) The composition according to any one of the appendices 1 to 7, wherein the sugar is a non-reducing sugar and / or a reducing sugar. (Note 9) The sugar derivative is selected from the group consisting of nucleosides, nucleotides, and sugar alcohols, and is a composition according to any one of the appendices 1 to 8. (Note 10) The composition according to Appendix 9, wherein the nucleoside comprises uridine and / or deoxyuridine. (Note 11) The composition according to Appendix 9 or 10, wherein the sugar alcohol is selected from the group consisting of sorbitol and xylitol. (Note 12) The surfactant is a composition according to any one of the appendices 1 to 11, comprising polyvinylpyrrolidone (PVP) and / or modified polyalkylene glycol. (Note 13) The modified polyalkylene glycol is the composition described in Appendix 12, comprising Soluplus. (Note 14) Containing the aforementioned sugar, The composition according to any one of the appendices 1 to 13, wherein the concentration of the sugar is 5-25% (w / v). (Note 15) The aforementioned sugars are monosaccharides, disaccharides, and / or trisaccharides. The composition according to any one of the appendices 1 to 14, wherein the concentration of the sugar is 5-25% (w / v). (Note 16) The aforementioned sugar derivative is a sugar alcohol, The composition according to any one of the appendices 1 to 15, wherein the concentration of the sugar alcohol is 1 to 20% (w / v). (Note 17) The composition according to any one of the appendices 1 to 16, wherein the concentration of the surfactant is 1 to 20% (w / v). (Note 18) Furthermore, a composition according to any one of the appendices 1 to 17, comprising an antioxidant. (Note 19) The composition according to Appendix 18, wherein the antioxidant is selected from the group consisting of glutathione, lipoic acid (alpha-lipoic acid, thioctic acid), ascorbic acid (vitamin C) or its derivatives, tocopherol (vitamin E) or its derivatives, acetylcysteine, and chelating agents. (Note 20) The composition described in Appendix 19, wherein the ascorbic acid derivative is selected from the group consisting of ascorbic acid 2-glucoside, ascorbic acid 2-phosphate, tetrahexyldecanoate ascorbyl, and 3-O-ethylascorbic acid. (Note 21) The composition according to Appendix 19 or 20, wherein the tocopherol derivative is selected from the group consisting of α-tocopheryl phosphate, tocopherol acetate, and tocopherol succinate. (Note 22) The antioxidant is a composition according to any one of the appendices 19 to 21, comprising lipoic acid and glutathione. (Note 23) The composition according to any one of the appendices 18 to 22, wherein the concentration of the antioxidant is 1 nmol / L to 100 mmol / L. (Note 24) Furthermore, a composition containing an amino acid, as described in any of the appendices 1 to 23. (Note 25) The composition described in Appendix 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. (Note 26) The composition according to Appendix 24 or 25, wherein the concentration of the amino acid is 0.01 to 100 mmol / L. (Note 27) Furthermore, the composition according to any one of the appendices 1 to 26, comprising an aqueous solvent. (Note 28) The aqueous solvent is selected from the group consisting of water, buffer solutions, infusion solutions, and culture media, as described in Appendix 27. (Note 29) The buffer solution is selected from the group consisting of Hanks equilibrium salt solution (HBSS), phosphate buffer, Dulbecco's phosphate-buffered saline, Hepes buffer solution, and amino acid buffer, as described in Appendix 28. (Note 30) A composition according to any one of the appendices 1 to 29, wherein the cell viability after a cell freeze-thaw test is 60% or more. (Note 31) A composition according to any one of the appendices 1 to 30, which is substantially free of organic solvent cell cryoprotective agents. (Note 32) The cell cryoprotective agent of the organic solvent is selected from the group consisting of dimethyl sulfoxide, propylene glycol, ethylene glycol, glycerol, and ethanol, as described in Appendix 31. (Note 33) A composition according to any one of the appendices 1 to 32, which is substantially free of animal-derived ingredients. (Note 34) The composition according to Appendix 33, wherein the animal-derived component is a protein, lipid, peptide, hormone, amino acid, and / or vitamin obtained from an animal raw material. (Note 35) A composition according to any one of the appendices 1 to 34, which is substantially free of serum and / or plasma. (Note 36) A composition that is chemically defined, as described in any of the appendices 1 to 35. <Methods for freezing cells> (Note 37) A method for freezing cells, comprising a freezing step of freezing the cells in the presence of any of the compositions described in Appendix 1 to 36. (Note 38) The freezing method according to Appendix 37, comprising a preservation step of storing the frozen cells in a frozen state. (Note 39) The freezing method according to Appendix 37 or 38, wherein the cells are primary cultured cells and / or cells isolated from living organisms, cultured in vitro. (Note 40) The freezing method according to any one of the appendices 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, osteocytes, chondrocytes, and insulin-producing cells. (Note 41) A freezing method described in any one of the appendices 37 to 40, performed in vitro or in vivo. <Cell preparation> (Note 42) A cell preparation comprising cells and a composition described in any of the appendices 1 to 36. (Note 43) A cell preparation as described in Appendix 42, which is frozen or solid. (Note 44) The cell preparation described in Appendix 42 or 43 is 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, osteocytes, chondrocytes, and insulin-producing cells. (Note 45) A cell preparation according to any of the appendices 42 to 44, obtained by any of the freezing methods described in appendices 37 to 41. <Treatment Method> (Note 46) A treatment method comprising an administration step of administering a cell preparation described in any of appendices 42 to 45 to the subject. (Note 47) The treatment method described in Appendix 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 explained above, this disclosure makes it possible to protect cells during freezing without substantially adding organic solvent cryoprotective agents. For this reason, this disclosure is extremely useful in fields such as cell therapy and regenerative medicine.

Claims

1. A composition for cryopreserving cells, comprising a sugar or a derivative thereof.

2. The cell composition according to claim 1, wherein the sugar is a monosaccharide, disaccharide, trisaccharide, and / or polysaccharide.

3. The composition according to claim 2, wherein the disaccharide is selected from the group consisting of sucrose, maltose, sucralose, lactose, cerbiose, lactobionic acid, lactulose, melibiose, and isomaltose.

4. The composition according to claim 2 or 3, wherein the disaccharide comprises sucrose.

5. Furthermore, the composition according to claim 1 or 2, comprising an antioxidant.

6. The composition according to claim 5, wherein the antioxidant comprises an antioxidant selected from the group consisting of glutathione, lipoic acid, ascorbic acid or its derivatives, tocopherol or its derivatives, acetylcysteine, and chelating agents.

7. The composition according to claim 5, wherein the antioxidant comprises lipoic acid and glutathione.

8. The composition according to claim 5, wherein the concentration of the antioxidant is 1 nmol / L to 100 mmol / L.

9. Furthermore, the composition according to claim 1 or 2, comprising an aqueous solvent.

10. The composition according to claim 9, wherein the aqueous solvent is selected from the group consisting of water, buffer, infusion solution, and culture medium.

11. The composition according to claim 1 or 2, wherein the cell viability after a cell freeze-thaw test is 60% or more.

12. The composition according to claim 1 or 2, which is substantially free of organic solvent cell cryoprotective agents.

13. The composition according to claim 12, wherein the organic solvent cell cryoprotectant is selected from the group consisting of dimethyl sulfoxide, propylene glycol, ethylene glycol, glycerol, and ethanol.

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

15. A cell preparation comprising cells and the composition according to claim 1 or 2.

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